THE MYCOTA 9

A Comprehensive Treatise on Fungi as Experimental Systems

for Basic and Applied Research
Series Editors: Dee Carter · Anuradha Chowdhary
Joseph Heitman · Ulrich Kück

Yen-Ping Hsueh
Meredith Blackwell Editors

Fungal
Associations
Third Edition
The Mycota

A Comprehensive Treatise on Fungi as

Experimental Systems for Basic and Applied
Research

Volume 9

Series Editors
Dee Carter, School of Life and Environmental Sciences, The University of
Sydney, Sydney, NSW, Australia
Anuradha Chowdhary, Department of Medical Mycology, VP Chest Institute,
University of Delhi, Delhi, India
Joseph Heitman, Duke University Medical Center, Durham, NC, USA
Ulrich Kück, General and Molecular Botany, Ruhr-University Bochum,
Bochum, Germany
The fungi represent a heterogenous assemblage of eukaryotic microorganisms
and have become favored organisms for research at the cellular and molecular
level. Such research involvement has been stimulated by interest in the
biotechnological application of fungi in processes related to industry, agricul-
ture and ecology. Considering both yeasts and mycelial fungi, THE
MYCOTA highlights developments in both basic and applied research and
presents an overview of fungal systematics and cell structure. Foremost
authorities in research on mycology have been assembled to edit and contrib-
ute to the volumes.
Yen-Ping Hsueh • Meredith Blackwell
Editors

Fungal Associations

Third Edition
Editors
Yen-Ping Hsueh Meredith Blackwell
Institute of Molecular Biology, Department of Biological Sciences
Academia Sinica Louisiana State University
Taipei, Taiwan Baton Rouge, LA, USA
Department of Biological Sciences
University of South Carolina
Columbia, SC, USA

ISSN 2945-8048 ISSN 2945-8056 (electronic)

The Mycota
ISBN 978-3-031-41647-7 ISBN 978-3-031-41648-4 (eBook)
https://doi.org/10.1007/978-3-031-41648-4

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Series Preface

The Mycota is an encyclopedic book series published by Springer as a

comprehensive treatise spanning the Fungal Kingdom through a focus on
fungi as experimental systems for basic and applied research. Articles in this
series present introductory information in a comprehensive manner to broaden
our general knowledge, along with state-of-the-art information on selected
topics in fungal biology. Thus, these articles serve as a timeless reference
source for historically relevant discoveries in fungal biology to drive further
advances in the field.
The Mycota was founded in 1994 by Karl Esser and Paul Lemke. Since
then 15 volumes have been published, with several now in their third edition.
The steadily growing interest in The Mycota, reflected in particular by the high
number of e-book downloads, encouraged Springer to continue this publica-
tion with a new international Series Editorial Board beginning in 2022.
Historically, the study of fungi originated as a sub-discipline of botany and
was a largely descriptive discipline until the early nineteenth century.
Subsequent experimental research has propelled the field of mycology, and
achievements in the genetics and molecular biology of fungi have benefited
studies in the related fields of fungal biochemistry and biophysics, plant
pathology, medical mycology, and systematics. Ground-breaking research
has been carried out using fungi as recognized by the receipt of nine Nobel
Prizes, including the revolutionary 1945 Nobel Prize in Physiology or Medi-
cine for the discovery of penicillin and the foundational 2006 Nobel Prize in
Chemistry for establishing the molecular basis of eukaryotic transcription. All
such studies expand our knowledge of fungi and of fungal processes and
improve our ability to understand, utilize, and control fungi for the benefit of
humankind.
Fungi have invaded every conceivable ecological niche. Saprobic forms
abound, especially in the decay of organic debris. Pathogenic forms exist with
both plant and animal hosts. Fungi even grow on other fungi. They are found in
aquatic as well as soil environments, and their spores have been found in the air
of extreme environments. Fungi can be variously associated with plants as
symbionts in the form of lichens, mycorrhizae, and endophytes and also
occur as overt pathogens. Association with animal systems varies; examples
include the predaceous fungi that trap nematodes, the micro-fungi that grow in
the anaerobic environment of the rumen, and medically important pathogens
afflicting humans. Currently, taxonomists have identified about 150,000

v
vi Series Preface

species, but it is thought that over 90% of fungal species remain undescribed,
with conservative estimates ranging from 2 to 5 million fungal species on earth.
From this perspective, there are new topics in fungal biology to explore,
which will expand the current volumes and take them in new directions.
Further, there will be new volumes in areas (e.g., Cryptomycota) that were
not covered before.
Our understanding of the evolution of fungi is still incomplete and mainly
based on species that can be grown in culture. But recent environmental DNA
analyses have revealed a highly diverse form of eukaryotic life that branches
with the Fungi, and thus the resulting and highly diverse clades were named
the cyptomycota. The discovery of novel intermediate forms will redefine the
fungal tree of life. Other topics will consider the adaptation of fungi to climate
changes, the occurrence of fungal pathogens in the environment, and the
dispersal of fungi during global pandemics.
Fungal constituents of the microbiome have received much less attention
thus far, yet recent findings from clinical and animal studies clearly establish
fungi as a significant component of the oral, gastrointestinal, pulmonary, and
skin microbiomes. Finally, the relevance of fungal biology to society is
reflected by the increasing number of fungal related human diseases.
In the history of pharmacology, fungi have always been sources of useful
molecules for humans, but the diversity of molecules that can be obtained
from fungi is still largely under explored. However, efforts in fungal genomics
provide resources for data-driven genome mining and large-scale
comparisons to explore the molecular repertoires produced by fungi. The
result will be new compounds with applications in the pharmaceutical and
agricultural science industries.
Fungi can also help the entire planet, and may for example be relevant in
specific sectors, such as that linked to pollution from plastic. Fungi produce a
wide range of enzymes that have the potential to break down the chemical
bonds of plastic polymers, and in this context, the potential role of marine
fungi in plastic degradation may be of major relevance. Finally, new
biomaterials from fungal species may open the door to alternatives to fossil-
based materials, and thus reduce environmental pollution.
For consistency throughout this series of volumes, the names adopted for
major groups of fungi should be followed according to the following paper,
which gives an overview of all of the orders in the fungal kingdom: https://
pubmed.ncbi.nlm.nih.gov/32660385/
We are grateful to Springer for continuing The Mycota series and are
especially thankful to all the volume editors in selecting topics and assembling
experts from diverse fields of fungal biology.

Sydney, Australia Dee Carter

Delhi, India Anuradha Chowdhary
Durham, USA Joseph Heitman
Bochum, Germany Ulrich Kück
July 15th, 2022
Volume Preface to the Third Edition

Fungi are heterotrophic eukaryotes with chitinous cell walls; some species are
single-celled flagellates, yeasts, or yeast-like cells, and many species have
complex structures composed of filaments known as hyphae. Except for
spore-bearing mushrooms and other large reproductive structures, mycology’s
charismatic megamycota (Blackwell 2011), most fungi go unseen except for
their traces of deterioration and disease. Other characteristics of fungi include
osmotropic nutrition whereby enzymes exported to the substrate dissolve
organic nutrients that then are absorbed into the organism. Fungi also produce
vitamins, sterols, and aromas, often important in their interactions and com-
munication with other organisms. Fungi are an extraordinarily diverse group,
in part because of myriad associations (Vega and Biedermann 2019) with
almost all kinds of organisms including other fungi.
About a billion years ago populations of ancestral eukaryotic single-celled
organisms diverged into fungi and animals, two dominant megadiverse
groups we recognize today. Together with plants, fungi and animals constitute
the “fauna, flora, and funga,” a triumvirate of the most prominent organisms
on Earth (Pfister 2020; Berbee and Blackwell 2023). Fungal associations
range widely from simple co-occurrence, perhaps the result of only accidental
spore dispersal, to complex multipartite symbioses in the broad sense, includ-
ing mutualistic, commensalistic, and parasitic lifestyles.
This volume looks at the results of a billion years of fungal associations
revealed by current methods:
• Many animals and plants are protected from fungi by preadapted and
evolved defenses.
• Some fungi have been able to have such intimate contact with other
organisms that their DNA has been exchanged.
• The dogma that obligately symbiotic fungi cannot be grown in axenic
culture is being investigated with the help of genomes.
• Aromatic volatile compounds lure or repel other organisms in what would
seem to be a confusing mélange of bacterial, animal, and plant compounds
in the environment.
• Examples of mycoviruses and their influence on fungal phenotypes are
remarkable; additional associations and their effects await discovery.
[Meredith Blackwell was especially interested to see a reference to virus-

vii
viii Volume Preface to the Third Edition

infected chestnut trees in Michigan, perhaps those discovered by an under-

graduate at Hope College, Holland, Michigan, where she once taught.]
• Classic textbook examples of fungal associations have been completely
updated by the application of traditional and new molecular techniques.
These chapters cover lichens, including a model lichen, arbuscular mycorrhi-
zal fungi, ectomycorrizal fungi, and an endophyte rendered much less toxic.
• Also included are fungal predators of nematodes, fungi that control the
minds of insects, and even more fungal killers of insects.
• The volume closes with a pharmacopeia of secondary metabolites
synthesized by sclerotium-producing Xylarias in termite galleries—
metabolites with pharmaceutical potential.
Development of new technologies precedes advancements in our science.
Evidence of progress brought about by the polymerase chain reaction (PCR),
especially applied to fungi (White et al. 1990), and all the resulting
improvements in molecular biology are evident in the research discussed in
this volume. As we completed editing these chapters, artificial intelligence
(AI) crept up and suddenly burst forth into the scientific literature and mass
media, even into the hearing rooms and statutes of governemental bodies. The
study of fungal associations and related fields is poised to adopt the use of AI
through today’s increased access to supercomputers, ever-expanding
databases (including the proliferation of scientific literature), and a need to
recognize complex patterns; perhaps AI even engenders a willingness to have
complicated decisions made for us and eases the demands to undertake
repetitive tasks. We anticipate that as successive technologies are developed
and improved, future studies of fungi and the organisms with which they
associate will continue to reap benefits.
We appreciate the help of our colleagues Donald H. Pfister, Joseph
W. Spatafora, and Marc Rapport for editorial assistance. We are especially
grateful to our contacts at Springer Nature, Sabine Schwarz, Executive Editor,
Life Sciences & Biomedicine Books, and Bibhuti Bhusan Sharma, Project
Coordinator (Books), for their unlimited support from beginning to end during
the preparation of Mycota 9: Fungal Associations. Without them two
neophytes would not have managed. Our helpful and patient families are of
course gratefully acknowledged.

Columbia, SC Meredith Blackwell

Taipei, Taiwan Yen-Ping Hsueh
11 June 2023

Literature
Blackwell M (2011) The Fungi: 1, 2, 3, . . . 5.1 million species? Biodiversity
special issue. Am J Bot 98:426–438. doi: 10.3732/ajb.1000298
Berbee M, Blackwell M (2023) The origin of fungi: A billion years of
functional and morphological diversification. In: Harper D, Seberg O
(eds) The origins of all things. Munksgaard, Copenhagen, pp 231–277
Volume Preface to the Third Edition ix

Pfister DR (2020) Fantastic Funga: why language matters. Deep Funga Blog.
https://fundis.org/resources/blog/127-fantastic-funga-why-language-
matters
Vega FE, Biedermann PH (2020) On interactions, associations, mycetangia,
mutualists and symbioses in insect-fungus symbioses. Fungal Ecol 44:
100909. doi:10.1016/j.funeco.2019.100909
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis
MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to
methods and applications. Academic, New York
Contents

1 When Plants and Animals First Met Fungi: Insights

from the Evolution of Host Immune Systems . . . . . . . . . . . . . 1
Emily D. Trudeau and Mary L. Berbee
2 Metabolic Constraints and Dependencies Between
“Uncultivable” Fungi and Their Hosts . . . . . . . . . . . . . . . . . . 33
Saleh Rahimlou, C. Alisha Quandt, and Timothy Y. James
3 Horizontal Gene Transfer in Fungi and Its Ecological
Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Carla Gonçalves, Chris Todd Hittinger, and Antonis Rokas
4 An Overview of Fungal Volatile Organic
Compounds (VOCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Samantha Lee, Richard Hung, and Joan W. Bennett
5 Viruses that Affect Phenotype and Fitness of Fungi . . . . . . . . 113
Bradley I. Hillman and Massimo Turina
6 Lichens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Martin Grube
7 Umbilicaria muhlenbergii: A Model for Studying
Molecular Mechanisms Regulating Initial Fungal
Symbiotic Interactions with Algal Cells . . . . . . . . . . . . . . . . . 181
Yanyan Wang, Xinli Wei, Yuting Hu, and Jin-Rong Xu
8 After Air, Light, and Water, the Next Most Important
Thing Is Grass: An Introduction to the Epichloë–Grass
Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Daniel A. Bastías, Emma R. Applegate, Pedro E. Gundel,
Linda J. Johnson, Wade J. Mace, Christina D. Moon,
Sushma Prakash, and Stuart D. Card
9 Signals and Host Cell Remodeling in Arbuscular
Mycorrhizal Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Andrea Genre, Serena Capitanio, and Paola Bonfante

xi
xii Contents

10 Masters of Manipulation: How Our Molecular

Understanding of Model Symbiotic Fungi and
Their Hosts Is Changing the Face of “Mutualism” . . . . . . . . . 249
Jonathan M. Plett, Annegret Kohler, and Francis Martin
11 Nematode-Trapping Fungi and Caenorhabditis elegans
as a Model System for Predator–Prey Interactions . . . . . . . . . 273
Guillermo Vidal-Diez de Ulzurrun, Sheng-Chian Juan,
Tzu-Hsiang Lin, and Yen-Ping Hsueh
12 When a Mind Is Not Its Own: Mechanisms of Host
Behavior Control by Parasitic Fungi . . . . . . . . . . . . . . . . . . . 293
Carolyn Elya
13 Genetics and Infection Biology of the
Entomopathogenic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Song Hong, Junmei Shang, Yanlei Sun, and Chengshu Wang
14 Xylaria Sclerotia Formed Within Termite Nests:
A Review of Their Biology and Human Uses . . . . . . . . . . . . . 333
Huei-Mei Hsieh and Yu-Ming Ju
Editors and Contributors

About the Series Editors

Dee Carter is Professor of Microbiology at the

University of Sydney. She has a degree in micro-
biology and biochemistry from Otago University,
New Zealand, and a PhD from Imperial College,
London, UK. After postdoctoral fellowships in
Montpellier, France, and UC Berkeley,
California, USA, she moved to Australia in
1995 to take up a lectureship at the University
of Sydney. Dee’s research has encompassed
(1) the population genetics and ecology of medi-
cally important fungi, including yeast and mold
pathogens; (2) responses of fungi to host and
antifungal stress, including the production of var-
iant morphological forms; and (3) the
transcriptome and proteome response to antifun-
gal therapy, particularly during synergistic
interactions between antifungals and natural
products. As well as research, Dee teaches micro-
biology including mycology, epidemiology, and
molecular biology at Sydney University.

Anuradha Chowdhary is Professor of Medical

Mycology at the Vallabhbhai Patel Chest Insti-
tute, University of Delhi, India. She received her
medical degree in 1992 and MD (Microbiology)
degree in 1996 from the University of Delhi,
India, and PhD from the Faculty of Medical
Sciences, Radboud University Medical Center,
Netherlands. Dr Chowdhary’s research interests
include molecular ecology and population genet-
ics of pathogenic fungi, antifungal drug resistance

xiii
xiv Editors and Contributors

mechanisms, especially Aspergillus and Candida

spp., and the epidemiology of systemic mycoses.
She is currently working on molecular epidemi-
ology of Candida auris, terbinafine-resistant
dermatophytes, and azole-resistant Aspergillus
fumigatus.

Joseph Heitman is James B. Duke Professor

and Chair, Department of Molecular Genetics and
Microbiology, Duke University, Durham, NC,
USA. His research studies model and pathogenic
fungi addressing fundamental questions of scien-
tific and medical importance. Pioneering studies
with Baker’s yeast revealed how immunosup-
pressive natural products interdict signaling
cascades via FKBP12-drug complexes, and dis-
covered TOR as a globally conserved nutrient
sensor targeted by the immunosuppressive,
antiproliferative drug rapamycin. His research
discovered unisexual reproduction of pathogenic
microbes, with implications for pathogen emer-
gence, how sex generates diversity, and how sex
evolved. Dr Heitman’s lab has further developed
genetic and genomic approaches elucidating
molecular principles of fungal virulence,
identifying therapeutic targets, and illustrating
convergent evolution of fungal mating-type loci
with mammalian, insect, and plant sex
chromosomes, defined the calcium-activated pro-
tein phosphatase calcineurin as a globally
conserved fungal virulence factor, and elucidated
functions of RNAi in microbial pathogen genome
integrity, hypervirulent outbreak lineages, and
drug resistance via epimutation.

Ulrich Kück is Professor of General and

Molecular Botany at the Ruhr-University in
Bochum, Germany. He graduated in Biology
and Chemistry, and has a long-standing experi-
ence in the molecular biology of fungi, algae, and
plants. His research with fungi has focused on
two general aspects: first, genetic engineering of
the secondary metabolism of biotechnically rele-
vant filamentous fungi, including functional
Editors and Contributors xv

genomics and proteomics, and second, molecular

genetic analysis of cellular growth in filamentous
fungus, with a focus on sexual development. In
particular, he is interested in the function of
mating-type loci, and the involvement of
conserved signaling complexes, such as the
striatin-interacting phosphatases and kinases
(STRIPAK) complex, in the control of cellular
and developmental processes.

About the Volume Editors

Yen-Ping Hsueh was born in Taipei, Taiwan.

Biology has always been her favorite subject in
school so she followed her interest and majored in
Plant Pathology and Microbiology at the National
Taiwan University as an undergraduate. She
received BSc (2001) and MSc (2003) degrees,
and moved to Duke University to pursue her
PhD under the guidance of Dr Joseph Heitman.
Her PhD work mainly focused on the sexual cycle
of the human fungal pathogen Cryptococcus
neoformans. After receiving her PhD in Molecu-
lar Genetics and Microbiology from Duke Uni-
versity in 2008, she continued to work with
Heitman briefly and then moved to Caltech to
start her postdoctoral training with Dr Paul
W. Sternberg in 2010. Her postdoctoral work
was an investigation of the interactions between
nematode-trapping fungi and the model nematode
C. elegans.
In 2015, Hsueh returned to Taiwan, to begin
her independent career at the Institute of Molecu-
lar Biology in Academia Sinica where she is
currently Associate Research Fellow. Her labora-
tory studies the predator–prey interactions
between carnivorous fungi and the model nema-
tode Caenorhabditis elegans. Members of the
Hsueh lab explore different aspects of the biology
of these predator–prey systems and are striving to
understand the mechanisms that underlie their
interactions and coevolution. They hope to har-
ness the nematocidal potential of the carnivorous
xvi Editors and Contributors

fungi, which will facilitate the discovery of a

novel treatment or biocontrol method to combat
parasitic nematode infections in plants, animals,
and humans in the future. Because of her original
research, Hsueh has been recognized as an
EMBO Global Investigator, EMBO Young
Investigator, and more recently, a Taiwan Out-
standing Women in Science Young Talent Award
and is an active participant in the international
scientific community.

Meredith Blackwell born in Abbeville,

Louisiana, spent her early years on the banks of
the Vermilion Bayou and attended public schools
in Louisiana. As an undergraduate pursuing lib-
eral arts interests, a deep-sea fishing trip led her to
become a biology major (BS, University of
Louisiana, 1961). She studied ichthyology at the
University of Alabama (MS, 1963) and began a
PhD at the University of Texas at Austin in 1964.
At Texas, she eventually came under the guidance
of C. J. Alexopoulos, and her enduring interest in
Mycology developed (PhD, 1973). During post-
doctoral research with Henry Aldrich and James
Kimbrough at the University of Florida, she was
introduced to insect-associated fungi. She spent
the first 6 years of her career primarily in a teach-
ing position at Hope College, Holland, Michigan.
The next 33 years (1981–2014) were spent in
the Department of Biological Sciences, Louisiana
State University, where she attained the rank of
Boyd Professor. Her research supported by NSF
included studies of desert myxomycetes, phylog-
eny of Laboulbeniomycetes and other insect
ectoparasites, and the taxonomic and geographic
distribution of insect-associated yeasts. Since
2014, she has lived in Columbia, South Carolina,
where she is an affiliate in the Department of
Biological Sciences at the University of South
Carolina. Blackwell is a former president of the
Mycological Society of America (MSA) and the
International Mycological Association (IMA).
She is a fellow of the British Mycological Asso-
ciation, MSA, IMA, and the American Academy
of Arts and Sciences. She has received several
mycological awards and is proud to share her
name with a number of fungal taxa.
Editors and Contributors xvii

Contributors

Emma Applegate Resilient Agriculture, AgResearch Limited, Grasslands

Research Centre, Palmerston North, New Zealand
Daniel Bastias Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Joan W. Bennett Department of Plant Biology, Rutgers, The State Univer-
sity of New Jersey, New Brunswick, NJ, USA
Mary Berbee Department of Botany, University of British Columbia,
Vancouver, BC, Canada
Paola Bonfante Dipartimento di Scienze della Vita e Biologia dei Sistemi,
Università di Torino, Torino, Italy
Stuart Card Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Serena Capitanio Dipartimento di Scienze della Vita e Biologia dei Sistemi,
Università di Torino, Torino, Italy
Carolyn Elya Department of Organismic and Evolutionary Biology,
Harvard University, Cambridge, MA, USA
Andrea Genre Dipartimento di Scienze della Vita e Biologia dei Sistemi,
Università di Torino, Torino, Italy
Carla Gonçalves Department of Biological Sciences and Evolutionary Stud-
ies Initiative, Vanderbilt University, Nashville, TN, USA
Martin Grube Institute of Biology, University of Graz, Graz, Austria
Pedro Gundel Instituto de Ciencias Biológicas, Talca University, Talca,
Chile
Bradley Hillman Plant Biology, Rutgers University, New Brunswick, NJ,
USA
Chris Todd Hittinger Laboratory of Genetics, DOE Great Lakes Bioenergy
Research Center, Center for Genomic Science Innovation, Wisconsin Energy
Institute, J.F. Crow Institute for the Study of Evolution, University of
Wisconsin-Madison, WI, USA
Song Hong Laboratory of Insect Developmental and Evolutionary Biology,
CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of
Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai,
China
Huei-Mei Hsieh Institute of Plant and Microbial Biology, Academia Sinica,
Nankang, Taipei, Taiwan
Yen-Ping Hsueh Institute of Molecular Biology, Academia Sinica,
Nankang, Taipei, Taiwan
xviii Editors and Contributors

Richard Hung Mycorrhizal Applications, St. Louis, MO, USA

Timothy Y. James Department of Ecology and Evolutionary Biology, Uni-
versity of Michigan, Ann Arbor, MI, USA
Linda Johnson Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Yu-Ming Ju Institute of Plant and Microbial Biology, Academia Sinica,
Nankang, Taipei, Taiwan
Sheng-Chian Juan Institute of Molecular Biology, Academia Sinica,
Nankang, Taipei, Taiwan
Annegret Kohler UMR 2UMR INRA/UHP 1136, Interactions Arbres/
Micro-organismes, Centre INRA de Nancy, Champenoux, France
Samantha Y. Lee Graduate Women in Science, Mullica Hill, NJ, USA
Tzu-Hsiang Lin Institute of Molecular Biology, Academia Sinica, Nankang,
Taipei, Taiwan
Wade Mace Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Francis Martin 2UMR INRA/UHP 1136, Interactions Arbres/Micro-
organismes, Centre INRA de Nancy, Champenoux, France
Christina Moon Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Jonathan M. Plett Hawkesbury Institute for the Environment, Western
Sydney University, Richmond, NSW, Australia
Sushma Prakash Resilient Agriculture, AgResearch Limited, Grasslands
Research Centre, Palmerston North, New Zealand
Alisha Quandt Department of Ecology and Evolutionary Biology, Univer-
sity of Colorado, Boulder, CO, USA
Saleh Rahimlou Department of Ecology and Evolutionary Biology, Univer-
sity of Michigan, Ann Arbor, MI, USA
Antonis Rokas Department of Biological Sciences and Evolutionary Studies
Initiative, Vanderbilt University, Nashville, TN, USA
Junmei Shang Laboratory of Insect Developmental and Evolutionary Biol-
ogy, CAS Center for Excellence in Molecular Plant Sciences, Shanghai
Institute of Plant Physiology and Ecology, Chinese Academy of Sciences,
Shanghai, China
Yanlei Sun Laboratory of Insect Developmental and Evolutionary Biology,
CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of
Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai,
China
Editors and Contributors xix

Emily Trudeau Department of Botany, University of British Columbia,

Vancouver, BC, Canada
Massimo Turina IPSP-CNR, Torino, Italy
Guillermo Vidal-Diez de Ulzurrun Institute of Molecular Biology, Acade-
mia Sinica, Nankang, Taipei, Taiwan
Chengshu Wang Key Laboratory of Insect Developmental and Evolution-
ary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shang-
hai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences,
Shanghai, China
School of Life Science and Technology, ShanghaiTech University, Shanghai,
China
Yan-Yan Wang State Key Laboratory of Mycology, Institute of Microbiol-
ogy, Chinese Academy of Sciences, Beijing, China
Jin-Rong Xu Department of Botany and Plant Pathology, Purdue Univer-
sity, West Lafayette, IN, USA
 When Plants and Animals First Met
Fungi: Insights from the Evolution 1
of Host Immune Systems

Emily D. Trudeau and Mary L. Berbee

Abstract interactions between host proteins and patho-

Fungi interact with plants and animals, leading gen molecules. We contend that phylogenetic
variously to mutually beneficial partnerships ages of the host receptors provide a window,
or to devastating disease. Reflecting the impor- imperfect but intriguing, into the origins of the
tance of plant–fungal and animal–fungal host systems that have been detecting fungi
interactions, the innate immune systems of over hundreds of millions of years of
terrestrial plants and animals detect fungi geological time.
using inherited recognition receptors. Upon
recognition, the receptor proteins stimulate Keywords
signaling pathways that foster intimate Innate immunity · Plant–fungus interactions ·
mutualisms or trigger robust, multi-level Animal–fungus interactions · Microbe-
innate defenses against parasitism. To provide associated molecular patterns · Host pattern-
points of discussion about how the evolution- recognition receptor evolution · LysM
ary history of host recognition may have domain · GH19 chitinase · C-type lectin-like
unfolded, we analyze the phylogenetic receptor · Geological time · Fungal cell wall
distributions of pattern-recognition receptors
important in plant and animal immunity, and
of two groups of enzymes important in 1.1 Introduction
defenses: chitinases in plants and
β-glucanases in animals. Our analysis takes Reconstructing evolutionary histories of plant and
advantage of recently sequenced genomes of animal proteins involved in sensing and
early-diverging host plants and animals. We responding to fungi provides a genomic basis
draw upon experimental studies using model upon which to speculate about how and when
organisms that reveal how some host plants fungi first interacted with plants and animals.
and animals perceive fungi. To infer functional When fungi meet plant or animal hosts, myriad
homology, we explore recent studies using complex mechanisms mediate interactions. Host
experimental approaches to analyze binding receptors recognize fungal molecules, setting in
motion complex signaling cascades to mount a
E. D. Trudeau · M. L. Berbee (✉) defense or to initiate mutualistic symbiosis.
Department of Botany, University of British Columbia, The mechanisms underlying fungus–plant or
Vancouver, BC, Canada fungus–animal interactions have important
e-mail: [email protected]; mary. implications, both positive and negative. Fungal
[email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 1

Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_1
2 E. D. Trudeau and M. L. Berbee

pathogens devastate over 125 million metric tons last a few months or a lifetime, but it is not passed
of major crop plants including rice, wheat, and to the next generation.
maize every year and account for over 25% of Innate immunity, the subject of this chapter, is
crop losses worldwide (Boddy 2016). Arbuscular based on genetically inherited receptors that rec-
mycorrhizal symbiosis, thought to have evolved ognize and bind ligands, which are molecules
concurrently with the first land plants, likely had a characteristic of various microbes. Distinguishing
role in helping early plants to become established among molecules from interacting microbes
on land, thereby shaping the first terrestrial enables eukaryotic hosts to initiate intracellular
ecosystems (Delaux and Schornack 2021). signaling, leading to partnerships with mutualists,
Fungi in humans range from causative agents tolerance of commensals, or the detection and
of life-threatening, systemic diseases to destruction of pathogens. In vertebrates, response
commensals, such as Candida spp., which can to microbes involves coordination between innate
be asymptomatic colonizers of mucous and adaptive immune systems. However, the vast
membranes. Some mammals, such as ruminants majority of eukaryotes including plants and
and other herbivores, derive benefits from a mutu- invertebrates have only the more ancient innate
alistic relationship with gut fungi. These fungi immune systems.
possess the remarkable ability to break down We focus on a first line of innate defense that
plant cellulose, which is typically indigestible, starts when host proteins, pattern-recognition
into digestible sugars (Gordon and Phillips receptors at the cell surface, detect microbe-
1998). Mammals and birds are protected from associated molecular patterns (Sanchez-Vallet
most potentially deadly fungal pathogens by et al. 2014; Hatinguais et al. 2020). The hosts’
body temperatures above 35 °C, by slightly alka- receptors typically target microbial cell
line body fluids (most fungi prefer slightly acidic coverings. Their ability to distinguish among
substrates), and by skin surfaces that resist fungal microbes usually involves their binding
attachment (Casadevall 2005). However, selec- preferences. Cell coverings—chitin, mannose,
tion for additional layers of antifungal protection and β-1,3-1,6-glucan in fungi (Gow et al. 2017);
must have exerted continuing force through ani- lipopolysaccharides and peptidoglycans in Gram-
mal evolution. This is evident from medical negative bacteria; and peptidoglycans and
records and laboratory experiments showing that teichoic acid in Gram-positive bacteria (Fisher
pathogenic fungi rage out of control when our and Mobashery 2020)—are ancient molecules,
layers of immune protection are compromised but their organization and components vary and
(Brown et al. 2018). Especially in parts of the overlap, due in part to continuing selective pres-
world where availability of sophisticated antiviral sure from pathogens and symbionts. Here, we
drugs is limited, fungal infections kill 500,000 focus on fungal recognition, but the same or sim-
HIV/AIDS patients per year (Limper et al. 2017) ilar host receptors may also detect oomycetes,
because virus-induced faults in adaptive immu- nematodes, and other organisms that share similar
nity reduce antifungal protection. cell surface biochemicals.
Immune systems vary across groups of Many of the receptors important in
organisms. Adaptive immunity is widely shared recognizing fungi and other microbes are multi-
by vertebrates and appears to have originated domain proteins with an extracellular lectin
among early jawed fishes (Buchmann 2014; domain. Lectin domains are non-catalytic and
Rombout et al. 2005). Adaptive immunity results are defined by function; they bind ligands, usually
when an individual’s exposure to an antigen sugars, without altering them (De Schutter and
creates a “molecular memory,” which can take Van Damme 2015). Lectins are perhaps most
the form of antibodies specific to the antigen. familiar as tools used in cell biology to identify
Vaccination or exposure to infection triggers polysaccharides through their binding
adaptive immunity. The molecular memory may specificities, as when the lectin wheat germ
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 3

agglutinin, labeled appropriately and added to 2018; Amses et al. 2022). To specify particular
fungal cells, reveals through its binding the loca- proteins, GenBank accession numbers are given
tion of chitin in fungal walls (James and Berbee in parentheses in the text or are included in
2012). In animal and plant cells, receptors bearing figures.
different lectin domains can distinguish patho- Immunology as a field has burgeoned, and
genic or non-pathogenic microbes by their differ- astounding progress brings with it specialized
ent cell wall components. Enzymes, in contrast to terminology that can be a barrier to understanding
lectins, bind and alter their substrates. Among for those from other disciplines. We minimized
enzymes, some, such as chitinases in plants and our use of acronyms and abbreviations,
animals and β-glucanases in insects, have roles apologizing to colleagues who prefer the more
both in detecting and in destroying pathogens. concise notation. Our chapter now focusses on
After a host protein recognizes a microbe- phylogenetic interpretation of proteins with roles
associated molecular pattern, host responses usu- in recognizing fungal cell walls.
ally involve cellular signaling, leading variously
to phagocytosis or agglutination of the microbe,
to the secretion of antimicrobial toxins, or to 1.2 How Plants Detect
programmed cell death that limits a pathogen’s and Respond to Fungi
growth by killing its host cell. This chapter does
not explore signaling or how a host ultimately In this section, we speculate on the origins of
disarms a microbe. For reviews that use elegant chitin perception and chitin-degrading enzymes
diagrams to explain host processes, see Erwig and in plants (Fig. 1.1). We review how green algae
Gow (2016) (mammals); Hatinguais et al. (2020) (chlorophyte and streptophyte algae), bryophytes,
(mammals); Hoffmann (2003) (Drosophila); and vascular plants perceive fungi, and we
Ngou et al. (2022) (plants); and Gauthier et al. explore the diversity in structure and function of
(2010) (comparing selected eukaryotes). plant chitinases—the defense-related enzymes
Because host receptors and enzymes important that target the fungal cell wall. We present a
in innate immunity are inherited vertically, they phylogenetic analysis of one family (glycoside
can be analyzed phylogenetically. We chose a hydrolase 19) of plant chitinases, reconstructing
few of the many host proteins with experimen- the evolutionary history of this family spanning
tally demonstrated roles in fungal recognition. bacterial, algal, and plant lineages.
Experimental data on the molecular basis for
microbial detection come largely from studies of
plant models such as Arabidopsis and rice, animal 1.2.1 Plants Detect Fungi via Pattern-
models including mice, and human cell cultures. Recognition Receptors that
In the following pages, we discuss the appearance Contain Extracellular Lysin
of these proteins through host evolution using Motifs (LysM): Lectin Domains
published phylogenies where applicable. We that Bind Chitin
then use BLAST searches, comparison of protein
domain structures, information on ligand binding The plant immune system allows plants to per-
sites, and some original phylogenetic analyses to ceive, initiate defense responses, and mount a
apply newly available data to inferences about resistance to fungal pathogens. Plants evolved to
when proteins with homologous function may recognize fungi by detecting chitin fragments
have originated. We use published ages of host as microbe-associated molecular patterns. Chitin
clades to infer protein ages, with the caveat that is an insoluble long-chain polymer of
estimates of clade ages vary across studies, espe- N-acetylglucosamines that can be degraded by
cially for unicellular, early-diverging taxa without chitinases to produce soluble fragments of eight
rich fossil records (Morris et al. 2018; Chang et al. or fewer N-acetylglucosamine residues (Buendia
2015, 2019; Nelsen et al. 2020; Lutzoni et al. et al. 2018). Other microbe-associated molecular
4 E. D. Trudeau and M. L. Berbee

Fig. 1.1 Overview of

distributions of chitin,
chitin recognition receptors,
and defense-related
proteins across lineages that
are reviewed over the next

Ferns
Fungi

Animals
Bacteria

Diatoms

Chlorophyte algae

Angiosperms
Gymnosperms
Bryophytes
Streptophyte algae

Lycophytes
pages. Icons created with
BioRender.com

1 1

2 2 2

5 1 Chitin
2 Chitin synthase
4 4 4
3 GH18 chitinase
4 GH19 chitinase
5 LysM receptor-like
kinase
3

patterns perceived by plants include peptidogly- kinase domain needed for signal transduction
can from bacterial cell walls, a polymer made up (Shiu and Bleecker 2003). LysM domains can
of alternating residues of N-acetylglucosamine detect and bind to not only chitin fragments but
and N-acetylmuramic acid. In hosts, primary also bacterial peptidoglycan. They were first dis-
defense responses such as cell wall alterations, covered in peptidoglycan-hydrolyzing enzymes,
production of reactive oxygen species, and and the name originates from the bacterial cell
expression of defense-related genes like lysis that results (Buendia et al. 2018; Willmann
chitinases are initiated by recognition of chitin et al. 2011).
fragments (Khokhani et al. 2021). A second LysM domains of around 40–50 amino acid
layer of defense is triggered upon recognition of residues that belong to the carbohydrate-binding
fungal effectors, which are secreted by some module family 50 are found in most organisms,
fungi to interfere with plant immune responses, according to the Carbohydrate-Active Enzymes
ultimately resulting in hypersensitive responses database (www.cazy.org) (Drula et al. 2022).
and programmed cell death, as reviewed by de They are not highly conserved in terms of primary
Wit (2007). The focus of this section will be on sequence, although cysteine pairs separated by
primary immune responses to fungi. one amino acid are found between the three
LysM-containing receptors can be receptor- LysM domains of most plant LysM receptor-like
like kinases, with a cytoplasmic kinase domain kinases and receptor-like proteins, and are
that can signal when the LysM domains have required for disulfide bridging between domains
bound to chitin oligomers (Fig. 1.2a), or (Lefebvre et al. 2012). Secondary and tertiary
receptor-like proteins that lack the cytoplasmic structures of LysMs are, however, highly
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 5

Fig. 1.2 Structure of LysM receptor-like kinases. (a) (D) residues indicative of RD kinase activity are denoted
Domain architecture of CERK1 LysM receptor-like kinase by an asterisk. Threonine (T) residues essential for chitin
(SP signal peptide, LysM1-3 LysM domains, TM trans- signaling are denoted by a number sign. LYK5 from
membrane domain). (b) Partial alignment of kinase Arabidopsis and LYK3 from Chara lack these
domains in LysM receptor-like kinases from plants and residues. Darker shading indicates more highly conserved
streptophyte algae. Arginine (R) and aspartic acid positions

conserved, consisting of two α-helices stacked serves as the high-affinity chitin-binding receptor.
onto two antiparallel β-sheets (Buendia et al. AtCERK1 can bind chitin but shows only modest
2018). chitin binding compared to AtLYK5. AtCERK1
provides kinase activity and is required for chitin-
1.2.1.1 Chitin Perception in Land Plants triggered immune responses. Chitin oligomers
Typically Involves Complexes of induce heterodimerization between AtLYK5 and
Two or More LysM-Containing AtCERK1, and AtLYK5 is required for
Receptors at the Plasma subsequent AtCERK1 homodimerization
Membrane (Fig. 1.3a) (Gong et al. 2020). AtCERK1
These complexes are typically composed of two phosphorylates the cytoplasmic kinase PBL27,
receptor-like kinases, or one receptor-like kinase which induces MAPK cascades and immune
and one receptor-like protein. One receptor usu- responses.
ally has higher chitin-binding affinity, while the The first plant chitin receptor to be identified—
other has an active kinase domain that is neces- OsCEBiP—was cloned from rice in 2006 (Kaku
sary for signal transduction (Fig. 1.3). In general, et al. 2006). OsCEBiP is a LysM receptor-like
plant LysM-containing receptors have three protein that is anchored to the plasma membrane
extracellular LysM domains, with one or more by a glycophosphatidylinositol. It binds chitin but
domains binding chitin directly. In Arabidopsis lacks the cytoplasmic kinase domain needed for
thaliana, chitin oligomers are detected by a recep- signal transduction (Jiang et al. 2019). Two
tor complex made up of AtCERK1 and AtLYK5 CEBiP monomers homodimerize to bind the
(Fig. 1.3a). AtLYK5 is a LysM receptor-like same chitin oligomer, showing preference for
kinase, but has an inactive kinase domain, and longer chitin fragments consisting of about seven
6 E. D. Trudeau and M. L. Berbee

Fig. 1.3 LysM receptor-like kinases and receptor-like phosphorylate a cytoplasmic kinase (RLCK185), activate
proteins detect chitin and initiate immune responses. (a) a MAPK cascade, and induce defense responses. (c) In
In A. thaliana receptor-like kinases CERK1 and LYK5 the moss P. patens, CERK1 activates MAPK signaling
form a complex to bind chitin, phosphorylate a cytoplas- cascades. (d, e) Streptophyte algal genomes have CERK1
mic kinase (PBL27), activate a mitogen-activated protein or LYK5 homologs with untested roles in chitin percep-
kinase (MAPK) cascade, and induce defense-related gene tion and defense. (d) Chara braunii’s genome encodes
expression. (b) In rice, O. sativa, CERK1 and receptor-like seven LysM receptor-like kinases. (e) Nitella mirabilis’s
protein CEBiP form a complex to bind chitin, transcriptome encodes a CERK1 homolog

to eight N-acetylglucosamine residues (Fig. 1.3b). kinase function to the receptor complex (Shimizu
OsCEBiP then heterodimerizes to form a com- et al. 2010). OsCERK1 phosphorylates a
plex with the LysM receptor-like kinase receptor-like cytoplasmic kinase (OsRLCK185),
OsCERK1, which cannot bind chitin but provides which in turn activates the MAPK cascade
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 7

resulting in immune responses and defense sig- mediating interactions between land plants and
naling (Sanchez-Vallet et al. 2014). fungi.
In the moss Physcomitrium patens (Fig. 1.3c), For Arabidopsis AtCERK1, mutational analy-
PpCERK1, an ortholog to AtCERK1, binds chitin sis showed that kinase function, chitin signaling,
and then initiates MAPK cascade activation and defense-related gene expression required
(Bressendorff et al. 2016). Physcomitrium phosphorylation sites at threonine (T) residues
patens’s three LYK5 genes are homologs to T479 and T573 (Fig. 1.2b) (Suzuki et al. 2016).
AtLYK5 (Fig. 1.3a), and silencing of LYK5 CERK1 bears an RD kinase, having a conserved
genes in P. patens renders plants chitin insensi- arginine (R) residue immediately preceding a
tive (Orr et al. 2020). Additionally, one PpLYK5 conserved aspartic acid (D) residue in the cata-
was shown to be highly expressed upon infection lytic loop of the kinase domain (Greeff et al.
with the fungus Botrytis cinerea (Reboledo et al. 2012). These T, R, and D residues are conserved
2021; Orr et al. 2020). in CERK1 receptors with active kinases in
Representing the streptophyte algae, the clos- Arabidopsis (NP_566689), Oryza
est relatives to land plants, the genome of Chara (XP_015650771), Selaginella (XP_002977121),
braunii encodes seven LysM receptor-like and Physcomitrium (XP_024388364) (Fig. 1.2b).
kinases (LYK1–7) (Fig. 1.3d) and the In the CERK1 homolog from N. mirabilis
transcriptome of Nitella mirabilis encodes a (JV789971), T, R, and D residues are similarly all
homolog to CERK1 from land plants (Fig. 1.3e). conserved.
Arabidopsis LysM receptor-like kinases with-
1.2.1.2 LysM Receptor-Like Kinases out kinase activity such as AtLYK5 (AAB80675)
Evolved in the Common lack the homologous T, R, and D residues. Rais-
Ancestor of Land Plants and ing questions about functions of its LysM
Streptophyte Algae receptor-like kinases, all seven of the genes in
Members of the CERK1 clade of LysM receptor- C. braunii have a valine (V), not a T that would
like kinases are as yet known only from align at site T573 of AtCERK1, as shown by
streptophyte algae and land plants (Figs. 1.2 and CbLYK3 (GBG90350) in Fig. 1.2b. All but one
1.3). Some chlorophyte, red, and glaucophyte of the LysM receptor-like kinase genes in
algae have LysM domain-containing proteins of C. braunii lack the R and/or D residues character-
uncharacterized function, but proteins that have istic of RD kinases. Gene family expansion of
both the kinase and the LysM domain are as yet LysM receptor-like kinases occurred indepen-
unknown in these lineages (Van Holle and Van dently in C. braunii and land plants, and future
Damme 2019; Delaux and Schornack 2021). Dis- experimental testing of the C. braunii receptors
tantly related LysM domains are also found may give fascinating results (Nishiyama et al.
across bacteria, fungi, and animals, implying 2018). As Nishiyama et al. (2018) point out, the
that the LysM domain is even more ancient than C. braunii receptors’ expansion may have
the divergence of eukaryotes from bacteria, allowed adaption to a range of interactions
estimated to have occurred over three billion with chitin- and peptidoglycan-containing
years ago (Battistuzzi et al. 2004; Knoll 2014). organisms.
Receptors like CERK1 with active kinase
domains are conserved in land plants, with a 1.2.1.3 Plant Chitin Receptors Can
possible ortholog in the streptophyte alga Nitella Initiate Both Immune and
mirabilis (Fig. 1.3). Nitella, in Charophyceae, Symbiotic Responses
diverged from the ancestor of land plants 635 mil- The common ancestor to all land plants may have
lion years ago (Ma) (estimated from Fig. 1.3 in been preadapted for symbiosis with mycorrhizal
Morris et al. 2018), suggesting that CERK1 may fungi, based on the many genes important in
have been present before plant terrestrialization. fungal colonization that are shared between land
That it is highly conserved implies importance for plants and the streptophyte algae (Delaux et al.
8 E. D. Trudeau and M. L. Berbee

2015). Since microbial symbionts—namely land plants and are represented by 407 Ma fossils.
mycorrhizal fungi—produce chitin oligomers, Ectomycorrhizae, restricted to seed plants, are
perception of these microbe-associated molecular known only from 50 Ma fossils, and the root-
patterns does not inevitably trigger host defense associated nitrogen-fixing bacteria, restricted to
responses. Longer chitin oligomers of six to eight flowering plants, have a fossil record dating
residues can initiate both immune and symbiotic back to ~85 Ma (Bonfante and Requena 2011;
responses but are stronger activators of host van Velzen et al. 2019).
immune responses than are short-chain oligomers Arbuscular mycorrhizal fungi produce a com-
(Khokhani et al. 2021). The legume Medicago bination of chitin oligomers and LCOs during
truncatula uses the receptor complex MtLYK9/ colonization of their host plants. Symbiotic
MtCERK1 in both its symbiotic and immune outcomes are favored when LCOs suppress the
responses, and either response can be activated plant immune responses triggered by long-chain
by four- or eight-residue chitin oligomers (Feng chitin oligomers (Maillet et al. 2011). Upon LCO
et al. 2019). Similarly, in rice, the receptor-like detection, the host initiates signaling pathways
kinase OsCERK1 can detect short-chain that increase the number of lateral roots—the
oligomers in arbuscular mycorrhizal signaling, most common sites of arbuscular fungal coloni-
suggesting a dual role in immunity and symbiosis zation. Many of the signaling proteins are shared
(Carotenuto et al. 2017). Discriminating between by bryophytes and vascular plants (Delaux and
symbiotic hyphae and pathogenic appressoria Schornack 2021), implying that they were already
may involve additional factors such as plant nutri- in place when these clades of land plants diverged
ent status and perception of mechanical stimuli ~515–474 Ma (Morris et al. 2018).
(Khokhani et al. 2021). Evidently, plants are bal- Production of LCOs may represent a fungal
ancing activation of immune signaling and sym- pre-adaptation to interactions with plants because
biotic signaling to respond appropriately to chitin it is not restricted to mycorrhizal fungi. Rush et al.
oligomers. (2020) showed that fungi in five phyla, including
saprotrophs, parasites, and mutualists, produce
LCOs. They demonstrated experimentally that
1.2.2 Lipo-Chitooligosaccharides LCOs can function as growth regulating
(LCOs), Chitin-Based Signaling hormones in ascomycetes and basidiomycetes
Molecules, Are Widely (Rush et al. 2020). A role in regulating fungal
Distributed Among Fungi development may well be ancestral because LCO
production, based on its distribution across
The fungal LCOs modulate plant immune chytrids, Blastocladiomycota, and Dikarya,
responses when produced by mycorrhizal fungi originated some 800 Ma (Chang et al. 2019),
and nitrogen-fixing bacteria. Like chitin hundreds of millions of years before land plants.
oligomers, LCOs are perceived by LysM receptor
kinases that activate downstream signaling
cascades (Wanke et al. 2020). First characterized 1.2.3 Plant Perception of Other
in nitrogen-fixing bacteria that form root nodules Fungal Molecules Is Not as Well
in legumes (Roche et al. 1996), LCOs were sub- Characterized
sequently shown to initiate signaling for mutual-
istic symbioses in hosts of arbuscular β-1,3-glucan and β-1,6-glucan are other major
mycorrhizae (Maillet et al. 2011) and components of the fungal cell wall, together
ectomycorrhizae (Cope et al. 2019). Land plant making up around half of its dry weight. Wanke
mutualisms via perception of LCOs arose et al. (2020) found that response to β-glucans,
convergently. Arbuscular mycorrhizae are the polymers of glucose, varies among plant species
oldest of the mutualisms with LCO recognition; and that the degree of polymerization plays a
they involve the earliest-diverging lineages of major role in recognition and response. In
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 9

Arabidopsis, perception of short-chain β-glucans 19 (GH18 and GH19) (Drula et al. 2022). GH18
requires AtCERK1. In contrast in rice, perception and GH19 chitinases show no evidence of homol-
of long-chain β-glucans requires OsCERK1 ogy (Hamel et al. 1997) as they exhibit different
(Wanke et al. 2020). catalytic mechanisms and differ in their domain
Arabidopsis can also recognize other fungal architectures and 3D structures (Cheng et al.
microbe-associated molecular patterns, for exam- 2021). While GH18 chitinases are widely
ple producing ethylene in response to xylanases distributed across plant, bacterial, animal, and
secreted by fungi and bacteria to degrade hemi- fungal lineages, GH19 chitinases are mainly
cellulose. Arabidopsis recognizes the fungal found in plants and bacteria (Sharma et al.
endopolygalacturonase enzymes that target pectin 2020), although GH19 chitinase function has
in plant cell walls. These molecular patterns are been experimentally characterized in
detected at the plasma membrane by receptor-like Microsporidia (Han et al. 2016), and using
proteins that contain leucine-rich repeats (Anand BLAST searches, we found putative GH19
et al. 2021; Zeilinger et al. 2016). chitinases in some fungi (e.g., GenBank
Leucine-rich repeats also occur in nucleotide- accessions AWI66960 and KAG9575357). Plant
binding, leucine-rich repeat (NLR) receptors in chitinases were further divided into seven classes,
plants. Well-characterized plant NLR receptors prior to their classification into GH18 and GH19
are important in detecting fungal molecules but families (Fig. 1.4). GH18 includes classes III
being cytoplasmic rather than at cell surfaces and V, while GH19 includes classes I, II, IV,
(Maekawa et al. 2011; Martinon and Tschopp VI, and VII (Adrangi and Faramarzi 2013).
2005); they are beyond the topics of this chapter. Classes were defined based on protein cata-
We discuss the roles of leucine-rich repeats in lytic domains, sequence lengths, and the presence
animal toll-like receptors in the upcoming section or absence of non-catalytic chitin-binding
on detection of fungi by animals. domains (CBDs) or carbohydrate-binding
modules that promote the association of the
enzyme with the substrate (Fig. 1.4) (Kitaoku
1.2.4 Chitinases in Plant Defenses et al. 2022). An alternative framework classifies
Against Fungi GH19 chitinases into “loopful” (class I and II)
and “loopless” (class IV) chitinases based on the
Plants defend themselves against fungi and other presence or absence of up to six loop regions in
microbes by expressing pathogenesis-related the catalytic domain (Hoell et al. 2006; Taira et al.
proteins including chitinases (Collinge et al. 2011). Recognizing that classes failed to fully
1993; Pusztahelyi 2018). Plant chitinases com- reflect phylogeny or capture variation within clas-
promise fungal cell wall integrity by degrading ses, Orlando et al. (2021) applied a clustering
chitin polymers through the hydrolytic cleavage system to divide the catalytic domains of GH19
of glycosidic bonds between β-1,4-linked N- chitinases into two subfamilies consisting of
acetylglucosamine monomers (Punja and Zhang 51 groups.
1993). Through this process, chitin oligomers are There will, however, always be exceptions to
released, stimulating host immune responses the generalized rules involved in classification of
(Sanchez-Vallet et al. 2014). Many plant patho- multi-domain proteins that evolve with domain
genic fungi make their first contact with the host shuffling. For example, chitinases in a subclass
in the apoplast. The apoplast, the extracellular of class III in the horsetail Equisetum arvense and
space beyond the plasma membrane, contains fern Pteris ryukyuensis (Inamine et al. 2015;
hydrolytic enzymes and toxins that inhibit fungal Onaga and Taira 2008) contain LysM domains.
growth. This type of LysM–chitinase conjugate is not
The Carbohydrate-Active Enzymes database usually found in plants, but is common in fungal
(www.cazy.org) classifies plant chitinases into chitinases (Kitaoku et al. 2022). With BLAST
two families: glycoside hydrolases 18 and searches, we found a putative GH18 class V
10 E. D. Trudeau and M. L. Berbee

Fig. 1.4 Domain architecture characteristic of classes with a C-terminal extension are transmitted to the vacuole,
within GH19 and GH18 chitinases. Classes I, II, IV, VI, while those without are transmitted extracellularly to the
and VII are GH19 chitinases that differ in length and apoplast (Renner and Specht 2012). Classes III and V
presence or absence of a chitin binding domain (CBD), are GH18 chitinases that share a catalytic domain with a
hinge region, and C-terminal extension (Passarinho and de conserved DxDxE sequence motif but show little sequence
Vries 2002; Orlando et al. 2021). CBDs promote the similarity, and generally lack CBDs (Ohnuma et al. 2011)
association of the enzyme with the substrate. Chitinases

chitinase in the spikemoss Selaginella resistance to the fungal pathogen Botrytis cinerea
moellendorffii (XP002976579) with a predicted resulted when Arabidopsis was transformed with
CBD domain more commonly found in class I a class IV chitinase from mulberry that is
and IV chitinases of family GH19. upregulated upon B. cinerea infection (Xin et al.
2021). A class VI chitinase in cashew degraded
1.2.4.1 Biological Functions and colloidal chitin and inhibited the growth of the
Expression Patterns of Plant fungus Lasiodiplodia theobromae in vitro by
Chitinases Vary Among Classes altering hyphal morphology (Oliveira et al.
and Species 2020). Class III chitinases from Populus
Since plants do not synthesize chitin, their trichocarpa acted on both chitin and chitosan,
chitinases can play roles in defense against the deacetylated form of chitin (Zhang et al.
chitin-containing pathogens such as fungi without 2022).
compromising their own cellulosic cell walls Many plant chitinases can also contribute to
(Taira et al. 2009). Much research implicates regulating developmental processes such as seed
plant chitinases in defense responses against germination and response to abiotic stresses such
fungi (Kasprzewska 2003; van Loon et al. 2006; as freezing and low nutrient conditions (González
Passarinho and de Vries 2002). Some chitinases et al. 2015). A class II chitinase from rye (Secale
are synthesized constitutively irrespective of cereale AAG53610) plays a role in extracellular
stress conditions, while others can be induced frost resistance (Yeh et al. 2000). Plant chitinases
by pathogen infection (Xin et al. 2021). The might be involved in developmental regulation by
class I chitinase from rye (Secale cereale acting on endogenous arabinogalactan proteins
BAB18519) is an extracellular inhibitor of fungal that contain N-acetylglucosamines (van Hengel
growth (Yamagami and Funatsu 1993). Improved et al. 2001), but this has not yet been confirmed
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 11

experimentally (Hromadova et al. 2021). A role from land plants, green algae, bacteria, and
in senescence was suggested when ethylene diatoms. Glaucophyte algae and red algae
(a plant hormone associated with senescence) appeared to lack homologs.
increased expression of a class I chitinase in Figure 1.5 clade 1 shows that angiosperm
Arabidopsis (Passarinho and de Vries 2002). An GH19 sequences previously placed in classes I,
ethylene-dependent signaling pathway was II, and VI together form a monophyletic
required for induction of this chitinase in plants group including the seedless vascular plants,
infected with B. cinerea (Thomma et al. 1999). bryophytes, streptophyte algae, chlorophyte
This class I chitinase might also serve in host algae, and a bacterium, Chitinophaga pinensis.
defense as it inhibited growth of the fungus Most of these sequences have the chitin-binding
Trichoderma reesei in vitro. sites predicted by Orlando et al. (2021). Some do
not, e.g., Secale cereale (AAG53610), and com-
parison of function of proteins with and without
1.2.5 Evolutionary History of Plant predicted binding sites may be useful in further
Chitinases refining bioinformatic predictions. Denser sam-
pling of bacterial chitinases would be needed to
Since GH18 chitinases can be found across plant, test the significance of their relationship to
animal, bacterial, and fungal lineages, and since eukaryotic proteins. Together, class I and class
plant GH18 chitinases are more similar to bacte- II proteins form the well-supported clade
rial and fungal GH18 chitinases than other plant 2 (Fig. 1.5), which also appeared in Oliveira
chitinases, it follows that GH18 chitinases or et al.’s (2020) analysis of angiosperm sequences.
“protochitinases” could have been present in the From our broader sampling, clade 2 sequences
common ancestor of bacteria and eukaryotes. appear to have originated before the first
GH19 chitinases, on the other hand, are known divergences of vascular plants 451–431 Ma
mostly from plants and bacteria, although fewer (Morris et al. 2018), as they encompass flowering
bacterial sequences than plant sequences have plants and Selaginella. Within clade 2, class I and
been experimentally characterized (Ohno et al. class II sequences are not separated, indicating
1996; Sharma et al. 2020; Yano et al. 2021). that chitin-binding domains (Fig. 1.5) are gained
According to an analysis by Orlando et al. or lost convergently through domain shuffling.
(2021), GH19 chitinases can be divided into a A good example of lack of monophyly of class I
mostly bacterial clade and a mostly plant vs class II comes from Secale cereale because
clade at the basal node of the gene tree. The one of its class II sequences (BAB18520) is
focus of our analysis is on plant GH19 chitinases, more closely related to its class I sequence
and Orlando et al. (2021) should be consulted for (BAB18519), with significant bootstrap support,
insights into the phylogeny of bacterial GH19 than it is to another of its class II sequences
chitinases. (AAG53610) (Fig. 1.5).
Our analysis expands the clade of known
1.2.5.1 Phylogenetic Analysis of GH19 relatives of class VI chitinases from angiosperms
Chitinases to include other seed plants, bryophytes, and
Previous phylogenies of chitinases emphasized seedless vascular plants, estimated to have
seed plants (Taira et al. 2011; Oliveira et al. diverged starting 515–474 Ma (Morris et al.
2020), but increasingly comprehensive data 2018) (Fig. 1.5, clade 3). Chitinase function has
from chlorophyte algae, streptophyte algae, been lost in some angiosperms in class VI,
bryophytes, and seedless vascular plants allow e.g., Arabidopsis (AAL37737) (Fig. 1.5). The
us to explore the deeper origins of GH19 chitinases from the seedless plants have retained
chitinases. To reconstruct the evolutionary his- a glutamic acid residue in the enzyme’s active site
tory of GH19 chitinases (Fig. 1.5), we analyzed that was lost from the corresponding angiosperm
sequences homologous to Arabidopsis chitinases proteins, consistent with possible conservation of
12 E. D. Trudeau and M. L. Berbee

Fig. 1.5 GH19 chitinases in plants have origins in the lycophyte, and fern genomes using different classes of
common ancestor of chlorophyte and streptophyte algae, Arabidopsis chitinases as queries. The tree was rooted
showing gene duplications in early land plants and reten- with the bacterial clade as the outgroup. Bacterial
tion of gene copies. In this maximum likelihood tree from sequences nested within plant clades are boxed in red.
GH19 catalytic domains, species names are followed by Bootstrap values greater than 50 are indicated above the
GenBank accession numbers, and Roman numerals in nodes. Numbered nodes 1–4 denote well-supported clades
parentheses indicate class as assigned by previous litera- that are further discussed in the text. Icons created with
ture. Sequences without an associated class number were BioRender.com
obtained through BLAST searches of algal, bryophyte,

function of class VI chitinases through early land Retention of genes implies continued selection
plant evolution. Classes IV and VII form clade for a diversity of chitinases in land plants, and
4 (Fig. 1.5), consisting of seed plants that initially therefore of defense-related enzymes that target
diverged ~365–331 Ma (Morris et al. 2018). We chitin-containing cell walls of organisms like
found class IV among flowering plant sequences, fungi. Our analysis supports the presence of at
and class VII only from gymnosperms, as in least one GH19 chitinase in chlorophyte algae
Oliveira et al. (Table S2, 2020). and in the common ancestor of streptophyte
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 13

algae and land plants. Gene duplications in early 2005), homology between plant and animal
land plants are implied by supported clades that receptors may be at the level of individual
group angiosperms and Selaginella (class I and conserved domains but is not retained across the
II); angiosperms, seedless plants, and bryophytes whole of the multi-domain receptor proteins.
(class VI, clade 3); and angiosperms and Function constrains plant and animal receptors
gymnosperms (class VII and IV) (Fig. 1.5). to similar architecture. Many animal receptors,
Relationships of other sequences from like plant receptors, bind fungal cell wall
bryophytes, seedless vascular plants, diatoms, polysaccharides including β-glucan or chitin
and algae remain unresolved, leaving the earliest oligosaccharides using extracellular lectin
origins of the GH19 family shrouded in the mists domains. Upon binding, receptors typically form
of time. dimers or higher order polymers, and their cyto-
plasmic domains initiate signaling leading to an
1.2.5.2 Caveats to Reconstructing immune response.
Ancestral Chitin-Degrading
Abilities in Plants
The main target of chitinases in chlorophyte and 1.3.1 Evolution of Dectin-1, a C-Type
streptophyte algae in freshwater habitats may Lectin-Like Receptor
have been invertebrates and protists with cell with a Central Role in Fungal
walls of chitin or its deacetylated form chitosan Detection
(Mulisch 1993; Steinfeld et al. 2019) rather than
fungi. In a terrestrial environment, plant By binding β-glucans, the lectin dectin-1, short
chitinases may defend against insects or for “dendritic cell-associated C-type lectin-1,”
nematodes with chitinous mouthparts. Therefore, plays a key role in limiting initial invasion by
presence of chitinases in a genome cannot be fungi, and in activating human immune systems
strictly interpreted as a potential means for for further antifungal defense. Figure 1.6 shows
plant–fungal interactions. Some plant chitinases dectin-1 spanning the plasma membrane of
also display additional lysozyme-like activity and mobile amoeboid phagocytes including
can degrade peptidoglycan found in bacterial cell macrophages (Gantner et al. 2003). β-glucans
walls (Kasprzewska 2003). Furthermore, some bind at dectin-1’s extracellular C-type lectin
plant chitinases are solely involved in endoge- domain. C-type lectin domains, consisting of
nous developmental processes and therefore do about 120 amino acids, are rare in plant recogni-
not play a role in interactions with or response to tion receptors but common in animals. Most ani-
microbes. Experimental characterization of puta- mal genomes encode dozens of proteins equipped
tive chitinase genes is needed to confirm function with a domain of this type (Pees et al. 2016).
in response to fungi. Host responses after dectin-1 has bound
β-glucans can include ingestion and killing of
fungal cells by phagocytosis, damaging fungal
1.3 How Animals Detect and cells through the production of reactive oxygen
Respond to Fungi Using species, and secretion of cytokines leading to
Pattern-Recognition Receptors inflammation, increasing body temperature to dis-
advantage fungi (Gantner et al. 2003). Phagocytes
Pattern-recognition receptors in animals and can engage the adaptive immune system by
plants evolved convergently, with shuffling, presenting microbial antigens to T cells
recruitment, and re-assembly of ancient lectin (LeibundGut-Landmann et al. 2007).
and signaling domains leading to new, multi- Disruption of the CLEC7A gene encoding
domain receptor proteins. As shown by careful dectin-1 in mice reduced immune responses to
analysis (Urbach and Ausubel 2017; Ausubel the fungus Candida albicans and led to higher
14 E. D. Trudeau and M. L. Berbee

Fig. 1.6 Human pattern-recognition receptors on cells in dectins, toll-like receptors, langerin, lysMD3, and
the immune system, epithelium, or mucous membranes FIBCD1. The receptors differ in their binding affinities
initiate antifungal defenses including the itchy inflamma- for various polysaccharides, but a receptor that binds fungi
tion that is our response to fungal infections such as can usually also bind some kinds of bacteria. Upon bind-
athlete’s foot, ringworm, or candidiasis. As examples of ing wall components, receptors initiate signaling cascades
animal recognition of fungi, we focus on the evolution of leading to innate immune responses. The receptors also
receptor proteins diagrammed here. Cell wall contribute to activating the adaptive immune system for
polysaccharides from fungi or other microbes (left) are continuing or future defense against the microbes they
recognized by lectin domains of receptors on several have detected. Icons created with BioRender.com
human cell types (center). Receptor proteins include

fungal burdens and reduced survival (Taylor et al. first have appeared, we compared amino acid
2007). In addition to its proven role in fungal sequences from whole genomes in GenBank and
detection, dectin-1 has experimentally generated a likelihood phylogeny for its potential
demonstrated but still unexplained roles in orthologs.
elevating immune response to other microbial Dectin-1’s full 250 amino acids were alignable
pathogens that lack β-glucans, including for Amniota, the clade of terrestrial vertebrates
tuberculosis-causing species of Mycobacterium including reptiles, birds, and mammals. Within
(Yadav and Schorey 2006; Kalia et al. 2021). the C-lectin-like domain, Adachi et al. (2004)
used mutational analysis to show that a sequence
1.3.1.1 Comparative Analysis of Dectin- of three amino acids “WIH” is important in
1 Structure Across Vertebrates β-glucan binding (Fig. 1.7). We found this motif
Dectin-1 is clearly important in human and mouse only in mammals, although birds and alligators
recognition of fungi. To infer when dectin-1 may share similar amino acids in two of the three
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 15

Extracellular receptor,
ceptor, strength or specificity of binding to a polysaccha-
a C-type lectin domain
ride ligand.
ng
β-glucan bindin For our phylogenetic analysis of dectin-1’s
motif WIH
origins, we restricted our alignment to the
conserved ~120 amino acid C-type lectin domain
that appears to be homologous across
invertebrates and vertebrates. This meant exclud-
ing >100 additional but unalignable amino acids
from analysis. Figure 1.8 shows that the C-type
Stalk
lectin domain of human dectin-1 formed a
strongly supported group with vertebrates such
as alligators, birds, and mammals. Homologs
ITAM-like motif YTQL from snakes, amphibians, and fishes, and oddly,
Cytoplasmic
tail one purple urchin sequence clustered with dectin-
NH2 1 with 74% bootstrap support but roles of these
homologs are difficult to predict because their
Fig. 1.7 Dectin-1 proteins are pattern-recognition sequences lack the functionally important
receptors important in detecting fungi (Brown and Gordon
2001). Located on phagocytes, on dendritic cells, or on
“WIH” binding motif and the YxxL signaling
macrophages, the protein’s extracellular C-type lectin motif (Fig. 1.8). The phylogeny and conserved
domain binds β-glucans of fungal walls. Upon binding, binding and signaling motifs point to an ancestral
dectin-1 molecules cluster together, initiating phagocyto- dectin-1 that evolved to detect fungal β-glucans
sis of the fungus (Goodridge et al. 2011). The cytoplasmic
tail signals detection, leading to a multi-pronged innate
before the first Amniota diverged some
and adaptive immune response 332–318 Ma (dos Reis et al. 2015). Fungi are
the main β-glucan-containing terrestrial
heterotrophs. Dectin-1 likely evolved and
persisted over the course of evolution of the pre-
dominately terrestrial Amniota due to its impor-
homologous positions (WLQ in birds, WIQ in tance in protection against fungi.
alligators).
In host cell cytoplasm, a short N-terminal 1.3.1.2 Phylogeny of C-Type Lectin-Like
motif of the dectin-1 protein signals β-glucan Receptors Related to Dectin-1
binding (Rogers et al. 2005; Gantner et al. 2003) We included sequences from C-type lectin
(Fig. 1.7). The motif has a consensus sequence domains of dectin-2 (CLEC6A) and langerin
“YxxL,” where x is any amino acid. Categorized (CLECT4k) as examples of the several paralogs
as an “immunoreceptor tyrosine-based activation to human dectin-1 that participate in recognition
motif (ITAM)-like motif,” the motif is of fungal polysaccharides (Hatinguais et al. 2020)
phosphorylated and it then recruits a molecule (Fig. 1.6). C-type lectin domains of dectin-2-
of a spleen tyrosine kinase, initiating a signaling orthologs from amniotes also form a clade
cascade and an immune response (Rogers et al. (Fig. 1.8). Through its ability to bind mannan
2005). Members of the dectin-1 clade from disaccharides, dectin-2 detects bacteria and
mammals, birds, lizards, and an alligator share fungi (Feinberg et al. 2017).
the YxxL at a position homologous to the Langerin is expressed in mammalian
human protein. Stalks of dectin-1 orthologs from Langerhans cells, a population of phagocytes
birds, reptiles, and fish have predicted coiled-coil that originate in the bone marrow and migrate to
domains that are expected to interact, clustering the skin and mucous membranes (Fig. 1.6). In
the proteins into dimers or trimers. Here as in mammals, langerin serves as a receptor for
other lectins (Feinberg et al. 2010; Shrive et al. mannans, β-glucans, and keratan sulfate
2014), polymer formation may increase the (a polymer of alternating galactose and sulfated
16 E. D. Trudeau and M. L. Berbee

Fig. 1.8 C-type lectin-like receptors dectin-1 and its bacterial, and viral recognition. C-type lectin-like receptor
paralogs dectin-2 and langerin originated early in verte- domains in invertebrates, e.g., Drosophila or the spiny
brate evolution, playing important roles in fungal, starfish Acanthaster, are not supported as orthologs to
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 17

N-acetylglucosamine units). Langerin therefore responsible for signal transmission after ligand
detects HIV and other viruses, and bacterial and binding to the extracellular domain (Kawai and
fungal cells (Ota et al. 2018; de Jong et al. 2010; Akira 2010). These elements of protein architec-
Tateno et al. 2010). In a likelihood phylogeny, ture and function are analogous to some of the
langerin C-type lectin domains from mammals nucleotide-binding, leucine-rich repeat (NLR)
form a clade (Fig. 1.8). Mammalian langerin’s receptors in plants mentioned earlier (Maekawa
conserved “EPN” amino acid motif aligns with et al. 2011; Martinon and Tschopp 2005).
the “EPN” of dectin-2, possibly conferring the Metazoan toll-like receptors are found on
same preference for binding mannans (Drickamer mammalian phagocytic cells, neutrophils,
1992). Mammal and turtle sequences share an macrophages, and dendritic cells (Fig. 1.6)
N-terminal coiled-coil domain, consistent with (Romani 2011). Immunological consequences of
polymerization of langerin into a trimer stimulation of these receptors can include produc-
(Stambach and Taylor 2003). tion of antimicrobial molecules such as
Outside of mammals, langerin homologs differ phenoloxidase in insects and nitric oxide or reac-
in structure and predicted binding specificity, tive oxygen species in mammals (Thoma-
raising questions about functional homology. Uszynski et al. 2001; Gantner et al. 2003). Recep-
Turtle, lizard, and alligator orthologs may bind tor stimulation also leads to cytokine production
preferentially to galactose rather than mannose, and inflammation (Goodridge and Underhill
implied by their “QPD” motif instead of the 2008; Romani 2004).
“EPN” in mammals (Drickamer 1992). Lizard
and alligator proteins lacked a predicted 1.3.2.1 How Did Fungal Recognition
N-terminal coiled-coil domain. Evolve
Toll proteins are shared across most Metazoa, but
their functions vary (Brennan and Gilmore 2018;
1.3.2 Toll-Like Receptors of Ancient Gauthier et al. 2010). Toll was first discovered for
Origin Duplicated its role in establishing dorsoventral polarity in
and Specialized for Recognition Drosophila embryos, but it is also important in
of Varied Pathogen Drosophila immunity to fungi (Lemaitre et al.
and Symbiont Cell Wall 1996). Toll receptors are essential for early devel-
Polysaccharides opment in nematodes and in nerve regeneration in
annelids (Brennan and Gilmore 2018). Mamma-
Toll-like receptors (TLRs) have diverse functions lian toll seem to have lost any ancestral functions
including detection of fungi and a wide range of in development.
pathogens and other symbionts, and they have Roach et al. (2005, 2013) analyzed toll-like
been thoroughly reviewed (Romani 2004; Akira receptors phylogenetically. Humans have ten of
et al. 2006; Kawai and Akira 2010; Romani these proteins (e.g., NP_001305716) (Gantner
2011). Toll-like receptors have an extracellular et al. 2003; Roach et al. 2005). Toll proteins
ligand binding region that can be considered a duplicated prior to the radiation of vertebrates,
lectin, consisting of leucine-rich repeats. Upon giving rise to more than 8 paralogous clades
binding a ligand, these proteins typically dimer- (Roach et al. 2005). Families of orthologs have
ize. Their conserved intracellular portion is a TIR been retained through vertebrate evolution, and
(Toll–interleukin 1 (IL-1) receptor) domain that is each of 8 clades of toll paralogs (families 1–5 and

ä
Fig. 1.8 (continued) vertebrate receptor clades in a maxi- bootstrap proportions. “SS” designates predicted coiled-
mum likelihood analysis. Colored boxes enclose predicted coil regions potentially involved in polymerization of
orthologs to human receptors. GenBank accession num- receptors, increasing strength and specificity of ligand
bers follow genus names. Numbers at branches are binding
18 E. D. Trudeau and M. L. Berbee

7–9) includes an orthologous gene from a human 1.3.3 Chitinases and Chitin-Binding
and a fish (Roach et al. 2005). Proteins in Metazoan
Among clades, recognition specificities Responses to Fungi
diverged. Toll-like receptor families including
1, 2, 4, 6, and 9 in vertebrates may have evolved While proteins that detect chitin in metazoans
to detect bacterial ligands, lipopeptides, peptido- may be important in antifungal defense, the
glycan, or viral DNA (Akira et al. 2006), but they supporting evidence is less clear than it is in
also contribute to mammalian recognition of fun- plants. In animals, roles of chitin recognition
gal mannans and chitin oligosaccharides proteins include defense against non-fungal
(Hatinguais et al. 2020). Toward detection of parasites, nutrition, development, and modulation
fungi, Fuchs et al. (2018) showed that purified of immune responses.
chitin oligomers, chains of at least six N-
acetylglucosamine units, triggered an immune 1.3.3.1 Chitinases in Animal Immunity
response in human and mouse cells via the toll- Under some conditions, chitinases protect
like receptor 2. However, toll-like receptor 2 is animals from chitin-containing pathogens, from
also important in binding bacterial lipopeptides nematodes and fungi (Chang et al. 2020;
(Roach et al. 2005). Complicating phylogenetic Sutherland 2018). However, in mammals, when
interpretation of ancestral recognition function, produced over too long a time period, or at too
the binding specificity of toll depends on high a level, chitinase and chitinase-like proteins
interactions with other proteins. result in asthma, harmful inflammation, and dam-
Other proteins in the toll signal transduction age to hosts. Balance seems to be necessary so
pathway are more highly conserved than the toll that protection from parasites outweighs the dam-
receptors themselves. Interactions between the age to the host (Sutherland et al. 2014; Sutherland
TIR domain in toll with a second TIR domain in 2018).
an adaptor protein, MyD88 (myeloid differentia- Animals typically have orthologs to
tion primary response protein 88), initiate signal- chitotriosidase and acidic mammalian chitinase,
ing of ligand reception. TIR domains are ancient, two enzymatically active GH18 chitinases. In
distributed among bacteria, plants, and animals addition, animals have chitinase-like proteins
(Nimma et al. 2021), and MyD88 is shared by that evolved to become lectins, having lost enzy-
animals from sponges to mammals (Brennan and matic activity while retaining the ancestral chitin-
Gilmore 2018; Gauthier et al. 2010). The signal- binding ability (Bussink et al. 2007). In
ing cascade eventually activates nuclear factor mammals, chitotriosidase (e.g., human
κB, which is widely conserved across Metazoa, NP_003456) is secreted by macrophages and
functioning in activation of transcription of other cells important in immune responses.
inflammatory cytokine genes (Gauthier et al. Chitotriosidase is concentrated in lung and gut
2010). cells where encounters with pathogenic fungi are
The example of toll-like receptor 2, which frequent. In vivo, recombinant human
recognizes either bacterial or fungal ligands, chitotriosidase improved the survival of mice
together with the great age of signaling pathways, with compromised immune systems upon chal-
suggests that detection of fungi may originate lenge with pathogenic fungi, and in vitro, it
from the far older perception of varied stimuli in reduced growth of human-pathogenic fungi (van
an immune pathway that gains flexibility and Eijk et al. 2005). However, contrary to expecta-
adapts to new threats and opportunities through tion, deficiency in chitotriosidase production
the collaboration of multiple proteins. improved survival of mice to pathogens including
the fungus Cryptococcus neoformans (Wiesner
et al. 2015).
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 19

The acidic mammalian chitinases (e.g., human of muscle fibers (Graca et al. 2022). Whether and
NP_970615) protected against nematodes in the how FIBCD1 initiates cellular signaling is
gastrointestinal tract of mice (Vannella et al. unknown (Pilecki and Moeller 2020).
2016), but they have other roles that may be The conserved structure of FIBCD1 receptors
ancestral. Acidic chitinases seem to enable some originated in early vertebrates. The human
insectivorous mammals—bats, mice, and possi- FIBCD1 has an N-terminal cytoplasmic region,
bly small primates—to digest chitin from insect a transmembrane motif, and a fibrinogen-like rec-
exoskeletons for food (Ohno et al. 2016; Janiak ognition domain (Fig. 1.6) (Shrive et al. 2014).
et al. 2018; Strobel et al. 2013). Most vertebrate genomes have only a single
A chitinase-like protein, which has lost enzy- orthologous protein that aligns across the length
matic function, contributes to decreasing the par- of the human FIBCD1 protein. Amino acid
asite load in a mouse that is infected with a sequences in vertebrates from ray-finned fishes
nematode (Sutherland et al. 2014). Chitinase- to mammals were readily alignable over
like proteins also seem to moderate immune 464 sites. The fibrinogen domain of a jawless
responses and prolong macrophage life, increas- fish, a lamprey (represented by Petromyzon
ing survival in mice infected with the bacterium XP_032825085), is perhaps orthologous, but is
Streptococcus pneumoniae (Dela Cruz et al. more distant and its N-terminus is unalignable
2012). with the other vertebrates’ sequences. Our explo-
ration of related proteins supports Doolittle
1.3.3.2 Fibrinogen C Containing et al.’s (2012) inference that FIBCD1 may first
Domain 1 (FIBCD1) and have appeared in the most recent common ances-
Recognition of Chitin tor of jawless fishes and mammals, 534–459 Ma
Fibrinogen-related domains function as lectins, (dos Reis et al. 2015). Whatever its ancestral
some of them as immune receptors in Metazoa, function, FIBCD1’s ability to bind chitin suggests
(for reviews, see Thomsen et al. 2011; Doolittle that it could be adapted easily and repeatedly for
et al. 2012; Pilecki and Moeller 2020). Somewhat recognition of fungi. From its origin in early
confusingly, the name “fibrinogen” was first fishes, an FIBCD1 chitin recognition receptor
applied to vertebrate blood clotting proteins and may have allowed vertebrates to detect chitin-
then applied to a conserved domain shared by bearing parasitic arthropods, nematodes, or fungi.
many proteins with functions unrelated to blood. LysMD3 and Chitin Recognition LysMD3 is
Two clades of proteins, membrane-bound intriguing from an evolutionary perspective
FIBCD1, and ficolins from blood serum contain because of its central role in the chitin recognition
fibrinogen-related domains and have roles in receptors of plants. He et al. (2021) showed
mammalian immune recognition of fungi. that human LysMD3 (NP_938014) in lung epi-
Ficolins bind polysaccharides including N- thelial cells binds chitin oligomers and heat-
acetylglucosamine, N-acetylgalactosamine, treated Candida albicans cells, resulting in the
β-glucans, and peptidoglycans and then activate production of cytokines and inflammation. In
the complement cascade that results in the pro- mud crabs, LysMD3 orthologs bound bacterial
duction of antimicrobial peptides (Thomsen et al. peptidoglycans and lipopolysaccharides and
2011). contributed to killing bacteria (Wang et al.
The FIBCD1 protein plays multiple physio- 2021). No role in immunity was detected for
logical roles, but relevant to fungal recognition, LysMD3 in mice (Yokoyama et al. 2018) or fish
it binds chitin in human intestinal epithelial cells (Laroche et al. 2013).
(Schlosser et al. 2009) (Fig. 1.6). It reduces or The structure of orthologs to LysMD3 is
modulates fungal growth, and Moeller et al. conserved across animals, and the phylogenies
(2019) showed that its overexpression in mice of the proteins are largely congruent with the
alters the composition of fungi in mouse guts. organismal phylogeny, consistent with vertical
Its other varied roles include promoting growth transmission of a single conserved gene copy
20 E. D. Trudeau and M. L. Berbee

(Wang et al. 2020). The extracellular LysM diversified early. Being found in fungi and widely
domain serves as the receptor (Fig. 1.6). Some distributed among invertebrates, they may date
animal LysMD3 proteins have predicted coiled- back to the divergence of fungi from animals,
coil domains, suggesting that the monomers inter- 1481–1240 Ma (Parfrey et al. 2011). Aquatic
act to form dimers or higher order polymers, as in invertebrates including filter-feeding bivalves,
plant LysM proteins. The animal LysMD3 cyto- snails, crustaceans, and sea urchins make use of
plasmic C-terminus lacks the kinase domain that these enzymes for digestion when feeding on
is important in LysM signaling in plants, leaving organisms that use β-glucans as storage products
uncertainty about how signaling proceeds in or as cell wall materials, including brown or other
animals. heterokont algae, fungi, and some bacteria
(Fig. 1.9a, b) (Linton 2020; Julius 2018). Gain
and loss of carbohydrate-binding module
1.3.4 b-glucanases: Enzymes 39, another domain that increases the affinity of
of Digestion, Recruited by the enzyme for its substrate, has occurred repeat-
Invertebrates as Immune edly. One of many possible examples comes from
Pattern-Recognition Receptors, two β-glucanases from the purple sea urchin
First of Bacteria, and Later Strongylocentrotus purpuratus. The two proteins
of Fungi arose from recent duplication based on the 92%
identity of their glucanase domains, but one pro-
β-glucanases in family GH16, subfamily tein (NP_999681) has a carbohydrate-binding
4 (GH16_4), are carbohydrate-active enzymes module while the other, XP_030836386, does
important in digestion and in defense (Viborg not (Fig. 1.9a, b).
et al. 2019). Invertebrates lack dectin-1, the A role in immunity for GH16_4 glucanases
β-glucan-binding receptor protein discussed ear- was suggested when Unestam and Söderhäll
lier for its role in vertebrate recognition of fungi. (1977) demonstrated an increase in antimicrobial
Instead, invertebrates detect fungal β-glucans phenoloxidase in serum of crayfish treated with
with homologs of GH16_4 β-glucanases (Gottar sources of β-glucans including Saccharomyces
et al. 2006), an enzyme subfamily that is entirely cerevisiae cell wall fragments. One of the several
missing from vertebrates. paralogous glucanases in the beetle Tenebrio
The enzymes have endo-1,3-β-glucanase molitor (ACS36221) (Fig. 1.9a) hydrolyzes
(EC 3.2.1.39) activity and a catalytic site with the β-1,3- and β-1,6-glucans and kills yeast cells. It
amino acid sequence “ExDxxE” (Viborg et al. is produced in the beetle midgut, so the fungi may
2019) (Fig. 1.9). The GH16_4 β-glucanases be digested as food while being prevented from

ExDxxE
A. Digestion and GH16_4 glucanase
immunity
ExDxxE
B. Digestion and CBM39 GH16_4 glucanase
immunity
C. Immune receptor CBM39 GH16_4-like
Fungi, bacteria

Fig. 1.9 β-glucanases in the glycosyl hydrolase subfam- immune response. (c) In early winged insects, duplicated
ily 16_4 serve as digestive enzymes or immune receptors. genes lost the catalytic site as they evolved to specialize in
(a, b) Enzymes with catalytic site ExDxxE and with or immunity to bacteria or fungi. The CBM39 domain is
without a carbohydrate-binding module (CBM39) are responsible for recognizing the microbial cell walls. In
shared across invertebrates, functioning in digestion of green (left), signal peptide; in gray, region of unknown
algae or fungi for nutrition or as receptors to initiate an function
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 21

causing disease (Genta et al. 2009). Perovi- chitinases are ancient building blocks that bind
ć-Ottstadt et al. (2004) found that the sponge selectively to ligands including fungal or other
Suberites domuncula responded to glucan- microbial cell wall polysaccharides. Conserved
containing microbes with increasing synthesis of binding domains of modern plant or animal
a fibrinogen and an epidermal growth factor, receptors are variously combined with transmem-
proteins with possible roles in immune responses. brane domains, coiled-coil domains, kinase
domains, or TIR domains to form multi-domain
Specialized Pattern-Recognition Receptors proteins capable of transducing ligand binding
Evolved from GH16_4 Glucanases The into cellular responses such as antifungal
GH16_4 glucanases from winged insects that defenses.
retain the catalytic motif ExDxxE (Fig. 1.9a, b) We speculate that terrestrialization beginning
form a clade nested among other invertebrates. 450 Ma exposed plants, vertebrates, and insects to
(Hughes 2012). Also nested among other increased threats from fungal pathogens while
invertebrates is a second clade of insect GH16_4 offering increased opportunities for fungal
glucanase-like proteins that lost the catalytic mutualisms. Early plant and animal evolution
motif. Rather than β-glucan digestion, these took place in water, where selective pressure
proteins specialized as receptors that recognize from viruses, bacteria, and other eukaryotes may
microbial cell walls (Fig. 1.9c) (Hughes 2012; have been more significant than pressure from
Lee et al. 1996). The clade of receptor proteins fungi. This changed with the invasion of land.
spans Orthoptera (Timema shepardi The LysM receptor kinases shared among
CAD7264458) and Diptera (Drosophila streptophyte algae and land plants may have
melanogaster AAF49244), suggesting an age of evolved as part of an early system of defense
413–360 Ma (Misof et al. 2014). Among these against aquatic fungi, later bolstered among land
specialized receptors, a CBM39 domain has been plants by expansion of GH19 chitinases
shown to be essential for glucan binding (Lee paralleling the diversification of terrestrial fungi.
et al. 1996). Few aquatic fungi target vertebrates, and most
fungal pathogens of fishes are derived from ter-
Additional gene duplications among winged restrial ancestors (Gozlan et al. 2014). In early
insects gave rise to proteins with even more terrestrial vertebrates, specificity in fungal recog-
specialized functions. Drosophila Gram-negative nition increased with the origin of dectin-1 to bind
binding protein-3 (NP_523986) (Fig. 1.9c) pref- fungal β-glucans. In early winged insects, fungal
erentially binds fungal β-glucans (Gottar et al. recognition increased through adaptation of
2006). Gottar et al.’s (2006) experiments showed β-glucanases to detect β-glucans (Fig. 1.10).
that isolated protein-3 binds to β-glucans.
Overexpression of “protein-3” induced expres-
sion of drosomycin, an antimicrobial toxin. Flies 1.4.1 Limitations to Inferences About
with a null mutation in the binding protein were Functional Homology
more sensitive to fungal infection compared with
wild-type flies, producing lower levels of antimi- Inferences about ancestral ligand binding rely on
crobial proteins (Gottar et al. 2006). Whether knowledge of specificity of modern receptors.
related insects share an ability for specific detec- However, information on specificity of modern
tion of fungi is currently unclear. receptors is rarely available for a full range of
potential immunogenic compounds from fungi,
bacteria, or viruses. Whole genome sequences
1.4 Conclusions offer a terrific resource for tracking homologs to
well-studied proteins, but experimental evidence
As summarized in Fig. 1.10, domains including for homologous function in non-model species is
LysM, leucine-rich repeats, C-type lectins, and rarely available. We acknowledge limitations of
22 E. D. Trudeau and M. L. Berbee

Fig. 1.10 Origins of fungi and their recognition by plant chitooligosaccharides, after possible origin as fungal
or animal hosts. Arrows indicate age estimates based on hormones, were later recruited to signal plants to initiate
shared presence in clades, e.g., LysM motifs are shared by mycorrhizae. The rise of terrestrial fungi stimulated evolu-
and thus are inferred to have originated in a common tion of plant and animal recognition systems to detect new
ancestor of bacteria and eukaryotes more than three billion potential fungal pathogens and mutualists. (c) In the ances-
years ago (Battistuzzi et al. 2004; Knoll 2014). Dashed tor of streptophyte algae (in Charophyceae) and land
lines are confidence intervals, where available, on ages of plants, protein domain shuffling combined an ancient
taxon divergence from the literature. (a) Protein domains LysM domain with a kinase, resulting in LysM receptor
from these receptors and enzymes may have been present kinases that detect bacterial and fungal cell walls. New
in the common ancestor of cellular life. (b) Lipo- classes of chitinase paralogs (see Fig. 1.5) important in
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 23

this approach but hope for improvement. Mutant (Lu et al. 2020) and UniProt www.uniprot.org
studies to test gene function in non-model (UniProt Consortium 2021). We aligned the
organisms are becoming easier due to CRISPR- sequences using the L-INS-i model in MAFFT
Cas9 technology (Zhang et al. 2014). Increas- www.mafft.cbrc.jp/alignment/software (Katoh
ingly, sophisticated X-ray crystallographic stud- et al. 2019) with default settings and removing
ies of plant or mammal proteins provide sequences that could not be aligned.
information about amino acids in receptor The phylogenetic analysis included only the
proteins that bind fungal cell wall GH19 domains from the aligned sequences. To
polysaccharides. Glycan microarrays using trim the sequences, we used the hmmalign com-
libraries of sugar-containing molecules have mand with --trim option in HMMER 3.3.2 www.
worked well to test binding profiles of receptors hmmer.org against the GH19 HMM profile
such as human C-type lectins (Vendele et al. downloaded from the dbCAN2 HMM database
2020; Yamasaki et al. 2009). v.11 www.bcb.unl.edu/dbCAN2 (Zhang et al.
2018). We carried out a preliminary analysis
using IQTree www.iqtree.org (Minh et al. 2020)
1.5 Materials and Methods: Protein with 100 search replicates using the program’s
Sampling and Phylogenies model testing option. We excluded all but one
sequence from among clades of similar sequences
1.5.1 Phylogenetic Analysis of Plant from the same species, and carried out a final
GH19 Chitinases maximum likelihood analysis using IQTree with
100 search replicates and the WAG+I + G4
Our chitinase taxon sampling included plants, model, followed by ultrafast bootstrap analysis
multiple algal lineages, and bacteria. We started with 1000 bootstrap replicates.
by assembling representatives of seed plant GH19
chitinases from each class from among
characterized Arabidopsis sequences from the 1.5.2 Phylogenetic Analysis
Carbohydrate-Active Enzymes database www. of Animal C-Type Lectin-Like
cazy.org (Drula et al. 2022). To search for Domains, Taxon and Gene
homologs in glaucophyte, red, chlorophyte, and Sampling
streptophyte algae, and in bryophyte, lycophyte,
and fern embryophytes, we used BLAST searches We focused on dectin-1 receptors of β-1,3- and
with the Arabidopsis sequences as queries. We β-1,6-glucans due to their importance in fungal
also included characterized sequences from bac- recognition. To evaluate dectin-1’s monophyly,
teria and diatoms. For analysis, we selected we included sequences from two related proteins:
sequences from BLAST results using an e-value dectin-2 (CLEC6A) that binds mannose and from
cutoff of 1e-08 that had the appropriate domains langerin (CLEC4K) that binds mannose,
as predicted by CDD www.ncbi.nlm.nih.gov/cdd β-1,3-glucans, and N-acetylglucosamine (van

ä
Fig. 1.10 (continued) antifungal defenses arose as land vertebrates. Diptera and other flying insects recognize
plants evolved. (d) Vertebrate receptors for cell wall fungi with paralogs of β-glucanases that lost enzymatic
polysaccharides may have originated for detection of function. Sources of divergence dates for species trees are:
aquatic, non-fungal parasites, but proteins like FIBCD1 Fungi (Chang et al. 2015); plants and algae (Morris et al.
and Toll-like receptor 2 also adapted to detect fungal chitin 2018); vertebrates (dos Reis et al. 2015); insects (Misof
oligomers. Dectin-1 recognizes and signals detection of et al. 2014); and opisthokonts (Parfrey et al. 2011). Num-
fungal β-glucan. (e) Invertebrates recruited receptor bers are millions of years ago (Ma); Precambrian is not to
proteins from β-glucanases that had ancestral functions in scale with Paleozoic
digestion of food. β-glucanases were lost from early
24 E. D. Trudeau and M. L. Berbee

den Berg et al. 2012). This was not an exhaustive Acknowledgments Mary Berbee acknowledges funding
study of related proteins; e.g., we did not analyze from a Natural Sciences and Engineering Research Coun-
cil of Canada Discovery Frontiers Grant RGPIN-2022-
DC-SIGN or Mannose receptor (van den Berg 03813. We thank Carol Munro (University of Aberdeen)
et al. 2012). for a review of this manuscript, and Vivian Miao and
We used BLAST searches with the amino acid members of the Berbee lab for constructive comments.
sequences from pattern-recognition receptors
from well-characterized human or insect hosts as
queries. Our ingroup selection consisted of pro- References
tein sequences of taxa that shared domains and
motifs important in recognition function. Graphic Adachi Y, Ishii T, Ikeda Y, Hoshino A, Tamura H,
Aketagawa J, Tanaka S, Ohno N (2004) Characteriza-
representations, scores from the BLAST results, tion of β-glucan recognition site on C-type lectin,
and NCBI distance trees were helpful in dectin 1. Infect Immun 72(7):4159–4171. https://doi.
recognizing and selecting likely orthologous and org/10.1128/IAI.72.7.4159-4171.2004
paralogous sequences relative to the query. To Adrangi S, Faramarzi MA (2013) From bacteria to human:
a journey into the world of chitinases. Biotechnol Adv
track early origins of host recognition proteins, 31(8):1786–1795. https://doi.org/10.1016/j.
we repeated BLAST searches excluding succes- biotechadv.2013.09.012
sively broader clades of taxa, starting for example Akira S, Uematsu S, Takeuchi O (2006) Pathogen recog-
by excluding humans if the query was from a nition and innate immunity. Cell 124(4):783–801.
https://doi.org/10.1016/j.cell.2006.02.015
human, then excluding mammals, then excluding Amses KR, Simmons DR, Longcore JE, Mondo SJ,
amniotes, and so on. We searched for proteins in Seto K, Jerônimo GH, Bonds AE, Quandt CA, Davis
BLAST searches restricted to species to complete WJ, Chang Y (2022) Diploid-dominant life cycles
our datasets. As outgroups, we selected characterize the early evolution of Fungi. Proc Natl
Acad Sci U S A 119(36):e2116841119
paralogous proteins and proteins from distantly Anand G, Leibman-Markus M, Elkabetz D, Bar M (2021)
related organisms, usually invertebrates. Method for the production and purification of plant
We aligned amino acid sequences using immuno-active xylanase from Trichoderma. Int J Mol
MAFFT as described above. For domain structure Sci 22(8). https://doi.org/10.3390/ijms22084214
Ausubel FM (2005) Are innate immune signaling
predictions, we used NCBI CDD searches pathways in plants and animals conserved? Nat
(Lu et al. 2020) and InterPro https://www.ebi.ac. Immunol 6(10):973–979. https://doi.org/10.1038/
uk/interpro/ (Blum et al. 2021). We used ni1253
Waggawagga https://waggawagga.motorprotein. Battistuzzi FU, Feijao A, Hedges SB (2004) A genomic
timescale of prokaryote evolution: insights into the
de/ to predict coiled-coil protein structures (Simm origin of methanogenesis, phototrophy, and the coloni-
et al. 2015). Several of the immune receptors that zation of land. BMC Evol Biol 4(1):44. https://doi.org/
we analyzed were multi-domain proteins. We 10.1186/1471-2148-4-44
aligned only the conserved C-type lectin domain Blum M, Chang H-Y, Chuguransky S, Grego T,
Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T,
of vertebrate dectin-1 with distant homologs from Qureshi M, Raj S, Richardson L, Salazar GA,
invertebrates, excluding other domains of uncer- Williams L, Bork P, Bridge A, Gough J, Haft DH,
tain homology. We annotated positions of amino Letunic I, Marchler-Bauer A, Mi H, Natale DA,
acids that are functionally important in substrate Necci M, Orengo CA, Pandurangan AP, Rivoire C,
Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto
binding. SCE, Wu CH, Bateman A, Finn RD (2021) The
For phylogenetic analysis, we used raxmlGUI InterPro protein families and domains database:
2.0 (Edler et al. 2021), applying the program’s 20 years on. Nucleic Acids Res 49(D1):D344–D354.
model testing, and then using the selected model https://doi.org/10.1093/nar/gkaa977
Boddy L (2016) Pathogens of autotrophs. In: Watkinson
in a maximum likelihood analysis, followed by SC, Boddy L, Money NP (eds) The fungi, 3rd edn.
100 bootstrap replicates. Results were generally Academic Press, Waltham, MA, pp 245–292. https://
consistent with monophyly of dectin-1, dectin-2, doi.org/10.1016/b978-0-12-382034-1.00008-6
and langerin, and with the organismal phylogeny. Bonfante P, Requena N (2011) Dating in the dark: how
roots respond to fungal signals to establish arbuscular
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 25

mycorrhizal symbiosis. Curr Opin Plant Biol 14(4): Cheng H, Shao Z, Lu C, Duan D (2021) Genome-wide
451–457. https://doi.org/10.1016/j.pbi.2011.03.014 identification of chitinase genes in Thalassiosira
Brennan JJ, Gilmore TD (2018) Evolutionary origins of pseudonana and analysis of their expression under
Toll-like receptor signaling. Mol Biol Evol 35(7): abiotic stresses. BMC Plant Biol 21(1):87. https://doi.
1576–1587. https://doi.org/10.1093/molbev/msy050 org/10.1186/s12870-021-02849-2
Bressendorff S, Azevedo R, Kenchappa CS, Ponce de Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK,
Leon I, Olsen JV, Rasmussen MW, Erbs G, Newman Rasmussen U, Vad K (1993) Plant chitinases. Plant J
MA, Petersen M, Mundy J (2016) An innate immunity 3(1):31–40. https://doi.org/10.1046/j.1365-313x.1993.
pathway in the moss Physcomitrella patens. Plant Cell t01-1-00999.x
28(6):1328–1342. https://doi.org/10.1105/tpc.15. Cope KR, Bascaules A, Irving TB, Venkateshwaran M,
00774 Maeda J, Garcia K, Rush TA, Ma C, Labbe J, Jawdy S,
Brown GD, Gordon S (2001) A new receptor for Steigerwald E, Setzke J, Fung E, Schnell KG, Wang Y,
β-glucans. Nature 413(6851):36–37. https://doi.org/ Schlief N, Bucking H, Strauss SH, Maillet F, Jargeat P,
10.1038/35092620 Becard G, Puech-Pages V, Ane JM (2019) The
Brown GD, Willment JA, Whitehead L (2018) C-type ectomycorrhizal fungus Laccaria bicolor produces
lectins in immunity and homeostasis. Nat Rev lipochitooligosaccharides and uses the common sym-
Immunol 18(6):374–389. https://doi.org/10.1038/ biosis pathway to colonize Populus roots. Plant Cell
s41577-018-0004-8 31(10):2386–2410. https://doi.org/10.1105/tpc.18.
Buchmann K (2014) Evolution of innate immunity: clues 00676
from invertebrates via fish to mammals. Front de Jong MA, Vriend LE, Theelen B, Taylor ME,
Immunol 5. https://doi.org/10.3389/fimmu.2014. Fluitsma D, Boekhout T, Geijtenbeek TB (2010)
00459 C-type lectin Langerin is a β-glucan receptor on
Buendia L, Girardin A, Wang T, Cottret L, Lefebvre B human Langerhans cells that recognizes opportunistic
(2018) LysM receptor-like kinase and LysM receptor- and pathogenic fungi. Mol Immunol 47(6):1216–1225.
like protein families: an update on phylogeny and https://doi.org/10.1016/j.molimm.2009.12.016
functional characterization. Front Plant Sci 9:1531. De Schutter K, Van Damme EJM (2015) Protein-
https://doi.org/10.3389/fpls.2018.01531 carbohydrate interactions as part of plant defense and
Bussink AP, Speijer D, Aerts J, Boot RG (2007) Evolution animal immunity. Molecules 20(5):9029–9053. https://
of mammalian chitinase (-like) members of family doi.org/10.3390/molecules20059029
18 glycosyl hydrolases. Genetics 177(2):959–970. de Wit PJ (2007) How plants recognize pathogens and
https://doi.org/10.1534/genetics.107.075846 defend themselves. Cell Mol Life Sci 64(21):
Carotenuto G, Chabaud M, Miyata K, Capozzi M, 2726–2732. https://doi.org/10.1007/s00018-007-
Takeda N, Kaku H, Shibuya N, Nakagawa T, Barker 7284-7
DG, Genre A (2017) The rice LysM receptor-like Dela Cruz CS, Liu W, He CH, Jacoby A, Gornitzky A,
kinase OsCERK1 is required for the perception of Ma B, Flavell R, Lee CG, Elias JA (2012) Chitinase
short-chain chitin oligomers in arbuscular mycorrhizal 3-like-1 promotes Streptococcus pneumoniae killing
signaling. New Phytol 214(4):1440–1446. https://doi. and augments host tolerance to lung antibacterial
org/10.1111/nph.14539 responses. Cell Host Microbe 12(1):34–46. https://
Casadevall A (2005) Fungal virulence, vertebrate endo- doi.org/10.1016/j.chom.2012.05.017
thermy, and dinosaur extinction: Is there a connection? Delaux PM, Schornack S (2021) Plant evolution driven by
Fungal Genet Biol 42(2):98–106. https://doi.org/10. interactions with symbiotic and pathogenic microbes.
1016/j.fgb.2004.11.008 Science 371(6531). https://doi.org/10.1126/science.
Chang Y, Wang S, Sekimoto S, Aerts AL, Choi C, aba6605
Clum A, LaButti KM, Lindquist EA, Yee Ngan C, Delaux PM, Radhakrishnan GV, Jayaraman D, Cheema J,
Ohm RA (2015) Phylogenomic analyses indicate that Malbreil M, Volkening JD, Sekimoto H, Nishiyama T,
early fungi evolved digesting cell walls of algal Melkonian M, Pokorny L, Rothfels CJ, Sederoff HW,
ancestors of land plants. Genome Biol Evol 7(6): Stevenson DW, Surek B, Zhang Y, Sussman MR,
1590–1601. https://doi.org/10.1093/gbe/evv090 Dunand C, Morris RJ, Roux C, Wong GK, Oldroyd
Chang Y, Desiro A, Na H, Sandor L, Lipzen A, Clum A, GE, Ane JM (2015) Algal ancestor of land plants was
Barry K, Grigoriev IV, Martin FM, Stajich JE, Smith preadapted for symbiosis. Proc Natl Acad Sci U S A
ME, Bonito G, Spatafora JW (2019) Phylogenomics of 112(43):13390–13395. https://doi.org/10.1073/pnas.
Endogonaceae and evolution of mycorrhizas within 1515426112
Mucoromycota. New Phytol 222(1):511–525. https:// Doolittle RF, McNamara K, Lin K (2012) Correlating
doi.org/10.1111/nph.15613 structure and function during the evolution of
Chang D, Sharma L, Dela Cruz CS (2020) Chitotriosidase: fibrinogen-related domains. Protein Sci 21(12):
a marker and modulator of lung disease. Eur Respir 1808–1823. https://doi.org/10.1002/pro.2177
Rev 29(156):190143. https://doi.org/10.1183/ dos Reis M, Thawornwattana Y, Angelis K, Telford MJ,
16000617.0143-2019 Donoghue PCJ, Yang ZH (2015) Uncertainty in the
timing of origin of animals and the limits of precision
26 E. D. Trudeau and M. L. Berbee

in molecular timescales. Curr Biol 25(22):2939–2950. 519–533. https://doi.org/10.1111/j.1525-142X.2010.

https://doi.org/10.1016/j.cub.2015.09.066 00436.x
Drickamer K (1992) Engineering galactose-binding activ- Genta FA, Bragatto I, Terra WR, Ferreira C (2009) Purifi-
ity into a C-type mannose-binding protein. Nature cation, characterization and sequencing of the major
360(6400):183–186. https://doi.org/10.1038/360183a0 β-1,3-glucanase from the midgut of Tenebrio molitor
Drula E, Garron ML, Dogan S, Lombard V, Henrissat B, larvae. Insect Biochem Mol Biol 39(12):861–874.
Terrapon N (2022) The carbohydrate-active enzyme https://doi.org/10.1016/j.ibmb.2009.10.003
database: functions and literature. Nucleic Acids Res Gong BQ, Wang FZ, Li JF (2020) Hide-and-seek: Chitin-
50(D1):D571–D577. https://doi.org/10.1093/nar/ triggered plant immunity and fungal counterstrategies.
gkab1045 Trends Plant Sci 25(8):805–816. https://doi.org/10.
Edler D, Klein J, Antonelli A, Silvestro D (2021) 1016/j.tplants.2020.03.006
raxmlGUI 2.0: a graphical interface and toolkit for González LMG, El Kayal W, Morris JS, Cooke JEK
phylogenetic analyses using RAxML. Methods Ecol (2015) Diverse chitinases are invoked during the
Evol 12(2):373–377. https://doi.org/10.1111/ activity-dormancy transition in spruce. Tree Genet
2041-210X.13512 Genom 11:3. https://doi.org/10.1007/s11295-015-
Erwig LP, Gow NAR (2016) Interactions of fungal 0871-0
pathogens with phagocytes. Nat Rev Microbiol 14(3): Goodridge HS, Underhill DM (2008) Fungal recognition
163–176. https://doi.org/10.1038/nrmicro.2015.21 by TLR2 and dectin-1. In: Bauer S, Hartmann G (eds)
Feinberg H, Powlesland AS, Taylor ME, Weis WI (2010) Toll-like receptors (TLRs) and innate immunity.
Trimeric structure of langerin. J Biol Chem 285(17): Springer, Berlin, pp 87–109. https://doi.org/10.1007/
13285–13293. https://doi.org/10.1074/jbc.M109. 978-3-540-72167-3_5
086058 Goodridge HS, Reyes CN, Becker CA, Katsumoto TR,
Feinberg H, Jégouzo SA, Rex MJ, Drickamer K, Weis WI, Ma J, Wolf AJ, Bose N, Chan AS, Magee AS,
Taylor ME (2017) Mechanism of pathogen recognition Danielson ME, Weiss A, Vasilakos JP, Underhill DM
by human dectin-2. J Biol Chem 292(32): (2011) Activation of the innate immune receptor
13402–13414. https://doi.org/10.1074/jbc.M117. dectin-1 upon formation of a ‘phagocytic synapse’.
799080 Nature 472(7344):471–475. https://doi.org/10.1038/
Feng F, Sun J, Radhakrishnan GV, Lee T, Bozsoki Z, nature10071
Fort S, Gavrin A, Gysel K, Thygesen MB, Andersen Gordon GL, Phillips MW (1998) The role of anaerobic gut
KR, Radutoiu S, Stougaard J, Oldroyd GED (2019) A fungi in ruminants. Nutr Res Rev 11(1):133–168
combination of chitooligosaccharide and Gottar M, Gobert V, Matskevich AA, Reichhart J-M,
lipochitooligosaccharide recognition promotes Wang C, Butt TM, Belvin M, Hoffmann JA, Ferrandon
arbuscular mycorrhizal associations in Medicago D (2006) Dual detection of fungal infections in Dro-
truncatula. Nat Commun 10(1):5047. https://doi.org/ sophila via recognition of glucans and sensing of viru-
10.1038/s41467-019-12999-5 lence factors. Cell 127(7):1425–1437. https://doi.org/
Fisher JF, Mobashery S (2020) Constructing and 10.1016/j.cell.2006.10.046
deconstructing the bacterial cell wall. Protein Sci Gow NAR, Latge JP, Munro CA (2017) The fungal
29(3):629–646. https://doi.org/10.1002/pro.3737 cell wall: structure, biosynthesis, and function. Micro-
Fuchs K, Cardona Gloria Y, Wolz O-O, Herster F, biology. Spectrum 5:3. https://doi.org/10.1128/
Sharma L, Dillen CA, Täumer C, Dickhöfer S, microbiolspec.FUNK-0035-2016
Bittner Z, Dang T-M, Singh A, Haischer D, Schlöffel Gozlan RE, Marshall WL, Lilje O, Jessop CN, Gleason
MA, Koymans KJ, Sanmuganantham T, Krach M, FH, Andreou D (2014) Current ecological understand-
Roger T, Le Roy D, Schilling NA, Frauhammer F, ing of fungal-like pathogens of fish: What lies beneath?
Miller LS, Nürnberger T, LeibundGut-Landmann S, Front Microbiol 5:62. https://doi.org/10.3389/fmicb.
Gust AA, Macek B, Frank M, Gouttefangeas C, Dela 2014.00062
Cruz CS, Hartl D, Weber AN (2018) The fungal ligand Graca FA, Rai M, Hunt LC, Stephan A, Wang Y-D,
chitin directly binds TLR2 and triggers inflammation Gordon B, Wang R, Quarato G, Xu B, Fan Y (2022)
dependent on oligomer size. EMBO Rep 19(12): The myokine Fibcd1 is an endogenous determinant of
e46065. https://doi.org/10.15252/embr.201846065 myofiber size and mitigates cancer-induced myofiber
Gantner BN, Simmons RM, Canavera SJ, Akira S, atrophy. Nat Commun 13(1):1–22. https://doi.org/10.
Underhill DM (2003) Collaborative induction of 1038/s41467-022-30120-1
inflammatory responses by dectin-1 and toll-like recep- Greeff C, Roux M, Mundy J, Petersen M (2012) Receptor-
tor 2. J Exp Med 197(9):1107–1117. https://doi.org/10. like kinase complexes in plant innate immunity. Front
1084/jem.20021787 Plant Sci 3:209. https://doi.org/10.3389/fpls.2012.
Gauthier MEA, Du Pasquier L, Degnan BM (2010) The 00209
genome of the sponge Amphimedon queenslandica Hamel F, Boivin R, Tremblay C, Bellemare G (1997)
provides new perspectives into the origin of toll-like Structural and evolutionary relationships among
and interleukin 1 receptor pathways. Evol Dev 12(5): chitinases of flowering plants. J Mol Evol 44(6):
614–624. https://doi.org/10.1007/pl00006184
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 27

Han B, Zhou K, Li Z, Sun B, Ni Q, Meng X, Pan G, Li C, N (2006) Plant cells recognize chitin fragments for
Long M, Li T, Zhou C, Li W, Zhou Z (2016) Charac- defense signaling through a plasma membrane recep-
terization of the first fungal glycosyl hydrolase family tor. Proc Natl Acad Sci U S A 103(29):11086–11091.
19 chitinase (NbchiA) from Nosema bombycis (Nb). J https://doi.org/10.1073/pnas.0508882103
Eukaryot Microbiol 63(1):37–45. https://doi.org/10. Kalia N, Singh J, Kaur M (2021) The role of dectin-1 in
1111/jeu.12246 health and disease. Immunobiology 226(2):152071.
Hatinguais R, Willment JA, Brown GD (2020) PAMPs of https://doi.org/10.1016/j.imbio.2021.152071
the fungal cell wall and mammalian PRRs. Curr Top Kasprzewska A (2003) Plant chitinases – regulation and
Microbiol Immunol 425:187–223. https://doi.org/10. function. Cell Mol Biol Lett 8(3):809–824
1007/82_2020_201 Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online
He X, Howard BA, Liu Y, Neumann AK, Li L, Menon N, service: multiple sequence alignment, interactive
Roach T, Kale SD, Samuels DC, Li H, Kite T, Kita H, sequence choice and visualization. Brief Bioinform
Hu TY, Luo M, Jones CN, Okaa UJ, Squillace DL, 20(4):1160–1166. https://doi.org/10.1093/bib/bbx108
Klein BS, Lawrence CB (2021) LYSMD3: a mamma- Kawai T, Akira S (2010) The role of pattern-recognition
lian pattern recognition receptor for chitin. Cell Rep receptors in innate immunity: update on Toll-like
36(3). https://doi.org/10.1016/j.celrep.2021.109392 receptors. Nat Immunol 11(5):373–384. https://doi.
Hoell IA, Dalhus B, Heggset EB, Aspmo SI, Eijsink VG org/10.1038/ni.1863
(2006) Crystal structure and enzymatic properties of a Khokhani D, Carrera Carriel C, Vayla S, Irving TB,
bacterial family 19 chitinase reveal differences from Stonoha-Arther C, Keller NP, Ane JM (2021)
plant enzymes. FEBS J 273(21):4889–4900. https:// Deciphering the chitin code in plant symbiosis,
doi.org/10.1111/j.1742-4658.2006.05487.x defense, and microbial networks. Annu Rev Microbiol
Hoffmann JA (2003) The immune response of Drosophila. 75:583–607. https://doi.org/10.1146/annurev-micro-
Nature 426(6962):33–38. https://doi.org/10.1038/ 051921-114809
nature02021 Kitaoku Y, Taira T, Numata T, Ohnuma T, Fukamizo T
Hromadova D, Soukup A, Tylova E (2021) (2022) Structure, mechanism, and phylogeny of LysM-
Arabinogalactan proteins in plant roots - an update on chitinase conjugates specifically found in fern plants.
possible functions. Front Plant Sci 12:674010. https:// Plant Sci 321:111310. https://doi.org/10.1016/j.
doi.org/10.3389/fpls.2021.674010 plantsci.2022.111310
Hughes AL (2012) Evolution of the βGRP/GNBP/β-1, Knoll AH (2014) Paleobiological perspectives on early
3-glucanase family of insects. Immunogenetics 64(7): eukaryotic evolution. Cold Spring Harb Perspect Biol
549–558. https://doi.org/10.1007/s00251-012-0610-8 6(1):1–14. https://doi.org/10.1101/cshperspect.
Inamine S, Onaga S, Ohnuma T, Fukamizo T, Taira T a016121
(2015) Purification, cDNA cloning, and characteriza- Laroche F, Tulotta C, Lamers G, Meijer A, Yang P,
tion of LysM-containing plant chitinase from horsetail Verbeek F, Blaise M, Stougaard J, Spaink H (2013)
(Equisetum arvense). Biosci Biotechnol Biochem The embryonic expression patterns of zebrafish genes
79(8):1296–1304. https://doi.org/10.1080/09168451. encoding LysM-domains. Gene Expr Patterns 13(7):
2015.1025693 212–224. https://doi.org/10.1016/j.gep.2013.02.007
James TY, Berbee ML (2012) No jacket required–new Lee W-J, Lee J-D, Kravchenko VV, Ulevitch RJ, Brey PT
fungal lineage defies dress code: recently described (1996) Purification and molecular cloning of an induc-
zoosporic fungi lack a cell wall during trophic phase. ible gram-negative bacteria-binding protein from the
BioEssays 34(2):94–102. https://doi.org/10.1002/bies. silkworm, Bombyx mori. Proc Natl Acad Sci U S A
201100110 93(15):7888–7893. https://doi.org/10.1073/pnas.93.
Janiak MC, Chaney ME, Tosi AJ (2018) Evolution of 15.7888
acidic mammalian chitinase genes (CHIA) is related Lefebvre B, Klaus-Heisen D, Pietraszewska-Bogiel A,
to body mass and insectivory in primates. Mol Biol Herve C, Camut S, Auriac MC, Gasciolli V,
Evol 35(3):607–622. https://doi.org/10.1093/molbev/ Nurisso A, Gadella TW, Cullimore J (2012) Role of
msx312 N-glycosylation sites and CXC motifs in trafficking of
Jiang X, Bao H, Merzendorfer H, Yang Q (2019) Immune Medicago truncatula Nod factor perception protein to
responses of mammals and plants to chitin-containing plasma membrane. J Biol Chem 287(14):
pathogens. Adv Exp Med Biol 1142:61–81. https://doi. 10812–10823. https://doi.org/10.1074/jbc.M111.
org/10.1007/978-981-13-7318-3_4 281634
Julius ML (2018) Chapter 6 - Carbohydrate diversity LeibundGut-Landmann S, Groß O, Robinson MJ,
in microalgae: a phylogenetically arranged Osorio F, Slack EC, Tsoni SV, Schweighoffer E,
presentation. In: Levine IA, Fleurence J (eds) Tybulewicz V, Brown GD, Ruland J, Reis e Sousa C
Microalgae in health and disease prevention. Aca- (2007) Syk- and CARD9-dependent coupling of innate
demic, pp 133–144. https://doi.org/10.1016/B978-0- immunity to the induction of T helper cells that pro-
12-811405-6.00006-2 duce interleukin 17. Nat Immunol 8(6):630–638.
Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto- https://doi.org/10.1038/ni1460
Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya
28 E. D. Trudeau and M. L. Berbee

Lemaitre B, Nicolas E, Michaut L, Reichhart J-M, 216(12):2689–2700. https://doi.org/10.1084/jem.

Hoffmann JA (1996) The dorsoventral regulatory 20182244
gene cassette spätzle/Toll/cactus controls the potent Morris JL, Puttick MN, Clark JW, Edwards D, Kenrick P,
antifungal response in Drosophila adults. Cell 86(6): Pressel S, Wellman CH, Yang ZH, Schneider H,
973–983. https://doi.org/10.1016/S0092-8674(00) Donoghue PCJ (2018) The timescale of early land
80172-5 plant evolution. Proc Natl Acad Sci U S A 115(10):
Limper AH, Adenis A, Le T, Harrison TS (2017) Fungal E2274–E2283. https://doi.org/10.1073/pnas.
infections in HIV/AIDS. Lancet Infect Dis 17(11): 1719588115
e334–e343. https://doi.org/10.1016/S1473-3099(17) Mulisch M (1993) Chitin in protistan organisms. Eur J
30303-1 Protistol 29(1):1–18. https://doi.org/10.1016/s0932-
Linton SM (2020) Review: the structure and function of 4739(11)80291-9
cellulase (endo-β-1,4-glucanase) and hemicellulase (- Nelsen MP, Lücking R, Boyce CK, Lumbsch HT, Ree RH
β-1,3-glucanase and endo-β-1,4-mannase) enzymes in (2020) No support for the emergence of lichens prior to
invertebrates that consume materials ranging from the evolution of vascular plants. Geobiology 18(1):
microbes, algae to leaf litter. Comp Biochem 3–13
Physiol B: Biochem Mol Biol 240:110354. https:// Ngou BPM, Jones JDG, Ding P (2022) Plant immune
doi.org/10.1016/j.cbpb.2019.110354 networks. Trends Plant Sci 27(3):255–273. https://
Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, doi.org/10.1016/j.tplants.2021.08.012
Gonzales NR, Gwadz M, Hurwitz DI, Marchler GH, Nimma S, Gu W, Maruta N, Li Y, Pan M, Saikot FK, Lim
Song JS, Thanki N, Yamashita RA, Yang M, Zhang D, BYJ, McGuinness HY, Zaoti ZF, Li S, Desa S, Manik
Zheng C, Lanczycki CJ, Marchler-Bauer A (2020) MK, Nanson JD, Kobe B (2021) Structural evolution
CDD/SPARCLE: the conserved domain database in of TIR-domain signalosomes. Front Immunol 12.
2020. Nucleic Acids Res 48(D1):D265–d268. https:// https://doi.org/10.3389/fimmu.2021.784484
doi.org/10.1093/nar/gkz991 Nishiyama T, Sakayama H, de Vries J, Buschmann H,
Lutzoni F, Nowak MD, Alfaro ME, Reeb V, Saint-Marcoux D, Ullrich KK, Haas FB,
Miadlikowska J, Krug M, Arnold AE, Lewis LA, Vanderstraeten L, Becker D, Lang D, Vosolsobe S,
Swofford DL, Hibbett D (2018) Contemporaneous Rombauts S, Wilhelmsson PKI, Janitza P, Kern R,
radiations of fungi and plants linked to symbiosis. Heyl A, Rumpler F, Villalobos L, Clay JM,
Nat Commun 9(1):5451 Skokan R, Toyoda A, Suzuki Y, Kagoshima H,
Maekawa T, Kufer TA, Schulze-Lefert P (2011) NLR Schijlen E, Tajeshwar N, Catarino B, Hetherington
functions in plant and animal immune systems: so far AJ, Saltykova A, Bonnot C, Breuninger H,
and yet so close. Nat Immunol 12(9):817–826. https:// Symeonidi A, Radhakrishnan GV, Van
doi.org/10.1038/ni.2083 Nieuwerburgh F, Deforce D, Chang C, Karol KG,
Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Hedrich R, Ulvskov P, Glockner G, Delwiche CF,
Gueunier M, Cromer L, Giraudet D, Formey D, Petrasek J, Van de Peer Y, Friml J, Beilby M,
Niebel A, Martinez EA, Driguez H, Becard G, Denarie Dolan L, Kohara Y, Sugano S, Fujiyama A, Delaux
J (2011) Fungal lipochitooligosaccharide symbiotic PM, Quint M, Theissen G, Hagemann M, Harholt J,
signals in arbuscular mycorrhiza. Nature 469(7328): Dunand C, Zachgo S, Langdale J, Maumus F, Van Der
58–63. https://doi.org/10.1038/nature09622 Straeten D, Gould SB, Rensing SA (2018) The Chara
Martinon F, Tschopp J (2005) NLRs join TLRs as innate genome: secondary complexity and implications for
sensors of pathogens. Trends Immunol 26(8):447–454. plant terrestrialization. Cell 174(2):448–464 e424.
https://doi.org/10.1016/j.it.2005.06.004 https://doi.org/10.1016/j.cell.2018.06.033
Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B,
Woodhams MD, von Haeseler A, Lanfear R (2020) Mitsutomi M, Watanabe T (1996) A modular family
IQ-TREE 2: new models and efficient methods for 19 chitinase found in the prokaryotic organism Strep-
phylogenetic inference in the genomic era. Mol Biol tomyces griseus HUT 6037. J Bacteriol 178(17):
Evol 37(5):1530–1534. https://doi.org/10.1093/ 5065–5070. https://doi.org/10.1128/jb.178.17.
molbev/msaa015 5065-5070.1996
Misof B, Liu S, Meusemann K, Peters RS, Donath A, Ohno M, Kimura M, Miyazaki H, Okawa K, Onuki R,
Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG Nemoto C, Tabata E, Wakita S, Kashimura A,
(2014) Phylogenomics resolves the timing and pattern Sakaguchi M (2016) Acidic mammalian chitinase is a
of insect evolution. Science 346(6210):763–767. proteases-resistant glycosidase in mouse digestive sys-
https://doi.org/10.1126/science.1257570 tem. Sci Rep 6(1):1–9. https://doi.org/10.1038/
Moeller JB, Leonardi I, Schlosser A, Flamar A-L, srep37756
Bessman NJ, Putzel GG, Thomsen T, Hammond M, Ohnuma T, Numata T, Osawa T, Mizuhara M, Varum
Jepsen CS, Skjødt K, Füchtbauer E-M, Farber DL, KM, Fukamizo T (2011) Crystal structure and mode
Sorensen GL, Iliev ID, Holmskov U, Artis D (2019) of action of a class V chitinase from Nicotiana
Modulation of the fungal mycobiome is regulated by tabacum. Plant Mol Biol 75(3):291–304. https://doi.
the chitin-binding receptor FIBCD1. J Exp Med org/10.1007/s11103-010-9727-z
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 29

Oliveira ST, Azevedo MIG, Cunha RMS, Silva CFB, Reboledo G, Agorio AD, Vignale L, Batista-Garcia RA,
Muniz CR, Monteiro-Junior JE, Carneiro RF, Nagano Ponce De Leon I (2021) Transcriptional profiling
CS, Girao MS, Freitas CDT, Grangeiro TB (2020) reveals conserved and species-specific plant defense
Structural and functional features of a class VI responses during the interaction of Physcomitrium
chitinase from cashew (Anacardium occidentale L.) patens with Botrytis cinerea. Plant Mol Biol
with antifungal properties. Phytochemistry 180: 107(4–5):365–385. https://doi.org/10.1007/s11103-
112527. https://doi.org/10.1016/j.phytochem.2020. 021-01116-0
112527 Renner T, Specht CD (2012) Molecular and functional
Onaga S, Taira T (2008) A new type of plant chitinase evolution of class I chitinases for plant carnivory in
containing LysM domains from a fern (Pteris the Caryophyllales. Mol Biol Evol 29(10):2971–2985.
ryukyuensis): roles of LysM domains in chitin binding https://doi.org/10.1093/molbev/mss106
and antifungal activity. Glycobiology 18(5):414–423. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK,
https://doi.org/10.1093/glycob/cwn018 Smith KD, Hood LE, Aderem A (2005) The evolution
Orlando M, Buchholz PCF, Lotti M, Pleiss J (2021) The of vertebrate Toll-like receptors. Proc Natl Acad Sci U
GH19 engineering database: sequence diversity, sub- S A 102(27):9577. https://doi.org/10.1073/pnas.
strate scope, and evolution in glycoside hydrolase fam- 0502272102
ily 19. PLoS One 16(10):e0256817. https://doi.org/10. Roach JM, Racioppi L, Jones CD, Masci AM (2013)
1371/journal.pone.0256817 Phylogeny of toll-like receptor signaling: adapting the
Orr RG, Foley SJ, Sherman C, Abreu I, Galotto G, Liu B, innate response. PLoS One 8(1). https://doi.org/10.
Gonzalez-Guerrero M, Vidali L (2020) Robust 1371/journal.pone.0054156
survival-based RNA interference of gene families Roche P, Maillet F, Plazanet C, Debellé F, Ferro M,
using in tandem silencing of adenine Truchet G, Promé JC, Dénarié J (1996) The common
phosphoribosyltransferase. Plant Physiol 184(2): nodABC genes of Rhizobium meliloti are host-range
607–619. https://doi.org/10.1104/pp.20.00865 determinants. Proc Natl Acad Sci U S A 93(26):
Ota F, Hirayama T, Kizuka Y, Yamaguchi Y, Fujinawa R, 15305–15310. https://doi.org/10.1073/pnas.93.26.
Nagata M, Ismanto HS, Lepenies B, Aretz J, 15305
Rademacher C (2018) High affinity sugar ligands of Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O,
C-type lectin receptor langerin. Biochim Biophys Acta, Schweighoffer E, Williams DL, Gordon S, Tybulewicz
Gen Subj 1862(7):1592–1601. https://doi.org/10.1016/ VL, Brown GD, Reis e Sousa C (2005) Syk-dependent
j.bbagen.2018.04.004 cytokine induction by dectin-1 reveals a novel pattern
Parfrey LW, Lahr DJ, Knoll AH, Katz LA (2011) recognition pathway for C type lectins. Immunity
Estimating the timing of early eukaryotic diversifica- 22(4):507–517. https://doi.org/10.1016/j.immuni.
tion with multigene molecular clocks. Proc Natl Acad 2005.03.004
Sci U S A 108(33):13624–13629. https://doi.org/10. Romani L (2004) Immunity to fungal infections. Nat Rev
1073/pnas.1110633108 Immunol 4(1):11–24. https://doi.org/10.1038/nri1255
Passarinho PA, de Vries SC (2002) Arabidopsis Romani L (2011) Immunity to fungal infections. Nat Rev
chitinases: a genomic survey. Arabidopsis Book 1: Immunol 11(4):275–288. https://doi.org/10.1038/
e0023. https://doi.org/10.1199/tab.0023 nri2939
Pees B, Yang W, Zárate-Potes A, Schulenburg H, Rombout JHWM, Huttenhuis HBT, Picchietti S,
Dierking K (2016) Innate immune specificity through Scapigliati G (2005) Phylogeny and ontogeny of fish
diversified c-type lectin-like domain proteins in leucocytes. Fish Shellfish Immunol 19(5):441–455.
invertebrates. J Innate Immun 8(2):129–142. https:// https://doi.org/10.1016/j.fsi.2005.03.007
doi.org/10.1159/000441475 Rush TA, Puech-Pages V, Bascaules A, Jargeat P,
Perović-Ottstadt S, Adell T, Proksch P, Wiens M, Maillet F, Haouy A, Maes AQ, Carriel CC,
Korzhev M, Gamulin V, Müller IM, Müller WE Khokhani D, Keller-Pearson M, Tannous J, Cope
(2004) A (1-->3)-beta-D-glucan recognition protein KR, Garcia K, Maeda J, Johnson C, Kleven B,
from the sponge Suberites domuncula. Mediated acti- Choudhury QJ, Labbe J, Swift C, O'Malley MA, Bok
vation of fibrinogen-like protein and epidermal growth JW, Cottaz S, Fort S, Poinsot V, Sussman MR,
factor gene expression. Eur J Biochem 271(10): Lefort C, Nett J, Keller NP, Becard G, Ane JM
1924–1937. https://doi.org/10.1111/j.1432-1033. (2020) Lipo-chitooligosaccharides as regulatory
2004.04102.x signals of fungal growth and development. Nat
Pilecki B, Moeller JB (2020) Fungal recognition by mam- Commun 11(1):3897. https://doi.org/10.1038/s41467-
malian fibrinogen-related proteins. Scand J Immunol 020-17615-5
92(4):e12925. https://doi.org/10.1111/sji.12925 Sanchez-Vallet A, Mesters JR, Thomma BP (2014) The
Punja ZK, Zhang Y (1993) Plant chitinases and their roles battle for chitin recognition in plant-microbe
in resistance to fungal diseases. J Nematol 25(4): interactions. FEMS Microbiol Rev 39(2):171–183.
526–540 https://doi.org/10.1093/femsre/fuu003
Pusztahelyi T (2018) Chitin and chitin-related compounds Schlosser A, Thomsen T, Moeller JB, Nielsen O, Tornøe I,
in plant-fungal interactions. Mycology 9(3):189–201. Mollenhauer J, Moestrup SK, Holmskov U (2009)
https://doi.org/10.1080/21501203.2018.1473299 Characterization of FIBCD1 as an acetyl group-
binding receptor that binds chitin. J Immunol 183(6):
30 E. D. Trudeau and M. L. Berbee

3800–3809. https://doi.org/10.4049/jimmunol. Plant Cell Physiol 57(11):2312–2322. https://doi.org/

0901526 10.1093/pcp/pcw150
Sharma S, Singh R, Kaur R (2020) In silico characteriza- Taira T, Hayashi H, Tajiri Y, Onaga S, Uechi G,
tion of a unique plant-like “loopful” GH19 chitinase Iwasaki H, Ohnuma T, Fukamizo T (2009) A plant
from newly isolated Chitinophaga sp. YS-16. Curr class V chitinase from a cycad (Cycas revoluta): bio-
Microbiol 77(9):2248–2257. https://doi.org/10.1007/ chemical characterization, cDNA isolation, and post-
s00284-020-02022-2 translational modification. Glycobiology 19(12):
Shimizu T, Nakano T, Takamizawa D, Desaki Y, Ishii- 1452–1461. https://doi.org/10.1093/glycob/cwp119
Minami N, Nishizawa Y, Minami E, Okada K, Taira T, Mahoe Y, Kawamoto N, Onaga S, Iwasaki H,
Yamane H, Kaku H, Shibuya N (2010) Two LysM Ohnuma T, Fukamizo T (2011) Cloning and character-
receptor molecules, CEBiP and OsCERK1, coopera- ization of a small family 19 chitinase from moss
tively regulate chitin elicitor signaling in rice. Plant J (Bryum coronatum). Glycobiology 21(5):644–654.
64(2):204–214. https://doi.org/10.1111/j.1365-313X. https://doi.org/10.1093/glycob/cwq212
2010.04324.x Tateno H, Ohnishi K, Yabe R, Hayatsu N, Sato T,
Shiu SH, Bleecker AB (2003) Expansion of the receptor- Takeya M, Narimatsu H, Hirabayashi J (2010) Dual
like kinase/Pelle gene family and receptor-like proteins specificity of langerin to sulfated and mannosylated
in Arabidopsis. Plant Physiol 132(2):530–543. https:// glycans via a single C-type carbohydrate recognition
doi.org/10.1104/pp.103.021964 domain. J Biol Chem 285(9):6390–6400. https://doi.
Shrive AK, Moeller JB, Burns I, Paterson JM, Shaw AJ, org/10.1074/jbc.M109.041863
Schlosser A, Sorensen GL, Greenhough TJ, Holmskov Taylor PR, Tsoni SV, Willment JA, Dennehy KM,
U (2014) Crystal structure of the tetrameric fibrinogen- Rosas M, Findon H, Haynes K, Steele C, Botto M,
like recognition domain of fibrinogen C domain Gordon S, Brown GD (2007) Dectin-1 is required for
containing (FIBCD1) protein. J Biol Chem 289(5): β-glucan recognition and control of fungal infection.
2880–2887. https://doi.org/10.1074/jbc.M113.520577 Nat Immunol 8(1):31–38. https://doi.org/10.1038/
Simm D, Hatje K, Kollmar M (2015) Waggawagga: com- ni1408
parative visualization of coiled-coil predictions and Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT,
detection of stable single α-helices (SAH domains). Engele M, Sieling PA, Barnes PF, Rollinghoff M,
Bioinformatics 31(5):767–769. https://doi.org/10. Bolcskei PL, Wagner M, Akira S, Norgard MV,
1093/bioinformatics/btu700 Belisle JT, Godowski PJ, Bloom BR, Modlin RL
Stambach NS, Taylor ME (2003) Characterization of car- (2001) Induction of direct antimicrobial activity
bohydrate recognition by langerin, a C-type lectin of through mammalian toll-like receptors. Science
Langerhans cells. Glycobiology 13(5):401–410. 291(5508):1544–1547. https://doi.org/10.1126/sci
https://doi.org/10.1093/glycob/cwg045 ence.291.5508.1544
Steinfeld L, Vafaei A, Rosner J, Merzendorfer H (2019) Thomma BP, Eggermont K, Tierens KF, Broekaert WF
Chitin prevalence and function in bacteria, fungi and (1999) Requirement of functional ethylene-insensitive
protists. Adv Exp Med Biol 1142:19–59. https://doi. 2 gene for efficient resistance of Arabidopsis to infec-
org/10.1007/978-981-13-7318-3_3 tion by Botrytis cinerea. Plant Physiol 121(4):
Strobel S, Roswag A, Becker NI, Trenczek TE, 1093–1101. https://doi.org/10.1104/pp.121.4.1093
Encarnaçao JA (2013) Insectivorous bats digest chitin Thomsen T, Schlosser A, Holmskov U, Sorensen GL
in the stomach using acidic mammalian chitinase. (2011) Ficolins and FIBCD1: soluble and membrane
PLoS One 8(9):e72770. https://doi.org/10.1371/jour bound pattern recognition molecules with acetyl group
nal.pone.0072770 selectivity. Mol Immunol 48(4):369–381. https://doi.
Sutherland TE (2018) Chitinase-like proteins as regulators org/10.1016/j.molimm.2010.09.019
of innate immunity and tissue repair: helpful lessons Unestam T, Söderhäll K (1977) Soluble fragments from
for asthma? Biochem Soc Trans 46(1):141–151. fungal cell walls elicit defence reactions in crayfish.
https://doi.org/10.1042/BST20170108 Nature 267(5606):45–46. https://doi.org/10.1038/
Sutherland TE, Logan N, Rückerl D, Humbles AA, Allan 267045a0
SM, Papayannopoulos V, Stockinger B, Maizels RM, UniProt Consortium (2021) UniProt: the universal protein
Allen JE (2014) Chitinase-like proteins promote IL-17- knowledgebase in 2021. Nucleic Acids Res 49(D1):
mediated neutrophilia in a tradeoff between nematode D480–D489. https://doi.org/10.1093/nar/gkaa1100
killing and host damage. Nat Immunol 15(12): Urbach JM, Ausubel FM (2017) The NBS-LRR
1116–1125. https://doi.org/10.1038/ni.3023 architectures of plant R-proteins and metazoan NLRs
Suzuki M, Shibuya M, Shimada H, Motoyama N, evolved in independent events. Proc Natl Acad Sci U S
Nakashima M, Takahashi S, Suto K, Yoshida I, A 114(5):1063–1068. https://doi.org/10.1073/pnas.
Matsui S, Tsujimoto N, Ohnishi M, Ishibashi Y, 1619730114
Fujimoto Z, Desaki Y, Kaku H, Kito K, Shibuya N van den Berg LM, Gringhuis SI, Geijtenbeek TBH (2012)
(2016) Autophosphorylation of specific threonine and An evolutionary perspective on C-type lectins in infec-
tyrosine residues in Arabidopsis CERK1 is essential tion and immunity. Ann N Y Acad Sci 1253(1):
for the activation of chitin-induced immune signaling.
1 When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems 31

149–158. https://doi.org/10.1111/j.1749-6632.2011. 1,3-linked glucans is mediated by different receptor

06392.x systems. Plant J 102(6):1142–1156. https://doi.org/
van Eijk M, van Roomen CPAA, Renkema GH, Bussink 10.1111/tpj.14688
AP, Andrews L, Blommaart EFC, Sugar A, Verhoeven Wiesner DL, Specht CA, Lee CK, Smith KD,
AJ, Boot RG, Aerts JMFG (2005) Characterization of Mukaremera L, Lee ST, Lee CG, Elias JA, Nielsen
human phagocyte-derived chitotriosidase, a compo- JN, Boulware DR, Bohjanen PR, Jenkins MK, Levitz
nent of innate immunity. Int Immunol 17(11): SM, Nielsen K (2015) Chitin recognition via
1505–1512. https://doi.org/10.1093/intimm/dxh328 chitotriosidase promotes pathologic type-2 helper T
van Hengel AJ, Tadesse Z, Immerzeel P, Schols H, van cell responses to cryptococcal infection. PLoS Pathog
Kammen A, de Vries SC (2001) N-acetylglucosamine 11(3):e1004701. https://doi.org/10.1371/journal.ppat.
and glucosamine-containing arabinogalactan proteins 1004701
control somatic embryogenesis. Plant Physiol 125(4): Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D,
1880–1890. https://doi.org/10.1104/pp.125.4.1880 Tsuda K, Katagiri F, Fliegmann J, Bono JJ, Cullimore
Van Holle S, Van Damme EJM (2019) Messages from JV, Jehle AK, Gotz F, Kulik A, Molinaro A, Lipka V,
the past: new insights in plant lectin evolution. Front Gust AA, Nurnberger T (2011) Arabidopsis lysin-
Plant Sci 10:36. https://doi.org/10.3389/fpls.2019. motif proteins LYM1 LYM3 CERK1 mediate bacterial
00036 peptidoglycan sensing and immunity to bacterial infec-
van Loon LC, Rep M, Pieterse CM (2006) Significance of tion. Proc Natl Acad Sci U S A 108(49):19824–19829.
inducible defense-related proteins in infected plants. https://doi.org/10.1073/pnas.1112862108
Annu Rev Phytopathol 44:135–162. https://doi.org/ Xin Y, Wang D, Han S, Li S, Gong N, Fan Y, Ji X (2021)
10.1146/annurev.phyto.44.070505.143425 Characterization of the chitinase gene family in mul-
van Velzen R, Doyle JJ, Geurts R (2019) A resurrected berry (Morus notabilis) and MnChi18 involved in
scenario: single gain and massive loss of nitrogen- resistance to Botrytis cinerea. Genes (Basel) 13(1).
fixing nodulation. Trends Plant Sci 24(1):49–57. https://doi.org/10.3390/genes13010098
https://doi.org/10.1016/j.tplants.2018.10.005 Yadav M, Schorey JS (2006) The β-glucan receptor
Vannella KM, Ramalingam TR, Hart KM, de Queiroz PR, dectin-1 functions together with TLR2 to mediate mac-
Sciurba J, Barron L, Borthwick LA, Smith AD, rophage activation by mycobacteria. Blood 108(9):
Mentink-Kane M, White S, Thompson RW, Cheever 3168–3175. https://doi.org/10.1182/blood-2006-
AW, Bock K, Moore I, Fitz LJ, Urban JF Jr, Wynn TA 05-024406
(2016) Acidic chitinase primes the protective immune Yamagami T, Funatsu G (1993) Purification and some
response to gastrointestinal nematodes. Nat Immunol properties of three chitinases from the seeds of rye
17(5):538–544. https://doi.org/10.1038/ni.3417 (Secale cereale). Biosci Biotechnol Biochem 57(4):
Vendele I, Willment JA, Silva LM, Palma AS, Chai W, 643–647. https://doi.org/10.1271/bbb.57.643
Liu Y, Feizi T, Spyrou M, Stappers MH, Brown GD Yamasaki S, Matsumoto M, Takeuchi O, Matsuzawa T,
(2020) Mannan detecting C-type lectin receptor probes Ishikawa E, Sakuma M, Tateno H, Uno J,
recognise immune epitopes with diverse chemical, spa- Hirabayashi J, Mikami Y (2009) C-type lectin Mincle
tial and phylogenetic heterogeneity in fungal cell walls. is an activating receptor for pathogenic fungus,
PLoS Pathog 16(1):e1007927. https://doi.org/10.1371/ Malassezia. Proc Natl Acad Sci U S A 106(6):
journal.ppat.1007927 1897–1902. https://doi.org/10.1073/pnas.0805177106
Viborg AH, Terrapon N, Lombard V, Michel G, Yano S, Kanno H, Tsuhako H, Ogasawara S, Suyotha W,
Czjzek M, Henrissat B, Brumer H (2019) A subfamily Konno H, Makabe K, Uechi K, Taira T (2021) Clon-
roadmap of the evolutionarily diverse glycoside hydro- ing, expression, and characterization of a GH 19-type
lase family 16 (GH16). J Biol Chem 294(44): chitinase with antifungal activity from Lysobacter
15973–15986. https://doi.org/10.1074/jbc.RA119. sp. MK9-1. J Biosci Bioeng 131(4):348–355. https://
010619 doi.org/10.1016/j.jbiosc.2020.11.005
Wang Y, Wang X-P, Zhang B, Li Z-M, Yang L-G, Li X-C, Yeh S, Moffat BA, Griffith M, Xiong F (2000) Chitinase
Ma H (2020) A lysin motif-containing protein genes responsive to cold encode antifreeze proteins in
(SpLysMD3) functions as a PRR involved in the winter cereals. Plant Physiol 124(3):1251–1264.
antibacterial responses of mud crab, Scylla https://doi.org/10.1104/pp.124.3.1251
paramamosain. Fish Shellfish Immunol 97:257–267. Yokoyama CC, Baldridge MT, Leung DW, Zhao G,
https://doi.org/10.1016/j.fsi.2019.12.036 Desai C, Liu T-C, Diaz-Ochoa VE, Huynh JP,
Wang H, Chen X, Li S, Zhou C, Xu L, Wu Z, Chen X Kimmey JM, Sennott EL (2018) LysMD3 is a type II
(2021) Identification and expression analysis of membrane protein without an in vivo role in the
Langerhans cells marker Langerin/CD207 in response to a range of pathogens. J Biol Chem
grasscarp, Ctenopharyngodon idella. Gene 768: 293(16):6022–6038
145315. https://doi.org/10.1016/j.gene.2020.145315 Zeilinger S, Gupta VK, Dahms TE, Silva RN, Singh HB,
Wanke A, Rovenich H, Schwanke F, Velte S, Becker S, Upadhyay RS, Gomes EV, Tsui CK, Nayak SC (2016)
Hehemann JH, Wawra S, Zuccaro A (2020) Plant Friends or foes? Emerging insights from fungal
species-specific recognition of long and short beta-
32 E. D. Trudeau and M. L. Berbee

interactions with plants. FEMS Microbiol Rev 40(2): for automated carbohydrate-active enzyme annotation.
182–207. https://doi.org/10.1093/femsre/fuv045 Nucleic Acids Res 46(W1):W95–W101. https://doi.
Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome org/10.1093/nar/gky418
editing: progress, implications and challenges. Hum Zhang YJ, Ren LL, Lin XY, Han XM, Wang W, Yang ZL
Mol Genet 23(R1):R40–R46. https://doi.org/10.1093/ (2022) Molecular evolution and functional characteri-
hmg/ddu125 zation of chitinase gene family in Populus trichocarpa.
Zhang H, Yohe T, Huang L, Entwistle S, Wu P, Yang Z, Gene 822:146329. https://doi.org/10.1016/j.gene.
Busk PK, Xu Y, Yin Y (2018) dbCAN2: a meta server 2022.146329
 Metabolic Constraints
and Dependencies Between 2
“Uncultivable” Fungi and Their Hosts

Saleh Rahimlou, C. Alisha Quandt, and Timothy Y. James

Abstract themes across diverse fungi are the loss of

Many host-associated fungi are believed to be pathways for synthesis of long-chain fatty
uncultivable because they have thus far never acids, amino acids, and vitamins. Finally, we
been brought into culture. However, the discuss novel approaches and experimental
absence of success in bringing a fungus into systems that may be used to overcome the
cultivation in the laboratory should not be common barriers to cultivation.
interpreted as an indication that the fungus
cannot grow axenically without a living host. Keywords
In this chapter, we review the history of the Parasite · Biotrophic · Coevolution · Primary
cultivation of fungi and provide examples metabolites · Malassezia
where seemingly fastidious fungi have been
successfully cultivated, borrowing insights
from the literature on bacteria. We define a 2.1 Introduction
few of the hypotheses for why fungi become
uncultivable and suggest that only organisms 2.1.1 What Are Uncultivable Fungi?
with a highly predictable environment will
likely lose the ability to produce primary The cultivation of fungi has been essential in their
metabolites needed for making cells. Highly development as model organisms for the study of
host-specific parasites fit this description, mating, cell cycle regulation, genetic interactions,
while arbuscular mycorrhizae do not. As fun- and much more (Müller and Grossniklaus 2010).
gal genomics has blossomed, it provides an However, most model fungal systems, such as
unprecedented opportunity to investigate, Saccharomyces, Neurospora, or Coprinopsis,
from a genetic-biochemistry level, particular are organisms that are considered saprobic while
metabolic requirements that may provide many fungi are symbiotic and are reliant on other
insights into cultivation strategies. Common species for their nutrition. Because of these
nutritional dependencies many of these species
S. Rahimlou · T. Y. James (✉) of symbiotic fungi have never been brought into
Department of Ecology and Evolutionary Biology, culture, making them appear as if they are uncul-
University of Michigan, Ann Arbor, MI, USA tivable. However, there are multiple reasons to
e-mail: [email protected] believe that most of these fungi could actually be
C. A. Quandt brought into culture with the correct media, much
Department of Ecology and Evolutionary Biology, in the same way that tissue culturing of plants and
University of Colorado Boulder, Boulder, CA, USA

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 33

Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_2
34 S. Rahimlou et al.

animals with complex nutritional requirements is non-living organic material, i.e., as saprobes, or
also accomplished (Thorpe 2012; Yao and living tissues. Uncultivated fungi run the gamut
Asayama 2017). from symbionts to saprobes, and while there is a
Saprobes are defined as organisms using dead general tendency for species associated with liv-
organic material as food, and thus saprobic ing organisms, including pathogens and
growth is independent of a living host. Williams mutualists, to be unable to grow in artificial
(1992) has provided definitions and terminology media, some are readily amenable, for example,
for fungal cultivation. In summary, the term “axe- plant pathogens such as Magnaporthe grisea and
nic” should be used to refer to the growth of a Taphrina deformans. On the other hand, some
single species in the absence of whole, live saprobes have never been cultured, such as the
organisms, or living cells of any other species. chytridiomycete Rhizophydium keratinophilum
“Monoxenic” describes a culture containing and ascomycetes Rhytisma spp. In general, the
organisms or cells of two species (e.g., an term biotrophic is used to describe organisms
arbuscular mycorrhizal fungus and a soil bacte- that derive their nutrition from living host tissue
rium growing together on an agar plate). or cytoplasm (Jeffries and Young 1994), and this
“Dixenic” should be applied to a culture applies to species along the entirety of the symbi-
containing organisms or cells of three species, osis spectrum, such as endophytes and
etc. As stated by Williams (1992), the terms mycorrhizae as well as pathogens. As a result of
“aseptic,” “sterile,” “artificial,” “pure,” “bacte- long-term efforts to cultivate fungi, some
ria-free,” etc. are ambiguous or imprecise. Thus, biotrophic fungi can be grown with specific
uncultivable fungi are defined here as strains that nutritional or environmental requirements
have never been cultured in vitro, whether axenic, (Williams et al. 1966; Sugiura et al. 2020).
monoxenic, or polyxenic. In this chapter, we Examples of biotrophic fungi that are cultivated
attempt to make a distinction between uncultiva- axenically are haustorial mycoparasites such as
ble fungi and uncultivated fungi, the former refer- Dispira (Barker and Barnett 1973), nematode
ring to a group of fungi which could not be parasites such as Rhopalomyces (Ellis 1963),
cultivated under any conceivable conditions, and plant pathogens such as Phytophthora infestans,
the latter referring to those fungi which have corn smut Ustilago maydis, and leaf curl
never been cultivated thus far but may possibly Taphrina deformans (Mix 1949; Day and
be in the future. Anagnostakis 1971; Tumwine et al. 2000),
Finally, the speed at which particular fungi endophytes such as Epichlöe typhina (Bacon
grow impacts the assessment of their cultivability. et al. 1977), ectomycorrhizal fungi (EMF)
There are examples in the literature of very slow- (Vuorinen et al. 2015), lichens (Stocker-
growing fungi being cultured after months of Wörgötter 2001), and recently arbuscular mycor-
incubation, with fungi isolated from extreme rhizal fungi (Sugiura et al. 2020). Symbiotic fungi
environments being particularly well represented that are considered hemibiotrophic are those
(Gostinčar et al. 2022). How long researchers pathogens that start as biotrophs and then have a
should monitor cultures is not standardized—out- period of necrotrophy after the host cells have
side of clinical settings (Bosshard 2011)—and a been killed. Both hemibiotrophs and necrotrophs
systematic review of the literature on this has not are considered readily culturable. Heretical as it
been published. may seem, it is worth considering whether the
ability to grow in pure culture has any meaning.
For both Taphrina deformans and Ustilago
2.1.2 Major Groups maydis, it is the yeast phase that is readily
of Uncultivated Fungi cultured, a phase that is considered saprobic.
Therefore, the limitation to cultivation is not
Since fungi are chemoheterotrophic, they must hard-wired in the genome, but one of how the
derive their carbon compounds from either genes are being used and at what developmental
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 35

stage. Another point to consider is whether the 2.1.3 Where Do Uncultivated Fungi
ability to culture some biotrophic fungi, for exam- Grow and Should We View
ple, the amphibian pathogen Batrachochytrium Them as Uncultivable?
dendrobatidis, belies an innate saprobic ability
that is manifested in nature (Piotrowski et al. As seen above, the diversity of hosts of biotrophic
2004). In other words, how clear is the relation- fungi has a very broad range that includes other
ship between culturability and the ability to grow fungi, algae, protists, vascular plants,
on non-living nutritive sources? And does the invertebrates, and vertebrates. All biotrophs
ability to grow readily in culture necessarily sug- have a primary host in their life cycle, but some
gest some saprobic growth in nature? require a secondary (alternate) host to complete
It is also worth mentioning that many of the their life cycle when the primary host is unavail-
commonly used media for culturing fungi are able. For instance, some rust fungi may be
derived directly from complex and undefined restricted to a single host (autoecious) or may
plant-based sources (e.g., potato, corn, barley, infect alternate hosts (heteroecious). Puccinia
and cherry). This necessarily biases culture- graminis Pers. on barberry and cereal grains,
based techniques toward growing and identifying Cronartium ribicola Dietr. on pines and Ribes,
fungi which can regularly break down plant- and Gymnosporangium juniperi-virginianae
based sugars and may explain why plant Schw. on apple and cedar are leading examples
pathogens are much more readily “culturable” of heteroecious rusts, while Puccinia helianthi
than fungi which are pathogens and parasites of Schw. on sunflower and Uromyces striatus
other non-plant organisms, such as many Schroet. on alfalfa are autoecious species. The
cordycipitaceous fungi. roles of the alternate hosts in disease epidemiol-
Despite recent advances in culturing symbiotic ogy and pathogen variation vary greatly from
fungi, there are general patterns regarding culti- species to species and from region to region
vation that are apparent. Among the major fungal because of different climatic and cropping
groups, the following mutualists remain largely conditions (Zhao et al. 2016). The distribution
uncultivated: arbuscular mycorrhizas of obligate biotrophs is influenced by certain
(Glomeromycotina), mycorrhizal Endogonales, environmental and geographical factors; for
many ectomycorrhizas (including the genus example, some graminicolous rusts are negatively
Cortinarius), and most lichenized fungi. Of the affected by higher temperatures and increased
biotrophic plant pathogens, rusts (Pucciniales), concentrations of atmospheric CO2 (Helfer 2014).
powdery mildews (Erysiphales), and Physoderma As hosts for both symbionts such as mycorrhi-
(Blastocladiomycota) are mostly uncultured. The zal fungi and pathogens, vascular plants are the
algal parasites in Aphelidiomycota and some most common host for uncultivated fungi, but this
members of Chytridiomycota (e.g., likely reflects the rampant co-diversification of
Zygorhizidium) are uncultivated, in addition to fungi with plants as mutualists, pathogens, and
many mycoparasitic Zoopagomycota, saprobes (Lutzoni et al. 2018). Many plant spe-
Rozellomycota, and Caulochytrium cies are susceptible to infection by obligate
(Chytridiomycota). Uncultured animal symbionts biotrophs. For example, rusts are pathogenic on
are also common, such as Laboulbeniales, preda- vascular plants, including ferns, gymnosperms,
tory fungi in Zoopagales, some and most families of angiosperms. Powdery
Entomophthorales, the mammal pathogen mildews are among the most common plant
Pneumocystis, and insect pathogenic members diseases, infecting over 650 monocots and over
of Cordyceps s.l. and Blastocladiales, such as 9000 dicot species (Schulze-Lefert and Vogel
Coelomomyces. 2000). In addition, the majority of land plants
form symbiotic associations with mycorrhizal
fungi. Ectomycorrhizal fungi, which are mainly
36 S. Rahimlou et al.

associated with forest trees, are the most diverse org). While this is an impressive number, to for-
in temperate biomes, whereas the relative abun- mally catalog dark fungal taxa through culturing
dance and diversity of agriculturally important alone would take millennia or the underlying
arbuscular mycorrhizal fungi are higher in financial and labor resources would have to be
grasslands than in forests with unclear latitudinal increased by several orders of magnitude
patterns (Davison et al. 2015). The majority of (Lücking et al. 2021). To somehow overcome
mycorrhizal plants are arbuscular mycorrhizal this problem and facilitate the scientific commu-
(70%), whereas ectomycorrhizal plants, despite nication of massive eDNA data, the UNITE data-
being widely distributed (Read 1991), constitute base has emerged as the most widely used
only a tiny fraction of all vascular plant species resource for curated fungal ITS sequences,
(0.7%) (Soudzilovskaia et al. 2020). The orders including data from vouchered specimens and
Pezizales, Agaricales, Helotiales, Boletales, and reference sequences for so-called “species
Cantharellales include the largest number of hypotheses,” that is, phylogroups of sequences
ectomycorrhizal fungal lineages (Tedersoo et al. that are interpreted as species (Kõljalg et al.
2010). 2013). Currently, the UNITE database holds
Environmental metabarcoding has drastically around 290,000 species hypotheses with digital
changed our appreciation that a culture-based or object identifiers (DOIs) at a 98.5% similarity
specimen-based approach for fungal sampling threshold (https://unite.ut.ee), representing almost
may be missing the majority of fungal diversity. 10% of the estimated unrecognized fungi.
With environmental metabarcoding, taxonomy is
operating metaphorically in the dark, because
sequence-based identification cannot be verified 2.2 Why Do Symbionts Become
with voucher specimens, and culturing is neces- Uncultivable?
sary to bring these fungi “to life.” Culturing has a
long history of use in microbiology and mycol- We have tried to suggest a link between
ogy, but it faces various challenges. Since the culturability and symbiosis for fungi. At first
physiological requirements of dark taxa glance, this might seem to be a fundamental dif-
(unnamed sequences) are not known, culturing ference between prokaryotes and eukaryotes; i.e.,
requires resource-intensive approaches, a situa- the relationship between culturability and symbi-
tion that is comparable to prokaryote microbiol- osis is stronger for fungi than for bacteria,
ogy. For example, the cultivation of an Asgard because so-called free-living bacteria seem to be
archaeon took more than 5 years (Imachi et al. more difficult to cultivate than free-living fungi
2020), and initial cultivation of a bacterial strain (Solden et al. 2016). If so, this provides a major
may cost 10,000 US dollars (Overmann 2015). insight into the contrasting metabolic evolution
Even for fungi that can be readily cultured, the and ecosystem ecology of these lineages. What
quantity of predicted undiscovered species poses would drive the relationship between culturability
a logistical challenge. According to the World and symbiosis for fungi? We present four
dictionary of culture collections (http://www. non-mutually exclusive hypotheses for the rela-
wfcc.info/ccinfo/statistics), there are currently tionship between host-associated fungi and lack
about 892,000 deposited fungal cultures, of cultivability.
representing around 30,000 known species. This The most obvious hypothesis is that uncultiva-
number represents a maximum of 1.3% of the ble or fastidious fungi are metabolically in need
estimated 2.2–3.8 million fungal species of nutrients that are provided by the host, either in
(Tedersoo et al. 2014; Hawksworth and Lücking parasitism or mutualism. This predicts that fungi
2017; Kopytko et al. 2017; Lücking et al. 2021). that are endobiotic will be more likely to be
During the past two decades, about 10,000 new uncultivable than those that are epibiotic, because
fungi have been described based on cultures they are more influenced by the host. Facultative
(Index fungorum; http://www.indexfungorum. parasites, on the other hand, will be those least
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 37

subject to these constraints. Heritable bacteria with one another are more likely to lose function-
serve as an example of extreme metabolic depen- ality involved in the production of metabolites
dency. These bacteria are known as mutualistic that one host/partner is “providing.”
endosymbionts of insects that show transovarial A second hypothesis of why symbiotic fungi
inheritance and provide the host with essential become uncultivable is that the cultivation is only
amino acids (McCutcheon and Moran 2012). possible in a single life stage and the transition to
These associations are highly stable and, from that life stage cannot be triggered. For example,
the endosymbiont perspective, foster environ- Ustilago maydis can only be cultured as a yeast
mental predictability. An analogous situation from spores or galls. Isolation of dikaryotic
may be for the obligate parasite Pneumocystis, hyphae from leaf tissue has not been reported.
which is only known from the mammalian lung Similarly, spores of many ectomycorrhizal fungi
and has a highly reduced genome (Ma et al. will not readily germinate in culture but can be
2016). Rozellids and microsporidia are known cultured from colonized root tips or sporocarps.
as extremely morphologically reduced fungi The dimorphic facultative pathogen
lacking many of the basic primary metabolism Paracoccidioides loboi has never been cultivated,
pathways, such as nucleotide and amino acid leading Vilela et al. (2021) to hypothesize that the
biosynthesis (James et al. 2013; Keeling and inability to culture the fungus is related to the
Slamovits 2004; Dean et al. 2016; Quandt et al. inability of the fungus to transition from the path-
2017). These taxa also appear to have acquired ogenic (yeast form) to the saprobic hyphal form.
genes (encoding transferases and kinases This dimorphic inequality may be hard (also
involved in obtaining these building blocks and observed in nature) or technical (only related to
energy sources from the host) via horizontal current methods of culturing). A related phenom-
transfer from bacteria (Tsaousis et al. 2008; enon regarding life cycle transitions is one in
Alexander et al. 2016). Long-term endosymbiosis which the culturing environment does not allow
has led to the loss of genes to make primary a fungus to reproduce/sporulate, thus limiting
metabolites as they are now obtained from their proliferation in vitro. For example,
the host. monocentric chytrids are unicells that grow vege-
Using Escherichia coli as a model, D’Souza tatively and then convert completely into repro-
and Kost (2016) provided the first experimental ductive spores. Such a failure to sporulate could
evidence that when metabolites are available in result from either missing nutrients or missing
the environment, there is a selection for loss of the environmental cues.
ability to produce corresponding metabolites, and A third hypothesis is that host-associated fungi
these auxotrophic strains occurred much more require a chemical signal from a host because
frequently than by chance in enriched growth and specificity are tied together. For
environments than in “normal” ones. Specifically, example, Colletotrichum associated with post-
they grew E. coli in amino acid-rich environments harvest disease showed germination and appres-
and saw loss of genes encoding synthesis and sorium dependency on host ethylene production,
regulation of those amino acids, and, signifi- an indication of ripening (Flaishman and
cantly, they showed that the auxotrophs displayed Kolattukudy 1994). Chemical signals, mostly
a relative fitness advantage when grown in the undetermined, produced by plant hosts of both
presence of amino acids. In support of anecdotes AMF and EMF are associated with improved
concerning culturing of arbuscular mycorrhizae spore germination (Parniske 2008; Nara 2009).
fungi (AMF) in the presence of other fungi or The use of specific host-associated signals might
their filtrates (see Sect. 2.4), these authors also be desirable for host-specific plant pathogens
found that auxotrophic E. coli could derive amino such as powdery mildews and rusts; however,
acids from coexisting bacterial strains. This such a system could lead to rapid evolution of
strongly suggests that organisms growing regu- resistance genes in hosts. It is also hypothesized,
larly in close cellular contact (or intracellularly) with some evidence, that host recognition is
38 S. Rahimlou et al.

important for many insect pathogens to germinate Nitrate, ammonia, and amino acids are often
(Gao et al. 2011). Perhaps, without the correct available to fungi as a source of nitrogen, whereas
chemical cues from the host, spores may be nitrite is generally not available (Steinberg 1939).
unable to germinate in the culture. Yeast extract is used as a diverse form of nitrogen
Finally, physical cues and structure may be and trace vitamins suitable for many fungi.
missing in culture media. For example, Rozellids Unlike their animal counterparts, fungi are typi-
lack a cell wall in their endobiotic parasitic phase, cally able to make most of their growth factors,
allowing them to phagocytose host cytoplasm and including all of the amino acids, vitamins, fatty
to disperse within their coenocytic host, usually a acids, and nucleotides. Therefore, the nutrient
water mold (Powell et al. 2017). After consuming requirements for many species are considered
much of the host cytoplasm, Rozella forms minimal, and they may be cultured on a minimal
zoosporangia that are totally dependent on the medium with sources of carbon, nitrogen, phos-
presence of a host cell wall to generate turgor phate, sulfate, and trace elements (Howard 1900;
pressure for discharge. Rozella can use its chitin Machlis 1953). Essential vitamins are minimal
synthase genes to make a resting sporangium but most frequently involve biotin, thiamine,
wall; however, they are unassociated with their and pyridoxine (Steinberg 1950). Thiamine is
own zoosporangial wall (Held 1980). Haustorial one of the components of modified Melin-
parasites might similarly require physical Norkrans (MMN) media which is used to culti-
interactions that are missing in culture to produce vate ECM taxa. Thiamine biosynthesis is lost in
the proper assimilative structures. many biotrophs (Spanu 2012; Nagae et al. 2016),
Laboulbeniales, with their complex thallus mor- from the human-pathogenic Pneumocystis spp.
phology, may require the host exoskeleton for (Cisse et al. 2014) to the AMF Rhizophagus
development, but could also be subject to all of irregularis (Tisserant et al. 2013). Therefore, it
the above constraints on growth in culture. is not surprising perhaps that thiamine has long
been one of the most commonly added nutrients
to non-standard fungal culturing media (Robbins
2.3 The Evolving Approaches and Kavanagh 1938).
for Culturing Fungi In addition to thiamine, many biotrophic fungi
appear to be lacking genes involved in the assim-
Fungal cultivation dates back to the nineteenth ilatory sulfate reductase pathway (ASRP) and
century, contemporaneous with that of bacteria. some also lack the nitrogen reduction pathway
The development of pure culture techniques, as well. Ahrendt et al. (2018) recorded 7 unrelated
especially those derived from single cells, was biotrophic fungi missing all the genes in ASRP,
critical for the identification of etiological agents Duplessis et al. (2011) recorded them missing in
of disease, as required for fulfilling Koch’s the rust Puccinia graminis, and Cisse et al. (2014)
postulates (Hildebrand 1938). The most simple found ASRP missing in two species of
methods of cultivation rely on using extracts Pneumocystis. Whether deficiencies in ASRP
from plant products, such as malted barley, impact the ability of fungi to grow in axenic
potatoes, corn, and oats. Preparations of casein culture remains unknown, but the correlation
(milk protein) are particularly useful for growing between the perceived lack of cultivability of
some fungi; for example, the amphibian pathogen many fungi and loss of ASRP genes is now well
Batrachochytrium dendrobatidis can grow solely documented.
on tryptone, a digestion of casein. Numerous Currently, many investigators are focused on
early investigations with fungal cultures applying well-known media that work for
improved our understanding of fungal physiology described fungal species when cultivating new
and facilitated development of fully synthetic species. In this sense, exploration of fungal
media. Particular emphasis was placed on forms media for cultivating novel species is a less active
of nitrogen and carbon available for assimilation. area of research, and the exploration of fungal
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 39

diversity using cultivation approaches is biased plants (Schüβler and Walker 2011). It is accepted
toward recovering species that have already been that spores of AMF possess the suitable genetic
cultivated. It is easy to demonstrate such biases information and biosynthetic abilities to germi-
by comparison of culture-independent methods, nate once the appropriate, simple, physicochemi-
such as DNA metabarcoding or microscopy cal conditions of moisture, temperature, and pH
(Davis et al. 2018; Zhang et al. 2020; Hoyer and (Barea 1986) have been provided. Over 90% ger-
Hodkinson 2021), with culturing approaches. A mination can be obtained axenically on water
major example of bias in culturing is the example agar in the absence of any mineral or organic
from a very common lineage of soil fungi, the supply (Azcón-Aguilar et al. 1986). Many
Archaeorhizomycetes. These fungi were not hypotheses have been put forward to explain the
known or cultured until the twenty-first century failure of the continuous axenic growth of these
(Schadt et al. 2003; Porter et al. 2008), and in fungi (Hepper 1984; Siqueira 1987; Azcón-
particular, they were only cultured from a few of Aguilar et al. 1991; Bonfante-Fasolo and Perotto
many thousands of isolation attempts from indi- 1992; Williams 1992). In summary, the following
vidual root tips (Rosling et al. 2011). Culturing five hypotheses related to our previous mechanis-
fungal communities using rich media is a way to tic explanations of uncultivability have been
promote biases in growth rates and to favor postulated: (1) either the requirement for an
saprobes with general growth parameters over unknown essential metabolite(s) or the appropri-
symbionts which may have more specific nutrient ate form/amount has not yet been fulfilled; (2) the
limitations. Interestingly, in the case of continuous synthesis of enzymes or
Archaeorhizomyces, it is unclear whether or not macromolecules necessary during the early stages
the fungus is symbiotic. Archaeorhizomyces can of the germination process is blocked; (3) nuclear
readily grow, albeit at a slow rate, on a number of DNA replication during spore germination is
basic undefined media without complex absent, suggesting that there is no nuclear divi-
additions. This example shows that if one can sion in vitro; (4) the fungi have lost part of their
find these fungi and provide the correct cues, the genetic equipment involved in its free-living
limitation may not be the specifics of the growth growth during coevolution with the host plant
medium. On the other hand, as we discuss below, and, therefore, the corresponding gene function
very specific nutrient limitations, e.g., myristate, must be supplied by the host in the development
could be limiting factors that make fungi unculti- of biotrophic symbiosis; and (5) the intraradical
vated (Sugiura et al. 2020). One lesson from these feeding structure for the fungal mycelium can be
examples is that if these fungi that were probably crucial. This latter hypothesis would mean that
considered to be the least amenable to culture can only an active arbuscule would be able to absorb
actually be cultivated, could perhaps all fungi? In nutrients, possibly because of its structure and
the following, we will discuss some of the lessons permeability properties, its enzymatic equipment
learned from developing methods of culturing for nutrient transport, or the chemical and physi-
symbiotic fungi. cal environment where it is developed and posi-
tioned. It has been proposed that success in
culturing AM fungi would be achieved by defin-
2.4 Major Breakthroughs Cause ing the contribution of factors supplied by the
Reconsideration host plant to hyphal growth and supplying these
of Uncultivable Lineages factors axenically (Becard and Piche 1989).
The contribution of autotrophic higher plants
2.4.1 Big Spores, Big Needs: to the mutualistic relationships of the arbuscular
Arbuscular Mycorrhizal Fungi mycorrhiza (AM) appears to begin before the
AMF has been established in the root cortex
Arbuscular mycorrhizal fungi (AMF) are obligate (Barea 1986). Compounds released from plant
symbionts that cannot grow without their host roots seem to affect the establishment of the
40 S. Rahimlou et al.

association by acting on some pre-infection Using clues from the biochemistry and mor-
stages. Obviously, root exudation plays an impor- phology of AM symbioses helped uncover how
tant role in rhizosphere biology. Roots synthesize they may be cultured axenically. Extraordinarily
many organic compounds, some of which are large spores of AM fungi hinted that these species
released into the surrounding soil (Bowen 1980; have lost independent foraging for sugar acquisi-
Curl and Truelove 1986). The compounds tion because germinating spores must depend on
released by roots can be classified according to their own energy sources before a mycorrhizal
their mobility in the soils as follows: (1) diffusible, symbiosis can be established. AM fungi are also
water-soluble; (2) diffusible-volatile; and known to accumulate lipids that comprise
(3) non-diffusible compounds. Water-soluble 46–70% of the spore weight (Beilby and Kidby
exudates exert their influence at the root surface 1980). These high lipid contents are likely stored
and spread only a few millimeters into the soil, for germination and initial growth before success-
whereas volatile exudates act further away from ful infection. Observations of fatty acid importa-
the root surface, often several centimeters into the tion from host plants (Jiang et al. 2017;
soil (Koske and Gemma 1992). Becard and Piche Luginbuehl et al. 2017) and mycorrhizal defects
(1989) found that the presence of a growing root caused by lipid-related plant mutants (Bravo et al.
significantly stimulated the growth of the fungus 2017; Keymer et al. 2017) support the hypothesis
even when there was no root-fungal contact. They that AM fungi rely on their hosts for fatty acids.
suggested that the root may have exerted its influ- In a breakthrough paper, Sugiura et al. (2020)
ence by changing the composition of the agar determined addition of the fatty acid myristate
medium or the gaseous phase in the dual culture (C14:0) led to an increase in the biomass of
system. However, since active fungal growth Rhizophagus irregularis, inducing extensive
ceased with root removal, it appears that either hyphal growth and formation of infection-
the relevant compounds in root exudates were competent secondary spores (albeit smaller) in
unstable or they did not accumulate in sufficient asymbiotic cultures. Furthermore, it has been
quantities. Gianinazzi-Pearson et al. (1989) demonstrated that R. irregularis can take up
demonstrated that two flavanones (hesperetin fatty acids in its branched hyphae and use
and naringenin) and a flavone (apigenin), which myristate as a carbon and energy source, and
activate nod gene expression in Rhizobium, myristate also promoted the growth of
elicited mycelial growth responses from AMF Rhizophagus clarus and Gigaspora margarita
spores. They, therefore, suggested that the pres- (Sugiura et al. 2020). It has been reported that
ence of such molecules in root exudates may be the AM fungus Rhizophagus intraradices Sy167
involved in cell-to-cell contact between the can grow and develop fertile spores indepen-
mycorrhizal partners in the early pre-infection dently of a host plant in coculture with the bacte-
phases of the recognition phenomena between rium Paenibacillus Validus (Hildebrandt et al.
symbionts. Since root exudates are known to 2006). Kameoka et al. (2019) found that (S)-12-
stimulate microbial activity in the rhizosphere, methyltetradecanoic acid, a methyl branched-
they may exert an indirect activity on AMF due chain fatty acid isolated from Paenibacillus
to the associated microorganisms affecting spore Validus cultures, stimulates the branching of
germination (Barea 1986; Linderman 1992). In an hyphae germinated from mother spores and the
early study, Mosse (1959) found that spores of formation of secondary spores in axenic culture of
AMF that showed a low level of germination on the AM fungus Rhizophagus irregularis. Their
water agar could be stimulated to germinate in a extensive testing of fatty acids in R. irregularis
non-sterile, soil-water agar. She suggested that a revealed that palmitoleic acid induces more sec-
water-soluble, heat-labile, dialyzable product of ondary spores than the bacterial fatty acid.
microbial origin was responsible for the enhance- Although AM fungi accumulate lipids, many
ment of germination. ECM and saprobic fungi store carbohydrates such
as glycogen, trehalose, and mannitol as their
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 41

carbon sources (Martin et al. 1987; François and non-lipid-dependent lipophilic Malassezia spe-
Parrou 2001; Nehls 2008). AM fungi lack almost cies, which benefits from lipid addition to its
all the genes encoding polysaccharide hydrolases medium. This species differs from all other
that generate glucose except trehalase and glyco- Malassezia yeasts by the fact that lipid supple-
gen phosphorylase, whereas most of these poly- mentation is not an absolute requirement and
saccharide hydrolase genes are present in other growth does not depend on the presence of
phyto-biotrophic fungi (Kobayashi et al. 2018). long-chain fatty acid precursors in the medium.
Thus, AM fungi are hypothesized to have Human skin is frequently colonized by lipid-
completely abolished glucose acquisition except dependent Malassezia yeasts, but seldom by
for what is imported from their host plants. This M. pachydermatis. For cultivation of Malassezia
absolute sugar host dependency of AM fungi may species most investigators employed media
be reflective of its long symbiotic history. supplemented with olive oil, Tween 20, Tween
The growth of AM fungi is hypothesized to 80, or glycerol monostearate (Ushijima et al.
depend on many other compounds in addition to 1981). Furthermore, in almost all cases of isola-
lipids and carbohydrates. The absence of several tion of Malassezia, agar plates or slants were
metabolic pathways associated with the biosyn- overlaid with olive oil (Gordon 1951). It was
thesis of vitamin B6 derivatives and thiamine shown that the pathogenic yeast M. furfur (for-
synthesis has been confirmed (Kobayashi et al. merly Pityrosporum orbiculare and
2018). The missing thiamine synthetic pathway Pityrosporum ovale) grows in vitro only if fatty
suggests that AM fungi may have a non-thiamine- acids from C12 to C24 series are added to the
dependent infection system. Since these vitamins culture medium. Except for elaidinic and
are essential for events other than infection, the nervonic acids, all saturated and unsaturated
addition of these compounds to media may fatty acids tested support growth (Porro et al.
improve the culture efficiency of AM fungi. 1976). Until recently, it was assumed that
Malassezia evolved into a specialized and narrow
niche associated with the skin of mammalian
2.4.2 Malassezia: Another Lipid hosts. However, culture-independent studies of
Requiring Fungus fungi from environmental samples show that
Malassezia are exceedingly widespread and
Malassezia species are lipid-dependent and fas- ecologically diverse (Amend 2014). Recent stud-
tidious basidiomycetous yeasts that inhabit the ies in little-characterized marine environments
skin and mucosa of humans and other warm- point to extensive diversification of Malassezia-
blooded animals and are a major component of like organisms, providing exciting opportunities
the skin microbiome. They occur as skin to explore the ecology, evolution, and diversity of
commensals but are also associated with various this enigmatic group.
skin disorders and bloodstream infections. Under
certain circumstances, Malassezia species can be
involved in relatively common skin diseases, 2.4.3 Uredinales: Plant Pathogens
including pityriasis (tinea) versicolor, seborrheic Requiring Protein
dermatitis, and folliculitis. Their difficulty to cul-
ture axenically has long hampered studies of their Members of the Uredinales have usually been
systematics and diversity (Guého et al. 1996). considered classical obligate parasites among
Malassezia species are not able to grow on the fungal plant pathogens. However, there have
mycological media routinely used in clinical been successful attempts to germinate, grow,
laboratories because of their lipid dependency. It and maintain rust species. Attempts to culture
is therefore necessary to use specific media such rusts saprobically have been based on two
as Dixon agar or Leeming-Notman agar methods: firstly, the initiation of saprobic growth
containing lipids. M. pachydermatis is a from germinating uredospores and, secondly,
42 S. Rahimlou et al.

saprobic growth from mycelium formed in cultures of other cereal rusts have since been
infected host callus tissue. The original idea was attempted using the procedures described above.
to form callus tissue of a susceptible host with its It is noted that Bushnell and Stewart (cited in
rust fungus by aseptic transfer to culture media. Scott and Maclean 1969) tested 16 North Ameri-
Hotson and Cutter (1951) and Cutter (1959) can races of P. graminis tritici and obtained veg-
reported saprobic axenic culture from infected etative growth from 12 of these races. Most of
callus tissue of three rust species. Because of the these cultures survived initial subculturing, but
failure of others to repeat Cutter’s experiments, development from subsequent subcultures was
his work has not received the recognition it erratic. Macroscopic colonies, but no sporulation,
deserves. of three other Australian races of P. graminis
The greatest amount of work on the reaction of tritici and limited post-sporeling growth of
rust spores to nutrient media has been carried out P. graminis secalis and P. recondita tritici were
with uredospores. Among species of major inter- obtained (Scott and Maclean 1969). Singleton
est, they are the most prolific and uniformly pro- and Young (1968) reported vegetative growth of
duced spores and may be readily obtained by an American isolate of P. recondita, using the
reinfection of plants of the same host species. yeast extract-peptone medium. The dependence
While relatively little work has been done with on peptone, a digest of protein high in amino
the other spore forms, an overview of some of this acids, suggests that the rusts may be deficient in
work is given, since these forms may serve as production of specific amino acids that they may
starting materials for future culture experiments. scavenge from their hosts.
The first successful axenic culture of rust from Teliospores and basidiospores—Work on the
uredospores was reported by Williams et al. response of teliospores to nutrients, chemicals,
(1966, 1967) on an Australian isolate of wheat moisture, temperature, etc., has been mainly
stem rust. It was observed that septate vegetative concerned with overcoming dormancy (Arthur
mycelium was formed from uredospores of et al. 1929; Carleton 1903; Kucharek and Young
P. graminis var. tritici race 126-ANZ-6,7 1968; Thiel and Weiss 1920). If growth were to
incubated on an agar medium containing be obtained from teliospores presumably the
Czapek’s minerals, 0.1% yeast extract, and 3% promycelium would form basidiospores which
sucrose. In tracing the development of this myce- would germinate and grow, or else the
lium, it was observed that germination and germ promycelium itself would initiate saprobic
tube elongation had occurred after 2 days. Initial growth directly. It has been reported (Reed and
branching of germ tubes was observed 4 days Crabill 1915) that germinating teliospores of
after seeding, and this branching continued for Gymnosporangium juniperi-virginianae formed
3–4 weeks. In subsequent work (Williams et al. promycelia which occasionally produced hyphae
1967), it was observed that the addition of 0.1% without basidiospore formation. Bailey (1923)
Evan’s peptone to the medium enhanced vegeta- mentioned a frequent “indefinite growth” and
tive growth, and some cultures formed stromata branching of promycelia of Puccinia helianthi.
bearing uredospores and teliospores 2–4 weeks Other workers (Colley 1918; Spaulding 1922)
after inoculation. Bushnell (1968) has subse- described the formation of a narrow-twisted
quently repeated this work, using peptone hypha without basidiospore production when
medium containing glucose and the same teliospores of Cronartium ribicola were
Australian isolate of wheat stem rust. He noted germinated underwater. In light of these
that yeast extract medium without the addition of observations, promycelia may be useful material
peptone did not support growth. It has been for studies on the saprobic growth of some rusts.
observed that the replacement of sucrose with Aeciospores—Work on these spores has been
glucose and the addition of citrate to the yeast limited to studies of germination in distilled water
extract-peptone medium resulted in more consis- or host decoctions (Bailey 1923; Carleton 1903;
tent growth (Scott and Maclean 1969). Axenic Klebahn 1904; Reed and Crabill 1915; Spaulding
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 43

1922). These spores are often difficult to obtain in 2019). The validity of this approach was
abundance. demonstrated by culturing three diverse species
Spermatia—Although many workers failed in of TM7/Saccharibacteria along with their
attempts to germinate spermatia (De Bary 1887; interacting Actinobacteria hosts, including one
Reed and Crabill 1915), Plowright (1889) previously uncultured Actinomyces species. This
observed limited yeast-like budding when method, dubbed “reverse genomics,” exploits
spermatia of several rusts were placed on nutrient culture-independent sequence data for physical
solutions containing sucrose or honey. Other retrieval of specific microorganisms via antibody
workers (Brefeld 1981; Cornu 1876) observed affinity and does not depend on any physiological
similar phenomena. Carleton (1903) obtained selection criteria. Therefore, it should be broadly
germ tubes rather than budding, and their forma- applicable to any target organisms, as long as
tion was stimulated by sucrose or honey. robust cultivation conditions can be identified
Spaulding (1922) cited the unpublished work of for isolated cells. Antibody-isolated cells could
York and Overholts, who investigated the germi- also be funneled into other platforms that enable
nation of C. ribicola spermatia on various nutrient high-throughput media selection and optimiza-
solutions. It was noted that preincubation in the tion protocols, for example, microdroplets or
cold for up to 18 days was a necessary prerequi- ichips (Nichols et al. 2010; Berdy et al. 2017;
site for germination. Zengler et al. 2002).
Various approaches are used by
microbiologists to provide favorable conditions
2.5 Use of Genomic Data to Inform for bacteria to overcome limiting factors. In the
Cultivation Strategies following, we present three additional examples
of several successful experiments for cultivating
2.5.1 Examples from Bacteria fastidious bacteria:
It has been reported that He et al. (2015) have
Microbial diversity is unfathomably deep and been able to cultivate strain TM7x from the
getting deeper. Single-cell genomes and human oral cavity, which is the first taxon of the
metagenomes continue to increase the known candidate phylum Saccharibacteria (TM7). This
diversity of Bacteria and Archaea; however, was accomplished by plating saliva samples on a
while ‘omics can be used to infer physiological specialized oral culture medium (SHI medium)
or ecological roles for species in a community, and targeting the enrichment of TM7 taxa by
most of these hypothetical roles remain streptomycin selection. The successful enrich-
unvalidated. Genome-based metabolic ment of strain TM7x in streptomycin-added
reconstructions might provide insights that under- media might be explained by its association with
pin the cultivation of uncultured bacteria and the streptomycin-resistant Actinomyces
archaea, but so far this promise has only been odontolyticus XH001 species (Bor et al. 2016).
realized for a few species of microbes. Furthermore, a signaling interaction between the
The availability of metagenomics and two bacterial species has been confirmed by
metabarcoding means we know of the existence transcriptomic and metabolomic analyses. With
of certain taxa that have never been cultivated an extremely small cell size and reduced genome
(Jones et al. 2011; Tedersoo et al. 2017), and of 705 kb lacking the capacity to synthesize any
one aim is to cultivate those taxa from mixed amino acids, strain TM7x represents a classic
samples using our ever-improving understanding example of auxotrophy.
of translating genome into cellular phenotype. A Coxiella burnetii is the causative agent of Q
recently developed approach uses information fever, a worldwide zoonosis that causes polymor-
found in single-cell genomics and metagenomics phic clinical manifestations such as flu-like
sequences to design antibodies capable of selec- syndromes, pneumonia, endocarditis, meningitis,
tively isolating bacteria or archaea (Cross et al. and osteomyelitis (Raoult et al. 1990). This
44 S. Rahimlou et al.

microorganism has previously been isolated from (D’Onofrio et al. 2010; Lewis et al. 2010). Other
infected animal tissues and a tick in Montana growth-stimulatory factors include the quorum-
(Cox 1938a). Subsequently, this intracellular bac- sensing molecules acyl-homoserine lactones
terium was successfully cultivated in embryo- (AHL), which have been shown to facilitate the
nated eggs, mice, and guinea pigs (Cox 1938b). growth of an AHL-degrading novel
Coxiella burnetii could also be propagated in proteobacterium (Chan et al. 2009), and trace
HEL cells using a shell-vial assay (Raoult et al. elements at levels matching those found in the
1990), which is currently the method used for its source environment (Meyer-Dombard et al.
isolation from clinical samples (Gouriet et al. 2012).
2005). Recently, an axenic medium has been
described for C. Burnetii (Omsland et al. 2009,
2011). This medium was developed in three steps. 2.5.2 Examples from Fungi
First, metabolic pathway deficiencies were
identified in C. burnetii using in silico genomic Taking inspiration from genomics-based work in
analysis, and a medium was found that sustained bacterial systems (Omsland et al. 2013), Ahrendt
the metabolic activity of this organism, as et al. (2018) used information on the metabolic
measured through [35S] Cys-Met incorporation deficiencies inferred from the first fungal single-
(Omsland et al. 2008). In the second step, the cell genome sequencing to try to axenically cul-
transcript profiles and phenotype microarrays ture their target mycoparasitic species. The three
were analyzed, leading to the formulation of target species were all obligate parasites which
ACCM (acidified citrate cysteine medium) could only be grown in coculture with their fun-
(Omsland et al. 2009). This medium has an acidic gal hosts (Benny et al. 2016). Functional analysis
citrate buffer pH, is incubated in a 2.5% O2 atmo- of the genomes indicated certain commonalities
sphere, and is enriched with cysteine, among the uncultivable targets of the single-cell
neopeptone, and casamino acids. Optimization study. Specifically, these fungi lack the gene or
through the addition of methyl-β-cyclodextrin genes involved in production of thiamine,
has led to the formulation of ACCM 2 (Omsland spermidine, and biotin. Using media amended
et al. 2011), a medium that has allowed significant with three amino acids (cysteine, methionine,
progress in understanding C. burnetii and tryptophan), biotin, spermidine, thiamine,
pathogenesis. and magnesium sulfate, two of the three species
An effective and straightforward strategy for were able to grow axenically for the first time
stimulating the growth of uncultivated bacteria is (Ahrendt et al. 2018). One mycoparasite was
the supplementation of media with chemical still unable to grow successfully. While the
compounds that are likely to be required by the authors note that even those species which
bacteria in question. D’Onofrio et al. (2010) would grow axenically would still not sporulate
determined the identity of growth factors pro- in culture, this study represents a tale of modest
duced by “helper” strains that were responsible success in using genome-derived metabolic
for the growth of dependent uncultivated deficiencies as the basis for media creation and
organisms from marine sediment and were able new avenues for biotrophic fungal cultivability.
to stimulate the growth of the latter by Marine Malassezia species represent an inter-
supplementing media with the growth factors, esting case where the fungi have never been
namely iron-scavenging siderophores. Several cultivated, yet there are apparently distinct taxa
studies have demonstrated the growth-promoting that inhabit these ecosystems. One approach to
effect of siderophores on uncultivated and “diffi- cultivating these fungi would be to obtain a
cult-to-culture” bacteria (Guan and Kamino 2001; genome sequence first (Fig. 2.1). By comparison
Vartoukian et al. 2016). It has been suggested that of the marine metagenomes to genomes of terres-
the ability to autonomously produce siderophores trial species which have been successfully
may have been lost in uncultivated bacteria cultivated, corresponding nutrient deficiencies
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 45

Fig. 2.1 A schematic overview of how genomic data can sequence data, identifying Malassezia contigs from
be used to identify nutritional constraints in marine aquatic samples, and assessing its phylogenetic affinity to
Malassezia species. Sampling aquatic environments and skin-associated Malassezia, comparing genomic content
skins from mammals, Malassezia species associated with of aquatic and skin-associated Malassezia, culturing
the skin require lipids to grow, extracting DNA from both aquatic Malassezia by adding protein requirements to
samples, (meta)genome sequencing, assembling the media

can be investigated and added to the media as has

worked for bacterial species (Tyson et al. 2005). Coelomomyces on its hosts was accom-
plished with the discovery by Whisler
et al. (1975) that Coelomomyces has a com-
The Elusive Axenic Culture of Coelomomyces,
plex life cycle with alternation of
a Biotrophic Pathogen of Microarthropods
generations between a diploid stage
Coelomomyces (Blastocladiomycota) has
infecting mosquito larvae and a haploid
received much attention due to its role as a
stage infecting crustaceans, especially
primary pathogen of dipteran larvae.
copepods. Reliable cultures of
Because of the importance of mosquitoes
Coelomomyces can be maintained by grow-
as vectors of human disease, there has been
ing the mosquito larvae in the presence of
considerable interest in using
copepods such as Cyclops (Bland 1985).
Coelomomyces for biocontrol, with a few
The discovery of the copepod-infecting
reports of modestly successful application
phase helped to explain why infection of
(Scholte et al. 2004). Coelomomyces is gen-
mosquito larvae by field-collected diploid
erally resistant to culture, which has greatly
sporangia was unpredictable and reliant on
hindered its development as a biocontrol
addition of non-sterile substrata.
agent, though it may be readily cultured
Limited development in axenic culture
on its hosts (Bland 1985). A major break-
has been reported using a number of
through in the persistent cultivation of
(continued)
46 S. Rahimlou et al.

complex media (Shapiro and Roberts 1976; the draft genome for evidence of loss of
Castillo and Roberts 1980). For example, primary metabolism genes that may suggest
Shapiro and Roberts (1976) were able to dependencies on the hosts for supply. Con-
grow Coelomomyces axenically for trary to our expectations, these searches
2 years in a modified medium for myco- revealed the presence of fatty acid synthase
plasma. Importantly, Coelomomyces devel- homologs FAS1 and FAS2, suggesting the
opment in axenic culture has not yielded ability of Coelomomyces to perform fatty
mature sporangia capable of dehiscence acid elongation. However, the genome
which would be necessary for deployment lacked the genes necessary for synthesis of
as a biocontrol. Castillo and Roberts (1980) the vitamins thiamine and biotin and is
extensively studied the effects of various missing many of the biosynthesis pathways
vertebrate and invertebrate culture media for amino acids, such as histidine, trypto-
on the development of Coelomomyces phan, lysine, and arginine. Overall, these
punctatus. These media were only effective data are generally consistent with the previ-
at culturing hyphae from the sporophyte ous data on in vitro culturing of
stage and not the gametophyte, though Coelomomyces and suggest this approach
gametes were released in at least one could help with the culturing of the fungus.
medium condition. The media trials A major challenge will still be developing
performed by Castillo and Roberts shed methods to stimulate production of resting
some light on metabolic dependencies of sporangia or gametes which will be critical
Coelomomyces. First, conditioning of the for field deployment of a biocontrol.
media by the appropriate host cell line
(Anopheles stephensi) could stimulate
development of Coelomomyces, but condi-
tioning with non-hosts (A. gambiae and
2.6 The Evolution of Metabolic
A. aegypti) could not. Second, the addition
Constraints in Symbiosis is
of the tripeptide glycyl-L-histidyl-L-lysine
Shaped by the Host, Lifestyle,
improved Coelomomyces growth in some
and Infection Strategy
media, indicative of amino acid
dependencies. And third, brain heart infu-
Given that symbiotic fungi derive much of their
sion (BHI) was one of the better media for
nutrients from the host, it would stand to argue
cultivation. BHI is known to be rich in
that their primary metabolism should be greatly
lipids, fatty acids, sterols, and vitamins.
affected and predicted by the identity of the host
Importantly, Coelomomyces is known to
as well as the nature of the interaction. Obviously,
be associated with the fat bodies of the
not all symbionts are the same, and both
host and is assumed to consume them
mutualists and pathogens spend variable amounts
(Umphlett 1962; Couch 1972).
of energy building cells separate from their host.
Given these observations, it is likely that
Even if a parasite is primarily external to its host,
Coelomomyces has complex metabolic
it may yet be completely reliant on the host via
dependencies on its host, such as fatty
absorption through haustoria. Laboulbeniales, for
acids, vitamins, and amino acids. Very
example, are an uncultured group of ectoparasites
recently, the draft genomes of both gameto-
on arthropods with many haustorial species but
phyte and sporophyte stages have been pro-
with unclear metabolic dependencies on their
duced (Ettinger et al. 2023). The genome is
hosts (Haelewaters et al. 2022). Labouls are noto-
somewhat reduced, with 20–23 Mb and
rious for their high host specificity, meaning they
7416 protein-coding genes. We searched
may experience a highly predictable environment
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 47

which could facilitate loss of production of cer- reflected in genome sizes, primary metabolism
tain metabolites that could be sourced from the capabilities, and transporter proteins.
host (Benjamin and Shanor 1952). Genome How predictive can the type of host be in
sequencing is needed to address this question. patterning fungal symbiont primary metabolism,
The parasite-mutualist continuum imposes and can comparative genomics come to the aid of
variable demands on the host for nutrition this question? Specifically, when looking at a new
(Drew et al. 2021). Commensal species like genome from, for example, an uncultured fungus
Malassezia and Laboulbeniales may maintain known only from metagenomic sequencing, can
high metabolic flexibility as they source diverse we perform reverse ecology to determine what
nutrients from their hosts. Models of mutualism type of host it may associate with? Over the last
typically posit movement of sanctioned amounts two decades, comparative genomics of fungi has
of specific resources between host and symbiont sought the answer to this question by comparing
(Yoder and Tiffin 2017), whereas pathogens, and genomes of species that have different ecological
particularly necrotrophic ones, might be consid- niches (Stauber et al. 2020; Wu and Cox 2021;
ered less selective in the resources they obtain Romero-Olivares et al. 2021; Wang et al. 2022).
from their host. Where the host itself is a free- There are clear indications that animal pathogens
living organism such as a plant, consideration of and plant pathogens diverge, primarily in their
access to the host cell’s nutrients predicts that uses of proteinases and carbohydrate-active
necrotrophic and hemibiotrophic pathogens enzymes that may be at the frontline of extracting
would have the greatest access to resources, energy from their hosts (Wu and Cox 2021).
biotrophic pathogens would have less, and However, the basic building blocks of cells are
mutualists would have the least. Because of this, the same across the eukaryotes, with similar
mutualists should be the easiest to culture because membrane systems, nucleic acids, and proteins.
they are more dependent on resources from their These similarities allow fungi and other
hosts and are required to be able to synthesize a pathogens to switch quickly between hosts of
diversity of primary metabolites, and this different kingdoms (Quandt et al. 2015). On the
suggests that mycorrhizae, lichenized fungi, and other hand, pathogens of plants which lack essen-
endophytes will be culturable. Endobiotic tial vitamins and amino acids might be expected
mutualists, such as the highly obligate to more often evolve losses of primary metabo-
transovarian bacterial endosymbionts, go against lism genes more often than animals, whereas
this prediction (McCutcheon and Moran 2012). animal parasites may need to maintain them as
However, there may be a fundamental difference the host must acquire them from the environment.
between symbionts that are confined to vertical For example, the essential amino acids for
transmission within a host, versus those that have mammals, histidine, isoleucine, leucine, lysine,
shown horizontal transmission and require methionine, phenylalanine, threonine, trypto-
finding new partners (Law and Lewis 1983). phan, and valine, might be expected to be
This should also be reflected in the transporter maintained by their pathogens, while the other
protein families that should reflect the need to amino acids could potentially be taken from the
grow apart from the host, even if only momentar- host (Derilus et al. 2021). Other predictions based
ily. For example, the horizontally transferred on the host are that mycoparasites may be most
mycorrhizae should be expected to have a diverse likely to lose primary metabolism relative to ani-
set of transporter genes for supporting growth in mal pathogens, which in turn could happen more
the extraradical mycelium. Lichens that repro- than plant pathogens. This logic stems from phy-
duce with ascospores should similarly require a logenetic relatedness such that compatibility of
greater deal of transporter proteins than those that biochemical intermediates and even building
always co-disperse with their algal partners. blocks is likely increased as parasite and host
These predictions are testable by genome become more phylogenetically closely related.
sequencing and annotation as they should be
48 S. Rahimlou et al.

What do the genomic data suggest in light of flow, for example, retain and allow investigation
these predictions? We highlight trends by explor- of interspecies signaling and potential
ing primary metabolism in 8 symbiotic fungi with cross-feeding. By integrating multiple media and
the results summarized in Fig. 2.2. Firstly, cultivation conditions, this approach has been
genome size is variable across species and not a successful in expanding the repertoire of cultured
great indicator of whether primary metabolism species and strains from open environments and
genes will be lost. Most fungi maintain the ability human-associated microbiota. Restricting poten-
to synthesize most amino acids, with the excep- tial competition by stochastic dilutions of com-
tion of the water mold endoparasite Rozella plex microbial samples to a few or single cells,
allomycis and the lung pathogen Pneumocystis. isolated in micro-compartments, while exposing
Losses were not offset by the presence of addi- them to native environmental conditions through
tional amino acid permeases, indicating that para- porous membranes (Ichip and similar devices),
sitic fungi are likely using other pathways for has also resulted in pure cultures of microbes
scavenging amino acids, perhaps via peptidases. that were refractory to isolation using standard
Most fungi, however, retained the ability for techniques (Fig. 2.3).
uptake of ammonium, which could be used for To address each limiting factor, several inno-
building nitrogenous molecules, including amino vative methods have been developed. Kaeberlein
acids. The ability to synthesize the vitamins biotin et al. (2002) were among the first to propose the
and thiamine and the ability to make long-chain “simulated natural environment” concept. They
fatty acids were characteristics readily lost in the designed diffusion chambers within which
obligate symbiotic fungi, with the exception of organisms were seeded after extraction from
Puccinia graminis. The frequent loss of fatty acid marine sediment. The chambers were incubated
synthase in many symbiotic fungi is curious given under conditions mimicking the natural environ-
the diverse roles of these molecules in the cell ment, allowing the passage of growth-stimulatory
(Ciznár and Hostacká 1983); it may be that these chemical factors from the external environment
molecules are taken from the host as diverse across semi-permeable (0.03 μm-pore) mem-
sources of energy through the fatty acid degrada- brane walls of the chambers and resulting in the
tion pathway associated with production of growth, and ultimately pure culture, of previously
acetyl-CoA. Additional analyses of amino acid, uncultivated bacteria from the marine environ-
fatty acid, and other primary metabolite biosyn- ment (Fig. 2.3d). Bacteria of interest may also
thesis pathways are warranted to test some of the be exposed to signals or nutrients from the natural
predictions we have outlined. habitat, or engineered versions of the latter, after
encapsulation of single cells or subsets of the
microbial community as microdroplets using
2.7 Novel Experimental Systems microfluidic devices. Zengler et al. (2002)
for Cultivation of Fastidious encapsulated single bacterial cells in gel
Microbes microdroplets, incubated them in amended sterile
versions of the natural environmental medium,
The causes of microbial “uncultivability” are detected growth as microcolonies in the
numerous and have been associated with a microdroplets using flow cytometry, and success-
requirement for factors produced by other fully isolated and cultured several novel bacterial
microbes, strict interspecies interactions, slow strains following transfer to microtiter plates. The
growth, competition/inhibition, and dormancy. process of diluting mixed samples down to single
Various technical approaches have been devel- cells prior to attempted cultivation, termed “dilu-
oped to alleviate cultivation recalcitrance (many tion to extinction,” eliminates the potential draw-
of which were reviewed recently in Rämä and back of competition between bacteria in the
Quandt 2021 and Lewis et al. 2021). Encapsula- community and results ideally in pure cultures
tion and incubation in microdroplets under media (Connon and Giovannoni 2002). Park et al.
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 49

Puccinia graminis

Q
NH4 Saccharomyces cerevisiae
N R
E
C
S
D
W
H Rhizophagus irregularis N
NH4
Q
S E
P G T P
Y D R
V A C K
M NH4
H
G T W
L N E
I
Q V A Y
L S D R 12 mb F
L M
K K I
P
T
89 mb H
C
F W
G V

I
Y Malassezia globosa
138 mb M
F
A R NH4
Q
N
S E
P D H
C K
Y
Pneumocystis jirovecii M
9 mb I
G
V T W
F
S N L
W A
P Q Y

A C
R
F
Entomophaga maimaga
D
E L
NH4
8 mb P
G K
V K E Q

H
T
M
N
R H
Beauveria bassiana
C
I
S Y NH4
R N Q
S
D D
C E
K
T P
T H
G W
34 mb G
V
A
W F
Y
Rozella allomycis I
L M
P F
R
A
H Q
N
S
I M
D E V
C
phylogenetic distance to host

L
12 mb T 1.2 gb
K G A L ammonium permease

Y NH4
M I
V F
amino acid permease
W
thiamine

biotin
spatial separation from host long-chain fatty acids

Fig. 2.2 Simplified diagram of several metabolic exception of amino acids in Pneumocystis, which was
pathways and transport of nitrogen in fungal species of obtained from Ma et al. (2016). Synthesis of fatty acids
varying host dependency and phylogenetic distance from was assessed by BLAST search with Saccharomyces
host. Saccharomyces cerevisiae is shown as a saprobic cerevisiae FAS1 and FAS2 proteins. Presence of an
example. Data were based on genomic analyses using ammonium permease was assessed with BLAST using
predicted proteins of each species as found on the Joint S. cerevisiae MEP1. Amino acid permease numbers are
Genome Institute’s MycoCosm web page, accessed on taken from MycoCosm annotation pages under
03/26/2023 (Ahrendt et al. 2022). Production of amino orthologous gene clusters (KOGs) 1286 and 1287. Thia-
acids was assessed using a BlastKOALA annotation mine and biotin synthesis capacity was assessed by
using settings “Fungi” and “family_eukaryotes” BLAST searching for THI4 and THI6 and BIO2 and
(Kanehisa et al. 2016) with pathways reconstructed in BIO4, respectively, using S. cerevisiae as the source
KEGG Mapper (Kanehisa and Sato 2020), with the

(2011) co-cultured pairs of dependent organisms minimal medium was achieved only by
within oil-coated microdroplets; since the two co-cultivation, indicative of cross-feeding.
organisms in each symbiotic pair were auxotro- The filamentous nature of most fungi
phic for a different amino acid, growth in the complicates the methods described above, which
50 S. Rahimlou et al.

Fig. 2.3 Technological advances in isolating and observ- rate and branching of hyphae can be observed within a
ing fungi show promise for a better understanding of the microfluidic device that can be observed under a micro-
nutrient requirements, cultivability, growth kinetics, and scope (Uehling et al. 2019). A thin tube can be
interspecies interactions. (a) Microscale observation is manufactured in parallel arrays for replication with two
enabling investigation of the growth of fungi at scale. inoculation ports (I1 & I2), but one port could be used to
The Monorail Device (Berry et al. 2017) can be used to load metabolites. Not drawn to scale. (c) Dilution to
establish segregated coculture regions separated by a extinction method was successfully used to isolate 73 spe-
hydrogel (H) formed in a culture dish, such as a microwell cies of fungi from leaf litter. Homogenized leaf samples
plate. The hydrogel is loaded into a port (L) and forms a are size selected and diluted, and added to media such that
segregated set of chambers. The chambers can be loaded less than one colony-forming unit will grow per well. (d)
with one or more species or nutrient solutions, here In situ cultivation has been successful for some microbes
depicted as yeast species. Because the Monorail Device using cultivation chambers placed into the environment
can be used to set up many such chambers that fit into a but maintaining contact. In this example (Gavrish et al.
multiwell plate, the whole setup can be replicated allowing 2008), a metal washer with carbon-free agar is sandwiched
numerous nutrient solutions to be examined while moni- between two membranes, one which allows the ingrowth
toring growth via an inverted microscope. (b). The growth of bacteria and could be adapted for fungi

have been applied to bacteria as highlighted by with other organisms (Delgado-Ramos et al.
Rämä and Quandt (2021). Flow cytometry, 2014; Uehling et al. 2019; Samlali et al. 2022;
microdroplet-based, and microfluidic approaches Richter et al. 2022). The major advantages of
can be used to isolate individual fungal cells and these methods are the ability to visualize the
study their physiology, growth, and interactions growth of cells using a stereo- or compound
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 51

microscope, the ability to sterilize entire systems extracted from logical extension of how
depending on the material, and the small scale symbioses work. For mutualists, the reciprocal
allowing small amounts of experimental exchange of nutrients can be expected. While
chemicals to be used. Microfluidic and microscale this might satisfy energy or mineral requirements,
approaches show promise for growth of fastidious it does not generally lead to the expectation of
filamentous fungi and have been used to study the wholesale support of primary metabolism of one
response of hyphae to chemical stimuli and mutualist at the expense of another, except for the
interactions with other species observed using a case of ancient vertically transmitted
microscope (Richter et al. 2022) (Fig. 2.3a, b). endosymbionts. On the other hand, parasites are
However, these approaches are not yet amenable not constrained by the need for fair exchange.
to isolating fungal cells from complex Yet, the evidence points to the fact that they by
environments to use for culturing, and other and large need to retain many of their primary
approaches are necessary. Dilution to extinction biosynthesis. Evidence that even the rusts can be
uses the idea that the major obstacle for culturing cultivated axenically shows that most biotrophic,
is competition between fastidious species and obligate fungal pathogens may retain their pri-
more rapidly growing ones and has been used mary metabolic capacity. The more telling param-
successfully to cultivate a greater diversity of eter for determining loss of primary metabolism is
leaf litter fungi by isolating them into wells with environmental constancy, and this may be quite
<1 colony-forming units per well (Fig. 2.3c) different for heteroecious rusts, for example, in
(Collado et al. 2007). Such an approach can be comparison with the mammalian lung parasite
scaled up using equipment such as the Prospector Pneumocystis. AMF have complex life cycles
(Isolation Bio) that leverages nanoscale chambers that require exploration and proliferation in the
to grow microbes in very small volumes (Persyn soil matrix. For this reason, they have likely
et al. 2022). The power is in the scale; 6000 wells maintained the ability to produce most of their
can be automatically screened using fluorescent metabolites. Following breakthroughs in cultur-
reporters for growth. A final strategy that has been ing major symbiotic groups, nutritional
successful is to bring instrumentation into the dependencies do not seem to be the primary
field to conduct in situ baiting or trapping of obstacle. Nonetheless, many fungi remain uncul-
bacteria from soil or marine habitats (Gavrish tured. In part, this mostly reflects the lack of
et al. 2008; Berdy et al. 2017), where ingrowth effort, which is considerable, even for a single
of microbes occurs while maintaining exposure to microbial species.
the environment separated by a finely pored The rapid expansion of fungal genome
<0.03 μm membrane (Fig. 2.3d). Some efforts sequencing has led to opportunities for reverse
to implement this were accomplished for soil ecology or the use of genomes to understand
fungi in which chips are buried and colonized ecological strategies, particularly useful for
by fungi and other organisms in the soil matrix microbial species (Levy and Borenstein 2012).
(Mafla-Endara et al. 2021), and this method These same approaches can be used to determine
shows promise for the development of a strategy the metabolic dependencies of organisms based
to target filamentous fungi. on their genomes and to use this information to
improve culturing of these microbes (Tyson et al.
2005). One of the weakest links in this approach
2.8 Conclusions is that assumptions regarding protein function and
the nature of metabolic networks are extracted
The concept of metabolic dependencies of from model systems, yet we know that protein
symbionts on their hosts is well couched in functions and networks evolve (Wagner 2012).
examples but little in the way of theory. In this Beyond metabolic dependencies, there are
chapter, we have attempted to highlight some of other aspects of symbionts that may lead to their
the expectations and predictions that could be uncultivated status, which we outline and have
52 S. Rahimlou et al.

been discussed for bacteria (Lewis et al. 2021). fungus Glomus mosseae. Trans Br Mycol Soc 86:
They include the absence of physical or chemical 337–340
Azcón-Aguilar C, Garda-Garda F, Barea JM (1991)
cues and the inability to make life cycle Germinación y crecimiento axenico de los hongos
transitions in culture, specifically evidenced by formadores de micorrizas vesiculo-arbusculares. In:
dimorphic fungi that transition between saprobic LopezGorge J, Barea JM, Olivares J (eds) Fijación y
and pathogenic morphotypes, for example in the movilización biológiea de nutrientes, nuevas
tendencias. CSIC, Madrid, pp 129–147
uncultivated Paracoccidioides loboi (Vilela et al. Bacon CW, Porter JK, Robbins JD et al (1977) Epichloe
2021). P. loboi yeast cells cannot be transferred typhina from toxic tall fescue grasses. Appl Environ
from an infected human to a culture plate, yet the Microbiol 34(5):576–581
cells can readily be used to infect mice. It there- Bailey DL (1923) Sunflower rust. Minn Univ Agr Exp Sta
Tech Bull 16:1–31
fore appears that the major barrier to cultivating Barea JM (1986) Importance of hormones and root
this fungus is convincing it to switch its metabolic exudates in mycorrhizal phenomena. In: Gianinazzi-
state into a saprobic one, which is observed in Pearson V, Gianinazzi S (eds) Physiological and genet-
other Paracoccidioides species that are opportu- ical aspects of mycorrhizae. INRA, Paris, pp 77–187
Barker SM, Barnett HL (1973) Nitrogen and vitamin
nistic environmentally acquired infections requirements for axenic growth of the haustorial
(Rodrigues et al. 2023). Either an environmental mycoparasite, Dispira cornuta. Mycologia 65(1):
cue, physical stimulation, removal of a blocked 21–27
pathway, or nutritional requirements could Becard G, Piche Y (1989) New aspects on the acquisition
of biotrophic status by a vesicular arbuscular mycor-
explain the inability of the fungus to be rhizal fungus Gigaspora margarita. New Phytol 112:
cultivated. For P. loboi and other uncultivated 77–83
pathogens, complete genome sequences can help Beilby JP, Kidby DK (1980) Biochemistry of ungermi-
understand what their growth requirements are. nated and germinated spores of the vesicular-
arbuscular mycorrhizal fungus, Glomus caledonius:
However, a more complete picture may be needed changes in neutral and polar lipids. J Lipid Res 21(6):
that includes a picture of not only the genes in the 739–750
genome, but which ones are, and critically, which Benjamin RK, Shanor L (1952) Sex of host specificity and
ones are not expressed, so that such information position specificity of certain species of Laboulbenia
on Bembidion picipes. Am J Bot 39(2):125–131
can enlighten cultivation strategies. Benny GL, Smith ME, Kirk PM, Tretter ED, White MM
(2016) Challenges and future perspectives in the sys-
tematics of Kickxellomycotina, Mortierellomycotina,
References Mucoromycotina, and Zoopagomycotina. In: Dw L
(ed) Biology of Microfungi. Springer, Cham,
Heidelberg New York, Dordrecht, London, pp 65–126
Ahrendt SR, Quandt CA, Ciobanu D et al (2018) Berdy B, Spoering AL, Ling LL et al (2017) In situ
Leveraging single-cell genomics to expand the fungal cultivation of previously uncultivable microorganisms
tree of life. Nat Microbiol 3(12):1417–1428 using the ichip. Nat Protoc 12(10):2232–2242
Ahrendt SR, Mondo SJ, Haridas S et al (2022) Berry SB, Zhang T, Day JH et al (2017) Upgrading well
MycoCosm, the JGI’s fungal genome portal for com- plates using open microfluidic patterning. Lab Chip
parative genomic and multiomics data analyses. In: 17(24):4253–4264
Martin F, Uroz S (eds) Microbial environmental geno- Bland CE (1985) Culture. In: Couch JN, Bland CE (eds)
mics (MEG). Springer US, New York, pp 271–291 The Genus Coelomomyces. Academic, Orlando, FL,
Alexander WG, Wisecaver JH, Rokas A et al (2016) pp 349–359
Horizontally acquired genes in early-diverging patho- Bonfante-Fasolo P, Perotto S (1992) Plants and
genic fungi enable the use of host nucleosides and endomycorrhizal fungi: the cellular and molecular
nucleotides. PANS 113(15):4116–4121 basis of their interaction. In: Verma DPS
Amend A (2014) From dandruff to deep-sea vents: (ed) Molecular signals in plant-microbe
Malassezia-like fungi are ecologically hyper-diverse. communications. CRC, Boca Raton, FL, pp 445–470
PLoS Pathog 10(8):e1004277 Bor B, Poweleit N, Bois JS et al (2016) Phenotypic and
Arthur JC, Kern FD, Orton CR et al (1929) The plant rusts physiological characterization of the epibiotic interac-
(Uredinales). Wiley, New York tion between TM7x and its basibiont Actinomyces.
Azcón-Aguilar C, Diaz-Rodriguez RM, Barea JM (1986) Microb Ecol 71(1):243–255
Effect of soil microorganisms on spore germination Bosshard PP (2011) Incubation of fungal cultures: how
and growth of the vesicular-arbuscular mycorrhizal long is long enough? Mycoses 54(5):539–545
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 53

Bowen GD (1980) Misconceptions, concepts and Curl EA, Truelove B (1986) The rhizosphere. Springer,
approaches in rhizosphere biology. In: Ellwood DC, New York
Latham MJ, Hedger JN, Lynch JM, Slater JM (eds) Cutter VM Jr (1959) Studies on the isolation and growth of
Contemporary microbial ecology. Academic, London, plant rusts in host tissue cultures and upon synthetic
pp 283–304 media. I Gymnosporangium. Mycologia 51(2):
Bravo A, Brands M, Wewer V et al (2017) Arbuscular 248–295
mycorrhiza-specific enzymes FatM and RAM2 D’Onofrio A, Crawford JM, Stewart EJ et al (2010)
fine-tune lipid biosynthesis to promote development Siderophores from neighboring organisms promote
of arbuscular mycorrhiza. New Phytol 214(4): the growth of uncultured bacteria. Chem Biol 17(3):
1631–1645 254–264
Brefeld O (1981) Untersuchungen aus dem D’Souza G, Kost C (2016) Experimental evolution of
Gesammtgebiete der Mykologie. Heft 9, Die Hemiasci metabolic dependency in bacteria. PLoS Genet
und die Ascomyceten. Munster, p 159 12(11):e1006364
Bushnell WR (1968) In vitro development of an Australian Davis WJ, Picard KT, Antonetti J et al (2018) Inventory of
isolate of puccinia graminis f. sp. tritici. Phytopathol- chytrid diversity in two temporary forest ponds using a
ogy 58(4):526 multiphasic approach. Mycologia 110(5):811–821
Carleton MA (1903) Culture methods with Uredineae. J Davison J, Moora M, Öpik M et al (2015) Global assess-
Appl Micros Lab Methods 6(1):2109–2114 ment of arbuscular mycorrhizal fungus diversity
Castillo JM, Roberts DW (1980) In vitro studies of reveals very low endemism. Science 349(6251):
Coelomomyces punctatus from Anopheles 970–973
quadrimaculatus and Cyclops vernalis. J Invertebr Day PR, Anagnostakis SL (1971) Corn smut dikaryon in
Pathol 35(2):144–157 culture. Nat New Biol 231(18):19–20
Chan KG, Yin WF, Sam CK et al (2009) A novel medium De Bary A (1887) Comparative morphology and biology
for the isolation of N-acylhomoserine lactone- of the fungi, mycetozoa, and bacteria. Clarendon Press,
degrading bacteria. J Ind Microbiol Biotechnol 36(2): Oxford
247 Dean P, Hirt RP, Embley TM (2016) Microsporidia: why
Cisse OH, Pagni M, Hauser PM (2014) Comparative make nucleotides if you can steal them? PLoS Pathog
genomics suggests that the human pathogenic fungus 12(11):e1005870
Pneumocystis jirovecii acquired obligate biotrophy Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS et al
through gene loss. Genome Biol Evol 6(8):1938–1948 (2014) Flow cytometry of microencapsulated colonies
Ciznár I, Hostacká A (1983) Toxic properties of for genetics analysis of filamentous fungi. G3: Genes
Morganella morganii. Ceskoslovenska Epidemiol Genom Genet 4(11):2271–2278
Mikrobiol Imunol 32(3):126–133 Derilus D, Rahman MZ, Serrano AE et al (2021) Proteome
Collado J, Platas G, Paulus B et al (2007) High-throughput size reduction in Apicomplexans is linked with loss of
culturing of fungi from plant litter by a dilution-to- DNA repair and host redundant pathways. Infect Genet
extinction technique. FEMS Microbiol Ecol 60(3): Evol 87:104642
521–533 Drew GC, Stevens EJ, King KC (2021) Microbial evolu-
Colley RH (1918) Parasitism, morphology, and cytology tion and transitions along the parasite–mutualist con-
of Cronartium ribicola. J Agric Res 15(6):19–59 tinuum. Nat Rev Microbiol 19(10):623–638
Connon SA, Giovannoni SJ (2002) High-throughput Duplessis S, Cuomo CA, Lin YC et al (2011) Obligate
methods for culturing microorganisms in very-low- biotrophy features unraveled by the genomic analysis
nutrient media yield diverse new marine isolates. of rust fungi. PNAS 108(22):9166–9171
Appl Environ Microbiol 68(8):3878–3885 Ellis JJ (1963) A study of Rhopalomyces elegans in pure
Cornu M (1876) Ou doit-on chercher les organes culture. Mycologia 55(2):183–198
fécondateurs chez les urédinées et ustilaginées? Bull Ettinger CL, Ostovar T, Yacoub M et al (2023) Genomic
Soc Bot France 23(3):120–122 resources enable insight into the developmental
Couch JN (1972) Mass production of Coelomomyces, a transcriptome of the blastoclad fungus, Coelomomyces
fungus that kills mosquitoes. PNAS 69(8):2043–2047 lativittatus, an obligate parasite of mosquitoes and
Cox HR (1938a) A filter-passing infectious agent isolated microcrustaceans. bioRxiv 2023-01
from ticks. III. Description of organism and cultivation Flaishman MA, Kolattukudy PE (1994) Timing of fungal
experiments. Public Health Rep 53(52):2270–2276 invasion using host’s ripening hormone as a signal.
Cox HR (1938b) Use of yolk sac of developing chick PNAS 91(14):6579–6583
embryo as medium for growing rickettsiae of Rocky François J, Parrou JL (2001) Reserve carbohydrates
Mountain spotted fever and typhus groups. Public metabolism in the yeast Saccharomyces cerevisiae.
Health Rep 1896-1970:2241–2247 FEMS Microbiol Rev 25(1):125–145
Cross KL, Campbell JH, Balachandran M et al (2019) Gao Q, Jin K, Ying SH et al (2011) Genome sequencing
Targeted isolation and cultivation of uncultivated bac- and comparative transcriptomics of the model
teria by reverse genomics. Nat Biotechnol 37(11): entomopathogenic fungi Metarhizium anisopliae and
1314–1321 M. acridum. PLoS Genet 7(1):e1001264
54 S. Rahimlou et al.

Gavrish E, Bollmann A, Epstein S et al (2008) A trap for in barcoding reveals inter-plant variation in species rich-
situ cultivation of filamentous actinobacteria. J ness of endophytic root fungi in Elymus repens. J
Microbiol Methods 72(3):257–262 Fungi 7(6)
Gianinazzi-Pearson V, Branzanti B, Gianinazzi S (1989) Imachi H, Nobu MK, Nakahara N et al (2020) Isolation of
In vitro enhancement of spore germination and early an archaeon at the prokaryote–eukaryote interface.
hyphal growth of a vesicular arbuscular mycorrhizal Nature 577(7791):519–525
fungus by host root exudates and plant flavonoids. James TY, Pelin A, Bonen L et al (2013) Shared signatures
Symbiosis 7:243–255 of parasitism and phylogenomics unite Cryptomycota
Gordon MA (1951) Lipophilic yeastlike organisms and Microsporidia. Curr Biol 23(16):1548–1553
associated with tinea versicolor. J Invest Dermatol Jeffries P, Young TW (1994) Interfungal parasitic
17(5):267–272 relationships. Cab International, Wallingford
Gostinčar C, Zalar P, Gunde-Cimerman N (2022) No need Jiang Y, Wang W, Xie Q et al (2017) Plants transfer lipids
for speed: slow development of fungi in extreme to sustain colonization by mutualistic mycorrhizal and
environments. Fungal Biol Rev 39:1–14 parasitic fungi. Science 356(6343):1172–1175
Gouriet F, Fenollar F, Patrice JY et al (2005) Use of shell- Jones MD, Forn I, Gadelha C, Egan MJ et al (2011)
vial cell culture assay for isolation of bacteria from Discovery of novel intermediate forms redefines the
clinical specimens: 13 years of experience. J Clin fungal tree of life. Nature 474(7350):200–203
Microbiol 43(10):4993–5002 Kaeberlein T, Lewis K, Epstein SS (2002) Isolating
Guan LL, Kamino K (2001) Bacterial response to “uncultivable” microorganisms in pure culture in a
siderophore and quorum-sensing chemical signals in simulated natural environment. Science 296(5570):
the seawater microbial community. BMC Microbiol 1127–1129
1(1):1–11 Kameoka H, Tsutsui I, Saito K et al (2019) Stimulation of
Guého E, Midgley G, Guillot J (1996) The genus asymbiotic sporulation in arbuscular mycorrhizal fungi
Malassezia with description of four new species. by fatty acids. Nat Microbiol 4(10):1654–1660
Antonie Van Leeuwenhoek 69:337–355 Kanehisa M, Sato Y (2020) KEGG Mapper for inferring
Haelewaters D, Lubbers M, De Kesel A (2022) The haus- cellular functions from protein sequences. Protein Sci
torium as a driving force for speciation in thallus- 29(1):28–35
forming Laboulbeniomycetes. IMA Fungus 13(1):1 Kanehisa M, Sato Y, Morishima K (2016) BlastKOALA
Hawksworth DL, Lücking R (2017) Fungal diversity and GhostKOALA: KEGG tools for functional charac-
revisited: 2.2 to 3.8 million species. Microbiol Spectr terization of genome and metagenome sequences. J
5(4):5–4 Mol Biol 428(4):726–731
He X, McLean JS, Edlund A et al (2015) Cultivation of a Keeling PJ, Slamovits CH (2004) Simplicity and complex-
human-associated TM7 phylotype reveals a reduced ity of microsporidian genomes. Eukaryot Cell 3(6):
genome and epibiotic parasitic lifestyle. PNAS 1363–1369
112(1):244–249 Keymer A, Pimprikar P, Wewer V et al (2017) Lipid
Held AA (1980) Development of Rozella in Allomyces: a transfer from plants to arbuscular mycorrhiza fungi.
single zoospore produces numerous zoosporangia and elife 6:e29107
resistant sporangia. Canad J Bot 58(8):959–979 Klebahn H (1904) Die Wirtwechselnden Rostpilsen
Helfer S (2014) Rust fungi and global change. New Phytol XXXVII. Borntrager, Berlin
201(3):770–780 Kobayashi Y, Maeda T, Yamaguchi K et al (2018) The
Hepper CM (1984) Isolation and culture of VA mycorrhi- genome of Rhizophagus clarus HR1 reveals a common
zal (VAM) fungi. In: Powell CL, Bagyaraj DJ (eds) genetic basis for auxotrophy among arbuscular mycor-
VA mycorrhiza. CRC Press, Boca Raton, FL, pp rhizal fungi. BMC Genomics 19(1):1–11
95–112 Kõljalg U, Nilsson RH, Abarenkov K et al (2013) Towards
Hildebrand EM (1938) Techniques for the isolation of a unified paradigm for sequence-based identification of
single microorganisms. Bot Rev 4:627–664 fungi. Mol Ecol 21(22):5271–5277
Hildebrandt U, Ouziad F, Marner FJ et al (2006) The Kopytko M, Correa-Torres SN, Estévez-Gómez MJ
bacterium Paenibacillus validus stimulates growth of (2017) Biodegradación estimulada de los suelos
the arbuscular mycorrhizal fungus Glomus contaminados con pesticidas organoclorados. Revista
intraradices up to the formation of fertile spores. de investigación agraria y ambiental 8(1):119–130
FEMS Microbiol Lett 254(2):258–267 Koske RW, Gemma JN (1992) Fungal reactions to plants
Hotson HH, Cutter VM Jr (1951) The isolation and culture prior to mycorrhizal formation. In: Allen MJ
of Gymnosporangium juniperi-virgininae schw. upon (ed) Mycorrhizal functioning, an integrative plant fun-
artificial media. PNAS 37(7):400–403 gal process. Chapman and Hall, New York, pp 3–36
Howard A (1900) On a disease of tradescantia. Ann Bot Kucharek TA, Young HC (1968) Teliospore germination
14(53):27–38 of Puccinia recondita f. sp. tritici. In: Phytopathology
Hoyer AK, Hodkinson TR (2021) Hidden fungi: combin- (vol 58(8), 1056). 3340 Pilot Knob Road, St. Paul, MN
ing culture-dependent and-independent DNA 55121: American Phytopathological Society
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 55

Law R, Lewis DH (1983) Biotic environments and the Nara K (2009) Spores of ectomycorrhizal fungi: ecological
maintenance of sex–some evidence from mutualistic strategies for germination and dormancy. New Phytol
symbioses. Biol J Linn Soc 20(3):249–276 181:245–248
Levy R, Borenstein E (2012) Reverse ecology: from Nehls U (2008) Mastering ectomycorrhizal symbiosis: the
systems to environments and back. Evol Syst Biol impact of carbohydrates. J Exp Bot 59(5):1097–1108
751:329–345 Nichols D, Cahoon N, Trakhtenberg EM et al (2010) Use
Lewis K, Epstein S, D’onofrio A et al (2010) Uncultured of ichip for high-throughput in situ cultivation of
microorganisms as a source of secondary metabolites. J “uncultivable” microbial species. Appl Environ
Antibiot 63(8):468–476 Microbiol 76(8):2445–2450
Lewis WH, Tahon G, Geesink P et al (2021) Innovations Omsland A, Cockrell DC, Fischer ER et al (2008)
to culturing the uncultured microbial majority. Nat Rev Sustained axenic metabolic activity by the obligate
Microbiol 19(4):225–240 intracellular bacterium Coxiella burnetii. J Bacteriol
Linderman RG (1992) Vesicular-arbuscular mycorrhizal Res 190(9):3203–3212
and soil microbial interactions. In: Bethlenfalvay GJ, Omsland A, Cockrell DC, Howe D et al (2009) Host cell-
Linderman RG (eds) Mycorrhizae in sustainable agri- free growth of the Q fever bacterium Coxiella burnetii.
culture. ASA Special Publication, Madison, WI, pp PNAS 106(11):4430–4434
45–70 Omsland A, Beare PA, Hill J et al (2011) Isolation from
Lücking R, Aime MC, Robbertse B et al (2021) Fungal animal tissue and genetic transformation of Coxiella
taxonomy and sequence-based nomenclature. Nat burnetii are facilitated by an improved axenic growth
Microbiol 6(5):540–548 medium. Appl Environ Microbiol 77(11):3720–3725
Luginbuehl LH, Menard GN, Kurup S et al (2017) Fatty Omsland A, Hackstadt T, Heinzen RA (2013) Bringing
acids in arbuscular mycorrhizal fungi are synthesized Culture to the Uncultured: Coxiella burnetii and
by the host plant. Science 356(6343):1175–1178 Lessons for Obligate Intracellular Bacterial Pathogens.
Lutzoni F, Nowak MD, Alfaro ME et al (2018) Contem- PLoS Pathog 9(9):e1003540. https://doi.org/10.1371/
poraneous radiations of fungi and plants linked to journal.ppat.1003540
symbiosis. Nat Commun 9(1):5451 Overmann J (2015) Significance and future role of micro-
Ma L, Chen Z, Huang DW et al (2016) Genome analysis of bial resource centers. Syst Appl Microbiol 38(4):
three Pneumocystis species reveals adaptation 258–265
mechanisms to life exclusively in mammalian hosts. Park J, Kerner A, Burns MA et al (2011) Microdroplet-
Nat Commun 7(1):10740 enabled highly parallel co-cultivation of microbial
Machlis L (1953) Growth and nutrition of water molds in communities. PLoS One 6(2):e17019
the subgenus Euallomyces. I. Growth factor Parniske M (2008) Arbuscular mycorrhiza: the mother of
requirements. Am J Bot 40(3):189–195 plant root endosymbioses. Nat Rev Microbiol 6(10):
Mafla-Endara PM, Arellano-Caicedo C, Aleklett K et al 763–775
(2021) Microfluidic chips provide visual access to in Persyn A, Mueller A, Goormachtig S (2022) Drops join to
situ soil ecology. Commun Biol 4(1):889 make a stream: high-throughput nanoscale cultivation
Martin F, Ramstedt M, Söderhäll K (1987) Carbon and to grasp the lettuce root microbiome. Environ
nitrogen metabolism in ectomycorrhizal fungi and Microbiol Rep 14(1):60–69
ectomycorrhizas. Biochimie 69(6–7):569–581 Piotrowski JS, Annis SL, Longcore JE (2004) Physiology
McCutcheon JP, Moran NA (2012) Extreme genome of Batrachochytrium dendrobatidis, a chytrid patho-
reduction in symbiotic bacteria. Nat Rev Microbiol gen of amphibians. Mycologia 96(1):9–15
10(1):13–26 Plowright CB (1889) A monograph of the British
Meyer-Dombard DR, Shock EL, Amend JP (2012) Effects Uredineae and Ustilagineae. Kegan Paul, Trench &
of trace element concentrations on culturing Co., London
thermophiles. Extremophiles 16(2):317–331 Porro MN, Passi S, Caprilli F et al (1976) Growth
Mix AJ (1949) A monograph of the genus Taphrina. Univ requirements and lipid metabolism of Pityrosporum
Kans Sci Bull 33(1):3–167 orbiculare. J Invest Dermatol 66(3):178–182
Mosse B (1959) The regular germination of resting spores Porter TM, Schadt CW, Rizvi L et al (2008) Widespread
and some observations on the growth requirements of occurrence and phylogenetic placement of a soil clone
an Endogone sp. causing vesicular-arbuscular mycor- group adds a prominent new branch to the fungal tree
rhiza. Trans Br Mycol Soc 42:273–286 of life. Mol Phylogenet Evol 46(2):635–644
Müller B, Grossniklaus U (2010) Model organisms—a Powell MJ, Letcher PM, James TY (2017) Ultrastructural
historical perspective. J Proteome 73(11):2054–2063 characterization of the host–parasite interface between
Nagae M, Parniske M, Kawaguchi M et al (2016) The Allomyces anomalus (Blastocladiomycota) and Rozella
relationship between thiamine and two symbioses: allomycis (Cryptomycota). Fungal Biol 121(6–7):
root nodule symbiosis and arbuscular mycorrhiza. 561–572
Plant Signal Behav 11(12):e1265723 Quandt CA, Bushley KE, Spatafora JW (2015) The
genome of the truffle-parasite Tolypocladium
56 S. Rahimlou et al.

ophioglossoides and the evolution of antifungal Singleton LL, Young HC (1968) The in vitro culture of
peptaibiotics. BMC Genomics 16(1):1–14 Puccinia recondita f. sp. tritici. Phytopathology 58:
Quandt CA, Beaudet D, Corsaro D et al (2017) The 1068
genome of an intranuclear parasite, Siqueira JO (1987) Cultura axenicae monoxenica dos
Paramicrosporidium saccamoebae, reveals alternative fungos micorrizicos vesiculoarbusculares. In: II
adaptations to obligate intracellular parasitism. elife 6: Reuniao Brasileira sobre Micorrizas. Sao Paulo, 2:
e29594 44–70
Rämä T, Quandt CA (2021) Improving fungal cultivability Solden L, Lloyd K, Wrighton K (2016) The bright side of
for natural products discovery. Front Microbiol 12: microbial dark matter: lessons learned from the uncul-
706044 tivated majority. Curr Opin Microbiol 31:217–226
Raoult D, Vestris G, Enea M (1990) Isolation of 16 strains Soudzilovskaia NA, Vaessen S, Barcelo M et al (2020)
of Coxiella burnetii from patients by using a sensitive FungalRoot: global online database of plant mycorrhi-
centrifugation cell culture system and establishment of zal associations. New Phytol 227(3):955–966
the strains in HEL cells. J Clin Microbiol 28(11): Spanu PD (2012) The genomics of obligate (and
2482–2484 nonobligate) biotrophs. Annu Rev Phytopathol 50:
Read DJ (1991) Mycorrhizas in ecosystems. Experientia 91–109
47:376–391 Spaulding P (1922) Investigations of the white-pine blister
Reed HS, Crabill CH (1915) Cedar rust disease of apples rust. Washington, DC: US Department of Agriculture,
caused by Gymnosporangium Juniperi-virginianae Forest Service: Bulletin 957:1–100
Schw. Virginia Agr Expt Sta Tech Bull 9:1–106 Stauber L, Prospero S, Croll D (2020) Comparative geno-
Richter F, Bindschedler S, Calonne-Salmon M et al (2022) mics analyses of lifestyle transitions at the origin of an
Fungi-on-a-Chip: microfluidic platforms for single-cell invasive fungal pathogen in the genus Cryphonectria.
studies on fungi. FEMS Microbiol Rev 46(6):fuac039 MSphere 5(5):e00737–e00720
Robbins WJ, Kavanagh F (1938) Vitamin B1 or its Steinberg RA (1939) Growth of fungi in synthetic nutrient
intermediates and growth of certain fungi. Am J Bot solutions. Bot Rev 5:327–350
25(4):229–236 Steinberg RA (1950) Growth of fungi in synthetic nutrient
Rodrigues AM, Hagen F, Puccia R et al (2023) solutions. II Bot Rev 16(4):208–228
Paracoccidioides and Paracoccidioidomycosis in the Stocker-Wörgötter E (2001) Experimental lichenology
21st Century. Mycopathologia 12:1–5 and microbiology of lichens: culture experiments, sec-
Romero-Olivares AL, Morrison EW, Pringle A et al ondary chemistry of cultured mycobionts, resynthesis,
(2021) Linking genes to traits in fungi. Microb Ecol and thallus morphogenesis. Bryologist 104(4):
82(1):145–155 576–581
Rosling A, Cox F, Cruz-Martinez K et al (2011) Sugiura Y, Akiyama R, Tanaka S et al (2020) Myristate
Archaeorhizomycetes: unearthing an ancient class of can be used as a carbon and energy source for the
ubiquitous soil fungi. Science 333(6044):876–879 asymbiotic growth of arbuscular mycorrhizal fungi.
Samlali K, Alves CL, Jezernik M et al (2022) Droplet PNAS 117(41):25779–25788
digital microfluidic system for screening filamentous Tedersoo L, May TW, Smith ME (2010) Ectomycorrhizal
fungi based on enzymatic activity. Microsyst Nanoeng lifestyle in fungi: global diversity, distribution, and
8(1):123 evolution of phylogenetic lineages. Mycorrhiza 20:
Schadt CW, Martin AP, Lipson DA et al (2003) Seasonal 217–263
dynamics of previously unknown fungal lineages in Tedersoo L, Bahram M, Põlme S et al (2014) Global
tundra soils. Science 301(5638):1359–1361 diversity and geography of soil fungi. Science
Scholte EJ, Knols BG, Samson RA et al (2004) 346(6213):1256688
Entomopathogenic fungi for mosquito control: a Tedersoo L, Bahram M, Puusepp R et al (2017) Novel
review. J Insect Sci 4(1):19 soil-inhabiting clades fill gaps in the fungal tree of life.
Schulze-Lefert P, Vogel J (2000) Closing the ranks to Microbiome 5(1):1–10
attack by powdery mildew. Trends Plant Sci 5(8): Thiel AF, Weiss F (1920) The effect of citric acid on the
343–348 germination of the teliospores of Puccinia graminis
Schüβler A, Walker C (2011) Evolution of the 'Plant- tritici. Phytopathology 10:448–452
Symbiotic' Fungal Phylum, Glomeromycota. In: Thorpe T (2012) History of plant tissue culture. Plant Cell
Pöggeler S, Wöstemeyer J (eds) Evolution of fungi Cult Protoc 318:9–27
and fungal-like organisms. Springer, pp 164–185 Tisserant E, Malbreil M, Kuo A et al (2013) Genome of an
Scott KJ, Maclean DJ (1969) Culturing of rust fungi. Annu arbuscular mycorrhizal fungus provides insight into the
Rev Phytopathol 7(1):123–146 oldest plant symbiosis. PNAS 110(50):20117–20122
Shapiro M, Roberts DW (1976) Growth of Coelomomyces Tsaousis AD, Kunji ER, Goldberg AV et al (2008) A
psorophorae mycelium in vitro. J Invertebr Pathol novel route for ATP acquisition by the remnant
27(3):399–402 mitochondria of Encephalitozoon cuniculi. Nature
453(7194):553–556
2 Metabolic Constraints and Dependencies Between “Uncultivable”. . . 57

Tumwine J, Frinking HD, Jeger MJ (2000) Isolation Wang Y, Wu J, Yan J et al (2022) Comparative genome
techniques and cultural media for Phytophthora analysis of plant ascomycete fungal pathogens with
infestans from tomatoes. Mycologist 14(3):137–139 different lifestyles reveals distinctive virulence
Tyson GW, Lo I, Baker BJ et al (2005) Genome-directed strategies. BMC Genomics 23(1):34
isolation of the key nitrogen fixer Leptospirillum Whisler HC, Zebold SL, Shemanchuk JA (1975) Life
ferrodiazotrophum sp. nov. from an acidophilic micro- history of Coelomomyces psorophorae. Proc Natl
bial community. Appl Environ Microbiol 71(10): Acad Sci 72(2):693–696. https://doi.org/10.1073/
6319–6324 pnas.72.2.693
Uehling JK, Entler MR, Meredith HR et al (2019) Williams PG (1992) Axenic culture of arbuscular mycor-
Microfluidics and metabolomics reveal symbiotic rhizal fungi. In: Norris JR, Read DJ, Varma AK (eds)
bacterial–fungal interactions between Mortierella Methods in microbiology, vol 24. Academic, London,
elongata and Burkholderia include metabolite pp 203–220
exchange. Front Microbiol 10:2163 Williams PG, Scott KJ, Kuhl JL (1966) Vegetative Growth
Umphlett CJ (1962) Morphological and cytological of Puccinia graminis f. sp. tritici In Vitro. Phytopa-
observations on the mycelium of Coelomomyces. thology 56(12):1418
Mycologia 54(5):540–554 Williams PG, Scott KJ, Kuhl JL et al (1967) Sporulation
Ushijima T, Takahashi M, Ozaki Y (1981) Selective and and pathogenicity of Puccinia graminis f. sp. tritici
differential media for isolation and tentative identifica- grown on an artificial medium. Phytopathology 57(3):
tion of each species of Pityrosporum residing on nor- 326
mal or diseased human skin. Microbiol Immunol Wu B, Cox MP (2021) Comparative genomics reveals a
25(11):1109–1118 core gene toolbox for lifestyle transitions in
Vartoukian SR, Adamowska A, Lawlor M et al (2016) In Hypocreales fungi. Environ Microbiol 23(6):
vitro cultivation of ‘unculturable’ oral bacteria, 3251–3264
facilitated by community culture and media supple- Yao T, Asayama Y (2017) Animal-cell culture media:
mentation with siderophores. PLoS One 11(1): history, characteristics, and current issues. Reprod
e0146926 Med Biol 16(2):99–117
Vilela R, Huebner M, Vilela C et al (2021) The taxonomy Yoder JB, Tiffin P (2017) Sanctions, partner recognition,
of two uncultivated fungal mammalian pathogens is and variation in mutualism. Am Nat 190(4):491–505
revealed through phylogeny and population genetic Zengler K, Toledo G, Rappé M et al (2002) Cultivating the
analyses. Sci Rep 11(1):18119 uncultured. PNAS 99(24):15681–15686
Vuorinen I, Hamberg L, Müller M et al (2015) Develop- Zhang CB, Ren CH, Wang YL et al (2020) Uncovering
ment of growth media for solid substrate propagation fungal community composition in natural habitat of
of ectomycorrhizal fungi for inoculation of Norway Ophiocordyceps sinensis using high-throughput
spruce (Picea abies) seedlings. Mycorrhiza 25(4): sequencing and culture-dependent approaches. BMC
311–324 Microbiol 20(1):1–16
Wagner A (2012) Metabolic networks and their evolution. Zhao J, Wang M, Chen X et al (2016) Role of alternate
Evol Sys Biol 751:29–52 hosts in epidemiology and pathogen variation of cereal
rusts. Annu Rev Phytopathol 54:207–228
 Horizontal Gene Transfer in Fungi
and Its Ecological Importance 3
Carla Gonçalves, Chris Todd Hittinger, and Antonis Rokas

Abstract important conclusion that can be drawn from

Fungi are one of the most diverse kingdoms in studies of fungal HGT is that HGT rate varies
the tree of life, thrive in virtually every habitat extensively across lineages and that this varia-
on the planet, and are vital for a plethora of tion can generally be associated with distinc-
ecosystems and human-related activities. The tive ecologies, lifestyles, or genetic
advent of genomic technologies has backgrounds. Similarly, not all functional
revolutionized the study of fungal ecology categories of genes are equally likely to result
and evolution. Surprisingly, evolutionary in successful transfers. The factors underlying
genomic investigations have revealed that hor- both frequency and functional variations of
izontal gene transfer (HGT)—the movement HGT across fungi are thoroughly discussed.
of genetic material from one organism to Advances in genomic and synthetic biology
another by means other than sexual technologies will continue to shed light on
reproduction—is an important factor these questions and bring many other still
influencing the ecology and evolution of fun- unanswered questions into focus. Some of
gal genomes. This chapter uses an evolution- these questions and directions for future
ary framework to discuss the extent and research are highlighted at the end of this
distribution of HGT across fungal ecologies chapter.
and lifestyles, focusing on some of the best-
supported cases to address fundamental Keywords
questions such as frequencies, barriers, and Fungal ecology · Fungi · Horizontal gene
mechanisms for eukaryotic HGT. One transfer · Adaptive evolution · Budding yeasts ·
Fungal pathogens · Lateral gene transfer ·
C. Gonçalves (✉) · A. Rokas (✉) Gene cluster · Xenolog
Department of Biological Sciences and Evolutionary
Studies Initiative, Vanderbilt University, Nashville, TN,
USA 3.1 Introduction
e-mail: [email protected]; antonis.
[email protected]
Fungi populate virtually every habitat on the
C. T. Hittinger
Laboratory of Genetics, DOE Great Lakes Bioenergy planet. They play crucial roles in a variety of
Research Center, Center for Genomic Science Innovation, aquatic and terrestrial ecosystems and exhibit
Wisconsin Energy Institute, J.F. Crow Institute for the various lifestyles from saprobic to symbiotic or
Study of Evolution, University of Wisconsin-Madison, parasitic, engaging in diverse interactions with
Madison, WI, USA

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 59

Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_3
60 C. Gonçalves et al.

other microorganisms, algae, animals, or plants and Rokas 2011; Richards et al. 2009), quickly
(Selbmann et al. 2013; Stajich 2017; Coleine et al. followed.
2022; Hawksworth et al. 2017; Li et al. 2021; Despite the skepticism raised by a few
Rokas 2022). One key to their success is their surrounding eukaryotic HGT (discussed in Sect.
highly diverse genetic repertoire that translates 3.2), in light of the growing number of strongly
into extremely diversified biochemical abilities supported cases across diverse eukaryotic
and morphologies, turning fungi into one of the lineages, it is now unquestionable that genes do
most adaptable kingdoms in the tree of life. Fun- move horizontally into and even between
gal ecological and evolutionary diversity has been eukaryotes. HGT events have been convincingly
largely attributed to life cycles that entail a com- documented in multiple phylogenetically distant
bination of sexual and asexual reproduction cou- eukaryotic lineages ranging from algae
pled with major genomic evolutionary processes, (Schönknecht et al. 2013) to plants (Ma et al.
such as recombination (Heitman 2010; Magwene 2022) and animals (Dunning Hotopp 2011; Li
et al. 2011; Grandaubert et al. 2019), gene and et al. 2022). Reports of HGT in fungi have dra-
genome duplication (Wolfe 2015; Sinha et al. matically increased in recent years (Wisecaver
2017; Steenwyk and Rokas 2018; Todd and and Rokas 2015; Gonçalves et al. 2018; Shen
Selmecki 2020; Stalder et al. 2022), and intro- et al. 2018; Gonçalves and Gonçalves 2022;
gression and hybridization (Libkind et al. 2011; Bredeweg and Baker 2020), in part as an outcome
Stukenbrock 2016; Steensels et al. 2021). Yet, of the ever-growing number of available genomes
analyses of genomic data from certain major eco- that are empowering high-throughput evolution-
logical adaptations or lifestyle transitions (Rokas ary examinations of HGT (Marcet-Houben and
2022; Naranjo-Ortiz and Gabaldón 2019) suggest Gabaldon 2010; Hittinger et al. 2015; Shen et al.
that other mechanisms might also be important 2018).
contributors. Fungi are typically easy to grow under labora-
One such mechanism is horizontal gene trans- tory conditions and have relatively small
fer (HGT), which is defined as the movement of genomes that are nowadays straightforward to
genetic material through mechanisms distinct sequence and analyze. In addition, several species
from reproduction and vertical inheritance. The in diverse lineages are genetically tractable,
decoding of the genomes of numerous which allows for experimental validation of
prokaryotes more than two decades ago, coupled genotype–phenotype associations. These
with progress in phylogenetic methods, exposed characteristics turn fungi into exceptional models
HGT as a major driver of genome evolution in to address some of the major questions related to
Bacteria and Archaea, playing an indispensable eukaryotic HGT: how pervasive is it? What are
role in functional innovation and speciation and the mechanisms mediating it? Which factors con-
leading to a network of life (Doolittle 1999; tribute to its success? Is there variation in the
Dagan et al. 2008; Ochman et al. 2000; Arnold frequency of HGT across lineages, ecologies,
et al. 2022; Wagner et al. 2017). However, recog- and gene functions? What is the scale of its func-
nition of the importance and magnitude of HGT tional and ecological importance?
in the evolution of eukaryotes has only recently This chapter sheds light on some of these
gained momentum. Pioneering studies in protists questions with a special focus on the ecological
identified several instances of HGT involved in implications of HGT, showing how the advance
the evolution of organelles or adaptation to of high-throughput (gen)omics, supported by an
anaerobiosis (Richards et al. 2003; Archibald ever-growing body of experimental work, is giv-
et al. 2003; Andersson et al. 2003); the discovery ing rise to a new view where HGT is a major
of other instances of HGT in diverse eukaryotic mechanism of genome evolution in fungi. First,
lineages (Kondrashov et al. 2006; Richardson and we review the evidence that fungi have been
Palmer 2007), including fungi (Hall et al. 2005; acting as both donors and recipients of genetic
Slot and Hibbett 2007; Khaldi et al. 2008; Slot material with each other, as well as with other
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 61

eukaryotic and prokaryotic lineages. Using a their robust cell walls and absence of
state-of-the-art genome-scale phylogeny of the phagotrophy but are nevertheless quite permis-
fungal kingdom as a backbone, we draw and sive to HGT (Shen et al. 2018; Richards et al.
discuss a portrait of the distribution and relative 2011a).
quantification of HGT events. The frequency and Despite the growing number of strongly
success of these events seem to be generally supported cases, HGT in eukaryotes has not
governed by the same principles as in bacteria been free of controversy. The main arguments
(e.g., ecological overlap between donor and recip- that have been put forward against the occurrence
ient organisms and low interference with the of eukaryotic HGT, beyond those occurring
recipient gene network), but HGT rates are gen- within the context of endosymbiosis, are the
erally lower than in bacteria and vary substan- physical and molecular barriers inherent to the
tially across fungal lineages and among gene biology and genetics of eukaryotes, the lack of
functions—the most striking cases and possible information about the molecular mechanisms
reasons underlying those variations are discussed. mediating HGT, and the observation that eukary-
Using the most recent and well-supported studies ote pangenomes, which would be predicted to
as examples, this chapter discusses how several of result from the cumulative effects of ongoing
these HGT events were pivotal for adaptation to HGT over time, are smaller than prokaryote
new environments and for the transition and mod- pangenomes (Martin 2017). Additionally, the
ulation of lifestyles, further underscoring the con- occurrence of phylogenetic conflict between
tribution of HGT to the ecological success of gene trees and the species tree, the gold standard
fungi. for identifying HGT (Fitzpatrick 2012; Wisecaver
and Rokas 2015), is depicted as being either the
result of phylogenetic artifacts or a misinterpreta-
3.2 Mechanisms Mediating HGT tion of the alternative hypothesis of differential
in Eukaryotes loss (Martin 2017).
These arguments dissipate in the face of the
HGT requires both external environmental factors mounting evidence supporting opportunities and
and physical and molecular aspects intrinsic to the mechanisms mediating HGT in eukaryotes
host organism. A piece of foreign DNA must (Lacroix and Citovsky 2016; Sibbald et al.
enter the host cell, then the nucleus, and finally 2020), which do not substantially differ from
be stably integrated in the host genome; next, the those already established for bacteria. For
acquired DNA must spread and become fixed in instance, natural transformation has been
the population. The probability of each of these observed in both yeasts and filamentous fungi
events varies across time and lineages (Doolittle under certain environmental conditions (Nevoigt
1998; Huang 2013; Doolittle et al. 2003)—for et al. 2000; Morogovsky et al. 2022), and fusion
instance, between prokaryotes and eukaryotes, of asexual spores, which can result in the
but also among eukaryotes. While the existence exchange of genetic material, is known to occur
of a germ line is a major barrier for HGT in some between different species of filamentous fungi
eukaryotic lineages, access of foreign DNA to the (Mehta and Baghela 2021). Virus-mediated
nucleus is facilitated in species carrying transfers (Bian et al. 2020; Irwin et al. 2022),
endosymbionts (Doolittle 1998; Keeling and type IV secretion systems (Zoolkefli et al.
Palmer 2008; Husnik and McCutcheon 2017). 2021), and mobile genetic elements, such as
Environmental factors can also facilitate integra- transposons (Ropars et al. 2015; McDonald
tion of foreign DNA, as illustrated by bdelloid et al. 2019; Urquhart et al. 2022; Widen et al.
rotifer species frequently exposed to periods of 2022), have been shown to facilitate transfer of
desiccation (Nowell et al. 2018; Gladyshev et al. genetic material into eukaryotic cells. Once inside
2008; Eyres et al. 2015). Fungal cells can be the cell, integration of foreign DNA into the host
particularly impermeable to foreign DNA due to genome is commonly mediated by
62 C. Gonçalves et al.

non-homologous end joining but can also be While a newly transferred gene’s structural
facilitated by transposable elements (Ropars features are key for its successful expression, the
et al. 2015; McDonald et al. 2019; Urquhart protein product of the gene will also interact with
et al. 2022), homologous recombination with the resident proteins inside the cell; those
native homologs (Hao et al. 2010), or mediated interactions can sometimes be detrimental and
by microhomology segments (Ku et al. 2015). lead to its removal by purifying selection. This
After integration into the host genome, expres- in part explains why genes that have fewer
sion of the acquired gene(s) is key for its evolu- connections are more likely to be successfully
tionary retention. Some of the structural hurdles transferred (Cohen et al. 2011). Nonetheless,
to successful transcription and translation include some types of interacting partners can be
the presence or absence of introns, differences in co-transferred together. Proteins that interact
GC content and codon bias, differences in the with each other or take part in the same metabolic
genetic code or differences in transcriptional pathway are often physically close in fungal
promoters, transcription factor-binding sites, and genomes and are organized in gene clusters
ribosomal-binding sites. The magnitude of these (Rokas et al. 2018). In fact, horizontal transfers
structural differences is expected to be generally a of fungal metabolic gene clusters have been well
function of evolutionary distance. There are documented and have facilitated the acquisition
striking differences between bacterial and eukary- of new traits encoded by multiple genes (Khaldi
otic transcription and translation, suggesting that et al. 2008; Reynolds et al. 2018; Wisecaver and
HGT events between distantly related species are Rokas 2015; Urquhart et al. 2022). Advances in
less likely to be successful than those between long-read sequencing technologies are providing
closely related organisms (Andam and Gogarten evidence for the mechanisms mediating the trans-
2011). Nevertheless, HGT-derived genes can fer of some of these clusters, including
readily become functional, irrespective of evolu- demonstrating that they are sometimes embedded
tionary distance and even in the absence of selec- in massive transposable elements (Gluck-Thaler
tion (Li and Bock 2019). Gene expression can be et al. 2022). These mobile genetic entities can
accomplished by the capturing of native carry a diverse array of genes encoding different
promoters via genome rearrangements (Li and functions and are found in many fungi (Gluck-
Bock 2019), or by the presence of “leaky” Thaler et al. 2022). Further evidence of recent
promoters with flexible requirements for activity, intra-kingdom HGT events involving these
such as those found in certain transposable regions, coupled with experimental evidence of
elements in Drosophila (Palazzo et al. 2019). the underlying mechanisms (Urquhart et al.
Weak expression can also be mitigated by an 2023), reinforces the view that the actual fre-
increase in the gene copy number (Lind et al. quency of HGT between fungal species might
2010). Consistent with these mechanisms, evolu- be higher than current estimates.
tion of de novo promoters, acquisition of new Adding to the evidence deconstructing the
transcription and polyadenylation sites, and gene arguments denying the occurrence of eukaryotic
fusions have been specifically observed in func- HGT, pangenomes also occur in eukaryotes,
tional transfers of bacterial pathways into yeasts including fungi (Brockhurst et al. 2019;
(Gonçalves and Gonçalves 2019; Kominek et al. McCarthy and Fitzpatrick 2019; Richard 2020;
2019). Acquisition of introns (Fan et al. 2020; Fan et al. 2020; Barber et al. 2021). The acquired
Husnik and McCutcheon 2017) and GC content genes also often show levels of sequence similar-
(Zhang et al. 2019) or codon bias (Matriano et al. ity and other genomic signatures (e.g., retention
2021) changes have also been documented. of native genes) between donor and recipient
Although accomplishing expression is essential, lineages that are much higher than those expected
fine-tuning it in the new genomic context is also under a differential loss scenario (Slot and Rokas
needed to achieve proper protein translation 2011; Slot and Rokas 2010; Wisecaver and Rokas
(de Reus et al. 2019). 2015). Moreover, owing to the substantial
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 63

number of HGT involving donors and recipients are less likely to be successful than those between
that belong to distantly related lineages—such as closely related species (Andam and Gogarten
bacteria and eukaryotes (Husnik and 2011). The aforementioned methodological
McCutcheon 2017; Li et al. 2022; Gonçalves caveats notwithstanding, the preponderance of
et al. 2018; Kominek et al. 2019; Shen et al. bacterial genes transferred into fungi and other
2018)—and to the increasingly rich genome sam- eukaryotes might also suggest that transfers of
pling available, it is becoming more difficult to bacterial genes are more likely than eukaryotic
postulate differential gene loss as the most likely genes to be fixed in eukaryotic host genomes
explanation for the topology inconsistencies (Keeling and Palmer 2008). For instance, bacte-
observed between species trees and gene trees rial genes lack introns, which are generally
(Cote-L’Heureux et al. 2022). important for efficient gene expression in
eukaryotes. However, variations in intron patterns
can also hinder gene expression due to incorrect
3.3 Extent of HGT Involving Fungi splicing (Fitzpatrick 2012). Intron patterns vary
extensively across fungi (Lim et al. 2021), which
Fungi have been receiving and supplying genes could affect the success of some intra-kingdom
from and to various lineages for millions of years gene transfers as illustrated by the incorrect splic-
(Fig. 3.1). While fungi have mostly provided ing of Fusarium fujikuroi genes when introduced
genes to other eukaryotic lineages, many different into Aspergillus nidulans (de Reus et al. 2019).
fungal lineages have been recipients of both Due to their large evolutionary distance from
interdomain and intra-kingdom HGTs with fungi, bacteria might also harbor a wider reper-
impacts on a variety of biological processes toire of genes that confer ecologically relevant
(Wisecaver and Rokas 2015; Shen et al. 2018; functions or are less likely to disrupt the existing
Franco et al. 2022; Marsit et al. 2015; Coelho protein network. Perhaps more importantly, bac-
et al. 2013; Gonçalves et al. 2016, 2018; Khaldi teria are ubiquitous. Their effective population
et al. 2008; Luo et al. 2022). sizes are considerably larger than fungal or other
Although examples of HGT involving fungi eukaryotic populations, which means that fungi
abound, precise quantification of the frequency simply may encounter bacterial donors more fre-
and main sources of fungal HGT is challenging quently than fungal ones.
because the pipelines and criteria used often differ
across studies. The focus of these studies also
varies. For example, most studies have focused 3.3.1 Some Fungal Lineages Are
on inference of HGT in particular lineages or on More Prone to HGT
HGT from bacterial donors (Marcet-Houben and
Gabaldon 2010; Gonçalves et al. 2018; Shen et al. Rates of HGT vary widely across the fungal tree
2018) because such transfers are easier to detect, of life (Fig. 3.2). For instance, members of the
especially when employing high-throughput Pezizomycotina subphylum have long been
approaches. Therefore, our current appreciation thought to be particularly prone to HGT when
of the origin, distribution, and frequency of compared to their sister lineage, the yeasts in the
HGT across the fungal kingdom (Fig. 3.2) needs Saccharomycotina subphylum (Wisecaver and
to be interpreted in the light of these caveats. Rokas 2015; Marcet-Houben and Gabaldon
As observed in other eukaryotes (Gladyshev 2010), or to the Basidiomycota (Marcet-Houben
et al. 2008; Pauchet and Heckel 2013; Wybouw and Gabaldon 2010). Recently, a screen of HGT
et al. 2014; Schönknecht et al. 2013; Eme et al. across ~50 non-Dikarya species (i.e., from phyla
2017; Li et al. 2022), a significant fraction of the other than Ascomycota and Basidiomycota)
reported HGT events in fungi involve bacterial showed that the frequency of HGT also varies
sources (Fig. 3.1), which contrasts with the idea among other fungal phyla, being for instance
that HGT events between distantly related species particularly high in lineages of anaerobic gut
64 C. Gonçalves et al.

Bacteria

FUNGI
Basidioo
Asco
AAscoY
scoY
oY Non
o Dikarya
NNo
AscoFF
X

P ti t
Protists Insects
Plants

Fig. 3.1 Extent of HGT involving fungi. Fungal lineages oomycetes, although HGT events from fungi to other
were subdivided according to the wealth of information protists also have been documented (e.g., Matriano et al.
regarding HGT events. As such, fungal lineages belonging 2021). Directionality of HGT events involving fungi as
to the subkingdom Dikarya, for which more information is donors and recipients is represented by the arrows. The
publicly available, were subdivided as follows: Basidio thickness of the arrow represents a rough estimate of the
(representing the Basidiomycota), AscoY (representing relative number of genes inferred to be involved in HGT
ascomycete yeasts—Saccharomycotina), and AscoF events between the respective lineages, according to the
(representing ascomycete filamentous fungi— inspected literature. Examples of fungi-to-insect
Pezizomycotina). The Taphrinomycotina were not HGT (involving carotenoid biosynthetic genes)
included in this diagram because of their lower number (Nováková and Moran 2011; Moran and Jarvik 2010)
of species and scarce information regarding HGT (but see and fungi-to-plant HGT (involving glutathione
Slot and Rokas 2010). “Non Dikarya” represents fungal S-transferase Fhb7) (Wang et al. 2020) are
lineages other than the Ascomycota and the illustrated inside the grey circles
Basidiomycota. Protist lineages mostly refer to the

fungi (Ciach et al. 2021), such as the guts of herbivorous animals, these harsh
Neocallimastigomycota and Zoopagomycota conditions coexist with favorable ones, such as
(Wang et al. 2019; Murphy et al. 2019; Tabima the availability of large amounts of nutrients
et al. 2020). This is consistent with the high rates provided by plant material upon which herbivo-
of HGT found in bacteria thriving in the animal rous animals feed. Fungi belonging to the
gut (Groussin et al. 2021; Shterzer and Mizrahi Neocallimastigomycota play an important role in
2015). the degradation of this plant material, and the
Animal guts are particularly challenging acquisition of genes from neighboring rumen bac-
environments dominated by bacteria and teria might have impacted this symbiotic relation-
characterized by very low levels of oxygen. In ship (Fig. 3.3) (Murphy et al. 2019). Accounting
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 65

Malassezia

Basidiobolus Rhizophagus
Lorem ipsum

Neocallimastigomycota

W/S clade
Microsporidia

Metarhizium

Trichoderma

Fusarium Saccharomyces

Aspergillus

Penicillium

KEY:
Lineages Ecological associations Number HGT-derived genes
Microsporidia Human-related Beneficial qualitative
Non-Dikarya 1-10
Plant-related Pathogenic
11-20
Basidiomycota
Insect-related Non-pathogenic 21-50
Taphrinomycotina
Herbivore gut 51-100
Saccharomycotina
>100
Pezizomycotina Amphibian gut

Fig. 3.2 Distribution of HGT across the fungal kingdom. comparative genomics studies (Marcet-Houben and
Representation of the phylogenetic relationships among Gabaldon 2010; Shen et al. 2018; Ciach et al. 2021), and
fungi as adapted from the phylogenomic study of the gray rectangles when the reports result from isolated stud-
fungal kingdom from Li et al. (2021). Presence of ies. Older HGT events into entire lineages were not con-
HGT-derived genes is represented by the colored sidered for simplicity; however, many instances are
rectangles that form the outermost circle: rectangles with documented (Richards et al. 2011a). Ecological
different shades of green denote cases where quantitative associations, as well as the nature of such relationships
assessment was available in the literature from large (red being pathogenic, brown being non-pathogenic, and
66 C. Gonçalves et al.

for multiple duplications of acquired genes post- Pezizomycotina and Basidiomycota, notably,
transfer, approximately 2–3.5% of these species’ these transfers only involved genes originating
gene repertoire was inferred to be derived from in the Pezizomycotina. Similarly, closely related
HGT; this fraction is enriched in ecologically mycoparasite species belonging to the genus
relevant functions (Fig. 3.3), including monosac- Escovopsis, which seemingly feed exclusively
charide and polysaccharide degradation, which on Basidiomycota and exhibit a narrower
might have enabled these species to consume nutritional spectrum than Trichoderma, exhibit
new carbon sources (Murphy et al. 2019). Adap- negligible evidence for HGT (Druzhinina et al.
tation to the anaerobic lifestyle in the rumen gut 2018). This suggests that, although opportunity
was possibly also facilitated by the acquisition of (parasitism) can enable HGT, other genetic or
several genes involved in anaerobic nucleotide environmental factors also influence its success.
metabolism, fermentation pathways for energy For instance, genomic properties such as the
generation, and cofactor recycling (Murphy intron-rich nature of basidiomycetous genes
et al. 2019; Wiersma et al. 2020; Perli et al. might have hampered their successful
2021), functions that were previously shown to functionalization, which is consistent with the
be also involved in HGT in other anaerobic few reports of HGT involving Basidiomycota as
gut-dwelling microeukaryotes (Eme et al. 2017; donors to other fungal lineages (Slot and Hibbett
Andersson et al. 2003). 2007; Milner David et al. 2019).
The distantly related Basidiobolus Variation in HGT rates among clades is also
(Zoopagomycota), an endosymbiont that inhabits observed in yeasts belonging to the
the digestive tracts of amphibians, also shows Saccharomycotina subphylum (Ascomycota)
particularly high rates of HGT of genes involved (Shen et al. 2018) (Fig. 3.2). Species whose
in the production of secondary metabolites (SMs), nuclear genetic code differs from the universal
the biosynthesis of siderophores, and the synthe- one have significantly fewer HGT-acquired
sis of sexual reproduction precursor molecules; its genes (Shen et al. 2018), which supports the
high rates of HGT are hypothesized to have sig- hypothesis that altered genetic codes hamper
nificantly expanded the SM gene repertoire in a HGT. On the other end of the spectrum, a bud-
lineage that is otherwise a poor SM producer ding yeast lineage composed of species thriving
(Tabima et al. 2020). in the floral environment (belonging to the genera
Parasitic relationships might also favor HGT Wickerhamiella and Starmerella—also known as
owing to the close proximity between parasites the W/S clade) shows unexpectedly high rates of
and their hosts, as demonstrated for animals HGT originating in bacteria (Shen et al. 2018;
(Wijayawardena et al. 2013), plants (Yang et al. Gonçalves et al. 2018) and impacting multiple
2016), and oomycetes (Soanes and Richards metabolic pathways (Fig. 3.3) (Gonçalves et al.
2014). In mycoparasitic Trichoderma fungi 2018; Kominek et al. 2019; Gonçalves and
(Pezizomycotina), the repertoire of plant cell Gonçalves 2019). The reasons behind the partic-
wall-degrading enzymes almost doubled through ularly high incidence of HGT in the W/S clade
a series of gene acquisitions from their fungal remain obscure, but a turbulent evolutionary his-
prey species (Druzhinina et al. 2018). Although tory marked by numerous ancestral (gene) loss
Trichoderma fungi parasitize species from both

ä
Fig. 3.2 (continued) green being possibly beneficial), of tree, branches for which no information regarding HGT
the species/lineages discussed in this chapter or species for could be recovered from the literature are represented by
which particularly high rates of HGT were found are dashed lines
represented in the inner circle. In the fungal phylogenetic
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 67

Adaptation to the floral niche in yeasts

Sugar transport
Vitamin and metabolism
Iron
sequestration scavenging
Sucrose
Fructose extracellular invertase

Glucose Vitamin B1
Vitamin B1
pathway enzymes fructose transporter
derivatives
p
siderophores

active form
Vitamin B1
Ethanoll

Adaptation to the animal gut in anaerobic gut fungi

Carbohydrate utilization and fermentation Polymer degradation

Chitin

Carbohydrates Xylose Xylulose Xylulose 5P Cellulose CAZymes Peptidoglycan

Polymers Ribulose 5P GLYCOLISIS

Ribose Ribose 5P
Xyloglucan
ROS Starch
Galactose Galactose 1P Glucose 1P
Pyruvate Lactate
Galacto-, arabinose-, Rhamnose-
based polymers
AcetylCo-A Acetaldehyde Ethanol
Oxygen
Oxygen independent cofactor synthesis
O2 L-tryptophan
Spermine
O2O2
O2
coenzyme
c A L-aspartate quinolinate NAD+

Modulation of pathogenic lifestyles

Entomopathogenicity Phytopathogenicity Animal (human) pathogenicity

HOST CELL
lipid carriers
proteases
Nutrient Thymidine
+ TK TMP
Plant cell wall utilization and Host interation ATP
degradation transport NTT
ATP +
+ ADP
Tolerance plant defense CMP +
Generalism Supress plant defense GTP NTT GTP CMP kinase CDP DNA
Specialism Virulence factors NAD+ NAD+
RNA
MICROSPORIDIA CELL
protease
acetyltransferase

Enzymes Transporters Organization HGT-derived genes Sources HGT-derived genes

KEY: Functionally tested Functionally tested ‘Single’ genes Bacteria Insects
Function predicted Function predicted Operons/Clusters Pezizomycotina

Fig. 3.3 Impact of HGT across fungal ecologies and genes in adaptation to certain ecological niches or modu-
lifestyles. Pathways and functions impacted by HGT are lation of lifestyles can be envisioned. These examples
illustrated for select cases for which a role of HGT-derived include the adaptation of floral yeasts (Wickerhamiella/
68 C. Gonçalves et al.

events (Shen et al. 2018; Gonçalves et al. 2020) fungus Rhizophagus irregularis (Fig. 3.2) (Ciach
and their ecology provide some hints. et al. 2021; Li et al. 2018), which is an endosym-
Thriving in the floral niche (Lachance et al. biont of plants carrying its own bacterial
2001; de Vega et al. 2017), W/S-clade yeasts endosymbionts.
likely often travel across this environment in the In summary, the rate of HGT varies between
guts of flower-visiting insects. Both W/S species fungal lineages. But are all foreign genes equally
and other microbes thriving in the floral niche prone to HGT? In the following sections, the gene
show fingerprints of reductive evolution functions that seem to be more frequently
(Filannino et al. 2019), leading to the hypothesis involved in fungal HGT events are highlighted,
that both opportunity for HGT (e.g., in the insect and the possible factors driving the observed var-
gut) and selection might have acted upon the iation are discussed.
reacquisition and maintenance of ancestrally
lost, and ecologically relevant, traits (e.g.,
allowing the colonization of a wider range of 3.3.2 Genes and Functions
microhabitats within the floral environment). Recurrently Transferred into
Loss of specific DNA repair pathways has also Fungi
been associated with higher rates of successful
foreign DNA integration (Thomas and Nielsen The success of a given HGT depends on many
2005), but whether these and/or other molecular factors (see Sect. 3.2). A newly acquired gene that
particularities have facilitated HGT in these spe- is expressed can persist over a short period of time
cies awaits further investigations. Notably, there just by genetic drift (Gogarten and Townsend
is little evidence for ongoing HGT in this clade 2005), but selection can only act upon genes
(Gonçalves et al. 2020), suggesting that the that confer a fitness advantage (e.g., encode a
circumstances that once permitted extensive for- useful function). Genes with certain functions
eign DNA acquisition are no longer operating. seem to be more frequently transferred, such as
Carrying endosymbionts can also provide those involved in antibiotic resistance in bacteria
opportunities for HGT, as showcased across (Zhou et al. 2021).
eukaryotes (Qiu et al. 2013; Sloan et al. 2014; Many of the early reports of fungal HGT
Kondo et al. 2002). This model was proposed to involved nutrient transporters (Richards et al.
partially explain the high rates of HGT from 2006; Slot and Hibbett 2007; Hellborg et al.
bacteria and plants in the arbuscular mycorrhizal 2008; Galeote et al. 2010; Coelho et al. 2013),

ä
Fig. 3.3 (continued) Starmerella clade) to the floral niche degradation of insect cuticles (Zhang et al. 2019); modu-
impacted by the acquisition of several iron uptake, vitamin lation of phytopathogenicity across multiple lineages
scavenging, and sugar metabolism related genes impacted by the acquisition of genes generally involved
(Gonçalves et al. 2016, 2018; Gonçalves and Gonçalves in plant infection such as plant cell wall degradation
2019; Kominek et al. 2019); adaptation of anaerobic fungi enzymes, transport of nutrients, and production of viru-
from the Neocallimastigomycota to the gut of herbivorous lence factors and effector proteins (Soanes and Richards
animals assisted by the acquisition of genes involved in, 2014; McDonald et al. 2019; Yin et al. 2016; Qiu et al.
but not limited to, carbohydrate assimilation, fermentation 2016; Zhu et al. 2012; Tsavkelova et al. 2012; de Jonge
(Murphy et al. 2019), and oxygen-independent pathways et al. 2012); modulation of pathogenicity in members of
for cofactor biosynthesis (Perli et al. 2021) (oxygen- the unicellular animal parasites belonging to the
dependent pathways in other fungi are represented in Microsporidia aided by the acquisition of genes encoding
light gray to denote their absence in gut fungi); modula- nucleotide transporters (NTT) involved in ATP transport
tion of host shift (plants—phytopathogenicity to insects— and nucleotide transport, or enzymes (TK—thymidine
entomopathogenicity) and impact on the ability to infect a kinase, CMP kinase—cytidylate kinase) involved in
wider range of insect species (specialism to generalism) in nucleic acid synthesis and salvage (Alexander et al.
Metarhizium entomopathogens through the acquisition of 2016; Dean et al. 2018)
several bacterial and insect-derived genes involved in the
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 69

which were hypothesized to have been particu- 2014; Naranjo-Ortíz et al. 2016) have been fre-
larly relevant to the osmotrophic mode of fungal quently observed. For instance, distinct fungal
feeding (Richards and Talbot 2013; Richards lineages have independently acquired bacterial
et al. 2011a). Functional data provided further nitric oxide-detoxifying flavohemoglobins
support to the importance of these acquisitions (Yhb1) (Wisecaver et al. 2016), involved in
in shaping the nutritional versatility of fungi mediating nitric oxide resistance and nitrosative
(Coelho et al. 2013; Gonçalves et al. 2016; Marsit stress tolerance (Wisecaver et al. 2016; Ianiri
et al. 2015; Milner David et al. 2019). Interest- et al. 2020). In the basidiomycetous yeast
ingly, nutrient transporters seem to be so far Malassezia, two independent acquisitions of
mostly involved in intra-kingdom transfers, YHB1 were supported by both phylogenetic and
indicating that proper protein targeting to the synteny analyses, while functional experiments
membrane might be more difficult to accomplish suggest a possible role in the commensal lifestyle
when prokaryotic genes are involved. of Malassezia in the human skin (Ianiri et al.
Genes participating in diverse metabolic 2020).
functions are also commonly transferred into While some of these recurrent transfers might
fungi (Wisecaver and Rokas 2015; Richards encode new functions or enhance existing ones,
et al. 2011a), as illustrated by early reports of others are involved in the reacquisition of ances-
genes involved in pyrimidine (Gojković et al. trally lost traits. For instance, several yeasts
2004) or biotin (Hall et al. 2005) biosynthesis in belonging to the W/S clade, which had previously
Saccharomyces cerevisiae, and as more recently lost the entire alcoholic fermentation pathway
and broadly observed across different fungal (Gonçalves et al. 2018), later reacquired three
lineages (Wisecaver and Rokas 2015; Qiu et al. distinct alcohol dehydrogenase encoding genes
2016; Shen et al. 2018; Murphy et al. 2019; (ADH1) independently from distinct bacterial
Gonçalves et al. 2020). donors (Gonçalves et al. 2018, 2020). In one
The higher frequency of HGT involving genes species, the acquisition of this single gene, cou-
within certain functional categories might be pled with the co-option of a native enzyme, was
related to their low connectivity and ability to sufficient to reinstate alcoholic fermentation
function independently, meaning that a single (Gonçalves et al. 2018).
gene can by itself confer a new trait without Other metabolic functions might depend on
disrupting the extant protein network (Cohen the activity of multiple enzymes encoded by dif-
et al. 2011). Transporters are an emblematic ferent genes. Many of these multigenic metabolic
example of low connectivity proteins that can pathways are encoded in gene clusters, which can
alone impact the nutritional versatility of the be transferred across fungal species as a unit
host organism. Similarly, metabolic gene clusters (Wisecaver and Rokas 2015; Marcet-Houben
are also examples of standalone metabolic units and Gabaldón 2019), and have even been found
that can undergo HGT (Slot and Hibbett 2007; to do so recurrently.
Patron et al. 2007; Khaldi et al. 2008; Lind et al. For instance, the catabolic enzymes that are
2017; Reynolds et al. 2018; Slot and Rokas part of the Leloir pathway for the utilization of
2011). These examples suggest that function and galactose are encoded in GAL gene clusters in
connectivity are intertwined factors influencing ascomycetous yeasts, where they have been lost
the success of HGT. multiple times in different lineages (Harrison
The repeated involvement of certain genes et al. 2022). Interestingly, some of these lineages
and/or functions in fungal HGT suggests that reacquired GAL gene clusters via HGT from dis-
natural selection is involved in their maintenance. tantly related yeast species (Slot and Rokas 2010;
Recurrent transfers of genes involved in the same Haase et al. 2021), which in most cases resulted in
pathway within the same clade (Alexander et al. the reinstatement of the ability to utilize galactose
2016; Gonçalves et al. 2018; Shen et al. 2018) or (Haase et al. 2021). In the genus Torulaspora,
among distantly related lineages (Bruto et al. both species- and strain-level HGT events
70 C. Gonçalves et al.

involving GAL clusters might be related to adap- 3.3.3 Genes and Functions
tation to galactose-rich environments (Venkatesh Transferred from Fungi
et al. 2021; Silva et al. 2022).
Gene clusters have been commonly transferred Most known examples where fungi have been
across fungi, but their bacterial counterparts donors of genes involve other fungi as recipients.
(operons) rarely seem to transfer into eukaryotes However, fungal genes have occasionally jumped
even though they are recurrently transferred into the genomes of other eukaryotes,
among bacteria (Lawrence and Roth 1996). contributing to important evolutionary
Building on their high levels of HGT events innovations (Fig. 3.1). The highest rate of cross-
involving single genes, in the W/S yeast clade kingdom transfer involving fungi as donors prob-
two bacterial operons with distinct functions ably occurred in the oomycetes (Richards et al.
were also horizontally acquired (Kominek et al. 2006, 2011b), an independently evolved lineage
2019; Gonçalves and Gonçalves 2019). The first of osmophilic microbial eukaryotes. Both the
reported operon transfer introduced an entirely period of evolution in which these transfers took
new pathway for siderophore biosynthesis in the place, which coincides with the emergence of
common ancestor of this clade and is involved in phytopathogenicity in the oomycetes (Richards
iron scavenging in many extant species (Fig. 3.3) et al. 2011b), and the functions provided by the
(Kominek et al. 2019). Most species for which no acquired genes, which include nutrient transport,
siderophore production could be detected plant cell wall digestion, and interaction with or
underwent secondary losses of the operon adhesion to the host (Richards et al. 2006, 2011b;
(Kominek et al. 2019), possibly due to relaxation Soanes and Richards 2014; Savory et al. 2015),
of selective constraints. The other operon encodes hint at an important role of fungal genes in the
a vitamin B1 salvage and biosynthetic pathway evolution of oomycete plant pathogens.
and was acquired on two independent occasions Fungi-to-plant HGT might also have signifi-
from different bacterial donors by two yeast cantly contributed to plant evolution. Impacts on
subclades (Gonçalves and Gonçalves 2019). the transition from aquatic to terrestrial
Independent acquisition of additional genes environments (Ma et al. 2022) and on plant
expanded the spectrum of operon-encoded defense mechanisms (Liu et al. 2022) have been
functions, allowing for the scavenging of a hypothesized based on the predicted functions of
wider range of vitamin B1 derivatives (Fig. 3.3) the acquired genes. A striking example concerns
(Gonçalves and Gonçalves 2019). The absence of the HGT of a glutathione S-transferase (Fhb7)
native yeast orthologs from all the genomes from endophytic Epichloë fungi to a close relative
inspected suggests a loss-prior-to-acquisition sce- of wheat (Wang et al. 2020). The protein shares
nario, once again underscoring (recurrent) HGT ~97% of identity with those from Epichloë spe-
as an important compensatory mechanism for cies and displays a patchy distribution across the
ancestral pathway and trait loss. genus, suggesting that the acquisition of Fhb7
Although functional data are relatively scarce was fairly recent. Most notably, Fhb7 is involved
for a substantial fraction of HGT events involving in the detoxification of fungal-produced toxins,
fungi, acquired genes tend to generally maintain namely those produced by the phytopathogen
their original functions. Yet, in some cases Fusarium graminearum (Fig. 3.1) (Wang et al.
HGT-derived genes can functionally diverge 2020), indicating that the acquisition of this gene
(Savory et al. 2018) or be subsequently lost might be important for plant defense against fun-
(Campbell et al. 2012; Kominek et al. 2019). gal pathogens.
Insects also have horizontally acquired fungal
genes, and such events across insect lineages
might be substantially more widespread than pre-
viously anticipated (Li et al. 2022); however, for
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 71

most cases the functional role remains elusive. In 3.4 Ecological Impact
other cases, such as the 200-million-year-old of Fungal HGT
transfer of pectin-degrading enzymes from asco-
mycetous fungi to two herbivorous beetle Increasing experimental evidence has been instru-
lineages, the acquired genes might have mental in examining whether and how HGT
contributed to insect’s ability to degrade plant contributes to fungal adaptation in distinct
cell walls (Kirsch et al. 2014). Probably one of environments. As discussed in the previous sec-
the most iconic cases of fungi-to-insect HGT is tion, extensive and recurrent HGT can drive
the transfer of two carotenoid biosynthetic genes major ecological transitions by impacting several
to the pea aphid (Fig. 3.1) (Nováková and Moran aspects of the host biology (Fig. 3.3). Even in
2011; Moran and Jarvik 2010). Carotenoids are cases where the scale of HGT is small, its pheno-
essential to animals, being involved both in pig- typic consequences can be substantial. In some
mentation and in resistance to oxidative stress, cases, the independent emergence of traits in dis-
but they are exclusively obtained through their tantly related lineages can be promoted by HGT.
diet. Aphids circumvented this limitation by For instance, the independent acquisition of a
making use of carotenoid biosynthesis genes orig- single specific fructose transporter gene was
inally acquired from fungi, becoming therefore indispensable to the evolution of fructophily, the
able to produce their own carotenoids. Aphid preference for fructose over glucose, in two dis-
individuals with this fungal pathway have red tantly related Saccharomycotina yeast clades
body pigmentation instead of the typical green (Gonçalves et al. 2016). Similarly, the ability to
(Fig. 3.1), and this affects aphid susceptibility to sense gravity (gravitropism), which is important
predators and parasites (Moran and Jarvik 2010). to orient growth of fruiting bodies, is found across
The acquired genes currently display a typical the Basidiomycota and also in the distantly
aphid gene structure (e.g., longer introns), but related Mucorales (Nguyen et al. 2018). In the
their original gene orientation where the caroten- latter lineage, the acquisition of a bacterial protein
oid desaturase and the carotenoid synthase–carot- forming octahedral protein crystals might be at
enoid cyclase are divergently transcribed has the origin of the independent evolution of this
been preserved (Moran and Jarvik 2010), trait (Nguyen et al. 2018).
suggesting that this genetic makeup is important Fungal pathogens affecting humans, other
for concerted gene expression from a single pro- animals, and plants (Rokas 2022; Soanes and
moter region. Interestingly, the same genes were Richards 2014; Zhang et al. 2019) also show a
later detected in the genome of the spotted spider mosaic distribution across the fungal tree of life
mite, which also exhibits green and red (Fig. 3.2), indicating that pathogenicity evolved
colorations (Altincicek et al. 2012; Bryon et al. from non-pathogenic ancestors on multiple
2017). Whether these genes resulted from an occasions. This repeated evolution of pathogenic-
independent HGT event or from a single transfer ity has been driven by diverse genomic changes,
into the common ancestor of mites and aphids such as rearrangements or gene duplication and
followed by differential loss is unclear. loss (Rokas 2022; Möller and Stukenbrock 2017),
Finally, so far, HGT between bacteria and but certain aspects of fungal pathogenicity were
fungi seems to chiefly work in one direction shaped by HGT (Soanes and Richards 2014;
(from bacteria to fungi) since fungal or other Alexander et al. 2016; Zhang et al. 2019; Sun
eukaryotic genes have not been frequently found et al. 2016; de Vries et al. 2020; Corradi 2015;
in bacterial genomes. This might be because Gluck-Thaler and Slot 2015). In many instances,
opportunity and inherent mechanisms favor the acquired genes are encoded within gene
intradomain transfers in bacteria or because struc- clusters, massive transposable elements, or entire
tural barriers hinder eukaryote-to-prokaryote chromosomes encompassing single or multiple
transfers (Keeling and Palmer 2008). functional genes (Slot and Rokas 2011;
72 C. Gonçalves et al.

Wisecaver and Rokas 2015; Gluck-Thaler and 2015). This pattern suggests that, under similar
Slot 2015; Ma et al. 2010; McDonald et al. selective pressures, such as those encountered in
2019; Dhillon et al. 2015). the same ecological niche, HGT-derived genes
Several HGT events, involving both bacterial encoding similar functions tend to be retained.
and insect genes, contributed to the evolution of While some fungi can be pathogenic, others
entomopathogenicity in Metarhizium are beneficial for our daily lives. The most iconic
(Pezizomycotina). The acquired genes are gener- example is arguably the baker’s yeast Saccharo-
ally involved in the degradation of insect cuticles myces cerevisiae, which is involved in the pro-
and were shown to be functional and upregulated duction of a plethora of food products and
during cuticle penetration (Fig. 3.3) (Zhang et al. medicines. Several aspects of the metabolism of
2019). The HGT-derived repertoire varies among the baker’s yeast have been impacted by HGT,
species being larger in species that exhibit a such as the early acquisition of a bacterial URA1
broader host range (generalists). Particularly, an gene by a S. cerevisiae ancestor, which is thought
HGT-derived protease was experimentally shown to have facilitated growth under low-oxygen
to widen the host range of a specialist conditions (Hall et al. 2005), commonly faced in
Metarhizium species (Zhang et al. 2019), the natural environments where this organism
suggesting a role for HGT in shifting from a thrives. More recent events have influenced
specialist to a generalist lifestyle. other aspects of the metabolism of S. cerevisiae
Evolution of pathogenicity in the obligate strains associated with human-related activities.
intracellular parasite phylum of Microsporidia For example, S. cerevisiae strains associated with
was also fueled by several HGT events, mostly wine-making acquired three distinct regions from
impacting their ability to survive in and to scav- closely related yeast species; these regions
enge nucleotides and nucleosides from host cells encompass several genes that might provide a
(Corradi 2015; Alexander et al. 2016; Dean et al. selective advantage during wine fermentation,
2018). Some HGT-derived nucleotide such as sugar, vitamins, and amino acid or
transporters have diversified to import additional oligopeptide transporters (Novo et al. 2009;
substrates (Dean et al. 2018), while several Marsit et al. 2015). HGT-derived regions are par-
HGT-derived enzymes have expanded the range ticularly prevalent in other strains associated with
of usable nucleic acid components available in human-related environments, suggesting that they
host cells (Alexander et al. 2016), allowing for might be signatures of domestication (Barbosa
the scavenging of nucleic acid components in et al. 2018; Peter et al. 2018).
species seemingly unable to synthesize them de In cheese-making Penicillium species, multi-
novo. Some Microsporidia species also acquired ple large genomic regions, some of which embed-
genes from the host they parasitize (Pombert et al. ded in massive transposable elements (Gluck-
2015; Selman et al. 2011). Thaler et al. 2022), show evidence of interspecies
Generally, for many of the genes transferred HGT and might be involved in conferring a com-
into fungal pathogens, an association between the petitive advantage in the cheese environment
functions of acquired genes and the organism’s (Ropars et al. 2015). For example, genes involved
pathogenic lifestyle can be envisaged (Fig. 3.3). in lactose metabolism were found to be strongly
These functions are encoded in genes involved in upregulated during cheese rind maturation
specialized metabolism influencing interactions (Ropars et al. 2015). Lactose is especially abun-
with the host and environment (Wisecaver and dant in the first steps of cheese maturation when it
Rokas 2015; Sun et al. 2016; Ianiri et al. 2020), is readily converted into lactate by the lactic acid
host cell degradation (Zhang et al. 2019; Dhillon bacteria present; therefore, being able to rapidly
et al. 2015), scavenging of host resources utilize it would provide a competitive advantage.
(Alexander et al. 2016, Dean et al. 2018), viru- In summary, some of the HGTs into fungi are
lence (McDonald et al. 2019; Friesen et al. 2006), ancient, reflecting ancestral selective pressures
or stress resistance (Ianiri et al. 2020; Corradi that facilitated major evolutionary transitions
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 73

and affected entire lineages such as demonstrated acquisition of others? Are there other molecular
for some fungal pathogens; others are very recent, particularities that make these lineages more per-
reflecting short but intense periods of selection meable to HGT? Are some species naturally more
such as those imposed by recent human-related competent at DNA uptake than others? Which
activities. Finally, it is worth noting that ecologi- factors underlie the propensity to acquire
cal environments are complex and dynamic and foreign DNA?
that reproducing their key aspects in the labora-
tory is often impractical, hindering our ability to How Much HGT and from Whom? Bacterial
dissect the evolutionary forces that contributed to donors seem to be the most common source of
the acquisition and retention of HGT-derived foreign genes in fungal genomes, but nutrient
genes across fungi. transporters seem to be more frequently involved
in transfers within eukaryotes than in transfers
from bacteria. Are these observations resulting
3.5 Concluding Remarks from sampling biases in genome sequencing or
and Outstanding Questions methodological ascertainment biases, or do they
reflect biological factors? These questions would
We set out to discuss several major questions benefit from comparative genomic analyses. The
concerning HGT in eukaryotes based on what wealth of genomic data currently available for
has been learned from HGTs involving fungi. fungi (Shen et al. 2020; Li et al. 2021) provides
The mode, tempo, and magnitude of HGT in an encouraging launching pad for such analyses.
fungi seem to be, overall, a result of a complex So far, a handful of studies have focused on high-
equation involving environmental conditions, throughput inference of HGT from bacterial
cellular states (e.g., permissivity to foreign donors, yet the more challenging inference of
DNA), genetic backgrounds (e.g., gene content, HGT events between closely related species has
genetic code), and selective pressures. Although been less frequently addressed. This bias hinders
advances in fungal HGT in the last decade have our ability to evaluate the real extent of HGT
been tremendous, several outstanding questions across fungi or whether all lineages are equally
remain. likely to serve as donors. However, both rich
genome sampling and improved methodologies
Why Do Some Clades/Species Seem to Be can now be employed. For instance,
More Prone to HGT than Others? Species phylogenomic pipelines starting with fast screen-
thriving in certain ecological niches seem to be ing of potential candidates, such as the alien index
more prone to HGT, but the extent of the associa- scores (Gladyshev et al. 2008), followed by
tion between HGT and fungal ecology remains robust phylogenetic reconstruction using state-
unknown. The wealth of data from fungal species of-the-art statistical methods and meticulous spe-
with particularly high rates of HGT that are dis- cies tree and gene tree reconciliation analyses, can
tantly related and thrive in distinct ecological be employed for robust high-throughput HGT
contexts opens avenues for understanding the inference across fungi (Wisecaver and Rokas
relationship between fungal HGT and ecology. 2015; Gonçalves et al. 2018; Shen et al. 2018;
Examples of such lineages of particular interest Alexander et al. 2016).
for future research include the Wickerhamiella/
Starmerella clade of yeasts and the gut-dwelling What Happens Immediately After an HGT
fungi belonging to the Neocallimastigomycota Event? While some HGT events in fungi seem
because of the high percentages of foreign genes to have occurred rather recently, most of the cases
encoded in their genomes and their well-defined reported entail relatively old events, making it
ecologies. Can the repertoires of foreign and difficult to draw conclusions about, for instance,
native genes in these lineages provide hints? the processes involved in the accommodation of a
Can the acquisition of certain genes facilitate the
74 C. Gonçalves et al.

newly arrived gene to the new host cell. Which Conflict of Interest Statement A.R. is a scientific con-
mechanisms are more commonly used by fungi sultant for LifeMine Therapeutics, Inc.
when acquiring foreign DNA? What happens to
the genes immediately after being transferred?
Does the genome location where a foreign gene References
lands affect the success of an HGT event? How
Alexander WG, Wisecaver JH, Rokas A, Hittinger CT
does it impact the extant gene and protein net- (2016) Horizontally acquired genes in early-diverging
work? What is the relative role of selection and pathogenic fungi enable the use of host nucleosides
drift early in an HGT event? Does the donor affect and nucleotides. Proc Natl Acad Sci U S A 113:
the success of an HGT event? Although ambi- 4116–4121
Altincicek B, Kovacs JL, Gerardo NM (2012) Horizon-
tious, state-of-the-art approaches such as strain tally transferred fungal carotenoid genes in the
engineering and experimental evolution can help two-spotted spider mite Tetranychus urticae. Biol
address most of these questions. Lett 8:253–257
Andam CP, Gogarten JP (2011) Biased gene transfer in
microbial evolution. Nat Rev Microbiol 9:543–555
What Are the Population Dynamics of HGT? Andersson JO, Sjögren AM, Davis LA, Embley TM,
Population-level variability in the presence/ Roger AJ (2003) Phylogenetic analyses of diplomonad
absence of HGT-derived genes has only been genes reveal frequent lateral gene transfers affecting
studied in a few fungi (McDonald et al. 2019; eukaryotes. Curr Biol 13:94–104
Archibald JM, Rogers MB, Toop M, Ishida K-I, Keeling
Urquhart et al. 2022; Marsit et al. 2015). For PJ (2003) Lateral gene transfer and the evolution of
instance, certain HGT-acquired regions are only plastid-targeted proteins in the secondary plastid-
found in some S. cerevisiae populations. In some containing alga Bigelowiella natans. Proc Natl Acad
cases, very recent acquisitions might explain this Sci U S A 100:7678–7683
Arnold BJ, Huang IT, Hanage WP (2022) Horizontal gene
intraspecific patchy distribution, while in other transfer and adaptive evolution in bacteria. Nat Rev
cases, fluctuating selective pressures might either Microbiol 20:206–218
promote maintenance or gradual decay of previ- Barber AE, Sae-Ong T, Kang K, Seelbinder B, Li J,
ously acquired genes. These dynamics are still Walther G, Panagiotou G, Kurzai O (2021) Aspergillus
fumigatus pan-genome analysis identifies genetic
blurry, awaiting population-level studies, but variants associated with human infection. Nat
they constitute an interesting aspect that might Microbiol 6:1526–1536
inform us about the evolutionary forces that gov- Barbosa R, Pontes A, Santos RO, Montandon GG, de
ern HGT-derived genes, how they respond to Ponzzes-Gomes CM, Morais PB, Gonçalves P, Rosa
CA, Sampaio JP (2018) Multiple rounds of artificial
environmental changes, and how HGT impacts selection promote microbe secondary domestication—
(and is impacted by) fungal ecology at the popu- the case of cachaça yeasts. Genome Biol Evol 10:
lation level. 1939–1955
Bian R, Andika IB, Pang T, Lian Z, Wei S, Niu E, Wu Y,
Kondo H, Liu X, Sun L (2020) Facilitative and syner-
Acknowledgments Research in A.R.’s lab is supported gistic interactions between fungal and plant viruses.
by grants from the National Science Foundation Proc Natl Acad Sci U S A 117:3779–3788
(DEB-2110404), the National Institutes of Health/National Bredeweg EL, Baker SE (2020) Horizontal Gene Transfer
Institute of Allergy and Infectious Diseases (R56 in Fungi. In: Nevalainen H (ed) Grand challenges in
AI146096 and R01 AI153356), and the Burroughs fungal biotechnology. Springer International
Wellcome Fund. Research in C.T.H.’s lab is supported Publishing, Cham
by the National Science Foundation (DEB-1442148 and Brockhurst MA, Harrison E, Hall JPJ, Richards T,
DEB-2110403), in part by the DOE Great Lakes McNally A, MacLean C (2019) The ecology and evo-
Bioenergy Research Center (DOE BER Office of Science lution of pangenomes. Curr Biol 29:R1094–r1103
DE-SC0018409), the USDA National Institute of Food Bruto M, Prigent-Combaret C, Luis P, Moënne-Loccoz Y,
and Agriculture (Hatch Project 1020204), and an H. I. Muller D (2014) Frequent, independent transfers of a
Romnes Faculty Fellowship from the Office of the Vice catabolic gene from bacteria to contrasted filamentous
Chancellor for Research and Graduate Education with eukaryotes. Proc Biol Sci 281:20140848
funding from the Wisconsin Alumni Research Foundation. Bryon A, Kurlovs AH, Dermauw W, Greenhalgh R,
Riga M, Grbić M, Tirry L, Osakabe M, Vontas J,
Clark RM, Van Leeuwen T (2017) Disruption of a
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 75

horizontally transferred phytoene desaturase abolishes Microsporidia intracellular parasites. Nat Commun 9:
carotenoid accumulation and diapause in Tetranychus 1709
urticae. Proc Natl Acad Sci 114:E5871–E5880 Dhillon B, Feau N, Aerts AL, Beauseigle S, Bernier L,
Campbell MA, Rokas A, Slot JC (2012) Horizontal trans- Copeland A, Foster A, Gill N, Henrissat B, Herath P,
fer and death of a fungal secondary metabolic gene LaButti KM, Levasseur A, Lindquist EA, Majoor E,
cluster. Genome Biol Evol 4:289–293 Ohm RA, Pangilinan JL, Pribowo A, Saddler JN,
Ciach M, Pawłowska J, Muszewska A (2021) Horizontal Sakalidis ML, de Vries RP, Grigoriev IV, Goodwin
gene transfer in 44 early diverging fungi favors short, SB, Tanguay P, Hamelin RC (2015) Horizontal gene
metabolic, extracellular proteins from associated bac- transfer and gene dosage drives adaptation to wood
teria. bioRxiv 2021.12.02.471044 colonization in a tree pathogen. Proc Natl Acad Sci U
Coelho MA, Gonçalves C, Sampaio JP, Gonçalves P S A 112:3451–3456
(2013) Extensive intra-kingdom horizontal gene trans- Doolittle WF (1998) You are what you eat: a gene transfer
fer converging on a fungal fructose transporter gene. ratchet could account for bacterial genes in eukaryotic
PLoS Genet 9:e1003587 nuclear genomes. Trends Genet 14:307–311
Cohen O, Gophna U, Pupko T (2011) The complexity Doolittle WF (1999) Lateral genomics. Trends Cell Biol 9:
hypothesis revisited: connectivity rather than function M5–M8
constitutes a barrier to horizontal gene transfer. Mol Doolittle WF, Boucher Y, Nesbø CL, Douady CJ,
Biol Evol 28:1481–1489 Andersson JO, Roger AJ (2003) How big is the iceberg
Coleine C, Stajich JE, Selbmann L (2022) Fungi are key of which organellar genes in nuclear genomes are but
players in extreme ecosystems. Trends Ecol Evol 37: the tip? Philos Trans R Soc Lond Ser B Biol Sci 358:
517–528 39–57 discussion 57–8
Corradi N (2015) Microsporidia: eukaryotic intracellular Druzhinina IS, Chenthamara K, Zhang J, Atanasova L,
parasites shaped by gene loss and horizontal gene Yang D, Miao Y, Rahimi MJ, Grujic M, Cai F,
transfers. Annu Rev Microbiol 69:167–183 Pourmehdi S, Salim KA, Pretzer C, Kopchinskiy AG,
Cote-L’Heureux A, Maurer-Alcalá XX, Katz LA (2022) Henrissat B, Kuo A, Hundley H, Wang M, Aerts A,
Old genes in new places: a taxon-rich analysis of Salamov A, Lipzen A, LaButti K, Barry K, Grigoriev
interdomain lateral gene transfer events. PLoS Genet IV, Shen Q, Kubicek CP (2018) Massive lateral trans-
18:e1010239 fer of genes encoding plant cell wall-degrading
Dagan T, Artzy-Randrup Y, Martin W (2008) Modular enzymes to the mycoparasitic fungus Trichoderma
networks and cumulative impact of lateral transfer in from its plant-associated hosts. PLoS Genet 14:
prokaryote genome evolution. Proc Natl Acad Sci U S e1007322
A 105:10039–10044 Dunning Hotopp JC (2011) Horizontal gene transfer
de Jonge R, Peter van Esse H, Maruthachalam K, Bolton between bacteria and animals. Trends Genet 27:157–
MD, Santhanam P, Saber MK, Zhang Z, Usami T, 163
Lievens B, Subbarao KV, Thomma BPHJ (2012) Eme L, Gentekaki E, Curtis B, Archibald JM, Roger AJ
Tomato immune receptor Ve1 recognizes effector of (2017) Lateral gene transfer in the adaptation of the
multiple fungal pathogens uncovered by genome and anaerobic parasite Blastocystis to the gut. Curr Biol 27:
RNA sequencing. Proc Natl Acad Sci U S A 109: 807–820
5110–5115 Eyres I, Boschetti C, Crisp A, Smith TP, Fontaneto D,
de Reus E, Nielsen MR, Frandsen RJN (2019) Metabolic Tunnacliffe A, Barraclough TG (2015) Horizontal
and regulatory insights from the experimental horizon- gene transfer in bdelloid rotifers is ancient, ongoing
tal gene transfer of the aurofusarin and bikaverin gene and more frequent in species from desiccating habitats.
clusters to Aspergillus nidulans. Mol Microbiol 112: BMC Biol 13:90
1684–1700 Fan X, Qiu H, Han W, Wang Y, Xu D, Zhang X,
de Vega C, Albaladejo RG, Guzman B, Steenhuisen SL, Bhattacharya D, Ye N (2020) Phytoplankton
Johnson SD, Herrera CM, Lachance MA (2017) pangenome reveals extensive prokaryotic horizontal
Flowers as a reservoir of yeast diversity: description gene transfer of diverse functions. Science. Advances
of Wickerhamiella nectarea f.a. sp. nov., and 6:eaba0111
Wickerhamiella natalensis f.a. sp. nov. from Filannino P, Di Cagno R, Tlais AZA, Cantatore V,
South African flowers and pollinators, and transfer of Gobbetti M (2019) Fructose-rich niches traced the
related Candida species to the genus Wickerhamiella evolution of lactic acid bacteria toward fructophilic
as new combinations. FEMS Yeast Res 17 species. Crit Rev Microbiol 45:65–81
de Vries S, Stukenbrock EH, Rose LE (2020) Rapid evo- Fitzpatrick DA (2012) Horizontal gene transfer in fungi.
lution in plant-microbe interactions - an evolutionary FEMS Microbiol Lett 329:1–8
genomics perspective. New Phytol 226:1256–1262 Franco MEE, Wisecaver JH, Arnold AE, Ju Y-M, Slot JC,
Dean P, Sendra KM, Williams TA, Watson AK, Major P, Ahrendt S, Moore LP, Eastman KE, Scott K, Konkel Z,
Nakjang S, Kozhevnikova E, Goldberg AV, Kunji Mondo SJ, Kuo A, Hayes RD, Haridas S,
ERS, Hirt RP, Embley TM (2018) Transporter gene Andreopoulos B, Riley R, LaButti K, Pangilinan J,
acquisition and innovation in the evolution of Lipzen A, Amirebrahimi M, Yan J, Adam C,
76 C. Gonçalves et al.

Keymanesh K, Ng V, Louie K, Northen T, Drula E, Groussin M, Poyet M, Sistiaga A, Kearney SM, Moniz K,
Henrissat B, Hsieh H-M, Youens-Clark K, Lutzoni F, Noel M, Hooker J, Gibbons SM, Segurel L,
Miadlikowska J, Eastwood DC, Hamelin RC, Froment A, Mohamed RS, Fezeu A, Juimo VA,
Grigoriev IV, U’Ren JM (2022) Ecological generalism Lafosse S, Tabe FE, Girard C, Iqaluk D, Nguyen
drives hyperdiversity of secondary metabolite gene LTT, Shapiro BJ, Lehtimäki J, Ruokolainen L,
clusters in xylarialean endophytes. New Phytol 233: Kettunen PP, Vatanen T, Sigwazi S, Mabulla A,
1317–1330 Domínguez-Rodrigo M, Nartey YA, Agyei-Nkansah-
Friesen TL, Stukenbrock EH, Liu Z, Meinhardt S, Ling H, A, Duah A, Awuku YA, Valles KA, Asibey SO,
Faris JD, Rasmussen JB, Solomon PS, McDonald BA, Afihene MY, Roberts LR, Plymoth A, Onyekwere
Oliver RP (2006) Emergence of a new disease as a CA, Summons RE, Xavier RJ, Alm EJ (2021) Elevated
result of interspecific virulence gene transfer. Nat rates of horizontal gene transfer in the industrialized
Genet 38:953–956 human microbiome. Cell 184:2053–2067.e18
Galeote V, Novo M, Salema-Oom M, Brion C, Valério E, Haase MAB, Kominek J, Opulente DA, Shen XX, LaBella
Gonçalves P, Dequin S (2010) FSY1, a horizontally AL, Zhou X, DeVirgilio J, Hulfachor AB, Kurtzman
transferred gene in the Saccharomyces cerevisiae CP, Rokas A, Hittinger CT (2021) Repeated horizontal
EC1118 wine yeast strain, encodes a high-affinity fruc- gene transfer of GALactose metabolism genes violates
tose/H+ symporter. Microbiology 156:3754–3761 Dollo’s law of irreversible loss. Genetics 217
Gladyshev EA, Meselson M, Arkhipova IR (2008) Mas- Hall C, Brachat S, Dietrich FS (2005) Contribution of
sive horizontal gene transfer in bdelloid rotifers. Sci- horizontal gene transfer to the evolution of Saccharo-
ence 320:1210–1213 myces cerevisiae. Eukaryot Cell 4:1102–1115
Gluck-Thaler E, Slot JC (2015) Dimensions of horizontal Hao W, Richardson AO, Zheng Y, Palmer JD (2010)
gene transfer in eukaryotic microbial pathogens. PLoS Gorgeous mosaic of mitochondrial genes created by
Pathog 11:e1005156 horizontal transfer and gene conversion. Proc Natl
Gluck-Thaler E, Ralston T, Konkel Z, Ocampos CG, Acad Sci U S A 107:21576–21581
Ganeshan VD, Dorrance AE, Niblack TL, Wood Harrison M-C, LaBella AL, Hittinger CT, Rokas A (2022)
CW, Slot JC, Lopez-Nicora HD, Vogan AA (2022) The evolution of the GALactose utilization pathway in
Giant starship elements mobilize accessory genes in budding yeasts. Trends Genet 38:97–106
fungal genomes. Mol Biol Evol 39 Hawksworth DL, Lücking R, Heitman J, James TY (2017)
Gogarten JP, Townsend JP (2005) Horizontal gene trans- Fungal diversity revisited: 2.2 to 3.8 million species.
fer, genome innovation and evolution. Nat Rev Microbiology Spectrum, 5, 5.4.10
Microbiol 3:679–687 Heitman J (2010) Evolution of eukaryotic microbial
Gojković Z, Knecht W, Zameitat E, Warneboldt J, pathogens via covert sexual reproduction. Cell Host
Coutelis JB, Pynyaha Y, Neuveglise C, Møller K, Microbe 8:86–99
Löffler M, Piskur J (2004) Horizontal gene transfer Hellborg L, Woolfit M, Arthursson-Hellborg M, Piškur J
promoted evolution of the ability to propagate under (2008) Complex evolution of the DAL5 transporter
anaerobic conditions in yeasts. Mol Gen Genomics family. BMC Genomics 9:164
271:387–393 Hittinger CT, Rokas A, Bai FY, Boekhout T, Gonçalves P,
Gonçalves C, Gonçalves P (2019) Multilayered horizontal Jeffries TW, Kominek J, Lachance MA, Libkind D,
operon transfers from bacteria reconstruct a thiamine Rosa CA, Sampaio JP, Kurtzman CP (2015) Genomics
salvage pathway in yeasts. Proc Natl Acad Sci U S A and the making of yeast biodiversity. Curr Opin Genet
116:22219–22228 Dev 35:100–109
Gonçalves P, Gonçalves C (2022) Horizontal gene transfer Huang J (2013) Horizontal gene transfer in eukaryotes: the
in yeasts. Curr Opin Genet Dev 76:101950 weak-link model. BioEssays 35:868–875
Gonçalves C, Coelho MA, Salema-Oom M, Gonçalves P Husnik F, McCutcheon JP (2017) Functional horizontal
(2016) Stepwise functional evolution in a fungal sugar gene transfer from bacteria to eukaryotes. Nat Rev
transporter family. Mol Biol Evol 33:352–366 Microbiol 16:67
Gonçalves C, Wisecaver JH, Kominek J, Oom MS, Ianiri G, Coelho Marco A, Ruchti F, Sparber F, McMahon
Leandro MJ, Shen X-X, Opulente DA, Zhou X, Timothy J, Fu C, Bolejack M, Donovan O, Smutney H,
Peris D, Kurtzman CP, Hittinger CT, Rokas A, Myler P, Dietrich F, Fox D, LeibundGut-Landmann S,
Gonçalves P (2018) Evidence for loss and reacquisi- Heitman J (2020) HGT in the human and skin com-
tion of alcoholic fermentation in a fructophilic yeast mensal Malassezia: a bacterially derived
lineage. elife 7:e33034 flavohemoglobin is required for NO resistance and
Gonçalves P, Gonçalves C, Brito PH, Sampaio JP (2020) host interaction. Proc Natl Acad Sci 117:15884–15894
The Wickerhamiella/Starmerella clade—a treasure Irwin NAT, Pittis AA, Richards TA, Keeling PJ (2022)
trove for the study of the evolution of yeast metabo- Systematic evaluation of horizontal gene transfer
lism. Yeast 37(4):313–320 between eukaryotes and viruses. Nat Microbiol 7:
Grandaubert J, Dutheil JY, Stukenbrock EH (2019) The 327–336
genomic determinants of adaptive evolution in a fungal Keeling PJ, Palmer JD (2008) Horizontal gene transfer in
pathogen. Evol Lett 3:299–312 eukaryotic evolution. Nat Rev Genet 9:605–618
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 77

Khaldi N, Collemare J, Lebrun M-H, Wolfe KH (2008) Lim CS, Weinstein BN, Roy SW, Brown CM (2021)
Evidence for horizontal transfer of a secondary metab- Analysis of fungal genomes reveals commonalities of
olite gene cluster between fungi. Genome Biol 9:R18 intron gain or loss and functions in intron-poor species.
Kirsch R, Gramzow L, Theißen G, Siegfried BD, Ffrench- Mol Biol Evol 38:4166–4186
Constant RH, Heckel DG, Pauchet Y (2014) Horizon- Lind PA, Tobin C, Berg OG, Kurland CG, Andersson DI
tal gene transfer and functional diversification of plant (2010) Compensatory gene amplification restores fit-
cell wall degrading polygalacturonases: key events in ness after inter-species gene replacements. Mol
the evolution of herbivory in beetles. Insect Biochem Microbiol 75:1078–1089
Mol Biol 52:33–50 Lind AL, Wisecaver JH, Lameiras C, Wiemann P, Palmer
Kominek J, Doering DT, Opulente DA, Shen XX, Zhou X, JM, Keller NP, Rodrigues F, Goldman GH, Rokas A
DeVirgilio J, Hulfachor AB, Groenewald M, McGee (2017) Drivers of genetic diversity in secondary meta-
MA, Karlen SD, Kurtzman CP, Rokas A, Hittinger CT bolic gene clusters within a fungal species. PLoS Biol
(2019) Eukaryotic Acquisition of a Bacterial Operon. 15:e2003583
Cell 176:1356–1366.e10 Liu Y, Wang S, Li L, Yang T, Dong S, Wei T, Wu S,
Kondo N, Nikoh N, Ijichi N, Shimada M, Fukatsu T Liu Y, Gong Y, Feng X, Ma J, Chang G, Huang J,
(2002) Genome fragment of Wolbachia endosymbiont Yang Y, Wang H, Liu M, Xu Y, Liang H, Yu J, Cai Y,
transferred to X chromosome of host insect. Proc Natl Zhang Z, Fan Y, Mu W, Sahu SK, Liu S, Lang X,
Acad Sci U S A 99:14280–14285 Yang L, Li N, Habib S, Yang Y, Lindstrom AJ,
Kondrashov FA, Koonin EV, Morgunov IG, Finogenova Liang P, Goffinet B, Zaman S, Wegrzyn JL, Li D,
TV, Kondrashova MN (2006) Evolution of glyoxylate Liu J, Cui J, Sonnenschein EC, Wang X, Ruan J, Xue
cycle enzymes in Metazoa: evidence of multiple hori- J-Y, Shao Z-Q, Song C, Fan G, Li Z, Zhang L, Liu J,
zontal transfer events and pseudogene formation. Biol Liu Z-J, Jiao Y, Wang X-Q, Wu H, Wang E, Lisby M,
Direct 1:31–31 Yang H, Wang J, Liu X, Xu X, Li N, Soltis PS, Van de
Ku C, Nelson-Sathi S, Roettger M, Garg S, Hazkani- Peer Y, Soltis DE, Gong X, Liu H, Zhang S (2022) The
Covo E, Martin WF (2015) Endosymbiotic gene trans- Cycas genome and the early evolution of seed plants.
fer from prokaryotic pangenomes: inherited chimerism Nat Plants 8:389–401
in eukaryotes. Proc Natl Acad Sci 112:10139–10146 Luo H, Hallen-Adams HE, Lüli Y, Sgambelluri RM, Li X,
Lachance MA, Starmer WT, Rosa CA, Bowles JM, Barker Smith M, Yang ZL, Martin FM (2022) Genes and
JS, Janzen DH (2001) Biogeography of the yeasts of evolutionary fates of the amanitin biosynthesis path-
ephemeral flowers and their insects. FEMS Yeast Res way in poisonous mushrooms. Proc Natl Acad Sci 119:
1:1–8 e2201113119
Lacroix B, Citovsky V (2016) Transfer of DNA from Ma LJ, van der Does HC, Borkovich KA, Coleman JJ,
bacteria to eukaryotes. mBio 7 Daboussi MJ, Di Pietro A, Dufresne M, Freitag M,
Lawrence JG, Roth JR (1996) Selfish operons: horizontal Grabherr M, Henrissat B, Houterman PM, Kang S,
transfer may drive the evolution of gene clusters. Shim WB, Woloshuk C, Xie X, Xu JR, Antoniw J,
Genetics 143:1843–1860 Baker SE, Bluhm BH, Breakspear A, Brown DW,
Li Z, Bock R (2019) Rapid functional activation of a Butchko RA, Chapman S, Coulson R, Coutinho PM,
horizontally transferred eukaryotic gene in a bacterial Danchin EG, Diener A, Gale LR, Gardiner DM,
genome in the absence of selection. Nucleic Acids Res Goff S, Hammond-Kosack KE, Hilburn K,
47:6351–6359 Hua-Van A, Jonkers W, Kazan K, Kodira CD,
Li M, Zhao J, Tang N, Sun H, Huang J (2018) Horizontal Koehrsen M, Kumar L, Lee YH, Li L, Manners JM,
gene transfer from bacteria and plants to the arbuscular Miranda-Saavedra D, Mukherjee M, Park G, Park J,
mycorrhizal fungus Rhizophagus irregularis. Front Park SY, Proctor RH, Regev A, Ruiz-Roldan MC,
Plant Sci 9 Sain D, Sakthikumar S, Sykes S, Schwartz DC,
Li Y, Steenwyk JL, Chang Y, Wang Y, James TY, Stajich Turgeon BG, Wapinski I, Yoder O, Young S,
JE, Spatafora JW, Groenewald M, Dunn CW, Hittinger Zeng Q, Zhou S, Galagan J, Cuomo CA, Kistler HC,
CT, Shen X-X, Rokas A (2021) A genome-scale phy- Rep M (2010) Comparative genomics reveals mobile
logeny of the kingdom Fungi. Curr Biol 31:1653– pathogenicity chromosomes in Fusarium. Nature 464:
1665.e5 367–373
Li Y, Liu Z, Liu C, Shi Z, Pang L, Chen C, Chen Y, Pan R, Ma J, Wang S, Zhu X, Sun G, Chang G, Li L, Hu X,
Zhou W, Chen X-X, Rokas A, Huang J, Shen X-X Zhang S, Zhou Y, Song C-P, Huang J (2022) Major
(2022) HGT is widespread in insects and contributes to episodes of horizontal gene transfer drove the evolu-
male courtship in lepidopterans. Cell 185(16):2975- tion of land plants. Mol Plant 15:857–871
2987 Magwene PM, Kay{kç{ Ö, Granek JA, Reininga JM,
Libkind D, Hittinger CT, Valério E, Gonçalves C, Dover J, Scholl Z, Murray D (2011) Outcrossing, mitotic
Johnston M, Gonçalves P, Sampaio JP (2011) Microbe recombination, and life-history trade-offs shape
domestication and the identification of the wild genetic genome evolution in Saccharomyces cerevisiae. Proc
stock of lager-brewing yeast. Proc Natl Acad Sci U S A Natl Acad Sci 108:1987–1992
108:14539–14544
78 C. Gonçalves et al.

Marcet-Houben M, Gabaldon T (2010) Acquisition of intra-domain horizontal gene transfer of d-amino acid
prokaryotic genes by fungal genomes. Trends Genet metabolism enzymes in eukaryotes. Front Microbiol 7
26:5–8 Nevoigt E, Fassbender A, Stahl U (2000) Cells of the yeast
Marcet-Houben M, Gabaldón T (2019) Evolutionary and Saccharomyces cerevisiae are transformable by DNA
functional patterns of shared gene neighbourhood in under non-artificial conditions. Yeast 16:1107–1110
fungi. Nat Microbiol 4:2383–2392 Nguyen TA, Greig J, Khan A, Goh C, Jedd G (2018)
Marsit S, Mena A, Bigey F, Sauvage FX, Couloux A, Evolutionary novelty in gravity sensing through hori-
Guy J, Legras JL, Barrio E, Dequin S, Galeote V zontal gene transfer and high-order protein assembly.
(2015) Evolutionary advantage conferred by an PLoS Biol 16:e2004920
eukaryote-to-eukaryote gene transfer event in wine Nováková E, Moran NA (2011) Diversification of genes
yeasts. Mol Biol Evol 32:1695–1707 for carotenoid biosynthesis in aphids following an
Martin WF (2017) Too much eukaryote LGT. BioEssays ancient transfer from a fungus. Mol Biol Evol 29:
39:1700115 313–323
Matriano DM, Alegado RA, Conaco C (2021) Detection Novo M, Bigey F, Beyne E, Galeote V, Gavory F,
of horizontal gene transfer in the genome of the Mallet S, Cambon B, Legras J-L, Wincker P,
choanoflagellate Salpingoeca rosetta. Sci Rep 11:5993 Casaregola S, Dequin S (2009) Eukaryote-to-eukary-
McCarthy CGP, Fitzpatrick DA (2019) Pan-genome ote gene transfer events revealed by the genome
analyses of model fungal species. Microb Genom 5: sequence of the wine yeast Saccharomyces cerevisiae
e000243 EC1118. Proc Natl Acad Sci U S A 106:16333–16338
McDonald MC, Taranto AP, Hill E, Schwessinger B, Nowell RW, Almeida P, Wilson CG, Smith TP,
Liu Z, Simpfendorfer S, Milgate A, Solomon PS, Fontaneto D, Crisp A, Micklem G, Tunnacliffe A,
Pietro AD (2019) Transposon-mediated horizontal Boschetti C, Barraclough TG (2018) Comparative
transfer of the host-specific virulence protein ToxA genomics of bdelloid rotifers: insights from desiccating
between three fungal wheat pathogens. MBio 10: and nondesiccating species. PLoS Biol 16:e2004830
e01515–e01519 Ochman H, Lawrence JG, Groisman EA (2000) Lateral
Mehta N, Baghela A (2021) Quorum sensing-mediated gene transfer and the nature of bacterial innovation.
inter-specific conidial anastomosis tube fusion between Nature 405:299–304
Colletotrichum gloeosporioides and C. siamense. IMA Palazzo A, Lorusso P, Miskey C, Walisko O, Gerbino A,
Fungus 12:7 Marobbio CMT, Ivics Z, Marsano RM (2019) Tran-
Milner David S, Attah V, Cook E, Maguire F, Savory scriptionally promiscuous “blurry” promoters in
Fiona R, Morrison M, Müller Carolin A, Foster Tc1/mariner transposons allow transcription in dis-
Peter G, Talbot Nicholas J, Leonard G, Richards tantly related genomes. Mob DNA 10:13
Thomas A (2019) Environment-dependent fitness Patron NJ, Waller RF, Cozijnsen AJ, Straney DC,
gains can be driven by horizontal gene transfer of Gardiner DM, Nierman WC, Howlett BJ (2007) Origin
transporter-encoding genes. Proc Natl Acad Sci 116: and distribution of epipolythiodioxopiperazine (ETP)
5613–5622 gene clusters in filamentous ascomycetes. BMC Evol
Möller M, Stukenbrock EH (2017) Evolution and genome Biol 7:174
architecture in fungal plant pathogens. Nat Rev Pauchet Y, Heckel DG (2013) The genome of the mustard
Microbiol 15:756–771 leaf beetle encodes two active xylanases originally
Moran NA, Jarvik T (2010) Lateral transfer of genes from acquired from bacteria through horizontal gene trans-
fungi underlies carotenoid production in aphids. Sci- fer. Proc R Soc B Biol Sci 280:20131021
ence 328:624–627 Perli T, Vos AM, Bouwknegt J, Dekker WJC, Wiersma
Morogovsky A, Handelman M, Kandil AA, Shadkchan Y, SJ, Mooiman C, Ortiz-Merino RA, Daran JM, Pronk
Osherov N, Goldman GH (2022) Horizontal gene JT (2021) Identification of oxygen-independent
transfer of triazole resistance in Aspergillus fumigatus. pathways for pyridine nucleotide and coenzyme A
Microbiol Spectr 0:e01112–e01122 synthesis in anaerobic fungi by expression of candidate
Murphy CL, Youssef NH, Hanafy RA, Couger MB, genes in yeast. MBio 12:e0096721
Stajich JE, Wang Y, Baker K, Dagar SS, Griffith Peter J, De Chiara M, Friedrich A, Yue J-X, Pflieger D,
GW, Farag IF, Callaghan TM, Elshahed MS (2019) Bergström A, Sigwalt A, Barre B, Freel K, Llored A,
Horizontal gene transfer as an indispensable driver for Cruaud C, Labadie K, Aury J-M, Istace B,
evolution of neocallimastigomycota into a distinct Lebrigand K, Barbry P, Engelen S, Lemainque A,
gut-dwelling fungal lineage. Appl Environ Wincker P, Liti G, Schacherer J (2018) Genome evo-
Microbiol 85 lution across 1,011 Saccharomyces cerevisiae isolates.
Naranjo-Ortiz MA, Gabaldón T (2019) Fungal evolution: Nature 556:339–344
major ecological adaptations and evolutionary Pombert JF, Haag KL, Beidas S, Ebert D, Keeling PJ
transitions. Biol Rev 94:1443–1476 (2015) The Ordospora colligata genome: evolution
Naranjo-Ortíz MA, Brock M, Brunke S, Hube B, Marcet- of extreme reduction in microsporidia and host-to-par-
Houben M, Gabaldón T (2016) Widespread inter- and asite horizontal gene transfer. MBio 6
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 79

Qiu H, Yoon HS, Bhattacharya D (2013) Algal Banfield JF, Garavito RM, Carr K, Wilkerson C,
endosymbionts as vectors of horizontal gene transfer Rensing SA, Gagneul D, Dickenson NE,
in photosynthetic eukaryotes. Frontiers. Plant Sci 4 Oesterhelt C, Lercher MJ, Weber APM (2013) Gene
Qiu H, Cai G, Luo J, Bhattacharya D, Zhang N (2016) transfer from bacteria and archaea facilitated evolution
Extensive horizontal gene transfers between plant of an extremophilic eukaryote. Science 339:1207–
pathogenic fungi. BMC Biol 14:41 1210
Reynolds HT, Vijayakumar V, Gluck-Thaler E, Korotkin Selbmann L, Egidi E, Isola D, Onofri S, Zucconi L, de
HB, Matheny PB, Slot JC (2018) Horizontal gene Hoog GS, Chinaglia S, Testa L, Tosi S, Balestrazzi A,
cluster transfer increased hallucinogenic mushroom Lantieri A, Compagno R, Tigini V, Varese GC (2013)
diversity. Evol Lett 2:88–101 Biodiversity, evolution and adaptation of fungi in
Richard GF (2020) Eukaryotic Pangenomes. In: extreme environments. Plant Biosyst 147:237–246
Tettelin H, Medini D (eds) The pangenome: diversity, Selman M, Pombert JF, Solter L, Farinelli L, Weiss LM,
dynamics and evolution of genomes. Springer, Cham Keeling P, Corradi N (2011) Acquisition of an animal
Richards TA, Talbot NJ (2013) Horizontal gene transfer in gene by microsporidian intracellular parasites. Curr
osmotrophs: playing with public goods. Nat Rev Biol 21:R576–R577
Microbiol 11:720–727 Shen XX, Opulente DA, Kominek J, Zhou X, Steenwyk
Richards TA, Hirt RP, Williams BA, Embley TM (2003) JL, Buh KV, Haase MAB, Wisecaver JH, Wang M,
Horizontal gene transfer and the evolution of parasitic Doering DT, Boudouris JT, Schneider RM, Langdon
protozoa. Protist 154:17–32 QK, Ohkuma M, Endoh R, Takashima M, Manabe RI,
Richards TA, Dacks JB, Jenkinson JM, Thornton CR, Cadez N, Libkind D, Rosa CA, DeVirgilio J,
Talbot NJ (2006) Evolution of filamentous plant Hulfachor AB, Groenewald M, Kurtzman CP,
pathogens: gene exchange across eukaryotic Hittinger CT, Rokas A (2018) Tempo and mode of
kingdoms. Curr Biol 16:1857–1864 genome evolution in the budding yeast subphylum.
Richards TA, Soanes DM, Foster PG, Leonard G, Cell 175:1533–1545.e20
Thornton CR, Talbot NJ (2009) Phylogenomic analy- Shen X-X, Steenwyk JL, LaBella AL, Opulente DA,
sis demonstrates a pattern of rare and ancient horizon- Zhou X, Kominek J, Li Y, Groenewald M, Hittinger
tal gene transfer between plants and fungi. Plant Cell CT, Rokas A (2020) Genome-scale phylogeny and
21:1897–1911 contrasting modes of genome evolution in the fungal
Richards TA, Leonard G, Soanes DM, Talbot NJ (2011a) phylum Ascomycota. Sci Adv 6:eabd0079
Gene transfer into the fungi. Fungal Biol Rev 25:98– Shterzer N, Mizrahi I (2015) The animal gut as a melting
110 pot for horizontal gene transfer. Can J Microbiol 61:
Richards TA, Soanes DM, Jones MDM, Vasieva O, 603–605
Leonard G, Paszkiewicz K, Foster PG, Hall N, Talbot Sibbald SJ, Eme L, Archibald JM, Roger AJ (2020) Lat-
NJ (2011b) Horizontal gene transfer facilitated the eral gene transfer mechanisms and pan-genomes in
evolution of plant parasitic mechanisms in the eukaryotes. Trends Parasitol 36:927–941
oomycetes. Proc Natl Acad Sci U S A 108:15258– Silva M, Pontes A, Franco-Duarte R, Soares P, Sampaio
15263 JP, Sousa MJ, Brito PH (2022) A glimpse at an early
Richardson AO, Palmer JD (2007) Horizontal gene trans- stage of microbe domestication revealed in the variable
fer in plants. J Exp Bot 58:1–9 genome of Torulaspora delbrueckii, an emergent
Rokas A (2022) Evolution of the human pathogenic life- industrial yeast. Mol Ecol
style in fungi. Nat Microbiol 7:607–619 Sinha S, Flibotte S, Neira M, Formby S, Plemenitaš A,
Rokas A, Wisecaver JH, Lind AL (2018) The birth, evo- Cimerman NG, Lenassi M, Gostinčar C, Stajich JE,
lution and death of metabolic gene clusters in fungi. Nislow C (2017) Insight into the recent genome dupli-
Nat Rev Microbiol 16:731–744 cation of the halophilic yeast Hortaea werneckii: com-
Ropars J, Rodríguez de la Vega RC, López- bining an improved genome with gene expression and
Villavicencio M, Gouzy J, Sallet E, Dumas É, chromatin structure. G3 (Bethesda, MD) 7:2015–2022
Lacoste S, Debuchy R, Dupont J, Branca A, Giraud T Sloan DB, Nakabachi A, Richards S, Qu J, Murali SC,
(2015) Adaptive horizontal gene transfers between Gibbs RA, Moran NA (2014) Parallel histories of
multiple cheese-associated fungi. Curr Biol 25:2562– horizontal gene transfer facilitated extreme reduction
2569 of endosymbiont genomes in sap-feeding insects. Mol
Savory F, Leonard G, Richards TA (2015) The role of Biol Evol 31:857–871
horizontal gene transfer in the evolution of the Slot JC, Hibbett DS (2007) Horizontal transfer of a nitrate
oomycetes. PLoS Pathog 11:e1004805 assimilation gene cluster and ecological transitions in
Savory FR, Milner DS, Miles DC, Richards TA (2018) fungi: a phylogenetic study. PLoS One 2:e1097
Ancestral function and diversification of a horizontally Slot JC, Rokas A (2010) Multiple GAL pathway gene
acquired oomycete carboxylic acid transporter. Mol clusters evolved independently and by different
Biol Evol 35:1887–1900 mechanisms in fungi. Proc Natl Acad Sci U S A 107:
Schönknecht G, Chen W-H, Ternes CM, Barbier GG, 10136–10141
Shrestha RP, Stanke M, Bräutigam A, Baker BJ,
80 C. Gonçalves et al.

Slot JC, Rokas A (2011) Horizontal transfer of a large and Wang Y, Youssef NH, Couger MB, Hanafy RA, Elshahed
highly toxic secondary metabolic gene cluster between MS, Stajich JE (2019) Molecular dating of the emer-
fungi. Curr Biol 21:134–139 gence of anaerobic rumen fungi and the impact of
Soanes D, Richards TA (2014) Horizontal gene transfer in laterally acquired genes. mSystems 4
eukaryotic plant pathogens. Annu Rev Phytopathol 52: Wang H, Sun S, Ge W, Zhao L, Hou B, Wang K, Lyu Z,
583–614 Chen L, Xu S, Guo J, Li M, Su P, Li X, Wang G, Bo C,
Stajich JE (2017) Fungal genomes and insights into the Fang X, Zhuang W, Cheng X, Wu J, Dong L, Chen W,
evolution of the kingdom. Microbiol Spectr 5 Li W, Xiao G, Zhao J, Hao Y, Xu Y, Gao Y, Liu W,
Stalder L, Oggenfuss U, Mohd-Assaad N, Croll D (2022) Liu Y, Yin H, Li J, Li X, Zhao Y, Wang X, Ni F, Ma X,
The population genetics of adaptation through copy Li A, Xu SS, Bai G, Nevo E, Gao C, Ohm H, Kong L
number variation in a fungal plant pathogen. Mol Ecol (2020) Horizontal gene transfer of Fhb7 from fungus
Steensels J, Gallone B, Verstrepen KJ (2021) Interspecific underlies Fusarium head blight resistance in wheat.
hybridization as a driver of fungal evolution and adap- Science 368
tation. Nat Rev Microbiol 19:485–500 Widen SA, Campo Bes I, Koreshova A, Krogull D, Burga
Steenwyk JL, Rokas A (2018) Copy number variation in A (2022) Virus-like transposons cross the species bar-
fungi and its implications for wine yeast genetic diver- rier and drive the evolution of genetic
sity and adaptation. Front Microbiol 9 incompatibilities. bioRxiv 2022.07.12.499685
Stukenbrock EH (2016) The role of hybridization in the Wiersma SJ, Mooiman C, Giera M, Pronk JT (2020)
evolution and emergence of new fungal plant Squalene-tetrahymanol cyclase expression enables
pathogens. Phytopathology 106:104–112 sterol-independent growth of Saccharomyces
Sun B, Li T, Xiao J, Liu L, Zhang P, Murphy RW, He S, cerevisiae. Appl Environ Microbiol 86
Huang D (2016) Contribution of multiple inter- Wijayawardena BK, Minchella DJ, DeWoody JA (2013)
kingdom horizontal gene transfers to evolution and Hosts, parasites, and horizontal gene transfer. Trends
adaptation of amphibian-killing chytrid, Parasitol 29:329–338
batrachochytrium dendrobatidis. Front Microbiol 7 Wisecaver JH, Rokas A (2015) Fungal metabolic gene
Tabima JF, Trautman IA, Chang Y, Wang Y, Mondo S, clusters-caravans traveling across genomes and
Kuo A, Salamov A, Grigoriev IV, Stajich JE, Spatafora environments. Front Microbiol 6:161
JW (2020) Phylogenomic analyses of non-dikarya Wisecaver JH, Alexander WG, King SB, Hittinger CT,
fungi supports horizontal gene transfer driving diversi- Rokas A (2016) Dynamic evolution of nitric oxide
fication of secondary metabolism in the amphibian detoxifying flavohemoglobins, a family of single-
gastrointestinal symbiont, basidiobolus. G3 Genes| protein metabolic modules in bacteria and eukaryotes.
Genomes|Genetics 10:3417–3433 Mol Biol Evol 33:1979–1987
Thomas CM, Nielsen KM (2005) Mechanisms of, and Wolfe KH (2015) Origin of the yeast whole-genome dupli-
barriers to, horizontal gene transfer between bacteria. cation. PLoS Biol 13:e1002221
Nat Rev Microbiol 3:711–721 Wybouw N, Dermauw W, Tirry L, Stevens C, Grbić M,
Todd RT, Selmecki A (2020) Expandable and reversible Feyereisen R, Van Leeuwen T (2014) A gene horizon-
copy number amplification drives rapid adaptation to tally transferred from bacteria protects arthropods from
antifungal drugs. elife 9 host plant cyanide poisoning. elife 3:e02365
Tsavkelova E, Oeser B, Oren-Young L, Israeli M, Yang Z, Zhang Y, Wafula EK, Honaas LA, Ralph PE,
Sasson Y, Tudzynski B, Sharon A (2012) Identifica- Jones S, Clarke CR, Liu S, Su C, Zhang H, Altman NS,
tion and functional characterization of indole-3-acet- Schuster SC, Timko MP, Yoder JI, Westwood JH,
amide-mediated IAA biosynthesis in plant-associated dePamphilis CW (2016) Horizontal gene transfer is
Fusarium species. Fungal Genet Biol 49:48–57 more frequent with increased heterotrophy and
Urquhart AS, Chong NF, Yang Y, Idnurm A (2022) A contributes to parasite adaptation. Proc Natl Acad Sci
large transposable element mediates metal resistance in U S A 113:E7010–E7019
the fungus Paecilomyces variotii. Curr Biol 32:937– Yin Z, Zhu B, Feng H, Huang L (2016) Horizontal gene
950.e5 transfer drives adaptive colonization of apple trees by
Urquhart AS, Vogan AA, Gardiner DM, Idnurm A (2023) the fungal pathogen Valsa mali. Sci Rep 6:33129
Starships are active eukaryotic transposable elements Zhang Q, Chen X, Xu C, Zhao H, Zhang X, Zeng G,
mobilized by a new family of tyrosine recombinases. Qian Y, Liu R, Guo N, Mi W, Meng Y, Leger RJS,
Proc Natl Acad Sci U S A 120 (15):e2214521120 Fang W (2019) Horizontal gene transfer allowed the
Venkatesh A, Murray AL, Coughlan AY, Wolfe KH emergence of broad host range entomopathogens. Proc
(2021) Giant GAL gene clusters for the melibiose- Natl Acad Sci 116:7982–7989
galactose pathway in Torulaspora. Yeast 38:117–126 Zhou H, Beltrán JF, Brito IL (2021) Functions predict
Wagner A, Whitaker RJ, Krause DJ, Heilers JH, van horizontal gene transfer and the emergence of antibi-
Wolferen M, van der Does C, Albers SV (2017) otic resistance. Science. Advances 7:eabj5056
Mechanisms of gene flow in archaea. Nat Rev Zhu B, Zhou Q, Xie G, Zhang G, Zhang X, Wang Y,
Microbiol 15:492–501 Sun G, Li B, Jin G (2012) Interkingdom gene transfer
may contribute to the evolution of phytopathogenicity
3 Horizontal Gene Transfer in Fungi and Its Ecological Importance 81

in Botrytis cinerea. Evol Bioinformatics Online 8:105– of donor chromosomal genes whose deficiency is
117 responsible for accelerating bacterial and trans-
Zoolkefli FIRM, Moriguchi K, Cho Y, Kiyokawa K, kingdom conjugations by IncP1 T4SS machinery.
Yamamoto S, Suzuki K (2021) Isolation and analysis Front Microbiol 12:620535–620535
 An Overview of Fungal Volatile Organic
Compounds (VOCs) 4
Samantha Lee, Richard Hung, and Joan W. Bennett

Abstract food and flavor properties as indirect

Fungi emit many volatile organic compounds indicators of microbial growth, to stimulate
(VOCs) as mixtures of low molecular mass plant growth, and to attract insect pests.
alcohols, aldehydes, esters, terpenoids, thiols, Because these compounds can diffuse a long
and other small molecules. The study of the way from their point of origin, they are excel-
gas-phase portion of a metabolome is some- lent chemical signaling molecules and facili-
times called “volatomics.” Chemical separa- tate the ability of fungi to engage in “chemical
tion and VOC identification usually rely on conversations.” Volatiles shape ecology at the
gas chromatography-mass spectrometry level of organisms and populations. The phys-
(GC-MS). Fungal VOC profiles are both com- iological effects of fungal VOCs in host–path-
plex and dynamic: the specific compounds ogen relationships and mediating interspecific
produced and their abundance vary with the associations in natural ecosystem functioning
producing species, the age of the colony, water are emerging frontiers in volatile research.
availability, the substrate, the temperature, the
presence of other organisms, and other Keywords
parameters. The single most reported volatile Developmental signals · Infochemical ·
from fungi is 1-octen-3-ol. It functions as a Molecular communication · Mushroom
hormone within many fungal species; serves alcohol · 1-octen-3-ol · Quorum sensing ·
as both an attractant and a deterrent for some Volatome/volatilome · Volatomics
species of arthropods; and exhibits toxicity at
relatively low concentrations in several model
systems. Fungal VOCs have been studied by 4.1 Introduction
scientists from a broad range of subdisciplines
in both theoretical and applied contexts. In In terrestrial ecosystems, fungi occur as
biotechnology, VOCs are exploited for their decomposers, symbionts, and pathogens living
in close association with plants, animals, and
S. Lee other microbes. In these complex, multi-organism
Graduate Women in Science, Mullica Hill, NJ, USA ecosystems, interspecific and interkingdom sig-
R. Hung naling is essential and there is growing scientific
St. Louis, MO, USA awareness that much of this signaling occurs in
J. W. Bennett (✉) the gas phase. Nevertheless, the study of fungal
Department of Plant Biology, Rutgers University, New volatiles in biological communication is an
Brunswick, NJ, USA

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 83

Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_4
84 S. Lee et al.

understudied area of mycology. This chapter basements are all due mainly to gas-phase micro-
provides a general overview of these small, easily bial metabolites.
volatilized molecules. In terrestrial habitats, espe- It is likely that interest in fungal VOCs may
cially in soils with low moisture content, volatiles have begun with fungal species that humans can
provide a way for microbes to communicate with smell (Morath et al. 2012). Many mushrooms
one another, both within and between species have distinctive odor profiles, and it is known
(Massalha et al. 2017). Our goal is to draw atten- that the distinct bouquets of edible mushrooms
tion to the volatile molecules associated with are due to complex blends of VOCs (Breheret
fungal metabolism, to document some of the et al. 1997; Tirillini et al. 2000; Cho et al.
biological versatility that these molecules display, 2008). For example, anise-like odors are charac-
and to highlight their role in fungal associations. teristic of Clitocybe odora, Lentinellus
In recent years, there has been an increasing cochleatus, and Agaricus essettei (Rapior et al.
amount of scientific interest in fungal volatiles, 2002). A “boiled potato” or “farm-feed” odor is
and several valuable reviews have been published characteristic of several species of Suillus
(Bitas et al. 2013; Hung et al. 2015; Li et al. 2016; (de Pinho et al. 2008). Sulfur-containing volatiles
Dickschat 2017; Lee et al. 2017; Piechulla et al. are a highly recognizable group of VOCs of
2017; Farh and Jeon 2020; Inamdar et al. 2020; which the best known are from plants of the
Guo et al. 2021; Vlot and Rosenkranz 2022). genus Allium (chives, garlic, onion, etc.), where
Volatile organic compounds (VOCs) are low they are valued for their flavors in cooking and for
molecular weight carbon-containing compounds their application in traditional medicine. Similar
that evaporate easily at normal temperatures and organosulfur “alliaceous” compounds are made
pressures (Hermann 2010). The best-known by shiitake mushroom (Lentinula edodes) and
volatiles are chemical solvents and other indus- species of Tricholoma and Marasmius (Bloch
trial compounds associated with modern life such and Deorazio 1994; Sneeden et al. 2004).
as paints, cleaning supplies, petroleum fuels, Several mushrooms have common names that
adhesives, photographic solutions, and the like. reflect their odors: the mousepee pinkgill,
Exposures to high concentrations of industrial Entoloma incanum, smells like mouse urine, the
volatiles such as benzene, formaldehyde, methy- garlic parachute Marasmius alliaceus smells like
lene chloride, toluene, and xylene are known to garlic, and the cucumber cap, Macrocystidia
have both short- and long-term negative effects cucumis, is said to smell like either cucumber
on human health (McFee and Zavon 1988; Do sandwiches or cod liver oil. Several Agaricus
et al. 2015). Far less is understood about the species are said to smell like almonds (Boniface
physiological impact of biogenic VOCs. 2020). Stinkhorns in the genus Phallus emit
Plants and microbes emit a wide range of dimethyl trisulfide and skatole, yielding a repul-
volatile acids, alkanes, alkenes, carbonyls, sive stench that combines the worst of carrion and
alcohols, esters, terpenoids, and other small feces (Borg-Karlson et al. 1994). Here we must
molecules into the biosphere (Kesselmeier and add an important caveat: broad person-to-person
Staudt 1999; Baldwin et al. 2006; Vivaldo et al. variability in odor perception is a hallmark of
2017). Of these, the plant isoprenoids are perhaps olfaction research. Not everyone perceives odors
the best known. In particular, monoterpenes such the same way (Gilbert 2008). For example, the
as limonene (“citrusy”), menthol (“minty”), and aromatic pinkgill mushroom, Entoloma
pinene (“resinous”) have familiar odors (Yuan pleopodium, is said to smell of “pear drops,
et al. 2009). Many non-isoprenoid microbial amyl acetate, bananas, or nail polish remover”
VOCs also have distinctive aromas: the earthy (Boniface 2020, p. 30).
smell of garden soil, the noxious stench of spoiled Microscopic fungi (molds and mildews) simi-
foods, the body odors of people who rarely bathe, larly emit complex VOC mixtures. Most people
and the characteristic mustiness of damp know the musty or moldy odors associated with
mold-contaminated damp basements and other
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 85

enclosed indoor spaces. The odor signatures of a

given species or strain of filamentous fungus will
vary depending on the substrate, the length of
incubation, the type of nutrients, temperature,
and other environmental parameters. Further-
more, they vary considerably between species
(Pasanen et al. 1997; Nilsson et al. 2004; Fiedler
et al. 2005). In Aspergillus and Penicillium spe-
cies, the concentration of many VOCs increases
during sporulation (Börjesson et al. 1993).
Mushroom smells have been reviewed by de
Pinho et al. (2008) and Fraatz and Zorn (2010).
For a general database of flavors and scents, see
Dunkel et al. (2009). Fungal odors are sometimes
used as identifying characters in taxonomy, but
the subjective nature of this phenotype makes
“sniffing taxonomy” a subjective discipline
(Larsen 1998). That said, the human nose is an
exquisitely sensitive detection system. Humans
can distinguish an astonishing range of odors
and, for certain odors, are more sensitive than
rodents and dogs (McGann 2017).
The structures of a few representative fungal Fig. 4.1 Chemical structures of some representative fun-
gal volatile organic compounds: (a) sabinene, (b) 1-octen-
VOCs are given in Fig. 4.1. The single best
3-ol, (c) geosmin, (d) 3-methyl-1-butanol, (e)
source resource for microbial volatiles, including 2-methylisoborneal, (f) 3-methyl butyl acetate (isoamyl
fungal volatiles, is the database provided by acetate)
Lemfack et al. (2018) currently called the
“mVOC 3.0 database.” This resource aggregates were usually isolated by steam distillation
a large amount of information about microbial followed by liquid–liquid extraction and concen-
VOCs from over one thousand species of bacteria tration of the organic extract (Kaminski et al.
and fungi and 2000 compounds. The database 1972; Cronin and Ward 1971). These approaches
provides five different search options such that it led to the isolation of several major fungal VOCs,
can be used to find information on specific VOCs, but the technical limitations of the methodology
chemical structures, scaffolds or mass spectra of gave an underestimate of their number. Since that
bacteria, molds, and macroscopic fungi, as well as time, with more sophisticated trapping, separa-
information on compounds and their volatiliza- tion, and identification techniques, approximately
tion, pathways and species-specific signatures, 300 distinct VOCs have been identified from
and relevant literature. fungi where they occur as mixtures of simple
hydrocarbons, heterocycles, alcohols, phenols,
thioalcohols, thioesters, and their derivatives
4.2 Analytical Processes (Chiron and Michelot 2005; Korpi et al. 2009;
for Extraction, Separation, Lemfack et al. 2018). Not all these VOCs have
Identification, odors that humans can detect. However, of those
and Quantification that do smell, many of the most familiar ones
have extremely low odor thresholds. The human
The first major review on fungal volatiles by olfactory system is able to identify geosmin,
Hutchinson (1973) focused almost entirely on which has an earthy smell, in the range of
carbon dioxide. During the 1970s, fungal VOCs 150–200 ng/m3. The mushroom-like odor of
86 S. Lee et al.

1-octen-3-ol is recognized at 10 μg/m3, while the be used. Matysik et al. (2009) found that this
musty odor of 2-octen-1-ol is recognized at 16 μg/ method was particularly suitable in epidemiologi-
m3 (Zogorski et al. 2006). In tests with mice, cal studies that attempt to correlate concentrations
1-octen-3-ol and 1-octen-3-one were detected at of specific VOCs and indoor mold exposure.
the part per trillion range. The authors suggested After separation, the individual constituents of
that the high olfactory sensitivity to mold- VOC mixtures usually are identified by mass
associated odorants was linked to the behavioral spectrometry. Comparison of mass spectra with
relevance of the odor stimuli, i.e., the avoidance library spectra or determination of chro-
of mold-infested foods (Peixoto et al. 2018). matographic retention indices, ideally in conjunc-
The large number of different VOCs, their tion with the parallel determination of authentic
generally low concentrations, and the fact that standards, is used to confirm identity (Stoppacher
they tend to occur in mixtures pose challenges et al. 2010).
for comprehensive sampling and analysis. Differ- The means of delivering the sample to the
ent approaches are utilized for sampling, sample vacuum chamber for analysis have also changed
preparation, separation, identification, and quan- and improved over time. Since its introduction in
tification, depending on the situation and intended the 1990s, solid-phase microextraction (SPME)
application. Traditionally, an initial step of sepa- has emerged as an efficient and popular method
ration into purer components was followed by for assessing VOCs in various contexts
identification and quantification. In recent (Ackerman 1990; Jeleń 2003). A fiber coated
decades, purging and trapping of headspace with different types of adsorbents is exposed to
gases have gained favor, especially in studies of the headspace atmosphere, and subsequently, the
odor formation in agricultural settings (Abramson trapped contents are analyzed by GC-MS. SPME
et al. 1980, 1983). Trapping of headspace gases has been applied widely in the flavor and fra-
also has been used to obtain VOC profiles of a grance industry for monitoring freshness,
number of fungal species grown under detecting fungal contamination in stored
standardized laboratory conditions (Mattheis and products, and the like (Soria et al. 2015). For
Roberts 1992; Börjesson et al. 1993). The absor- good reviews of methodologies for use for
bance materials used for trapping (e.g., charcoal, separating, identifying, and quantifying biogenic
Super Q, and Tenax) can select for compounds VOCs, see Zhang and Li (2010), Materić et al.
with particular binding properties. Similarly, the (2015), and Tholl et al. (2021). In recent years,
choice of organic solvents (e.g., dichloromethane there has been a trend toward developing more
and hexane) can affect the VOC profile obtained, portable and miniaturized detection methods that
sometimes yielding an insufficient resolution of can be used in “real-world” situations, such as the
highly volatile, early eluting compounds (Kai early detection of disease outbreaks in the field or
et al. 2009). Larsen and Frisvad (1995) showed mold contamination in storage (Materić et al.
differences in collecting methods led to vastly 2015). For example, Anishchenko et al. (2018)
distinct profiles of fungal VOCs. developed a miniature GC-differential mobility
Most determination (separation and identifica- spectrometry (GC-DMS) platform small enough
tion) of VOCs now relies on gas chromatography- to fit into a shoe box. Smartphone-based VOC
mass spectrometry (GC-MS) because it integrates sensors also are emerging (Li et al. 2019), and
powerful separation capability and facilitates using a small SPME device for environmental
quantification and identification. Depending on VOC sampling through aerial drones has been
the intended purpose, several methods are cur- developed (Ruiz-Jimenez et al. 2019).
rently available. Air sampling onto Tenax desorp- Analysis of human exhaled breath is a nonin-
tion tubes followed by thermodesorption allows vasive method that shows promise for early-stage
accurate sampling at one point in time. In order to diagnostic support in many fields of medicine.
determine VOCs over an extended time, passive Although the volatile analysis in clinical medi-
diffusion monitors onto charcoal adsorbents can cine has yet to reach its promise, continuing
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 87

research interest almost guarantees that some of metabolites are the enormous group of diverse
the best future volatile research will come from natural products not essential to growth, often of
the investigation of human diseases (Davis et al. highly unusual chemical structure, which almost
2020). For example, detection of VOCs emitted always are restricted in taxonomic distribution.
by A. fumigatus has been suggested as a way for Antibiotics such as cephalosporins, hallucinogens
early diagnosis of aspergillosis (Heddergott et al. such as the ergot alkaloids, and mycotoxins such
2014), and unexpectedly, it was shown that as the trichothecenes are well-known examples of
dimethyl sulfide emitted by Pseudomonas fungal secondary metabolites (Bennett 1983;
aeruginosa, a pathogenic bacterium that often Bennett and Bentley 1989; Cole and Schweikert
co-infects lungs of cystic fibrosis patients, could 2003). Secondary metabolites are biosynthesized
stimulate growth of A. fumigatus (Briard et al. by special pathways (e.g., polyketides,
2016; Scott et al. 2019). non-ribosomal peptides, and isoprenoids) and
“Artificial olfaction” is an area of sensor tech- constitute the bulk of the field known as natural
nology that has proven useful in certain applied products chemistry. Advances in genomics facili-
situations. Electronic noses (sometimes called tate the detection of their signature gene clusters
artificial noses) have been used to mimic mam- using bioinformatics approaches (Keller et al.
malian olfactory systems and can be applied in 2005; Rokas et al. 2020).
healthcare and other biomedical applications. Due to their low molecular weights and phase-
Interestingly, the diseases caused by specific dependent appearance, fungal VOCs as a group
microbial pathogens are associated with charac- sometimes are labeled as secondary metabolites.
teristic odiferous compounds, e.g., Pseudomonas However, there are problems with this simplistic
causes a “grape” odor of skin, while the sweat of categorization. Secondary metabolites usually are
patients with rubella is said to smell like “freshly made by only a taxonomically limited number of
plucked feathers” (Pavlou and Turner 2000). In producing species, while most VOCs are found
laboratory experiments, electronic noses were across a broad range of producing organisms. For
able to distinguish between uncontaminated example, the production of the toxic secondary
samples and those contaminated with dry-rot metabolite aflatoxin is restricted to a few species
wood decay fungi (Kuske et al. 2005). In a clini- within the genus Aspergillus, while the produc-
cal setting, E-nose technology was used to detect tion of the volatile 1-octen-3-ol is widespread
aspergillosis in patients with cystic fibrosis across many fungi as well as plants and animals
(De Heer et al. 2016). A comprehensive historical (Cole and Schweikert 2003; Chiron and Michelot
review of electronic nose technology is provided 2005; Combet et al. 2006; Pennerman et al.
by Wilson and Baietto (2009, 2011). 2022). Secondary metabolites are generally pro-
Finally, it should be noted that dogs can be duced by complex metabolic pathways encoded
trained to detect mold growth with 75–94% accu- by clusters of linked genes (Zhang et al. 2005;
racy (Griffith et al. 2007). Keller et al. 2005). Although less is known about
the pathways that produce VOCs, many are either
metabolic transformation products of lipids,
4.3 Classification of VOCs proteins, heterocyclic metabolites, and other
components of living tissues, or are degradation
The anabolic metabolic pathways of bacteria, end products (“waste products”) of fungal cata-
fungi, and plants can be divided into the dichoto- bolic pathways.
mous categories of primary and secondary metab- The common fungal VOC, 1-octen-3-ol, is a
olism. Primary metabolites are essential to the life good case in point. Although 1-octen-3-ol is
of the organism and represent the unity of bio- sometimes called a secondary metabolite, it is
chemistry. Examples include Krebs cycle derived from the enzymatic or non-enzymatic
intermediates, amino acids, lipids, and nucleic oxidative breakdown of linoleic acid (Pennerman
acids (Berg et al. 2007). In contrast, secondary et al. 2022). In Psalliota bispora, both 1-octen-3-
88 S. Lee et al.

ol and a less volatile 10-oxo-trans-8-decenoic parameters. Examples from this enormous and
acid are produced through the enzymatic oxida- scattered literature are given here.
tion and cleavage of linoleic (Wurzenberger and
Grosch 1984). In Pleurotus pulmonarius, two
separate lipoxygenases may be involved in the 4.4.1 Exploitation of Fungal VOCs:
production of 1-octen-3-ol and 10-oxo-trans-8- Flavors and as Indicators
decenoic acid (Assaf et al. 1997). In Podospora of Fungal Growth
anserina, it requires both cyclooxygenase and
lipoxygenase genes to produce C8 volatiles Biogenic VOCs can be detected in real time. They
(Ferrari et al. 2018). Enzymatic pathways prefer- provide a way to noninvasively monitor safety
ably yield the R enantiomer of 1-octen-3-ol, but applications within the food industry and for
auto-oxidation yields racemic mixtures potable water supplies. In drinking water, “off”
(Tsitsigiannis and Keller 2007; Lee et al. 2020). flavors sometimes are caused by very low
Another example is 6-pentyl pyrone (6-PP), a concentrations of microbial VOCs. Geosmin and
lactone responsible for a distinctive coconut 2-methylisoborneol (MIB) are two VOCs that are
aroma produced by certain Trichoderma species. among the most common compounds involved in
It is a degradation product of linoleic acid malodor problems in surface waters (Watson
(Zeilinger et al. 2016). et al. 2000). They give a musty or earthy odor;
In summary, not all the “small molecules” both have an exceptionally low odor threshold.
outside of the central pathways of intermediary MIB is also a factor in cork taints in winemaking.
metabolism are secondary metabolites. Therefore, Using isotopic labeling experiments, Bentley and
in this chapter, we do not classify fungal VOCs as Meganathan (1981) determined that geosmin is a
either primary or secondary metabolites. Instead, degraded sesquiterpene and MIB is a methylated
we define VOCs according to their number of monoterpene. With improved and inexpensive
carbons, their ring structures, and their substituent methods for monitoring VOCs, such as those
group and as acids, ketones, aldehydes, terpenes, associated with artificial nose technologies, elec-
and the like. tronic odor detection will likely become an
increasingly useful tool for checking food and
water safety.
4.4 Overview of Fungal Volatiles Mold ripened cheeses are among the best-
known food fermentations involving filamentous
Fungal VOCs are of both theoretical and practical fungi. Blue cheeses such as Gorgonzola, Roque-
significance within disparate scientific fort, and Stilton, and white cheeses such as Brie
disciplines. They have been studied for their fla- and Camembert, gain their distinctive flavors
vor properties, as indicators to detect the presence from methyl ketones and various alcohols pro-
of fungal growth, as possible contributors to “sick duced by fungal metabolism by species of Peni-
building syndrome,” and as signals for fungal cillium (Karahadian et al. 1985; Gallois and
development. Moreover, in recent years, the Langlois 1990). Fungi are also used in various
VOCs from endophytes have emerged as of par- commercial bioconversion products for making
ticular interest because some have shown antibi- flavor products that can be considered “natural
otic activity while others have the potential for aromas” (Berger et al. 1992; Schreier 1992). For
possible use as fuel compounds or “biodiesel.” example, Ischnoderma spp. and other white-rot
Numerous research studies have revealed that fungi can produce benzaldehyde, which has a
fungal VOC profiles are both complex and bitter almond flavor, while a variety of yeasts
dynamic: the compounds produced, and their and filamentous basidiomycetes have been used
abundance, vary with the producing species, the to produce clove (eugenol), jasmine (benzyl ace-
age of the fungal colony, moisture level, substrate tate), and rose-like (2-phenylethanol) odors
type, temperature, and other environmental (Copetti 2019).
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 89

The study of aroma compounds in beers, (Wilkins et al. 2000; Korpi et al. 2009; Claeson
wines, and other spirits and their relationship et al. 2002; Matysik et al. 2008). Fungus-like
with the perceived flavor of alcoholic beverages odors can be recognized at concentrations greater
represents a huge field that addresses the volatile than 0.035 μg/m3. Generally, the background
compounds associated with yeast fermentation. microbial VOC concentrations in mold-free
For introductions to this vast literature, see buildings are similar to those found in outdoor
(Meilgaard 1975a, b) and Robinson (2006). We air ranging between 2.2 and 8.8 μg/m3. VOC
will not attempt to cover food and flavor chemis- levels significantly higher than the background
try in this chapter. See Bennett and Feibleman ranges may indicate an increase in the active
(2001) for an earlier review on mixed bacterial– microbial production possibly associated with
fungal food fermentations. adverse health effects (Ström et al. 1994). Vola-
VOCs can be used as indirect indicators of tile sampling can be used to look for fungi in
mold growth, even in the absence of visible small openings in drywall or upholstered furni-
colonies. Methods for remote volatile capture ture and provides a noninvasive method for deter-
and analysis have allowed an expansion of mining the presence of fungi on valuable objects
applications. Considerable research has gone of cultural heritage such as tapestries, on the
into developing strategies that detect fungal wooden frameworks behind paintings, or in
volatiles as an indirect and noninvasive way to libraries (Micheluz et al. 2016). For example,
indicate the presence of fungal growth in agricul- Joblin et al. (2010) developed an index that was
ture, water-damaged buildings, and art used to compare the level of VOCs in the Lascaux
conservation. caves contaminated with Fusarium solani before
Fungal contamination of stored foods and and after antifungal treatment.
feeds is a global problem in agriculture. Fungi
decrease the nutritive value of stored foods,
spoil them by creating off flavors, and can pro- 4.4.2 Building-Related Illnesses
duce mycotoxins that render foods poisonous to
human beings and other animals. Compounds Occupants of damp indoor spaces often complain
such as geosmin, 1-octen-3-ol, 3-octanol, and of irritation of eyes and mucous membranes,
3-methyl-1-butanol are regularly found in associ- respiratory discomfort, malaise, headaches, gas-
ation with stored grains contaminated with fungi trointestinal disturbances, and a variety of other
(Börjesson et al. 1989, 1993; Mattheis and symptoms that are often lumped together and
Roberts 1992). Volatiles can be used to detect called “nonspecific building-related illness,” or
fungal contamination on agricultural products by the more controversial name of “sick building
(Wadood et al. 2020; He et al. 2022). Fungal syndrome.” Many scientists have hypothesized
VOCs have been used to monitor favorable and that this spectrum of adverse health is associated
unfavorable food quality (Karlshøj et al. 2007). with exposure to airborne bacteria and fungi,
With advancements in electronic nose technol- aerosolized toxins, or bacterial metabolites such
ogy, “mapping” of volatile compounds may pre- as endotoxins (Thorn 2001; Straus et al. 2003;
dict the levels of fungi found in agricultural Burge 2004; IOM 2004; Li and Yang 2004;
products and identify individual species WHO 2009). Mycotoxins have received most of
(Schnürer et al. 1999). the attention as the hypothetical cause of the
The VOC profiles of common molds grown on symptoms of sick building syndrome (Jarvis and
building materials have been analyzed in the lab- Miller 2005; Straus 2009). However, VOCs may
oratory (Sunesson et al. 1995, 1996). Mold VOC also contribute to the adverse health
profiles have also been studied in water-damaged consequences associated with damp indoor
buildings where compounds such as 2-methyl-1- spaces, especially in cases where there is no visi-
propanol and 3-methyl-1-butanol have been ble evidence of mold growth (Walinder et al.
suggested as useful indicators of mold growth 2005; Mølhave 2009). High vapor pressures,
90 S. Lee et al.

low to medium water solubility, and low molecu- fungal VOC decreased dehydrogenase activity as
lar weights allow both fungal and bacterial VOCs well as levels of nitric oxide but increased levels
to persist and migrate in the environment, and to of reactive oxygen species (ROS). It was
diffuse through enclosed wall cavities, air condi- hypothesized that the negative consequences of
tioning filters, and vapor barriers. The level of the exposures were related to necrotic
VOCs measured in the indoor air of mold- mechanisms and that they had implications for
infested building varies with the ventilation rate, human health (Macedo et al. 2022).
moisture levels, composition of mold population,
and area of the building/room and is in a state of
constant change. One of the highest reported 4.4.3 VOCs from Endophytes
concentrations for a single VOC, 1-octen-3-ol,
found in problem buildings is 900 μg/m3 or Endophytes are microorganisms that live
0.16 ppm (Morey et al. 1997). Moreover, intercellularly within plant tissues without caus-
1-octen-3-ol has been reported to be one of the ing any evident adverse effects (Bacon and White
major fungal VOCs emitted by various species of 2000). They have been found in almost every
fungi (Aspergillus, Cladosporium, Fusarium, plant species examined and are likely to play a
Penicillium, Stachybotrys, etc.) regularly found significant role in plant community structure
in moldy and water-damaged buildings (Rodriguez et al. 2009). Fungal endophytes have
(Sunesson et al. 1996) or from composting been studied as a source of novel secondary
facilities (Fischer et al. 1999). Studies in our metabolites (Tan and Zou 2001) and also have
laboratory have shown that gas-phase 1-octen-3- gained attention as producers of bioactive VOCs.
ol is more toxic to human embryonic stem cells Muscodor albus (“stinky white fungus”) is an
than toluene (Inamdar et al. 2011) and that it endophyte that produces a blend of inhibitory or
inhibits seed germination and plant growth in lethal VOCs to a wide range of bacteria and
Arabidopsis thaliana (Lee et al. 2016a). An pathogenic fungi. Using GC-MS, Strobel et al.
investigation of fungal VOCs from hospital air (2001) found that M. albus produced a mixture
showed that 2-heptanone and 2-methyl-1- of volatile acids, alcohols, esters, ketones, and
propanol, not 1-octen-3-ol, were the most fre- lipids, which individually had inhibitory but not
quent compounds isolated (Pantoja et al. 2016). lethal effects against test species such as Fusar-
Our laboratory has pioneered the use of the ium solani, Pythium ultimum, and Rhizoctonia
genetic model Drosophila melanogaster for solani. When applied collectively, these same
studying the physiological impact of fungal VOCs acted synergistically to kill a broad range
volatiles isolated from indoor environments. of plant-pathogenic fungi and bacteria (Strobel
When adult Drosophila flies were exposed for et al. 2001). Since the original isolation of
one week to low concentrations of chemical Muscodor albus from the cinnamon tree, several
standards of 2-octanone (0.5%); 2,5 other Muscodor strains and species that emit anti-
dimethylfuran (0.5%) (DMF), 3-octanol (0.5%), biotic mixtures of VOCs have been isolated
trans-2-octenal (0.5%), and 1-octen-3-ol (0.1%), (Atmosukarto et al. 2005; Zhang et al. 2010).
they exhibited 40%, 35%, 60% 50%, and 100% This selective antimicrobial effect can be
lethality, respectively (Inamdar et al. 2010). By harnessed against undesirable pathogens and has
exposing third instar D. melanogaster larvae, we been termed “mycofumigation.” Muscodor albus
developed an eclosion bioassay and successfully has been used for the biological control of
demonstrated the toxicity of VOCs from fungi damping-off of broccoli seedlings grown in
isolated from flooded homes in the aftermath of greenhouse soilless mix (Mercier and Jiménez
Hurricanes Katrina and Sandy (Inamdar and 2004; Mercier and Manker 2005). Oxyporus
Bennett 2015; Zhao et al. 2017). Other Drosoph- latemarginatus, an endophyte isolated from pep-
ila studies using chemical standards of 1-octen-3- per plants, has also shown a positive
ol showed that exposure of flies to this common mycofumigation ability against post-harvest
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 91

decay organisms (Lee et al. 2009). Using genetic pests by secreting active metabolites. Moreover, it
screens, Alpha et al. (2015) showed that the tox- is important to remember that ethanol is a VOC
icity of M. albus VOCs was caused by (Vajzovic et al. 2012) and that ethanol plays a
permeabilizing of the cell membrane and an inter- morphogenetic role in the yeast–hyphal transition
ference of DNA metabolism. Mycofumigation in Candida albicans (Chauhan et al. 2011).
agents have also been isolated from other While most mycologists are familiar with the
endophytes including Fusarium proliferatum, an role of fungi as endophytes of plants, we are less
unidentified species of Diaporthe, and familiar with the role of bacteria as residents of
Geotrichum candidum (Pangga 2021; Mitra fungi. It is becoming increasingly apparent that
et al. 2023). many fungi carry bacterial lineages that are trans-
VOCs from endophytes may have other bio- mitted vertically (Bonfante and Desirò 2017). The
technological applications. Growing fungi in the VOCs of these fungal endobacteria are beyond
presence of M. albus has been used as a selection the scope of this chapter, but because fungi and
tool to isolate other fungi that produce bioactive bacteria tend to live in close associations with
volatiles. Species resistant to the VOCs produced plants and each other, it is not always clear during
by M. albus are then screened for the activity of volatile analysis which domain of organisms is
their own VOC profile. Using this method, an producing a given metabolite.
endophytic species of Gliocladium was isolated
from a Patagonian species of Eucryphia. This
Gliocladium species produced a number of 4.4.4 Truffles
VOCs, of which one of the most interesting was
1,3,5,7 cyclooctatetraene or annulene, an unstable “Truffle” is the common name for a group of
and flammable compound used as a rocket pro- ascomycete fungi that form underground fruiting
pellant during World War II (Stinson et al. 2003). structures and emit aromatic compounds that
Strobel et al. (2008) coined the term attract animals which eat them and then, in turn,
“mycodiesel” when he found that the endophyte disperse their spores through their feces. Mention
Gliocladium roseum produced several VOCs nor- of these delectable but strange fungi goes back to
mally associated with diesel fuel. classical times (Ramsbottom 1953). A truffle is
Endophytic fungi in the genera Ascocoryne the reproductive structure of an ectomycorrhizal
(Griffin et al. 2010), Phoma (Strobel et al. fungus; see Hall et al. (2007) for a list of the plant
2011), and Phomopsis also synthesize VOCs species that support truffle formation. The most
that have fuel potential. Phomopsis species famous truffle species resemble small, misshapen
isolated from an orchid in Ecuador produces bicy- potatoes, hence their genus name Tuber, for
clic monoterpene sabinene, a monoterpene with a “lump” (Jeandroz et al. 2008). Despite their
peppery odor first isolated from plants (Singh undistinguished appearance, Tuber magnatum,
et al. 2011). Several related monoterpenes are the “white truffle,” and Tuber melanosporum,
being investigated as possible components of air- the “black truffle,” are deemed the most desirable
craft fuel (Rude and Schirmer 2009). For six of all edible fungi. In the world of gourmets,
species of oleaginous fungi studied, the resultant truffles are among the most expensive of food
biodiesel quality reflected the fatty acid profile of ingredients and therefore are often used as
the feedstock oil used (Shafiq and Chechan seasonings in other dishes, shaved into paper-
2019). thin slices, or minced as a garnish (Hall et al.
In recent years, there has been growing interest 1994). In addition to their reputation as the ulti-
in yeasts as endophytes. Species of Cryptococcus, mate gourmet fungus, these fungi are purported to
Debaryomyces, Sporobolomyces, and serve as aphrodisiacs (Ramsbottom 1953; Bone
Rhodotorula have been reported from many 2011). In her book simply titled Truffles,
plants (Doty 2013). These endophytic yeasts Elisabeth Luard writes that “when ripe and
improve plant vigor by helping host plants resist ready, the truffle reeks of sex” (p. 14) and goes
92 S. Lee et al.

on to say that “the perfume for which it was 2,4-dithiapentane is considered most important
valued was held to induce the kind of behavior (Niimi et al. 2021). In their meta-analysis of the
among celibate priests and nuns that could lead to data from the mVOC database, Elmassry et al.
the birth of children” (Luard 2006, p. 36). Other (2020) found that “volatile sulfur metabolism is
descriptions of the lascivious “intoxicating odor” dominated by bacteria rather than by fungi.”
of truffles and human reactions to this odor are Thus, it seems that the most famous odor
given in Mycophilia. Revelations from the Weird compounds attributed to fungi may be of bacterial
World of Mushrooms (Bone 2011). origin!
As with all complex aromas, the prized truffle
smell is caused by a mixture of individual volatile
molecules that vary with the producing species, 4.5 VOCs as Ecological Signaling
the age of the fruiting body, the weather, the Agents
general climate, the truffle microbiome, and
other environmental factors. Descriptors as varied With each passing year, there is growing recogni-
as “meaty, cheese-like, fruity green apple, malty, tion of the extent of chemical communication in
and mushroomy” have been used to describe the the biosphere and the role that volatile chemicals
different truffle odors. However, none of these play in biological signaling. Chemical signaling
pedestrian adjectives apparently come close to occurs within and between cells, between
capturing the alluring scent of a fresh truffle individuals of the same species, and also between
(Allen and Bennett 2021). Not surprisingly, truf- different species. Entomologists and chemical
fle VOCs have been the subject of intense scien- ecologists have been at the forefront of studying
tific interest and more than one hundred different chemical signaling molecules and have devel-
VOCs have been reported from a range of truffle oped an extensive vocabulary that includes
species using different extraction and identifica- terms such as “allelochemicals,” “infochemicals,”
tion techniques (Splivallo et al. 2007a; Vita et al. and “semiochemicals” (Table 4.1). Only some of
2015). They encompass many small hydrocarbon the infochemicals studied to date are found in the
molecules, but the most distinctive truffle VOCs gas phase, but the concepts and definitions are
are thiols (Mauriello et al. 2004; Splivallo and useful whatever the physical states of the signal-
Ebeler 2015; Mustafa et al. 2020). In other ing molecule.
words, the key aroma compounds that make Chemical signaling includes both long- and
truffles so commercially valuable all contain sul- short-range cellular communication. A major dis-
fur (Schmidberger and Schieberle 2017). VOC tinction is that pheromones mediate intraspecific
analysis of truffle fruiting bodies revealed that interactions, while allelochemicals mediate inter-
these thiol derivatives were exclusively specific interactions. However, these terms are
synthesized by the resident bacterial microbiome not mutually exclusive. A single chemical can
and not by the truffle. When fruiting bodies were function in more than one type of interaction
treated with antibacterial agents, the sulfur- (Nordlund and Lewis 1976). For example, the
containing volatiles were fully suppressed; in nearly ubiquitous fungal volatile, 1-octen-3-ol,
contrast, fungicide treatment had no inhibitory depending on the context and biological system,
effect (Splivallo et al. 2015). For Tuber borchii, is described as a hormone, pheromone,
thiophene derivatives are major contributors to semiochemical, or allelochemical.
the human-sensed aroma, with 3-methyl-4,5- The revolution in mammalian microbiome
dihydrothiophene as the single most important research has made all biologists aware of the
compound in terms of its contribution to the over- fact each macroscopic “higher organism” is a
all aroma (Splivallo and Ebeler 2015; Splivallo complex multi-organism. Similarly, metabolomic
and Culleré 2016). Of the more than 60 volatile research has broadened our awareness that many
compounds detected from Tuber magnatum, only metabolites used as interspecific signaling agents
11 contribute to the smell of which are in the gas phase (Nicholson et al. 2005;
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 93

Table 4.1 Technical terms frequently encountered in discussions of chemical signaling compounds
Term with definition and citations
Allelochemical—Broadly, a compound that mediates chemical interactions between organisms (Hooper and Pickett
2004). Allelochemicals were originally termed “allelochemics” by Whittaker and Feeny (1971) to describe toxic
chemicals made by a plant to defend itself against competing plants.
Allomone—An allelochemical that only benefits the organism that emits the chemical signal (Hooper and Pickett 2004)
“Originally defined as a chemical substance produced or acquired by an organism, which, when it contacts an
individual of another species in the natural context, evokes in the receiver a behavioral or physiological reaction
adaptively favorable to the emitter” (Brown 1968; Nordlund and Lewis 1976).
Kairomone—A controversial term for an allelochemical which benefits only the receiving organism, but not the
producer; i.e., the emitter does not benefit from the interaction (Nordlund and Lewis 1976; Hooper and Pickett 2004).
Synomone—An allelochemical which benefits both the producing and receiving organisms.
Autoinducer—A compound used by quorum sensing bacteria to monitor cell density.
Auto-inhibitor (“self-inhibition”)—a term introduced by Allen (1957) to describe the inhibition of fungal spore
germination when spores are overcrowded, a process thought to ensure efficient substrate colonization.
Crowding effect—The delay and inhibition of spore germination resulting from high spore concentrations (Trinci and
Whittaker 1968).
Growth regulating substances (GRS)—A term sometimes used as a synonym for “plant hormone.” Examples include
auxins and gibberellins in plants and possibly 10-oxo-trans-8-decenoic acid in fungi (Takahashi 1986; Mau et al. 1992).
Chemical ecology—The field that studies how plants, animals, and microbes use chemicals to communicate in natural
ecosystems.
Hormone—A term originally used by endocrinologists to describe chemicals secreted by a gland or group of cells in
vertebrates to regulate specific physiological processes within the organism. The definition has been expanded to
describe chemicals in other organisms that control and regulate growth and development within the same organism as
the compound is secreted.
Infochemical—Information-conveying chemical. “A chemical that, in the natural context, conveys information in an
interaction between two individuals, evoking in the receiver a behavioral or physiological response that is adaptive to
either one of the interactants or to both” (Dicke and Sabelis 1988).
Mushroom alcohol—The common name of 1-octen-3-ol, one of the most prevalent fungal VOCs. Another synonym is
matsutake alcohol.
PGPB—Plant growth-promoting bacteria.
PGPF—Plant growth-promoting fungi.
Psi factors—“Precocious sexual inducers” first identified from A. nidulans by Champe et al. (1987). Champe and
el-Zayat (1989) also used the term “sexual sporulation hormone.” These oxylipin compounds are involved in several
fungal developmental cascades and are classified according to the fatty acids from which they are derived (Tsitsigiannis
and Keller 2007; Brodhun and Feussner 2011).
Quorum sensing—Ability of bacterial populations to communicate and coordinate their behavior mediated by the use
of chemical signaling molecules (Fuqua et al. 1994; Bassler and Losick 2006). Quorum sensing was originally called
“autoinduction” (Nealson and Hastings 1979).
1-oceten-3-ol—An oxylipid C-8 alcohol that is produced by numerous fungi, as well as by many plants, and that can
also be emitted by certain arthropods and mammals. It regularly acts as a semiochemical. Depending on the scientific
context and literature, it is variously called a pheromone, a kairomone, a synomone, or by other jargon terms.
Oxylipin—A large family of structurally related oxygenated polyenoic fatty acids and the metabolites derived from
them. Many oxylipins have physiological activities. They are abundant in mammals but also served as signals of intra-
and intercellular communication in other vertebrates, invertebrates, plants, and fungi. In microbes, they regulate
growth, differentiation, and apoptosis in addition to the development of the infectious processes caused by some
pathogenic microorganisms (Noverr and Erb-Downward 2003; La Camera et al. 2004; Tsitsigiannis and Keller 2007).
Pheromone—An “ecto-hormone,” a chemical signal mediating interactions between organisms of the same species
(Hooper and Pickett 2004). A “substance that is secreted by an organism to the outside and causes a specific reaction in
a receiving organism of the same species” (Karlson and Luscher 1959; Nordlund and Lewis 1976). Examples include
the trail pheromones and sex pheromones that have been well documented in insects.
Semiochemical—“A chemical involved in the chemical interaction between organisms” (Nordlund and Lewis 1976).
Volatilome (sometimes “volatome”)—The complete collection of volatile metabolites from a given source under a
specific set of environmental parameters, sometimes called the “volatile metabolome” (Kimball 2016).
Volatomics—The volatile part of a metabolome; the study of the kinds and concentrations of volatile organic
compounds (VOCs) emitted from a biological system and the study of how changes in metabolism can affect their
makeup.
94 S. Lee et al.

Dietert and Silbergeld 2015). For example, in a et al. 2010; Hossain et al. 2014). VOC-mediated
review of soil microbiomes, Raza et al. (2021) signaling is believed to help plants defend against
proposed “that soil microbe-plant communities pathogen attacks (Raza et al. 2021).
could be viewed as ‘metarhizobiomes,’ where When Elmassry et al. (2020) used the mVOC
VOC-mediated interactions extend the plant database of microbial volatiles to identify phylo-
rhizobiome further out through interconnected genetic signals, they found that the bacterial
microbial metapopulation networks.” Volatile- domain contained 33 phylogenetically conserved
phase signaling molecules allow organisms to VOCs. The following compounds tended to be
work together at a distance. from bacteria: acetic acid, dimethyl disulfide,
dimethyl trisulfide, indole, isovaleric acid,
2-tridecanone, propanoic acid, and
4.5.1 Bacterial VOCS 2-undecanone. When these VOCs were found in
a fungal volatome, it was hypothesized that they
Bacterial species, like fungi, produce complex were due to the presence of “resident” bacteria
cocktails of VOCs, often in close collaboration growing on the fungi. Geosmin was one of
with fungi (e.g., see the section on truffles above). 11 VOCs that were conserved from both bacteria
As with fungi, VOC profiles from various bacte- and fungi (Elmassry et al. 2020).
ria have been analyzed. For example, twenty-six
species in the genus Streptomyces revealed
mixtures of alcohols, alkanes, alkenes, ketones, 4.5.2 Quorum Sensing
terpenoids, and thiols in a range of concentrations
and combinations (Schöller et al. 2002). Forty- Quorum sensing refers to cellular communication
two different volatiles were found from systems that generate a population response.
Myxococcus xanthus (Dickschat et al. 2005b), Quorum sensing allows microorganisms to com-
and the volatile blend of a marine Streptomyces municate and coordinate their behavior via the
species exhibited antibiotic properties (Dickschat secretion of signaling molecules that accumulate
et al. 2005a). See Schulz and Dickschat (2007), during their growth and then act in a population-
Audrain et al. (2015), Schulz-Bohm et al. (2017), dependent manner. Quorum sensing was origi-
and Weisskopf et al. (2021) for comprehensive nally studied in bacteria where the molecules
surveys of bacterial VOCs. used to monitor cell density were named
Bacteria communicate with one another using “autoinducers.” The concentration of
many molecular mechanisms and have interactive autoinducers increased as a function of rising
effects on the organisms with which they share bacterial population density; when a minimum
ecological niches, including plants, animals, threshold of autoinducers was detected, the bac-
fungi, and other bacteria. Bacterial volatiles teria responded by altering gene expression
have been studied for their uses in foods, particu- (Bassler and Losick 2006; Camilli and Bassler
larly in the dairy industry; their production of 2006). Quorum sensing indicated that certain
“off” odors in food and water supplies; and genes were expressed only when bacteria are
other applied areas. Soil bacteria associated with crowded together (von Bodman et al. 2003).
the rhizosphere have been investigated for their Overall, the study of quorum sensing has
growth-promoting activities. There is an exten- transformed our view of “single cell” organisms.
sive literature on plant growth-promoting bacteria The first quorum sensing system described at
(PGPB) (Glick 2020). Similarly, there are plant the molecular level was in Vibrio fischeri, a bio-
growth-promoting fungi (PGPF) which include luminescent marine bacterium in which the bac-
species of Aspergillus, Fusarium, Trichoderma, teria grow to high cell density within the light
Penicillium, Piriformospora, Phoma, and Rhizoc- organ of the Hawaiian squid (Nealson et al.
tonia, which have the natural ability to stimulate 1970; Nealson and Hastings 1979). When
various growth-related traits of plants (Shoresh V. fischeri cells are free-living, they do not
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 95

luminesce. However, in the nutrient-rich environ- regulating the expression of most of the traits
ment of the squid’s light organ, they grow to a needed for infection and virulence (Flavier et al.
high concentration and produce an acyl- 1997; von Bodman et al. 2003). In environments
homoserine lactone which acts as an autoinducer such as soil and plant surfaces, opportunities for
(Eberhard et al. 1981). The population-dependent signaling in the liquid phase are limited. There-
release of autoinducer leads to transcription of the fore, it is highly likely that many other volatile
luciferase gene and bioluminescence (Kaplan and autoinducers will be discovered and that “signal-
Greenberg 1985). The V. fischeri system has ing in the gas phase may be one of the next
become a paradigm for quorum sensing in important frontiers in quorum sensing” (Horswill
Gram-negative bacteria; in addition, many other et al. 2007).
Gram-negative bacteria have cell-to-cell signal- Quorum sensing molecules, both volatile and
ing systems that use fatty acid derivatives as non-volatile, have also been identified in fungi
autoinducers (Whitehead et al. 2001; Bassler (Hornby et al. 2001), where they can participate
and Losick 2006). Quorum sensing also occurs in cross-kingdom communication (Dixon and
widely in Gram-positive bacteria, where the best Hall 2015). The best studied system is Candida
studied autoinduction signals are oligopeptides albicans, a dimorphic fungus, which grows as a
(Waters and Bassler 2005; Shapiro 1998). commensal on humans in yeast form but
Once thought to be rare, quorum sensing functions as an opportunistic pathogen with
systems are now known to be common. They hyphal morphology. Hornby et al. (2001) showed
are involved in a large number of complex envi- that farnesol, an acyclic sesquiterpene alcohol,
ronmental responses that include virulence and modulates the yeast-to-hyphal transition in Can-
biofilm formation in Pseudomonas aeruginosa dida; this yeast to hyphal transition is essential for
(Van Delden and Iglewski 1998; Rumbaugh causing disseminated disease (Hogan 2006).
et al. 2000), fruiting body production in There is increasing evidence that quorum sensing
Myxococcus (Shimkets 1999), antibiotic and pig- is widespread in fungi and that in addition to
ment production in Serratia (Thomson et al. farnesol many of these systems oxylipins such
2000), pathogenicity in plant-pathogenic bacteria as 1-octen-3-ol play an important role (Kües and
(von Bodman et al. 2003), and many other impor- Navarro-González 2009; Wongsuk et al. 2016).
tant phenomena that allow bacterial colonies to Aromatic alcohols are involved in quorum sens-
behave in a fashion similar to multicellular ing in Saccharomyces cerevisiae (Chen and Fink
organisms. These bacterial systems are almost 2006). Quorum sensing mechanisms in medically
always studied in the context of aqueous important fungal species have received the most
environments. In the laboratory, the approach is attention and have been reviewed (Tian et al.
to use shaken liquid-batch cultures whereby the 2021). In particular, farnesol and other quorum
quorum sensing response is detected at a specific sensing molecules are involved in the yeast-to-
point in the growth curve, coinciding with a hyphal morphological changes that are important
threshold concentration of the signal (Horswill in pathogenesis (Chow et al. 2021). C. albicans
et al. 2007). Because of the dominant aqueous makes aliphatic alcohols (e.g., ethyl alcohol,
experimental model, it is not surprising that only isoamyl alcohol, 1-dodecanol, 2-dodecanol, and
a few bacterial quorum sensing signals that oper- nerolidol) and aromatic alcohols (e.g.,
ate through the gas phase have been discovered. 2-phenylethyl alcohol and tryptophol) that inhibit
Nevertheless, some cases have been reported. filamentation (Chauhan and Mohan Karuppayil
Ralstonia (Pseudomonas) solanacearum is a 2021).
Gram-negative soil-borne plant pathogen that Specialized terminology tends to be discipline
causes bacterial wilt in a wide variety of impor- specific and hinders effective communication
tant crops, including eggplant, potato, and between scientists from different fields. For
tobacco (Agrios 2008). It uses the volatile signal, instance, the jargon used to describe the cell
3-hydroxyl palmitic acid methyl ester, for density-dependent signaling systems important
96 S. Lee et al.

in fungal spore germination and fruit body devel- VOCs are involved in the formation of well-
opment was developed independently from terms known symbioses between yeasts and insects
used by bacteriologists to describe bacterial quo- (Stefanini 2018) or with other putative hosts
rum sensing systems (Table 4.1). Many fungal (Ling et al. 2020). Moreover, many VOCs are
processes, such as morphogenesis, mating, bio- involved in arthropod defense. For example, a
film development, and apoptosis, are farnesol combination of 1-octen-3-ol and geosmin func-
mediated (Tian et al. 2021). Hence, more and tioned as defensive allomones in millipedes
more mycologists have adopted the quorum sens- (Ômura et al. 2002). 1-octen-3-ol also interrupts
ing terminology to describe the molecules that the response of beetles to their aggregation hor-
function as virulence factors, regulate fungal mor- mone (Poland et al. 2009). Most true bugs
phological change in fungi, or otherwise influ- (Heteroptera) have scent glands in what has
ence fungal behaviors (Wongsuk et al. 2016; been described as “overwhelming chemical forti-
Tian et al. 2021). fication” (Aldrich 1988). Moreover, many species
secrete attractant pheromones. For example, vari-
ous bugs secrete (E)-2-hexanal, hexanal,
4.5.3 Entomology hexanoic acid, 2-butyl-2-octenal, β-pinene, limo-
nene, and farnesene as alarm or trail pheromones
Entomologists have been pioneers of chemical for defense against their main predators (Aldrich
ecology, so it is not surprising that the best- 1988). For those wishing an introduction to the
known cases of VOC signaling involve fascinating world of insect defensive secretions,
arthropods, often studied under “pheromone biol- many of which utilize noxious smelling VOCs
ogy.” Many VOCs elicit specific behaviors such such as caprylic acid, we recommend Thomas
as insect mating. A complete survey of this topic Eisner’s beautifully illustrated book For Love of
would require an encyclopedia of its own; only a Insects (Eisner 2003).
few examples are given here as an introduction to For a long time, it has been known that fungal
this fascinating field. The articles cited give odors attract insects that live on fungi or on
access to the extensive literature describing chem- substrates decayed by fungi (Fäldt et al. 1999).
ical communication with an emphasis on fungal In one interesting case in point, female houseflies
gas-phase molecules. do not lay their eggs on animal feces that have
Many fungal volatiles, particularly eight- been colonized by Fusarium, Phoma, Rhizopus,
carbon alcohols and ketones, act as and other fungi. Using chemical standards, six
semiochemicals and function in insect attraction fungal VOCs that consistently inhibit oviposition
and deterrence (Pierce et al. 1991a, 1991b; Dowd were determined: dimethyl disulfide,
and Bartelt 1991; Luntz 2003; Nilssen 1998; phenylacetaldehyde, 2-phenylethanol, citronellal,
Ômura et al. 2002; Ramoni et al. 2001; Davis and norphytone (Lam et al. 2010).
et al. 2013). Many blood-feeding insects have When the chemical composition of insect
highly developed olfactory systems whereby vol- attractants is known, in some cases effective arti-
atile semiochemicals enter the antennae and later ficial pheromones can be prepared to trap insect
bind to olfactory receptor neurons (Weeks et al. pests. Moreover, “elucidation of chemical
2011). C-8 alcohols emitted by decay fungi may messengers for predatory and weed feeding bugs
serve as trophic signals for conifer feeding bark may lead to pheromonal husbandry of these ben-
beetle prey and as bypass trophic signals for their eficial insects” (Aldrich 1988). Fungal volatiles
predators (Zhang and Schlyter 2004). As assayed may provide innovative, safe pest management
using a pitfall olfactometer test, grain beetles strategies for economically problematic insect
showed strong attraction to 1-octen-3-ol (Pierce species (Hayashi 2015; Holighaus and Rohlfs
et al. 1991b). 2016).
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 97

4.5.4 Developmental Signals showed that volatiles from a given bacterial strain
for Plant Growth cause different responses in different fungi; i.e.,
associations of fungi and bacteria interact in dif-
Plant growth promotion by microbial species has ferent ways. The VOCs of PGPR are associated
been studied in several contexts. Microbes influ- with induced systemic resistance (Farag et al.
ence plant growth by reducing levels of disease 2006). Furthermore, VOCs also lead to an
(e.g., by antibiosis or competition with enhancement of aroma compounds in basil
pathogens), stimulating growth, and (Banchio et al. 2009).
biofertilization. When scientists study these Certain bacterial volatiles promote plant health
interactions, beneficial effects are usually easiest by inhibiting the growth of fungal pathogens.
to demonstrate in the laboratory, with decreasing Negative effects on fungi include the inhibition
success in greenhouses, and only a few microbes of both sporulation and spore germination. Posi-
functioning successfully in field situations tive effects on sclerotial and fruit body develop-
(Lugtenberg and Kamilova 2009). ment were often observed (Vespermann et al.
Bacterial volatiles have received the most 2007; Mackie and Wheatley 1999; Fernando
attention. Many growth-promoting rhizobacteria et al. 2005). Plants live with complex microbial
produce volatile organic compounds that have a consortia that include both bacterial and fungal
positive impact on plant growth (Vespermann species. The VOCs emitted from these consortia
et al. 2007). For example, strains of Bacillus frequently serve as the communication signals
subtilis, Bacillus amyloliquefaciens, and used within and between kingdoms (Schulz-
Enterobacter cloacae promoted plant growth by Bohm et al. 2017; Heenan-Daly et al. 2021).
releasing several volatiles of which acetoin and Just as bacterial VOCs can enhance or inhibit
2,3-butanediol gave the highest level of growth plant growth, fungal VOCs also have many
promotion (Ryu et al. 2003, 2005). Exposure to effects on plant growth and development (Lee
B. subtilis volatiles induces beneficial effects, et al. 2015; Sánchez-López et al. 2016). For
including increases in plant size and weight; mod- example, Fusarium oxysporum and Verticillium
ulation of root-system architecture; and differen- dahliae both emit volatiles that increase
tial expression of numerous plant genes (Zhang Arabidopsis thaliana tolerance to salt stress
et al. 2007; Xie et al. 2009; Gutiérrez-Luna et al. (Li and Kang 2018; Li et al. 2018). Exposure to
2010). Photosynthetic capacity is also increased volatiles from Alternaria alternata, Penicillium
with higher chlorophyll content and upregulation charlesii, and Penicillium aurantiogriseum
of genes encoding chloroplast proteins (Zhang increases starch accumulation in leaves of
et al. 2008; Xie et al. 2009). VOCs stimulate the Arabidopsis, barley, maize, potato, tomato, and
synthesis of indole-3-acetic acid, cytokinin, and tobacco (Ezquer et al. 2010). VOCs from
gibberellins. Moreover, genes involved in auxin Alternaria alternata upregulate sucrose, starch,
biosynthesis are upregulated in plants exposed to and glucose transport and modification enzymes,
VOCs from growth-promoting rhizobacteria while VOCs from phytopathogenic Rhizoctonia
(Ryu et al. 2003). solani increase primary root growth in the turnip
When plant growth-promoting bacteria Brassica rapa (Moisan et al. 2021).
(PGPR) interact with plants, VOCs are important Our lab has studied the effect of volatiles from
components of the signaling process several Trichoderma species on the plant genetic
(Vespermann et al. 2007). Several bacterial model Arabidopsis thaliana. Hung et al. (2013)
volatiles, including alcohols, ammonia, HCN, found that plants grown in the presence of
and phenazine-1-carboxylic acid, have antifungal volatiles from T. viride, without any direct physi-
properties that contribute to the biocontrol cal contact between the plants and the fungi, were
properties of PGPR (Choudhary et al. 2008; Kai larger, had more chlorophyll, and bloomed earlier
et al. 2009; Whipps 2001). Kai et al. (2009) than controls, while other volatiles such as
1-octen-3-ol caused negative effects (Hung et al.
98 S. Lee et al.

2014). In similar studies, A. thaliana was grown

in the presence of twenty different biocontrol
strains of Trichoderma. Co-growth with nine of
these strains yielded plants with increased bio-
mass and chlorophyll concentration (Lee et al.
2016b). Arabidopsis also was grown in the pres-
ence of physiological concentrations of chemical
standards of 26 VOCs previously identified from
Trichoderma (Lee et al. 2016a). Increases in fresh
weight and chlorophyll contents were observed
by exposure to seven of these volatiles: 2-methyl-
1-butanol, 3-methyl-1-butanol, 2-methyl-1-
propanol, 2-heptylfuran, 1-decene, 1-octen-3-ol,
and 2-heptanone. Of compounds tests, plants
exposed to vapors of 1-decene had the greatest
increase in fresh weight and chlorophyll content.
Transcriptome analysis of plants exposed to
1-decene revealed that 123 genes were differen-
tially expressed, including genes involved in cell
wall modification, auxin induction, stress, and
defense responses (Lee et al. 2019).
Below-ground volatiles produced by a strain
of Trichoderma viride improved plant growth in
okra (Abelmoschus esculentus) and induced
defense enzyme activities, resulting in reduced
lipid peroxidation and cell death. In addition,
these VOCs inhibited the growth of Sclerotium
rolfsii, a rot pathogen. VOCs detected from
T. viride included 2-butyl-1-octanol, methyl- Fig. 4.2 The natural product 1-octen-3-ol is produced by
bicyclooctan-7-ol, 1-methyl cycloundecanol, many fungal and plant species. This eight-carbon volatile
causes myriad stimulatory and inhibitory responses in
tetradecanoic acid, benzestrol, oleic acid, fungi, plants, and animals. Specific effects are mediated
benzenediol, 9-tricosene, hexadecanoic acid, and in a species-specific and concentration-dependent manner
ascaridole (Singh et al. 2021).
“auto-inhibition.” These processes detect spore
over-crowding and maximize the chances of sur-
4.5.5 Developmental Signals vival and colony formation once the spore has
for Fungal Spore Germination germinated. High spore concentrations inhibit
and Growth germination; with lower spore concentrations,
germination proceeds. In general, these effects
Many biological activities, including chemotropic are reversible and do not affect later mycelial
interactions, growth coordination, and inhibition growth. Auto-inhibition has been well studied in
of the growth of other fungi, are mediated by Colletotrichum, where several germination self-
volatile and non-volatile signaling metabolites. inhibitors have been chemically characterized as
See Leeder et al. (2011) for a useful review. non-volatile indole compounds and where these
Figure 4.2 illustrates some of the interactions compounds are specific for the producing species
associated with 1-octen-3-ol. (Lax et al. 1985; Tsurushima et al. 1995). The cell
The fungal sensing processes that prevent pre- density-dependent germination inhibitor systems
mature germination are called “self-inhibition” or
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 99

in fungi resemble quorum sensing systems Trichoderma, very low concentrations of

described for bacteria (see above). 1-octen-3-ol, 3-octanol, and 3-octanone induced
The single most commonly reported volatile conidiation. However, at the highest
from both macroscopic and microscopic fungi is concentrations tested for 1-octen-3-ol (500 μM)
1-octen-3-ol which is also called “mushroom and 3-octanol and 3-octanone (both 1000 μM),
alcohol” or “matsutake alcohol” because it was both conidiation and growth were inhibited
first identified from Tricholoma matsutake (Nemcovic et al. 2008). In another study
(Wood and Fesler 1986). In the straw mushroom, identifying and profiling the naturally occurring
Volvariella volvacea, for example, 1-octen-3-ol volatile metabolites of Trichoderma atroviride,
accounts for the majority (72–83%) of the total 1-octen-3-ol and 3-octanone reached their maxi-
volatiles detected (Mau et al. 1997). Mushroom mum concentrations simultaneously to
alcohol occurs in two enantiomer forms, (R)-(-)- conidiation (Stoppacher et al. 2010).
1-octen-3-ol and (S)-(+)-1-octen-3-ol, of which Similar effects of C-8 compounds have been
the most abundant naturally occurring isomer is observed in Aspergillus nidulans (Herrero-Garcia
the R enantiomer, sometimes called “roctenol.” et al. 2011). High conidial densities yielded auto-
Roctenol is produced commercially where it is inhibition of germination, which was associated
used as an insect attractant (Bohbot and Dickens with 1-octen-3-ol produced in association with
2009; Kline et al. 2007). (R)- (-)-1-octen-3-ol aerial hyphae. The inhibition effect was revers-
has a characteristic “mushroomy” odor, while ible; conidiospores germinated after 1-octen-3-ol
(S)-(+)-1-octen-3-ol is more grassy or moldy; was removed. The closely related compound
(R)-(-)-1-octen-3-ol is the predominant naturally 3-octanone enhanced sporulation responses in
occurring enantiomer of 1-octen-3-ol (Mosandl aerial cultures (Herrero-Garcia et al. 2011). It
et al. 1986). It is of interest that the phylogenetic also is of interest that several C-18 oxylipins
analysis of microbial VOCs by Elmassry et al. derived from linoleic acid (psi factors) can repress
(2020) showed that 1-octen-3-ol, 3-octanone, conidiation and induce premature sexual sporula-
aristolochene, and 2-pentylfuran are primarily tion in A. nidulans (Champe et al. 1987; Champe
synthesized by fungi and not by other microbes. and el-Zayat 1989). Oxylipins are broadly
Mushroom alcohol is produced with involved in the developmental regulation of
10-oxo-trans-8-decenoic acid by the enzymatic aspergilli (Tsitsigiannis and Keller 2007), and
breakdown of linoleic acid. Both compounds 10-oxo-trans-8-decenoic acid stimulates mycelia
inhibit the mycelial growth of Penicillium growth, stipe lengthening, and the initiation of
expansum at low (1.25 mM) concentrations fruiting in Agaricus bisporus (Mau et al. 1992).
(Okull et al. 2003). In Penicillium paneum, Oxylipins have been found from fungal
dense suspensions of conidia show poor germina- pathogens in both plants and animals. While the
tion; again the auto-inhibitor is 1-octen-3-ol oxylipin chemical end products are similar, the
(Chitarra et al. 2004). Volatile compounds pro- fungal enzymes involved in their production are
duced by P. paneum under high spore density different from those isolated from plants and
conditions also inhibit the mycelial growth of animals. Several lines of evidence suggest that
other fungal species belonging to a variety of plant oxylipins are able to mimic fungal
genera, suggesting a range of actions beyond oxylipins, suggesting a form of “reciprocal
auto-inhibition. It has been hypothesized that cross-talk” in certain host–pathogen relationships
1-octen-3-ol interferes, in a reversible manner, (Brodhagen et al. 2008). See Brodhun and
with essential metabolic processes involved in Feussner (2011) for a comprehensive review of
the swelling and germination of the conidia fungal oxylipins and the enzymes involved in
(Chitarra et al. 2005). their production.
The effect of 1-octen-3-ol is dependent on its Various pheromone communication systems
concentration and on the stage of fungal colony are used to coordinate colony formation, fruit
formation. In dark-grown cultures of body development, and reproduction in higher
100 S. Lee et al.

fungi (Kües and Navarro-González 2009). The (Kishimoto et al. 2007). In addition, some soil
factors that influence sporocarp development in bacteria emit antifungal VOCs (Liu et al. 2008).
the cultivated mushroom Agaricus bisporus have Numerous fungal species engage in symbiotic
been studied widely. Primordium formation interactions with plants such as mycorrhizae,
depends on both the presence of a “casing layer” endophytes, and lichens, while a large number
containing appropriate bacteria and on sufficient are plant and insect pathogens. Although fewer
ventilation. The air exchange is important fungi are mammalian pathogens, the species that
because it removes inhibitory VOCs, especially affect humans are becoming increasingly impor-
1-octen-3-ol produced by A. bisporus, but also tant in modern medicine. In all these interspecific
3-ethyl-1-hexanol produced by the rye on which interactions, chemical conversations are taking
the A. bisporus spawn is grown. Pseudomonad place, many of them facilitated by VOCs.
populations in the casing metabolize 3-ethyl-1- Because volatile compounds can be sensed at a
hexanol, thus removing its inhibitory effect distance, they are ideal for serving as intra- and
(Noble et al. 2009). interspecific chemical signals or “infochemicals.”
Phytopathologists have long recognized the
importance of molecular signaling during plant–
4.5.6 Other Associations pathogen interactions. In the rhizosphere, multi-
ple species of bacteria and fungi interact with
Many organisms produce metabolites that are plant roots and their exudates. Moreover,
used in deterring competitors. Humans categorize non-pathogenic microbes such as nitrogen-fixing
some of these chemical defenses as antibiotics bacteria and mycorrhizal fungi that establish ben-
(if they selectively kill microbes that we wish to eficial relationships with plants rely on complex
see dead) or toxins (if they kill us or our favored signaling networks (Oldroyd and Downie 2004;
plant and animal species). Well-known plant sec- Harrison 2005). Many of the other chapters in this
ondary metabolites such as alkaloids are thought book describe classical and molecular approaches
to have evolved as defense mechanisms. Plants to studying such fungal associations, and Ortiz-
have exquisitely refined chemical defense Castro et al. (2009) have reviewed the role of
mechanisms against insect herbivory, including microbial signals in plant growth and
indirect defenses whereby they emit volatiles development.
that attract natural enemies (Howe and Jander The soil remains an underexplored habitat that
2008). A survey of 1500 fungal secondary contains a rich diversity of microbial life forms.
metabolites published between 1993 and 2001 The relevance of secondary metabolites and
showed that more than half had antibacterial, volatiles in soil ecosystems has been reviewed
antifungal, or antitumor activity (Pelaez 2005). in the monograph edited by Karlovsky (2008). It
The literature on fungal secondary metabolites is has been estimated that a single gram of soil may
extensive and has been reviewed elsewhere contain tens of thousands of different fungal,
(Turner 1971; Turner and Aldridge 1983; Cole bacterial, archaeal, and protist species. The soil
and Schweikert 2003). Far less is known about properties such as nutrient and oxygen availabil-
the deterrence activity of plant, fungal, and bacte- ity and the physiological state of the microbial
rial VOCs. Nevertheless, certain fungal VOCs are population make dynamic and unique soil-
known to have growth inhibitory effects on plants specific communities (McNeal and Herbert
(Splivallo et al. 2007b). When A. thaliana is 2009). Multispecies microbial populations play a
exposed to 1-octen-3-ol, the defense genes that key role in sustaining soil microcosms. Commu-
are associated with wounding or ethylene and nication within rhizosphere bacterial populations,
jasmonic acid signaling are turned on (Kishimoto within mycelial colonies, and between fungi and
et al. 2007). Moreover, plants exposed to 1-octen- bacteria is mediated by signaling molecules, but
3-ol can inhibit the expansion of the pathogen, we are only beginning to learn the specifics of this
Botrytis cinerea, on the infected leaves chemical information exchange. Gas-phase
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 101

molecules are essential components of these pathogen interactions and non-pathogen ecologi-
chemical conversations. These compounds can cal contexts is an emerging frontier for future
diffuse a long way from their point of origin, research. VOCs produced by a given microbial
and they can persist and migrate in soil species can have multiple effects on other
environments, areas of dense vegetation, and microbes and organisms and can be used for
other microhabitats that harbor interacting defense, environmental monitoring, and nutrient
populations of bacteria and fungi. acquisition (Wheatley et al. 1997; Bruce et al.
Much of the molecular work on biofilm forma- 2004; Minerdi et al. 2009). We agree with Tarkka
tion has been conducted in laboratory settings in and Piechulla (2007) that it is likely that many
the context of quorum sensing research using more multifunctional and multi-organismic vola-
single-species biofilms. However, biofilms tile-based interactions exist in ecosystems that
found in nature are multispecies communities have been envisioned in the past. As the analyti-
that can harbor several hundred species where cal methods for assaying VOCs improve, we will
various forms of intercellular signaling are being find increasing evidence of the crucial importance
used. By definition, the specialized vocabulary of of VOC-mediated cross-talk between species, not
“autoinducers” is not appropriate in biofilm only for fungal–bacterial associations but also for
communities. Nevertheless, many of the same fungal–plant and fungal–animal associations.
signaling molecules and pathways are in use
(Kolter and Greenberg 2006).
The marine alga Pyropia haitanensis uses a 4.6 Summary
lipoxygenase enzyme to convert large quantities
of C20:4 fatty acids into 1-octen-3-ol (Luo et al. This essay has highlighted some of the major
2014). Production of this important signaling ways microbial VOCs have been studied to date
molecule may act as a messenger that induces a and gives examples of how communication in
“primed” state by upregulating the synthesis of biological systems is mediated chemically
methyl jasmonic acid, indole-3-acetic acid, and through the atmosphere. Numerous plant–fungal,
gibberellin A3 (Chen et al. 2019). insect–fungal, and bacterial–fungal interactions
In an unusual “Trojan horse” form of bacterial that involve VOC signaling have been discov-
pathogenesis against nematodes, Bacillus ered, but many more remain to be elucidated.
nematocida B16 lures nematodes by emitting Technological advances with respect to our abil-
several volatile organic compounds, including ity to detect VOCs and our capacity to monitor
benzaldehyde and 2-heptanone that are much their physiological effects in plant, animal, and
more attractive to worms than those from ordi- microbial systems will open the door to new
nary dietary bacteria. Once the bacteria enter the views of the complexities underlying chemical
intestine of nematodes, they secrete proteases that signaling across the biosphere.
preferentially target essential intestinal proteins, Microbial VOCs have been studied by
leading to nematode death (Niu et al. 2010). An scientists from a broad range of disciplines for
even more striking example of a volatile–nema- both theoretical and applied reasons. As a result,
tode interaction concerns the mushroom the published information about fungal VOCs is
Pleurotus ostreatus (the “oyster mushroom”), scattered widely among diverse scientific litera-
which for years has been known to be carnivo- ture. In general, entomologists do not cross-
rous. Lee et al. (2023) showed that 3-octanone reference the microbiological literature, nor do
accumulates in specialized cells called toxocysts food and flavor chemists pay attention to the
and that the use of chemically pure 3-octanone physiological activities of the compounds they
recapacitates the rapid paralysis, calcium influx, study. Nevertheless, in recent years, there has
and neuronal death arising from fungal contact. been an increased interdisciplinary interest in fun-
As these tantalizing cases illustrate, the effect gal volatile research as evidenced by the quantity
of bacterial and fungal VOCs in both host– of review articles on the subject and the breadth of
102 S. Lee et al.

applications and interactions that have emerged. References

By orchestrating these different kinds of knowl-
edge, it becomes ever more obvious that very Abramson D, Sinha RN, Mills JT (1980) Mycotoxin and
small gas-phase molecules from fungi and all odor formation in moist cereal grain during granary
storage. Cereal Chem 57:346–351
biogenic sources can have enormous physiologi- Abramson D, Sinha RN, Mills JT (1983) Mycotoxin and
cal impacts within and between organisms across odor formation in barley stored at 16 and 20% moisture
the major domains of life. VOCs facilitate the in Manitoba. Cereal Chem 60:350–355
formation and maintenance of mutualistic symbi- Ackerman D (1990) A natural history of the senses. Vin-
tage Books, Random House, New York, NY
otic relationships of fungi with plants, animals, Agrios GN (2008) Plant pathology, 5th edn. Elsevier, San
and/or bacteria. Nevertheless, their study remains Diego, CA
technically challenging. They are produced in Aldrich JR (1988) Chemical ecology of the heteroptera.
complex mixtures, and while the number of Annu Rev Entomol 33:211–238
Allen PJ (1957) Properties of a volatile fraction of
volatiles in a biogenic blend can be large, their uredospores of Puccinia graminis var. tritici affecting
individual abundance is generally low. their germination and development. I Biological activ-
The scientific community has just begun to ity. Plant Physiol 32:385–389
eavesdrop on the chemical discourse conducted Allen K, Bennett JW (2021) Tour of truffles: aromas,
aphrodisiacs, adaptogens, and more. Mycobiology
via gas-phase molecules. As we learn more about 49(3):201–212
chemical signaling, it is becoming increasingly Alpha CJ, Campos M, Jacobs-Wagner C, Strobel SA
apparent that the significance of biogenic VOCs, (2015) Mycofumigation by the volatile organic
including fungal VOCs, has been underestimated. compound-producing fungus Muscodor albus induces
bacterial cell death through DNA damage. Appl Envi-
It is our hope that this chapter will elicit the ron Microbiol 81(3):1147–1156
interest of fungal biologists to ask questions Anishchenko IM, McCartney MM, Fung AG, Peirano DJ,
such as: How do microbes use VOCs as intraspe- Schirle MJ, Kenyon NJ, Davis CE (2018) Modular and
cific developmental signals and as interspecific reconfigurable gas chromatography/differential mobil-
ity spectrometry (GC/DMS) package for detection of
signaling cues? In what way can we incorporate volatile organic compounds (VOCs). Int J Ion Mobil
the insights of the entomological community into Spectrom 21:125–136
the mindset of bacteriologists and mycologists? Assaf S, Hadar Y, Dosoretz CG (1997) 1-Octen-3-ol and
What kinds of technological tools are needed to 13-hydroperoxylinoleate are products of distinct
pathways in the oxidative breakdown of linoleic acid
expand the study of VOCs? By what means can by Pleurotus pulmonarius. Enzym Microb Technol 21:
VOC research provide a platform for productive 484–490
interdisciplinary collaborations? How many com- Atmosukarto I, Castillo U, Hess W, Sears J, Strobel G
mon volatile compounds like 1-octen-3-ol serve (2005) Isolation and characterization of Muscodor
albus I-41.3s, a volatile antibiotic producing fungus.
unsuspected physiological activities? In what way Plant Sci 169:854–861
will metabolomic research revolutionize the study Audrain B, Farag MA, Ryu CM, Ghigo JM (2015) Role of
of biogenic gas-phase molecules? bacterial volatile compounds in bacterial biology.
FEMS Microbiol Rev 39(2):222–233
Bacon CW, White JW (eds) (2000) Microbial endophytes.
Acknowledgments Research in the Bennett laboratory
Marcel Dekker, New York
has been funded by the Northeast Sustainable Agriculture
Baldwin IT, Halitschke R, Paschold A, von Dahl CC,
Research and Education Grant (GNE14-084-27806); the
Preston CA (2006) Volatile signaling in plant-plant
U.S. Fish & Wildlife Service Grant (WNS 2016-6);
interactions: “talking trees” in the genomics era. Sci-
USDA-ARS Cooperative Agreement (Grant
ence 311:812–815
No. 2-47012); the Rutgers University Research Fund;
Banchio E, Xie X, Zhang H, Paré PW (2009) Soil bacteria
and the Sloane and Eppley Foundations. JWB is grateful
elevate essential oil accumulation and emissions in
to the late Karl Esser for his mentorship and to Olivia
sweet basil. J Agric Food Chem 57:653–657
Froehlich, Arati Inamdar, Shannon Morath, Sally Padhi,
Bassler BL, Losick R (2006) Bacterially speaking. Cell
Kayla Pennerman, and Guohua Yin for years of fruitful
125:237–246
conversations and collaborations.
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 103

Bennett JW (1983) Differentiation and secondary metabo- Brodhagen M, Tsitsigiannis DI, Hornung E, Goebel C,
lism in mycelial fungi. In: Bennett JW, Ciegler A (eds) Feussner I, Keller NP (2008) Reciprocal oxylipin-
Secondary metabolism and differentiation in Fungi. mediated cross-talk in the Aspergillus–seed
Marcel Dekker, New York pathosystem. Mol Microbiol 67:378–391
Bennett JW, Bentley R (1989) What’s in a name? – Micro- Brodhun F, Feussner I (2011) Oxylipins in fungi. FEBS J
bial secondary metabolism. Adv Appl Microbiol 34:1– 278:1047–1063
28 Brown WL (1968) An hypothesis concerning the function
Bennett JW, Feibleman T (2001) Fungal bacterial of the metapleural glands in ants. Am Nat 102:188–
interactions. In: Hock B (ed) The Mycota, vol 191
IX. Springer-Verlag, Berlin Bruce A, Verrall S, Hackett CA, Wheatley RE (2004)
Bentley R, Meganathan R (1981) Geosmin and Identification of volatile organic compounds (VOCs)
methylisoborneol biosynthesis in streptomycetes: evi- from bacteria and yeast causing growth inhibition of
dence for an isoprenoid pathway and its absence in sapstain fungi. Holzforschung 58:193–198
non-differentiating isolates. FEBS Lett 125(2): Burge PS (2004) Studies on the role of fungi in sick
220–222 building syndrome. Occup Environ Med 61:185–190
Berg JM, Tymoch JL, Stryer L (2007) Biochemistry. WH Camilli A, Bassler BL (2006) Bacterial small-molecule
Freeman and Company, New York signaling pathways. Science 311:1113–1116
Berger RG, Drawert F, Tiefel P (1992) Naturally occuring Champe SP, el-Zayat AA (1989) Isolation of a sexual
flavours from fungi, yeasts, and bacteria. In: Patterson sporulation hormone from Aspergillus nidulans. J
RLS, Charlwood BV, MacLeod G, Williams AA (eds) Bacteriol 171:3982–3988
Bioinformation of flavours. The Royal Chemistry Champe SP, Rao P, Chang A (1987) An endogenous
Society, Cambridge, pp 1–20 inducer of sexual development in Aspergillus nidulans.
Bitas V, Kim H-S, Bennett J, Kang S (2013) Sniffing on J Gen Microbiol 133:1383–1387
microbes: diverse roles of microbial volatile organic Chauhan NM, Mohan Karuppayil S (2021) Dual identities
compounds in plant health. Mol Plant-Microbe Interact for various alcohols in two different yeasts. Mycology
26:835–843 12(1):25–38
Bloch E, Deorazio R (1994) Chemistry in a salad bowl: Chauhan NM, Raut JS, Karuppayil SM (2011) A morpho-
comparative organosulfur chemistry of garlic, onion, genetic regulatory role for ethyl alcohol in Candida
and shitake mushrooms. Pure Appl Chem 66:2205– albicans. Mycoses 54(6):e697–e703
2206 Chen H, Fink GR (2006) Feedback control of morphogen-
Bohbot JD, Dickens JC (2009) Characterization of an esis in fungi by aromatic alcohols. Genes Dev 20:
enantioselective odorant receptor in the yellow fever 1150–1161
mosquito Aedes aegypti. PLoS One 4:7032 Chen H, Yang R, Chen J, Luo Q, Cui X, Yan X, Gerwick
Bone E (2011) Mycophilia: revelations from the weird WH (2019) 1-Octen-3-ol, a self-stimulating oxylipin
world of mushrooms. Rodale Books messenger, can prime and induce defense of marine
Bonfante P, Desirò A (2017) Who lives in a fungus? The alga. BMC Plant Biol 19:37
diversity, origins and functions of fungal endobacteria Chiron N, Michelot D (2005) Odeurs de champignons:
living in Mucoromycota. ISME J 11(8):1727–1735 chimie et rôle dans les interactions biotiques — une
Boniface T (2020) The use of odours in the identification revue. Cryptogam Mycol 26:299–364
of mushrooms and toadstools. Field Mycol 21:28–30 Chitarra GS, Abee T, Rombouts FM, Posthumus MA,
Borg-Karlson A-K, Englund FO, Unelius CR (1994) Dijksterhuis J (2004) Germination of Penicillium
Dimethyl oligosulphides, major volatiles released paneum conidia is regulated by 1-octen-3-ol, a volatile
from Sauromatum guttatum and Phallus impudicus. self-inhibitor. Appl Environ Microbiol 70:2823–2829
Phytochemistry 35:321–323 Chitarra GS, Abee T, Rombouts FM, Dijksterhuis J (2005)
Börjesson T, Stöllman UM, Adamek P, Kaspersson A 1-octen-3-ol inhibits conidia germination of Penicil-
(1989) Analysis of volatile compounds for detection lium paneum despite of mild effects on membrane
of molds in stored cereals. Cereal Chem 66:300–304 permeability, respiration, intracellular pH, and changes
Börjesson T, Stöllman UM, Schnürer J (1993) the protein composition. FEMS Microbiol Ecol 54:67–
Off-odorous compounds produced by molds on 75
oatmeal agar: Identification and relation to other Cho IH, Namgung H-J, Choi H-K, Kim YS (2008)
growth characteristics. J Agric Food Chem 41:2104– Volatiles and key odorants in the pileus and stipe of
2111 pine-mushroom (Tricholoma matsutake Sing). Food
Breheret S, Talou T, Rapior S, Bessiere J-M (1997) Chem 106:71–76
Monoterpenes in the aromas of fresh wild mushrooms. Choudhary DK, Johri BN, Prakash A (2008) Volatiles as
J Agric Food Chem 45:831–836 priming agents that initiate plant growth and defence
Briard B, Heddergott C, Latge JP (2016) Volatile responses. Curr Sci 94(5):595–604
compounds emitted by Pseudomonas aeruginosa stim- Chow EWL, Pang LM, Wang Y (2021) From Jekyll
ulate growth of the fungal pathogen Aspergillus to Hyde: the yeast-hyphal transition of Candida
fumigatus. MBio 7:e00219 albicans. Pathogens 10(7):859
104 S. Lee et al.

Claeson A-S, Levin J-O, Blomquist GR, Sunesson A-L Dowd PF, Bartelt RJ (1991) Host-derived volatiles as
(2002) Volatile metabolites from microorganisms attractants and pheromone synergists for dried fruit
grown on humid building materials and synthetic beetle, Carpophilus hemipterus. J Chem Ecol 17:
media. J Environ Monit 4:667–672 285–308
Cole R, Schweikert M (2003) Handbook of secondary Dunkel M, Schmidt U, Struck S, Berger L, Gruening B,
fungal metabolites, vol 1-3. Academic Press, Hossbach J, Jaeger IS, Effmert U, Piechulla B,
Amsterdam Eriksson R, Knudsen J, Preissner R (2009)
Combet E, Henderson J, Eastwood DC, Burton KS (2006) SuperScent--a database of flavors and scents. Nucleic
Eight-carbon volatiles in mushrooms and fungi: Acids Res 37(Database issue):D291–D294
properties, analysis, and biosynthesis. Mycoscience Eberhard A, Burlingame AL, Eberhard C, Kenyon GL,
47:317–326 Nealson KH, Oppenheimer NJ (1981) Structural iden-
Copetti MV (2019) Fungi as industrial producers of food tification of autoinducer of Photobacterium fischeri
ingredients. Curr Opin Food Sci 25:52–56 luciferase. Biochemistry 20:2444–2449
Cronin DA, Ward MK (1971) The characterisation of Eisner T (2003) For love of insects. Harvard University
some mushroom volatiles. J Sci Food Agric 22:477– Press, Cambridge, MA
479 Elmassry MM, Farag MA, Preissner R, Gohlke BO,
Davis TS, Crippen TL, Hofstetter RW, Tomberlin JK Piechulla B, Lemfack MC (2020) Sixty-one volatiles
(2013) Microbial volatile emissions as insect have phylogenetic signals across bacterial domain and
semiochemicals. J Chem Ecol 39:840–859 fungal kingdom. Front Microbiol 11:557253
Davis C, Pleil J, Beauchamp J (eds) (2020) Breathborne Ezquer I, Li J, Ovecka M, Baroja-Fernández E, José
biomarkers and the human volatilome, 2nd edn. Muñoz F, Montero M, de Cerio J, Hidalgo M, Sesma
Elsevier MT, Behaji A, Etxeberria E (2010) Microbial volatile
De Heer K, Vonk SI, Kok M, Kolader M, Zwinderman emissions promote accumulation of exceptionally high
AH, Van Oers MHJ, Visser CE (2016) eNose technol- levels of starch in leaves in mono-and dicotyledonous
ogy can detect and classify human pathogenic molds plants. Plant Cell Physiol 51(10):1674–1693
in vitro: a proof-of-concept study of Aspergillus Fäldt J, Jonsell M, Nordlander G, Borg-Karlson A-K
fumigatus and Rhizopus oryzae. J Breath Res 10(3): (1999) Volatiles of bracket fungi Fomitopsis pinicola
036008 and Fomes fomentarius and their functions as insect
de Pinho PG, Ribeiro B, Goncalves RF, Baptista P, attractants. J Chem Ecol 25:567–590
Valentao P, Seabra RM, Andrade PB (2008) Correla- Farag MA, Ryu CM, Sumner LW, Pare PW (2006)
tion between the pattern volatiles and the overall aroma GC-MS SPME profiling of rhizobacterial volatiles
of wild edible mushrooms. J Agric Food Chem 56: reveals prospective inducers of growth promotion and
1704–1712 induced systemic resistance in plants. Phytochemistry
Dicke M, Sabelis MW (1988) Infochemical terminology: 67:2262–2268
based on cost-benefit analysis rather than origin of Farh ME, Jeon J (2020) Roles of fungal volatiles from
compounds? Funct Ecol 2:131–139 perspective of distinct lifestyles in filamentous fungi.
Dickschat JS (2017) Fungal volatiles–a survey from edible Plant Pathol J 36(3):193–203
mushrooms to moulds. Nat Prod Rep 34(3):310–328 Fernando WGD, Ramarathnam R, Krishnamoorthy AS,
Dickschat JS, Martens R, Brinkhoff T, Simon M, Schulz S Savchuk SC (2005) Identification and use of potential
(2005a) Volatiles releases by Streptomyces species bacterial organic antifungal volatiles in biocontrol. Soil
isolated from the North Sea. Chem Biodivers 2:837– Biol Biochem 37:955–964
865 Ferrari R, Lacaze I, Le Faouder P, Bertrand-Michel J,
Dickschat JS, Wenzel SC, Bode HB, Müller R, Schulz S Oger C, Galano J-M, Durand T, Moularat S, Tong
(2005b) Biosynthesis of volatiles by the LCH, Boucher C (2018) Cyclooxygenases and
Myxobacterium Myxococcus xanthus. Chembiochem lipoxygenases are used by the fungus Podospora
5:778–787 anserina to repel nematodes. Biochim Biophys Acta
Dietert RR, Silbergeld EK (2015) Biomarkers for the 21st (BBA)-Gen Subj 1862:2174–2182
century: listening to the microbiome. Toxicol Sci Fiedler N, Laumbach R, Kelly-McNeil K, Lioy P, Fan
144(2):208–216 Z-H, Zhang J, Ottenweller J, Ohman-Strickland P,
Dixon EF, Hall RA (2015) Noisy neighbourhoods: quo- Kipen H (2005) Health effects of a mixture of indoor
rum sensing in fungal-polymicrobial infections. Cell air volatile organics, their ozone oxidation products,
Microbiol 17:1431–1441 and stress. Environ Health Perspect 113:1542–1548
Do DH, Walgraeve C, Amare AN, Barai KR, Parao AE, Fischer G, Schwalbe R, Möller M, Ostrowski R, Dott W
Demeestere K, Van Langenhove H (2015) Airborne (1999) Species-specific production of microbial vola-
volatile organic compounds in urban and industrial tile organic compounds (MVOC) by airborne fungi
locations in four developing countries. Atmos Environ from a compost facility. Chemosphere 39:779–810
119:330–338 Flavier AB, Ganova-Raeva LM, Schell MA, Denny TP
Doty SL (2013) Endophytic yeasts: biology and (1997) Hierarchial autoinduction in Ralstonia
applications. In: Aroca R (ed) Symbiotic endophytes. solanacearum: control of actyl- homoserine lactone
Soil biology. Springer, Berlin, pp 335–343 production by a novel autoregulatory system
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 105

responsive to 30- hydroxypalmitic acid methyl ester. J transcriptional landscape of Solanum tuberosum in a
Bacteriol 179:7089–7097 passively ventilated growth system. Front
Fraatz MA, Zorn H (2010) Fungal flavours. In: Hofrichter Microbiol 1766
M (ed) The Mycota X: industial applications, vol Hermann A (2010) The chemistry and biology of volatiles.
X. industrial applications, 2nd edn. Springer, Berlin, Wiley, Hoboken, NJ, pp 1–428
Heidelberg, pp 249–264 Herrero-Garcia E, Garzia A, Cordobés S, Espeso EA,
Fuqua WC, Winans SC, Greenberg EP (1994) Quorum Ugalde U (2011) 8-Carbon oxylipins inhibit germina-
sensing in bacteria: the LuxR-LuxI family of cell tion and growth, and stimulate aerial conidiation in
density-responsive transcriptional regulators. J Bact Aspergillus nidulans. Fungal Biol 115:393–400
176:269–275 Hogan DA (2006) Talking to themselves: autoregulation
Gallois A, Langlois D (1990) New results in the volatile and quorum sensing in fungi. Eukaryot Cell 5:613–619
odorous compounds of French cheeses. Lait 70:89– Holighaus G, Rohlfs M (2016) Fungal allelochemicals in
106 insect pest management. Appl Microbiol Biotechnol
Gilbert A (2008) What the nose knows. The science of 100(13):5681–5689
scent in everyday life. Crown Pubs, New York, NY Hooper AM, Pickett JA (2004) Semiochemistry. In:
Glick BR (2020) Beneficial plant-bacterial interactions. Atwood JL, Steed JW (eds) Encyclopedia of supramo-
Springer lecular chemistry, vol 2. Marcel Dekker, New York, pp
Griffin MA, Spakowicz DJ, Gianoulis TA, Strobel SA 1270–1277
(2010) Volatile organic compound production by Hornby JM, Jensen EC, Lisec AD, Tasto JJ, Jahnke B,
organisms in the genus Ascocoryne and a Shoemaker R, Dussault P, Nickerson KW (2001) Quo-
re-evaluation of myco-diesel production by NRRL rum sensing in the dimorphic fungus Candida albicans
50072. Microbiology 156:3814–3829 is mediated by farnesol. Appl Environ Microbiol 67(7):
Griffith RT, Jayachandran K, Shetty KG, Whitstine W, 2982–2992
Furton KG (2007) Differentiation of toxic molds via Horswill A, Stoodley P, Stewart P, Parsek M (2007) The
headspace SPME-GC/MS and canine detection. effect of the chemical, biological, and physical envi-
Sensors 7:1496–1508 ronment on quorum sensing in structured microbial
Guo Y, Jud W, Weikl F, Ghirardo A, Junker RR, Polle A, communities. Anal Bioanal Chem 387:371–380
Benz JP, Pritsch K, Schnitzler J-P, Rosenkranz M Hossain MM, Sultana F, Miyazawa M, Hyakumachi M
(2021) Volatile organic compound patterns predict (2014) The plant growth promoting fungi Penicillium
fungal trophic mode and lifestyle. Commun Biol 4:673 spp. GP15-1 enhances growth and confers protection
Gutiérrez-Luna FM, López-Bucio J, Altamirano- against damping-off and anthracnose in the cucumber.
Hernández J, Valencia-Cantero E, Cruz HR, Macías- J Oleo Sci 63(4):391–400
Rodríguez L (2010) Plant growth-promoting Howe GA, Jander G (2008) Plant immunity to insect
rhizobacteria modulate root-system architecture in herbivores. Annu Rev Plant Biol 59:41–66
Arabidopsis thaliana through volatile organic com- Hung R, Lee S, Bennett JW (2013) Arabidopsis thaliana
pound emission. Symbiosis 51:75–83 as a model system for testing the effect of Trichoderma
Hall IR, Brown G, Byars J (1994) The black truffle: its volatile organic compounds. Fungal Ecol 6:19–26
history, uses and cultivation. New Zealand Institute for Hung R, Lee S, Bennett JW (2014) The effects of low
Crop & Food Research Limited, Christchurch concentrations of the enantiomers of mushroom alco-
Hall IR, Brown GT, Zambonelli A (2007) Taming the hol (1-octen-3-ol) on Arabidopsis thaliana. Mycology
truffle. the history, lore, and science of the ultimate 5:73–80
mushroom. Timber Press, Portland, OR Hung R, Lee S, Bennett JW (2015) Fungal volatile organic
Harrison MJ (2005) Signaling in the arbuscular mycorrhi- compounds and their role in ecosystems. Appl
zal symbiosis. Annu Rev Microbiol 59:19–42 Microbiol Biotechnol 99:3395–3405
Hayashi H (2015) Frontier studies on highly selective Hutchinson SA (1973) Biological activities of volatile
bio-regulators useful for environmentally benign agri- fungal metabolites. Annu Rev Phytopathol 11:223–
cultural production. Biosci Biotechnol Biochem 79: 246
877–887 Inamdar AA, Bennett JW (2015) Volatile organic
He P, Hassan MM, Tang F, Jiang H, Chen M, Liu R, Chen compounds from fungi isolated after Hurricane Katrina
Q (2022) Total fungi counts and metabolic dynamics induce developmental defects and apoptosis in a Dro-
of volatile organic compounds in paddy contaminated sophila melanogaster model. Environ Toxicol 30(5):
by Aspergillus niger during storage employing gas 614–620
chromatography-ion mobility spectrometry. Food Inamdar AA, Masurekar P, Bennett JW (2010) Neurotox-
Anal Methods 15(6):1638–1651 icity of fungal volatile organic compounds in Drosoph-
Heddergott C, Calvo AM, Latgé JP (2014) The volatome ila melanogaster. Toxicol Sci 117:418–426
of Aspergillus fumigatus. Eukaryot Cell 13:1014–1025 Inamdar AA, Moore JC, Cohen RI, Bennett JW (2011) A
Heenan-Daly D, Coughlan S, Dillane E, Doyle Prestwich model to evaluate the cytotoxicity of the fungal volatile
B (2021) Volatile compounds from Bacillus, Serratia, organic compound 1-octen-3-ol in human embryonic
and Pseudomonas promote growth and alter the stem cells. Mycopathologia 173:13–20
106 S. Lee et al.

Inamdar AA, Morath S, Bennett JW (2020) Fungal volatile freshwater swamp in Florida, U.S.A. Med Vet Entomol
organic compounds: More than just a funky smell? 21:323–331
Annu Rev Microbiol 74:101–116 Kolter R, Greenberg EP (2006) Microbial sciences: the
IOM - Institute of Medicine (2004) Damp indoor spaces superficial life of microbes. Nature 441:300–302
and health. National Academies Press, Korpi A, Jarnberg J, Pasanen A-L (2009) Microbial vola-
Washington, DC tile organic compounds. Crit Rev Toxicol 39:139–193
Jarvis BB, Miller JD (2005) Mycotoxins as harmful indoor Kües U, Navarro-González M (2009) Communication of
air contaminants. Appl Microbiol Biotechnol 66:367– fungi on individual, species, kingdom, and above king-
372 dom levels. In: Anke T, Weber D (eds) Physiology and
Jeandroz S, Murat C, Wang Y, Bonfante P, Tacon FL genetics. The Mycota XV. Springer, pp 79–106
(2008) Molecular phylogeny and historical biogeogra- Kuske M, Romain A-C, Nicolas J (2005) Microbial vola-
phy of the genus Tuber, the ‘true truffles’. J Biogeogr tile organic compounds as indicators of fungi. Can an
35(5):815–829 electronic nose detect fungi in indoor environments?
Jeleń HH (2003) Use of solid phase microextraction Build Environ 40:824–831
(SPME) for profiling fungal volatile metabolites. Lett La Camera S, Gouzerh G, Sandrine D, Laurent H,
Appl Microbiol 36:263–267 Bernard F, Michel F, Thierry H (2004) Metabolic
Joblin Y, Moularat S, Anton R, Bousta F, Orial G, reprogramming in plant innate immunity: the
Robine E, Picon O, Bourouina T (2010) Detection of contributions of phenylpropanoid and oxylipin
moulds by volatile organic compounds; application to pathways. Immunol Rev 198:267–284
heritage conservation. Int Biodeterior Biodegrad 64: Lam K, Tsang M, Labrie A, Gries R, Gries G (2010)
210–217 Semiochemical-mediated oviposition avoidance by
Kai M, Haustein M, Molina F, Petri A, Scholz B, Piechulla female house flies, Musca domestica, on animal feces
B (2009) Bacterial volatiles and their action potential. colonized with harmful fungi. J Chem Ecol 36:141–
Appl Microbiol Biotechnol 81:1001–1012 147
Kaminski E, Libbey LM, Stawicki S, Wasowicz E (1972) Larsen TO (1998) Volatiles in fungal taxonomy. In:
Identification of the predominant volatile compounds Frisvad JC, Bridge PD, Arora DK (eds) Handbook of
produced by Aspergillus flavus. Appl Microbiol 24: applied mycology, Chemical fungal taxonomy, vol
721–726 6. Marcel Dekker, New York, NY, pp 263–287
Kaplan HB, Greenberg EP (1985) Diffusion of Larsen TO, Frisvad JC (1995) Comparison of different
autoinducer is involved in regulation of the Vibrio methods for collection of volatile chemical markers
fischeri luminescence system. J Bacteriol 163:1210– from fungi. J Microbiol Methods 24:135–144
1214 Lax AR, Templeton GE, Meyer WL (1985) Isolation,
Karahadian C, Josephson DB, Lindsay RC (1985) Contri- purification, and biological activity of a self -inhibotor
bution of Penicillium sp. to the flavour of Brie and from conidia of Colletotrichum gloeosporioides. Phy-
Camembert cheese. J Dairy Sci 68:1865–1877 topathology 75:386–390
Karlovsky P (ed) (2008) Secondary metabolites in soil Lee SO, Kim HY, Choi GJ, Lee HB, Jang KS, Choi YH,
ecology. Springer, Berlin Kim JC (2009) Mycofumigation with Oxyporus
Karlshøj K, Nielsen PV, Larsen TO (2007) Fungal latemarginatus EF069 for control of postharvest
volatiles biomarkers of good and bad food quality. In: apple decay and Rhizoctonia root rot on moth orchid.
Dijksterhuis J, Samson RA (eds) Food mycology. CRC J Appl Microbiol 106:1213–1219
Press, Boca Raton, FL, pp 279–302 Lee S, Hung R, Yap M, Bennett JW (2015) Age matters:
Karlson P, Luscher M (1959) ‘Pheromones’: a new term the effects of volatile organic compounds emitted by
for a class of biologically active substances. Nature Trichoderma atroviride on plant growth. Arch
183:55–56 Microbiol 197:723–727
Keller NP, Turner G, Bennett JW (2005) Fungal secondary Lee S, Hung R, Yin G, Klich MA, Grimm C, Bennett JW
metabolism - from biochemistry to genomics. Nat Rev (2016a) Arabidopsis thaliana as bioindicator of fungal
Microbiol 3:937–947 VOCs in indoor air. Mycobiology 44:162–170
Kesselmeier J, Staudt M (1999) Biogenic volatile organic Lee S, Yap M, Behringer G, Hung R, Bennett JW (2016b)
compounds (VOC): An overview on emission, physi- Volatile organic compounds emitted by Trichoderma
ology and ecology. J Atmos Chem 33:23–88 species mediate plant growth. Fungal Biol Biotechnol
Kimball BA (2016) Volatile metabolome: problems and 3:1–14
prospects. Bioanalysis 8(19):1987–1991 Lee S, Yin G, Bennett JW (2017) Airborne signals:
Kishimoto K, Matsui K, Ozawa R, Takabayashi J (2007) volatile-mediated communication between plants,
Volatile 1-octen-3-ol induces a defensive response in fungi, and microorganisms. In: Dighton J, White JF
Arabidopsis thaliana. J Gen Plant Pathol 73:35–37 (eds) The fungal community. Its organization and role
Kline D, Allan SA, Bernier UR, Welch CH (2007) Evalu- in the ecosystem, 4th edn. CRC Press, Taylor &
ation of the enantiomers of 1-octen-3-ol and 1-octyn-3- Francis, pp 521–538
ol as attractants for mosquitoes associated with a Lee S, Behringer G, Hung R, Bennett J (2019) Effects of
fungal volatile organic compounds on Arabidopsis
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 107

thaliana growth and gene expression. Fungal Ecol 37: Macedo GE, Vieira PDB, Rodrigues NR, Gomes KK,
1–9 Rodrigues JF, Franco JL, Posser T (2022) Effect of
Lee NY, Choi DH, Kim MG, Jeong MJ, Kwon HJ, Kim fungal indoor air pollutant 1-octen-3-ol on levels of
DH, Kim YG, di Luccio E, Arioka M, Yoon HJ, Kim reactive oxygen species and nitric oxide as well as
JG (2020) Biosynthesis of (R)-(-)-1-octen-3-ol in dehydrogenases activities in Drosophila melanogaster
recombinant Saccharomyces cerevisiae with males. J Toxic Environ Health A 85(14):573–585
lipoxygenase-1 and hydroperoxide lyase genes from Mackie AE, Wheatley RE (1999) Effects and incidence of
Tricholoma matsutake. J Microbiol Biotechnol 30: volatile organic compound interactions between soil
296–305 bacterial and fungal isolates. Soil Biol Biochem 31:
Lee CH, Lee YY, Chang YC, Pon WL, Lee SP, Wali N, 375–385
Nakazawa T, Honda Y, Shie JJ, Hsueh YP (2023) A Massalha H, Korenblum E, Tholl D, Aharoni A (2017)
carnivorous mushroom paralyzes and kills nematodes Small molecules below-ground: the role of specialized
via a volatile ketone. Science. Advances 9(3):eade4809 metabolites in the rhizosphere. Plant J 90:788–807
Leeder AC, Palma-Guerrero J, Glass NL (2011) The social Materić D, Bruhn D, Turner C, Morgan G, Mason N,
network: deciphering fungal language. Nat Rev Gauci V (2015) Methods in plant foliar volatile organic
Microbiol 9:440–451 compounds research. Appl Plant Sci 3(12):1500044
Lemfack MC, Gohlke BO, SMT T, Preissner S, Mattheis JP, Roberts RG (1992) Identification of geosmin
Piechulla B, Preissner R (2018) mVOC 2.0: a database as a volatile metabolite of Penicillium expansum. Appl
of microbial volatiles. Nucleic Acids Res 46(D1): Environ Microbiol 58:3170–3172
D1261–D1265 Matysik S, Herbarth O, Mueller A (2008) Determination
Li N, Kang S (2018) Do volatile compounds produced by of volatile metabolites originating from mould growth
Fusarium oxysporum and Verticillium dahliae affect on wall paper and synthetic media. J Microbiol
stress tolerance in plants? Mycology 9:166–175 Methods 75:182–187
Li DW, Yang CS (2004) Fungal contamination as a major Matysik S, Herbarth O, Mueller A (2009) Determination
contributor to sick building syndrome. Adv Appl of microbial volatile organic compounds (MVOCs) by
Microbiol 55:31–112 passive sampling onto charcoal sorbents. Chemosphere
Li N, Alfiky A, Vaughan M, Kang S (2016) Stop and smell 76:114–119
the fungi: fungal volatile metabolites are overlooked Mau J-L, Beelman RB, Ziegler GR (1992) Effect of
signals involved in fungal interaction with plants. Fun- 10-oxo-trans-8-decenoic acid on growth of Agaricus
gal Biol Rev 30:134–144 bisporus. Phytochemistry 31:4059–4064
Li N, Wang W, Bitas V, Subbarao K, Liu X, Kang S Mau J-L, Chyau C-C, Li J-Y, Tseng Y-H (1997) Flavor
(2018) Volatile compounds emitted by diverse compounds in straw mushrooms Volvariella volvacea
Verticillium species enhance plant growth by harvested at different stages of maturity. J Agric Food
manipulating auxin signaling. Mol Plant-Microbe Chem 45:4726–4729
Interact 31:1021–1031 Mauriello G, Marino R, D'Auria M, Cerone G, Rana GL
Li Z, Paul R, Tis TB, Saville AC, Hansel JC, Yu T, (2004) Determination of volatile organic compounds
Ristaino JB, Qingshan W (2019) Non-invasive plant from truffles via SPME-GC-MS. J Chromatogr Sci 42:
disease diagnostics enabled by smartphone-based fin- 299–305
gerprinting of leaf volatiles. Nat Plants 5:856–866 McFee DR, Zavon P (1988) Solvents. In: Plog BA
Ling L, Tu Y, Ma W, Feng S, Yang C, Zhao Y, Wang N, (ed) Fundamentals of industrial hygiene, 3rd edn.
Li Z, Lu L, Zhang J (2020) A potentially important National Safety Council, Chicago, pp 95–121
resource: endophytic yeasts. World J Microbiol McGann JP (2017) Poor human olfaction is a 19th-century
Biotechnol 36:1–7 myth. Science 356(6338):eaam7263
Liu W, Mu W, Zhu B, Liu F (2008) Antifungal activities McNeal KS, Herbert BE (2009) Volatile organic
and components of VOCs produced by Bacillus metabolites as indicators of soil microbial activity and
subtilis G8. Curr Res Bacteriol 1:28–34 community composition shifts. Soil Sci Soc Am J 73:
Luard E (2006) Truffles. Frances Lincoln Limited, Childs 579–588
Hill, London Meilgaard MC (1975a) Flavor chemistry of beer. I. Flavor
Lugtenberg B, Kamilova F (2009) Plant-growth-promot- interaction between principal volatiles. Technical
ing rhizobacteria. Annu Rev Microbiol 63:541–556 Quarterly, Master Brewers Association of America
Luntz AJ (2003) Arthropod semiochemicals: mosquitoes, 12:107–117
midges and sealice. Biochem Soc Trans 31(Pt 1): Meilgaard MC (1975b) Flavor chemistry of beer. II. Flavor
128–133 and threshold of 239 aroma volatiles. Technical Quar-
Luo QJ, Zhu ZG, Zhu ZJ, Yang R, Qian J, Chen HM, Yan terly, Master Brewers Association of America 12:151–
XJ (2014) Different responses to heat shock stress 168
revealed heteromorphic adaptation strategy of Pyropia Mercier J, Jiménez JI (2004) Control of fungal decay of
haitanensis (Bangiales, Rhodophyta). PLoS One 9(4): apples and peaches by the biofumigant fungus
e94354 Muscodor albus. Postharvest Biol Technol 31:1–8
108 S. Lee et al.

Mercier J, Manker D (2005) Biocontrol of soil-borne aroma is explained by quantitative differences in key
diseases and plant growth enhancement in greenhouse volatile compounds. New Phytol 230(4):1623–1638
soilless mix by the volatile-producing fungus Nilssen AC (1998) Effect of 1-octen-3-ol in field trapping
Muscodor albus. Crop Prot 24:355–362 Aedes spp. (Dipt., Culicidae) and Hybomitra spp.
Micheluz A, Manente S, Rovea M, Slanzi D, Varese GC, (Dipt., Tabanidae) in subartic Norway. J Appl Entomol
Ravagnan G, Formenton G (2016) Detection of vola- 122:465–468
tile metabolites of moulds isolated from a Nilsson A, Kihlstrom E, Lagesson V, Wessen B,
contaminated library. J Microbiol Methods 128:34–41 Szponar B, Larsson L, Tagesson C (2004)
Minerdi D, Bossi S, Gullino ML, Garibaldi A (2009) Microorganisms and volatile organic compounds in
Volatile organic compounds: a potential direct long- airborne dust from damp residences. Indoor Air 14:
distance mechanism for antagonistic action of Fusar- 74–82
ium oxysporum strain MSA 35. Environ Microbiol 11: Niu Q, Huang X, Zhang L, Xu J, Yang D, Wei K, Niu X,
844–854 An Z, Bennett JW, Zou C, Yang J, Zhang KQ (2010) A
Mitra M, Venkatesh P, Ghissing U, Biswas A, Mitra A, Trojan horse mechanism of bacterial pathogenesis
Mandal M, Mishra HN, Maiti MK (2023) Fruity- against nematodes. Proc Natl Acad Sci U S A 107:
scented antifungal volatiles from endophytic 16631–16636
Geotrichum candidum PF005: broad-spectrum bioac- Noble R, Dobrovin-Pennington A, Hobbs PJ, Pederby J,
tivity against stored grain pathogens, mode of action Rodger A (2009) Volatile C8 compounds and
and suitable formulation for mycofumigation. Biol pseudomonads influence primordium formation of
Control 177:105129 Agaricus bisporus. Mycologia 101:583–591
Moisan K, Raaijmakers JM, Dicke M, Lucas-Barbosa D, Nordlund DA, Lewis WJ (1976) Terminology of chemical
Cordovez V (2021) Volatiles from soil-borne fungi releasing stimuli in intraspecific and interspecific
affect directional growth of roots. Plant Cell Environ interactions. J Chem Ecol 2:211–220
44:339–345 Noverr MC, Erb-Downward JR (2003) Production of
Mølhave L (2009) Volatile organic compounds and the eicosanoids and other oxylipins by pathogenic eukary-
sick building syndrome. In: Lippmann M otic microbes. Clin Microbiol Rev 16:517–533
(ed) Environmental toxicants: human exposures and Okull DO, Beelman RB, Gourama H (2003) Antifungal
their health effects, 3rd edn. Wiley-Interscience, activity of 10-oxo-trans-8-decenoic acid and 1-octen-
New York, pp 241–256 3-ol against Penicillium expansum in potato dextrose
Morath SU, Hung R, Bennett JW (2012) Fungal volatile agar medium. J Food Prot 66:1503–1505
organic compounds: a review with emphasis on their Oldroyd GED, Downie JA (2004) Calcium, kinases and
biotechnological potential. Fungal Biol Rev 26(2–3): nodulation signalling in legumes. Nat Rev Mol Cell
73–83 Biol 5:566–576
Morey P, Wortham A, Weber A, Horner E, Black M, Ômura H, Kuwahara Y, Tanabe T (2002) 1-Octen-3-ol
Muller W (1997) Microbial VOCs in moisture dam- together with geosmin: new secretion compounds
aged buildings. Healthy Build 1:245–250 from a polydesmid millipede, Niponia nodulosa. J
Mosandl A, Heusinger G, Gessner M (1986) Analytical Chem Ecol 28:2601–2612
and sensory differentiation of 1-octen-3-ol Ortiz-Castro R, Contreras-Cornejo H, Macias-Rodriguez-
enantiomers. J Agric Food Chem 34:119–122 L, Lopez-Bucio J (2009) The role of microbial signals
Mustafa AM, Angeloni S, Nzekoue FK, Abouelenein D, in plant growth and development. Plant Signal Behav
Sagratini G, Caprioli G, Torregiani E (2020) An over- 4:701–712
view on truffle aroma and main volatile compounds. Pangga IB (2021) Mycofumigation with the endophytic
Molecules 25(24):5948 fungi Fusarium proliferatum (Matsushima) Nirenberg
Nealson KH, Hastings JW (1979) Bacterial biolumines- and Diaporthe sp. for the control of banana and mango
cence: its control and ecological significance. anthracnose. Annal Trop Res 43(2):13–24
Microbiol Rev 43:496–518 Pantoja LDM, do Nascimento RF, de Araújo Nunes AB
Nealson KH, Platt T, Hastings JW (1970) Cellular control (2016) Investigation of fungal volatile organic
of the synthesis and activity of the bacterial lumines- compounds in hospital air. Atmos Pollut Res 7(4):
cent system. J Bacteriol 104:313–322 659–663
Nemcovic M, Jakubikova L, Viden I, Farkas V (2008) Pasanen P, Korpi A, Kalliokosi P, Pasanen AL (1997)
Induction of conidiation by endogenous volatile Growth and volatile metabolite production of Aspergil-
compounds in Trichoderma spp. FEMS Microbiol lus versicolor in house dust. Environ Int 23:425–432
Lett 284:231–236 Pavlou AD, Turner AP (2000) Sniffing out the truth:
Nicholson JK, Holmes E, Wilson ID (2005) Gut clinical diagnosis using the electronic nose. Clin
microorganisms, mammalian metabolism and Chem Lab Med 38:99–112
personalized health care. Nat Rev Microbiol 3:431– Peixoto L, Salazar LTH, Laska M (2018) Olfactory sensi-
438 tivity for mold-associated odorants in CD-1 mice and
Niimi J, Deveau A, Splivallo R (2021) Geographical- spider monkeys. J Comp Physiol A 204:821–833
based variations in white truffle Tuber magnatum
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 109

Pelaez F (2005) Biological activities of fungal growth in Arabidopsis. Proc Natl Acad Sci U S A 100:
metabolites. In: An Z (ed) Handbook of industrial 4927–4932
mycology. Marcel Dekker, pp 49–92 Ryu C-M, Farag MA, Paré PW, Kloepper JW (2005)
Pennerman KK, Yin G, Bennett JW (2022) Eight-carbon Invisible signals from the underground: Bacterial
volatiles: prominent fungal and plant interaction volatiles elicit plant growth promotion and induce sys-
compounds. J Exp Bot 73(2):487–497 temic resistance. Plant Pathol J 21:7–12
Piechulla B, Lemfack MC, Kai M (2017) Effects of dis- Sánchez-López ÁM, Baslam M, De Diego N, Muñoz FJ,
crete bioactive microbial volatiles on plants and fungi. Bahaji A, Almagro G, Ricarte-Bermejo A, García-
Plant Cell Environ 40(10):2042–2067 Gómez P, Li J, Humplík JF, Novák O (2016) Volatile
Pierce AM, Pierce HD, Borden JH, Oehlschlager AC compounds emitted by diverse phytopathogenic
(1991a) Fungal volatiles: semiochemicals for stored- microorganisms promote plant growth and flowering
product beetles (Coleoptera: Cucujidae). J Chem Ecol through cytokinin action. Plant Cell Environ 39(12):
3:567–579 2592–2608
Pierce AM, Pierce HD, Oehlschlager AC, Borden JH Schmidberger PC, Schieberle P (2017) Characterization of
(1991b) 1-octen-3-ol, attractive semiochemical for for- the key aroma compounds in white Alba truffle (Tuber
eign grain beetle, Ahasverus adevna (Waltl) (Coleop- magnatum pico) and Burgundy truffle (Tuber
tera: Cucujidae). J Chem Ecol 3:567–579 uncinatum) by means of the sensomics approach. J
Poland TM, Pureswaran DS, Ciaramitaro TM, Borden JH Agric Food Chem 65(42):9287–9296
(2009) 1-octen-3-ol is repellent to Ips pini (Coleoptera: Schnürer J, Olsson J, Börjesson T (1999) Fungal volatiles
Curculionidae: Scolytinae) in the midwestern United as indicators of food and feeds spoilage. Fungal Genet
States. Can Entomol 141:158–160 Biol 27:209–217
Ramoni R, Vincent F, Grolli S, Conti V, Malosse C, Schöller CEG, Gürtler H, Petersen R, Molin S, Wilkins K
Boyer F, Nagnan-Le Meillour P, Spinelli S, (2002) Volatile metabolites from Actinomycetes. J
Cambillau C, Tegoni M (2001) The insect attractant Agric Food Chem 50:2615–2621
1-octen-3-ol Is the natural ligand of bovine odorant- Schreier P (1992) Bioflavours: AN overview. In: Patterson
binding protein. J Biol Chem 276:7150–7155 RLS, Charlwood BV, MacLeod G, Williams AA (eds)
Ramsbottom J (1953) Mushrooms & toadstools: a study of Bioinformation of flavours. The Royal Society of
the activities of fungi. London, Collins, St. James’s Chemistry, Cambridge, pp 1–20
Place Schulz S, Dickschat JS (2007) Bacterial volatiles: the
Rapior S, Breheret S, Talou T, Pelissier Y, Bessiere JM smell of small organisms. Nat Prod Rep 24:814–842
(2002) The anise-like odor of Clitocybe odora, Schulz-Bohm K, Lara M-S, Paolina G (2017) Microbial
Lentinellus cochleatus, and Agaricus essettie. volatiles: Small molecules with an important role in
Mycologia 94:373–376 intra- and inter-kingdom interactions. Front Microbiol
Raza W, Wei Z, Jousset A, Shen Q, Friman VP (2021) 8:2484
Extended plant metarhizobiome: understanding vola- Scott J, Sueiro-Olivares M, Ahmed W, Heddergott C,
tile organic compound signaling in plant-microbe Zhao C, Thomas R, Bromley M, Latgé J-P,
metapopulation networks. Msystems 6(4):e00849– Krappmann S, Fowler S, Bignell E, Amich J (2019)
e00821 Pseudomonas aeruginosa-derived volatile sulfur
Robinson J (ed) (2006) The Oxford companion to wine, compounds promote distal Aspergillus fumigatus
3rd edn. Oxford University Press, Oxford growth and a synergistic pathogen-pathogen interac-
Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) tion that increases pathogenicity in co-infection. Front
Fungal endophytes: diversity and functional roles. Microbiol 10:2311
New Phytol 182:314–330 Shafiq SA, Chechan RA (2019) Influence of different
Rokas A, Mead ME, Steenwyk JL, Raja HA, Oberlies NH natural media on production of myco-diesel. IOP
(2020) Biosynthetic gene clusters and the evolution of Conf Ser Earth Environ Sci 388:012054
fungal chemodiversity. Nat Prod Rep 37:868–878 Shapiro JA (1998) Thinking about bacterial populations as
Rude MA, Schirmer A (2009) New microbial fuels: a multicellular organisms. Annu Rev Microbiol 52:81–
biotech perspective. Curr Opin Microbiol 12:274–281 104
Ruiz-Jimenez J, Zanca N, Lan H, Jussila M, Hartonen K, Shimkets LJ (1999) Intercellular signaling during fruiting-
Riekkola ML (2019) Aerial drone as a carrier for body development of Myxococcus xanthus. Annu Rev
miniaturized air sampling systems. J Chromatogr A Microbiol 53:525–549
1597:202–208 Shoresh M, Harman GE, Mastouri F (2010) Induced sys-
Rumbaugh KP, Griswold JA, Hamood AN (2000) The temic resistance and plant responses to fungal biocon-
role of quorum sensing in the in vivo virulence of trol agents. Annu Rev Phytopathol 48:21–43
Pseudomonas aeruginosa. Microbes Infect 2:1721– Singh SK, Strobel GA, Knighton B, Geary B, Sears J, Ezra
1731 D (2011) An endophytic Phomopsis sp. possessing
Ryu C-M, Farag MA, Hu C-H, Reddy MS, Wei H-X, Paré bioactivity and fuel potential with its volatile organic
PW, Kloepper JW (2003) Bacterial volatiles promote compounds. Microb Ecol 61:729–739
110 S. Lee et al.

Singh J, Singh P, Vaishnav A, Ray S, Rajput RS, Singh active volatile compounds having fuel potential. FEMS
SM, Singh HB (2021) Belowground fungal volatiles Microbiol Lett 320:87–94
perception in okra (Abelmoschus esculentus) facilitates Ström G, West J, Wessen B, Palmgren U (1994) Health
plant growth under biotic stress. Microbiol Res 246: implications of fungi in indoor environments: quanti-
126721 tative analysis of microbial volatiles in damp Swedish
Sneeden EY, Harris HH, Pickering J, Prince RC, houses. Air Qual Monogr 2:291–305
Johnson S, Li X, Block E, George GH (2004) The Sunesson AL, Vaes WHJ, Nilsson CA, Blomquist G,
sulfur chemistry of shitake mushroom. J Am Chem Andersson B, Carlson R (1995) Identification of vola-
Soc 126:458–459 tile metabolites from five fungal species cultivated on
Soria AC, García-Sarrió MJ, Sanz ML (2015) Volatile two media. Appl Environ Microbiol 61:2911–2918
sampling by headspace techniques. TrAC Trends Sunesson AL, Nilsson CA, Andersson B, Blomquist G
Anal Chem 71:85–99 (1996) Volatile metabolites produced by two fungal
Splivallo R, Culleré L (2016) The smell of truffles: from species cultivated on building materials. Ann Occup
aroma biosynthesis to product quality. True truffle Hyg 40(4):397–410
(tuber spp.) in the world: Soil ecology, systematics Takahashi N (1986) Chemistry of plant hormones. CRC
and biochemistry, pp 393–407 Press, Boca Raton, FL
Splivallo R, Ebeler SE (2015) Sulfur volatiles of microbial Tan RX, Zou WV (2001) Endophytes: a rich source of
origin are key contributors to human-sensed truffle functional metabolites. Nat Prod Rep 18:448–459
aroma. Appl Microbiol Biotechnol 99:2583–2592 Tarkka MT, Piechulla B (2007) Aromatic weapons:
Splivallo R, Bossi S, Maffei M, Bonfante P (2007a) Dis- truffles attack plants by the production of volatiles.
crimination of truffle fruiting body versus mycelial New Phytol 175:381–383
aromas by stir bar sorptive extraction. Phytochemistry Tholl D, Hossain O, Weinhold A, Röse USR, Wei Q
68:2584–2598 (2021) Trends and applications in plant volatile sam-
Splivallo R, Novero M, Bertea CM, Bossi S, Bonfante P pling and analysis. Plant J 106:314–325
(2007b) Truffle volatiles inhibit growth and induce an Thomson NR, Crow MA, McGowan SJ, Cox A, Salmond
oxidative burst in Arabidopsis thaliana. New Phytol GPC (2000) Biosynthesis of carbapenem antibiotic and
175:417–424 prodigiosin pigment in Serratia is under quorum sens-
Splivallo R, Deveau A, Valdez N, Kirchhoff N, Frey- ing control. Mol Microbiol 36:539–556
Klett P, Karlovsky P (2015) Bacteria associated with Thorn J (2001) The inflammatory response in humans after
truffle-fruiting bodies contribute to truffle aroma. Envi- inhalation of bacterial endotoxin: a review. Inflamm
ron Microbiol 17(8):2647–2660 Res 50:254–261
Stefanini I (2018) Yeast-insect associations: it takes guts. Tian X, Ding H, Ke W, Wang L (2021) Quorum sensing in
Yeast 35:315–330 fungal species. Annu Rev Microbiol 75:449–469
Stinson M, Ezra D, Hess WM, Sears J, Strobel G (2003) Tirillini B, Verdelli G, Paolocci F, Ciccioli P, Frattoni M
An endophytic Gliocladium sp. of Eucryphia (2000) The volatile organic compounds from the
cordifolia producing selective volatile antimicrobial mycelium of Tuber borchii Vitt. Phytochemistry 55:
compounds. Plant Sci 165:913–922 983–985
Stoppacher N, Kluger B, Zeilinger S, Krska R, Trinci APJ, Whittaker C (1968) Self-inhibition of spore
Schuhmacher R (2010) Identification and profiling of germination in Aspergillus nidulans. Trans Brit Mycol
volatile metabolites of the biocontrol fungus Soc 51:594–596
Trichoderma atroviride by HS-SPME-GC-MS. J Tsitsigiannis DI, Keller NP (2007) Oxylipins as develop-
Microbiol Methods 81:187–193 mental and host-fungal communication signals. Trends
Straus DC (2009) Molds, mycotoxins, and sick building Microbiol 15:109–118
syndrome. Toxicol Ind Health 25:617–635 Tsurushima T, Ueno T, Fukami H, Irie H, Inoue M (1995)
Straus DC, Cooley JD, Wong WC, Jumper CA (2003) Germination self-inhibitors from Colletotrichum
Studies on the role of fungi in sick building syndrome. gloeosporioides f.sp jussiaea. Mol Plant-Microbe
Arch Environ Health 58:475–478 Interact 8:652–657
Strobel GA, Dirkse E, Sears J, Markworth C (2001) Vola- Turner WB (1971) Fungal metabolites. Academic, London
tile antimicrobials from Muscodor albus a novel endo- Turner WB, Aldridge DC (1983) Fungal metabolites
phytic fungus. Microbiology 147:2943–2950 II. Academic, London
Strobel GA, Knighton B, Kluck K, Ren Y, Livinghouse T, Vajzovic A, Bura R, Kohlmeier K, Doty SL (2012) Novel
Griffin M, Spakowicz D, Sears J (2008) The produc- endophytic yeast Rhodotorula mucilaginosa strain
tion of myco-diesel hydrocarbons and their derivatives PTD3 II: production of xylitol and ethanol in the pres-
by the endophytic fungus Gliocladium roseum (NRRL ence of inhibitors. J Ind Microbiol Biotechnol 39(10):
50072). Microbiology 154:3319–3328 1453–1463
Strobel G, Singh SK, Riyaz-Ul-Hassan S, Mitchell AM, Van Delden C, Iglewski BH (1998) Cell-to-cell signaling
Geary B, Sears J (2011) An endophytic/pathogenic and Pseudomonas aeruginosa infections. Emerg Infect
Phoma sp. from creosote bush producing biologically Dis 4(4):551–560
4 An Overview of Fungal Volatile Organic Compounds (VOCs) 111

Vespermann A, Kai M, Piechulla B (2007) Rhizobacterial Wongsuk T, Pumeesat P, Luplertlop N (2016) Fungal
volatiles affect the growth of fungi and Arabidopsis quorum sensing molecules: role in fungal morphogen-
thaliana. Appl Environ Microbiol 73(17):5639–5641 esis and pathogenecity. J Basic Microbiol 56:440–447
Vita F, Taiti C, Pompeiano A, Bazihizina N, Lucarotti V, Wood WF, Fesler M (1986) Mushroom odors: Student
Mancuso S, Alpi A (2015) Volatile organic synthesis of the odoriferous compounds of the matsu-
compounds in truffle (Tuber magnatum Pico): compar- take mushroom. J Chem Educ 63:92
ison of samples from different regions of Italy and from Wurzenberger M, Grosch W (1984) The formation of
different seasons. Sci Rep 5:1–15 1-octen-3-ol from the 10-hydroperoxide isomer of
Vivaldo G, Masi E, Taiti C, Caldarelli G, Mancuso S linoleic acid by a hydroperoxide lyase in mushrooms
(2017) The network of plants volatile organic (Psalliota bispora). Biochim Biophys Acta - Lipids
compounds. Sci Rep 7(1):11050 Lipid Metab 794:25–30
Vlot AC, Rosenkranz M (2022) Volatile compounds-the Xie X, Zhang H, Pare P (2009) Sustained growth promo-
language of all kingdoms? J Exp Bot 73(2):445–448 tion in Arabidopsis with long-term exposure to the
von Bodman SB, Bauer WD, Coplin DL (2003) Quorum beneficial soil bacterium Bacillus subtilis (GB03).
sensing in plant-pathogenic bacteria. Annu Rev Plant Signal Behav 4:948–953
Phytopathol 41:455–482 Yuan JS, Himanen SJ, Holopainen JK, Chen F, Stewart
Wadood SA, Guo B, Zhang X, Raza A, Wei Y (2020) CN (2009) Smelling global climate change: mitigation
Geographical discrimination of Chinese winter wheat of function for plant volatile organic compounds.
using volatile compound analysis by HS-SPME/GC- Trends Ecol Evol 24(6):323–331
MS coupled with multivariate statistical analysis. J Zeilinger S, Gruber S, Bansal R, Mukherjee PK (2016)
Mass Spectrom 55:e4453 Secondary metabolism in Trichoderma–chemistry
Walinder R, Ernstgard L, Johanson G, Venge P, meets genomics. Fungal Biol Rev 30(2):74–90
Wieslander G (2005) Acute effects of a fungal volatile Zhang Z, Li G (2010) A review of advances and new
compound. Environ Health Perspect 113:1775–1778 developments in the analysis of biological volatile
Waters CM, Bassler BL (2005) Quorum sensing: cell-to- organic compounds. Microchem J 95:127–139
cell communication in bacteria. Annu Rev Cell Dev Zhang QH, Schlyter F (2004) Olfactory recognition and
Biol 21:319–346 behavioural avoidance of angiosperm nonhost volatiles
Watson SB, Brownlee B, Satchwill T, Hargesheimer EE by conifer-inhabiting bark beetles. Agric For Entomol
(2000) Quantitative analysis of trace levels of geosmin 6:1–19
and MIB in source and drinking water using headspace Zhang Y-Q, Wilkinson H, Keller NP, Tsitsigiannis D
SPME. Water Res 34:2818–2828 (2005) Secondary metabolite gene clusters. In: An Z
Weeks EN, Birkett MA, Cameron MM, Pickett JA, Logan (ed) Handbook of industrial microbiology. Marcel
JG (2011) Semiochemicals of the common bed bug, Dekker, pp 355–386
Cimex lectularius L. (Hemiptera: Cimicidae), and their Zhang H, Kim M-S, Krishnamachari V, Payton P, Sun Y,
potential for use in monitoring and control. Pest Manag Grimson M, Farag M, Ryu C-M, Allen R, Melo I, Paré
Sci 67:10–20 P (2007) Rhizobacterial volatile emissions regulate
Weisskopf L, Schulz S, Garbeva P (2021) Microbial vola- auxin homeostasis and cell expansion in Arabidopsis.
tile organic compounds in intra-kingdom and inter- Planta 226:839–851
kingdom interactions. Nat Rev Microbiol 19:391–404 Zhang H, Xie X, Kim MS, Kornyeyev DA, Holaday S,
Wheatley R, Hackett C, Bruce A, Kundzewicz A (1997) Pare PW (2008) Soil bacteria augment Arabidopsis
Effect of substrate composition on production of vola- photosynthesis by decreasing glucose sensing and
tile organic compounds from Trichoderma spp. inhibi- abscisic acid levels in planta. Plant J 56:264–273
tory to wood decay fungi. Int Biodeterior Biodegrad Zhang CL, Wang GP, Mao LJ, Komon-Zelazowska M,
39(2–3):199–205 Yuan ZL, Lin FC, Druzhinina IS, Kubicek CP (2010)
Whipps JM (2001) Microbial interactions and biocontrol Muscodor fengyangensis sp. nov. from southeast
in the rhizosphere. J Exp Bot 52(suppl 1):487–511 China: morphology, physiology and production of vol-
Whitehead NA, Barnard ANL, Slater H, Simpson NJL, atile compounds. Fungal Biol 114:797–808
Salmond GPC (2001) Quorum-sensing in Gram- Zhao G, Yin G, Inamdar AA, Luo J, Zhang N, Yang I,
negative bacteria. FEMS Microbiol Rev 25:365–404 Buckley B, Bennett JW (2017) Volatile organic
Whittaker RH, Feeny PP (1971) Allelochemics: chemical compounds emitted by filamentous fungi isolated
interactions between species. Science 171:757–770 from flooded homes after Hurricane Sandy show tox-
WHO (2009) Guidelines for indoor air quality: Dampness icity in a Drosophila bioassay. Indoor Air 27:518–528
and mold. Druckpartner Moser, Rheinbach Zogorski JS, Carter JM, Ivahnenko T, Lapham WW,
Wilkins K, Larsen K, Simkus M (2000) Volatile Moran MJ, Rowe BL, Squillace PJ, Toccalino PL
metabolites from mold growth on building materials (2006) The quality of our nation’s waters – volatile
and synthetic media. Chemosphere 41:437–446 organic compounds in the nation’s ground water and
Wilson AD, Baietto M (2009) Applications and advances drinking-water supply wells. In: U.S. Geological Sur-
in electronic-nose technologies. Sensors 9:5099–5148 vey Circular 1292. USGS, Reston, VA
Wilson AD, Baietto M (2011) Advances in electronic-nose
technologies developed for biomedical applications.
Sensors 11:1105–1176
 Viruses that Affect Phenotype
and Fitness of Fungi 5
Bradley I. Hillman and Massimo Turina

Abstract Keywords
Mycoviruses are the viruses that infect fungi Mycovirus · Fitness · Hypovirulence · dsRNA ·
and the unrelated stramenopiles such as Biocontrol · Vegetative incompatibility · RNA
oomycetes that have morphologies and silencing
lifestyles similar to fungi. Our knowledge of
mycoviruses and their effects on the biology of
their hosts has exploded in recent years. 5.1 Introduction
Although the field only started to mature in
the 1980s and 1990s, advances in technology By the time of the discovery and initial character-
coupled with the promise of mycoviruses for ization of fungal viruses, research with viruses of
biological control of fungi pathogenic to vertebrates, invertebrates, plants, and bacteria
plants, animals, and humans led to increased were already well-developed sciences. Why the
attention and dedicated research. Mycoviruses delay? For one thing, not many capable
are now classified within at least 16 virus researchers were looking for them. Most fungal
families, and that number grows continually research was done by plant pathologists who were
as new viruses that do not fit within the current interested in fungi as the most important cause of
taxonomy are described. This chapter disease in plants; they were accustomed to seeing
highlights some of the most important fungal sectoring and phenotypic differences in many of
virus systems, and some of the advances in our their fungal cultures, but few followed up on
understanding of mycoviruses, especially in these aberrant phenotypes, especially given the
the context of their effects on host biology limited toolset available at the time. Furthermore,
and fitness. their plant virologist colleagues had their hands
full characterizing the many different viruses that
were themselves plant pathogens. The timeline
given below provides an overview of important
events in the progress of early mycovirus
research:
B. I. Hillman (✉)
Plant Biology, Foran Hall, Rutgers University, New • 1940s Viruses that infect filamentous fungi
Brunswick, NJ, USA
e-mail: [email protected]
were first identified in cultivated mushrooms
as the agent of “La France disease” (Hollings
M. Turina
IPSP-CNR, Torino, Italy
1962).
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 113
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_5
114 B. I. Hillman and M. Turina

• 1950s, 1960s Discovery of Penicillium fungus. Thus, when investigators asked

stoloniferum cultures at Lilly Laboratories questions from a virological perspective, they
lead to the characterization of double-stranded began to get answers. The authors of this
RNA as an inducer of interferon in mammals review studied with two of the most important
(Kleinschmidt 1988). figures in the development of C. parasitica as
• 1970s As the discovery of fungal viruses a system for investigation of pathogenic fungal
became an emerging science in the 1970s, the viruses: Drs. Neal Van Alfen and
dogma that most were asymptomatic in their Donald Nuss.
fungal host began to take hold (Ghabrial and
Suzuki 2009) and suppress interest in the sci- Over the last few years, our knowledge of
ence of fungal virology. fungal virology and virus systems has exploded.
• 1970s, 1980s Fungal virology elevated as a This expansion has largely been the result of two
science by technology. Virus discovery, par- connected factors: first, technology, particularly
ticularly viruses with positive-sense (+) RNA sequencing technology, has allowed for rapid and
or double-stranded (ds) RNA genomes became abundant characterization of many new viruses in
much easier when rapid methods to purify and cultured and uncultured fungi; and second, the
analyze dsRNA were developed (Morris and number of research groups that now have fungal
Dodds 1979; Jackson et al. 1971). While this virus research as at least one major focus area of
likely led to continued lack of awareness of the their research programs has grown immensely.
importance of negative-sense (-) RNA and Most fungi that have been studied in the context
perhaps single-stranded (ss) DNA viruses in of their viral partners have plant associations,
fungi, these methods have allowed for substan- often but not always pathogenic to the plant
tial screening of many fungal cultures and host. With the exception of a few examples, rela-
culture collections. tively little is known of virus impacts on free-
• 1980s, 1990s Another particularly important living soil fungi, endophytic fungi, and epiphytic
milestone in the development of fungal virol- fungi, for example, beyond metagenomic
ogy as a science was the recognition and char- searches (Roossinck 2014); (reviewed by Sutela
acterization of the role of viruses as potential et al. 2019), and these represent fertile ground for
biological control agents of chestnut blight further research.
fungus, Cryphonectria parasitica. For some This review will highlight some of the experi-
time, C. parasitica viruses served as the mentally tractable fungal systems in which
model system for fungal virus research viruses are well characterized, and their pheno-
(Hillman and Suzuki 2004). The fungus had typic effects on their hosts have been documented
many features that rendered it an attractive and investigated in some depth. Our use of the
research model: it caused a disease of historic term “fitness” in the title of the chapter is some-
and ongoing international significance and what loose: only in rare cases have the negative or
could be found in temperate forests world- positive effects of virus infection on a fungal host
wide; the fungus was easy to isolate and been unambiguously linked to fitness outcomes in
grow stably in culture; it produced conidia a natural or experimental field setting. Virus tax-
abundantly in culture and ascospores could onomy and nomenclature are undergoing rapid
also be produced in culture; the fungus could change, and the reader is directed to the excellent
be transformed easily using molecular website for the International Committee for the
methods developed for other filamentous Taxonomy of Viruses for most current taxonomy
fungi. Most importantly, viruses of and nomenclature information about individual
C. parasitica had a vivid and demonstrable viruses: https://ictv.global/.
effect on the colony morphology of the fungus
and on its virulence to the tree host of the
5 Viruses that Affect Phenotype and Fitness of Fungi 115

5.2 Diversity of Viruses Infecting (Li et al. 2020). With the same traditional
Fungi and Oomycetes approach and a well carried out molecular and
biological characterization, also members of the
Discovery of mycoviruses for many years relied hadakavirus (multisegmented, up to 11-naked ss
on particle observation at the electron micro- RNA genomes) (Sato et al. 2020) and
scopic level or the use of dsRNA extraction polymycovirus (infectious as dsRNA)
(a hallmark of most RNA virus infections) as a (Kanhayuwa et al. 2015) were described.
tool to find and characterize mycoviruses, thus After these traditional approaches, in the sec-
skewing the representation of the mycovirosphere ond half of the second decade of the twenty-first
toward dsRNA viruses (i.e., members of the century, both the use of small RNAs (sRNA)
families Reoviridae, Partitiviridae, Totiviridae, (generally enriched in virus-derived sRNA) and
Chrysoviridae) and positive (+)strand RNA ribosomal RNA-depleted total RNA for HTS
viruses that greatly accumulate their dsRNA rep- associated with specific bioinformatic pipelines
licative form (members of the families brought the discovery of a number of new
Hypoviridae, Narnaviridae, and mycovirus clades, greatly widening the
Endornaviridae). Up to the end of the twentieth mycovirosphere to new uncharted territories. In
century, there were no DNA or negative (-) this respect, after initial steps that still used small
strand ssRNA viruses associated with fungi, and RNA (Vainio et al. 2015; Donaire and Ayllon
only a few plus-strand RNA viruses in the 2017) or total RNA (Deakin et al. 2017) from
Flexiviridae family whose ssRNA genomes individual fungal isolates, others looked at
were encapsidated in a coat protein-based particle metatranscriptomic samples, greatly increasing
(Ghabrial and Suzuki 2009). Since the first the diversity of the viruses described (Marzano
decade of the twentieth century, high throughput and Domier 2016; Marzano et al. 2016). In par-
sequencing (HTS) approaches began to be ticular, application of ORFan detection pipelines
applied to the untargeted discovery of (ORFan-encoding RNA segments without
mycoviruses while other targeted approaches corresponding DNA) led to the discovery of
with characterization methodologies other than viruses with heretofore unseen genome
dsRNA extraction also achieved some interesting organizations: the splipalmiviruses (with the
milestones in describing mycovirus biodiversity. palm domain of the RNA-dependent RNA poly-
Evidence of minus-strand RNA mycoviruses merase, RdRP, split between two coding genomic
was first gathered from genomic analysis where segments); the ambiviruses (ssRNA viruses with
endogenous non-retrovirus elements included bicistronic, ambisense genomes) (Sutela et al.
fragments of RdRP that matched RdRP from 2020), and the first Mononegavirales with
negative-stranded RNA viruses (Kondo et al. ambisense orientation (now classified in the
2013a). A few years later, the first bona fide genus Penicillimonavirus in the family
minus-strand RNA virus was characterized and Mymonaviridae) (Chiapello et al. 2020). Of inter-
remains at the moment the best characterized est also is the first member of the Bunyavirales
negative-stranded mycovirus (Liu et al. 2014). with a tricistronic RNA that encodes both the
Another milestone was the discovery of the first RdRP and the nucleocapsid (Nc) protein on the
DNA mycovirus: it belongs to the CRESS same RNA segment (now in the family
ssDNA viruses, and at the time of discovery the Tulasviridae) (Sutela et al. 2020). Finally, another
closest relatives were plant geminiviruses group of RNA viruses, ormycoviruses, encodes
(Yu et al. 2010). Recently different examples of putative RdRPs that are so distantly related to
ssDNA mycoviruses with multipartite currently characterized RNA virus RdRPs that
(multisegmented; i.e., the complete viral genome they cannot be reliably positioned
divided among multiple unique segments of phylogenetically, and they display the unusual
nucleic acid) genomes were also characterized feature of diverse catalytic triads in the C
116 B. I. Hillman and M. Turina

subdomain of the palm domain (Forgia et al. determining specific effects of a particular virus
2022). on the host fungus using rigorous methodology.
Another group of mycoviruses that was greatly Consider the example of a fungus that is infected
expanded by HTS is that of members of the order in its natural setting. When brought into a labora-
Bunyavirales, where for the first time a nucleo- tory setting, formally disentangling the virus–fun-
capsid encoding segment was associated to an gus interaction involves isolating the infected
RdRP (Nerva et al. 2019) in the genus fungus in pure culture, freeing the fungus from
Orthodiscovirus in the family Discoviridae. its viral parasite, comparing isogenic virus-
Other recent findings about fungal viruses and containing and virus-free colonies, purifying the
replicating RNA elements have demonstrated virus, and putting it back into the virus-free fun-
that they contain catalytic RNA elements gal isolate to recapitulate effects observed in the
(ribozymes) in the same way that plants and initial comparisons. Any of these steps can be
animals do: plant viroids replicate and can be technically challenging, and the real goal of deter-
maintained in fungi (Wei et al. 2019), unique mining the effects of the virus on the fungal host
viroid-like elements (mycoviroids) are found in in its natural setting still may not be achieved.
fungi (Dong et al. 2023), and ambiviruses that With some of the most important fungal systems,
replicate through a rolling circle replication such as fungal endophytes, mycorrhizae, or
cycle that relies on ribozymes, has brought the biotrophic pathogens, these steps may constitute
world of ribozymes to the Mycota. Still at this monumental challenges, and avenues that provide
time, no bona fide dsDNA viruses have been useful, albeit imperfect information are often nec-
characterized in the fungi. essary. For example, if a fungus cannot be
cultured, comparing a number of different
biological samples and determining virus pres-
5.3 Establishing a Link Between ence by total nucleic acid sequencing for compar-
Mycovirus Infection and Fungal ison may be useful. Some of the methods
Phenotype: Clearing (Curing) commonly used to develop virus-free fungal
Infections and Infectivity isolates are summarized in Fig. 5.1.
Systems

One of the most difficult tasks in mycovirus 5.3.1 Clearing/Curing Infections

research is the association of a specific phenotype
with the presence or absence of a specific 5.3.1.1 Single Asexual Spore Isolation
mycovirus. In principle, such an association can Colonies derived from single asexual spores, for
be established when two isogenic strains differ example single conidia in ascomycetes or single
only in the presence or absence of a specific virus. zoospores in oomycetes, have the advantage in a
In common lab practice, currently two approaches research context because they represent isogenic
can be taken: obtaining a cured strain from an representatives of their parent cultures. Therefore,
infected isolate or infecting a recipient strain if virus (+) and virus (-) single conidium isolates
with the mycovirus under study. In both cases, it can be generated from an infected culture, direct
is important that the initial infected or virus-free comparison of virus effects on fungal biology
isolate contain a clonal population of nuclei. may be possible.
Clonality can be obtained through single conidia Sometimes, a virus-infected fungus isolated
in most ascomycetes, since a single conidium from its natural setting cannot be cleared of its
generally has a single nucleus. Sterile mycelia virus infection in order to complete Koch’s
(often the case in some fungi in culture) would postulates. The task is easier, however, if a virus
make this a more difficult task. has a profound and easily observable effect on the
One of the most important and often difficult fungal colony morphology. For example, the dra-
components of fungal virus research is matic white color observed in Cryphonectria
5 Viruses that Affect Phenotype and Fitness of Fungi 117

Fig. 5.1 Methods used to clear virus infection from fila- case, virus-free isogenic isolates are identified by colony
mentous fungi, with C. parasitica as examples. Top panel: morphology and confirmed by molecular methods. Bot-
CHV1-infected isolate may be cleared of virus by (a) tom panel: CHV1-infected isolate crossed with virus-free
liberating and plating conidia on agar; (b) isolating, isolate. In the resulting cross, single ascospore progeny are
regenerating, and plating protoplasts; or (c) excising and all virus free, but are genetically unique, not isogenic
plating hyphal tips, possibly with antibiotic (e.g., cyclo-
heximide) or antiviral (e.g., ribavirin) amendment. In each

hypovirus 1 (CHV1)-infected C. parasitica is (Oh 1995; Oh and Hillman 1995). Often, single
immediately discernable from the orange color of conidial isolation fails to yield any virus-free
its uninfected isogenic counterparts when a conidial colonies (Oh and Hillman 1995; Polashock and
suspension derived from CHV1-infected strain Hillman 1994; Polashock et al. 1997).
EP713 is spread on culture medium and colonies
emerge and grow from single conidia (Figs. 5.1 and 5.3.1.2 Single Sexual Spore Isolation
5.2). If there is no such profound effect of virus on Many fungal viruses are not passed through the
fungal phenotype, investigators are usually reliant sexual cycle, and this therefore represents a route
upon nucleic acid detection methods to discern through which to clear a virus from its infected
whether single spore-derived colonies contain host. Thus, a virus that is recalcitrant to curing by
virus or not. Assuming the viral sequence is other means may be eliminated by performing
known, any of the various polymerase chain reac- crosses and single spore (meiospore) isolations
tion (PCR)-based methods for virus identification, (ascospores, basidiospores, or oospores). Unfor-
including rapid methods, may be deployed tunately, resulting virus-free strains have
(Urayama et al. 2015) or small colony blot methods undergone recombination and are not isogenic
may be used to screen hundreds of colonies at once with the virus-infected parent. Such methods
118 B. I. Hillman and M. Turina

Fig. 5.2 Methods used to initiate virus infection in fila- cDNA construct from which infectious viral RNA is tran-
mentous fungi. Transfection of protoplasts can be initiated scribed in vivo (e) results in an infected fungal isolate that
with (a) purified virus particles; (b) purified viral nucleic also can transmit virus by anastomosis and can transmit
acid; (c) nucleic acid derived from traditional cDNA the viral gene itself to progeny in sexual crosses. Finally,
clones; or (d) chemically synthesized nucleic acid based fusion of protoplasts from virus-infected and virus-free
on sequence data alone. Stable nuclear transformation of strains (f) circumvents barriers of vegetative
protoplasts and regeneration of fungal colonies with a incompatibility

serve as a step toward fulfilling Koch’s 2009 for discussion). Like many areas of
Postulates, but other steps are required to obtain mycovirus research, this is one in need of
isogenic virus (+) and virus (-) isolates. further work.
When the infected individual serves as male in Deng and colleagues (2007a) demonstrated
crosses with an uninfected female partner, virus that two mycoreoviruses were transmitted at
transmission from the male gametangium to the rates of ~60% through ascospores of
female often does not occur, and when the C. parasitica when the infected partner served
infected partner is the female in matings, a virus as female in crosses; this situation is in contrast
may suppress female fertility to the point that with the known suppression of female fertility in
sexual spores are not produced (Anagnostakis the same host by different hypoviruses
1982). A limited number of studies aimed at (Anagnostakis 1982; Fulbright et al. 1988).
quantifying the transmission rates of specific Deng et al. (2007b) further showed that two
viruses in controlled crosses have been published, genes required for mating in C. parasitica, the
and it appears that some viruses may be transmit- sex pheromone gene Mf2/1 and the yeast Ste12-
ted to sexual spore progeny from either the male like transcription factor cpst12, were severely
or the female partner in matings (see Pearson et al. suppressed by hypovirus infection, but not by
5 Viruses that Affect Phenotype and Fitness of Fungi 119

mycoreovirus infection, revealing a likely mech- effort. Sun and colleagues were able to cure
anism for female fertility in the mycoreovirus- Lentinula edodes, the important edible Shiitake
infected isolates. The importance to virus trans- mushroom, of two viruses through a combination
mission is that a virus-infected female in sexual of mycelial fragmentation and ribavirin treatment
crosses may transmit virus to ascospore progeny, (Sun et al. 2022). When feasible, virus-free
whereas an infected male more often does not. isolates should be obtained from more than one
Despite the fact that they are not isogenic, curing event, since the use of antivirals can result
virus-free fungal isolates derived from single sex- in unwanted off target mutations on the fungal
ual spores have experimental value and may pro- genomes.
vide a useful picture of the biological effects of a
virus on its fungal host if data are sufficient for
such analysis. In the example given above for 5.3.2 Infectivity Systems
C. parasitica mycoreoviruses, virus-infected sin-
gle ascospore progeny of C. parasitica were eas- Hypovirulence in C. parasitica caused by CHV1
ily distinguishable visually from uninfected infection is the classic example used to illustrate
progeny (Deng et al. 2007a), and the infected the CHV1–C. parasitica interaction. The geneti-
and uninfected colony morphologies were consis- cally tractable fungus can be transformed with
tent. Thus, given sufficient numbers in such high efficiently by using a gene knock out method
systems, a subset of virus-containing and virus- and infectious cDNA clones that are available for
free progeny can serve as a substitute, albeit an CHV1. Today a number of infectious clones of
imperfect one, for isogenic isolates. other mycoviruses are available, and technologies
have been developed for many different fungi.
5.3.1.3 Antibiotic Treatment Here we summarize some of the approaches for
Antibiotics have been used to eliminate viruses controlled infectivity of a recipient fungal isolate,
selectively from infected fungal cultures, usually as depicted in Fig. 5.2:
in conjunction with hyphal tip isolation of fungal
cultures. Cycloheximide, produced by Streptomy- 5.3.2.1 Transfer of the Infectious Agent
ces spp., inhibits eukaryotic translation elonga- to a Recipient Strain Through
tion and has been shown to be an effective Anastomosis:
means of curing some virus-containing isolates. Initially, even without knowing the exact nature
After it had been shown to be effective in yeast, of the causal agent, a maternal genetic element
Fulbright used cycloheximide to cure was shown to be transmissible through anastomo-
C. parasitica of certain viruses (Fulbright 1984). sis, the process of hyphal fusion between two
The antiviral ribavirin, used as treatment against a fungal individuals of the same species (Fig. 5.3),
number of human viruses, is a guanosine analog and to be associated with hypovirulence. This
that interferes with nucleic acid synthesis and process allows passage of cytoplasmic elements
especially capping. Ribavirin was effective at to a recipient strain. Experimentally it is impor-
eliminating Phytophthora infestans RNA virus tant to distinguish the recipient strain with a
2 (PiRV2) (Cai et al. 2019a) and PiRV3 (Cai marker (e.g., antibiotic resistance, green fluores-
et al. 2013) from its oomycete host, allowing for cent protein (GFP) expression, auxotrophic
isogenic virus (+) and virus (-) isolates to be mutants) in order to ensure that, indeed, only the
compared. But ribavirin was not effective at recipient strain is retrieved after anastomosis.
eliminating PiRV1 or PiRV4 from the same host Such an approach was pivotal to demonstrate
(Cai et al. 2009, 2012). Notably, elimination of the role of a transmissible cytoplasmic element
PiRV2 and PiRV3 from P. infestans required up in determining hypovirulence for C. parasitica
to 6 months of Ribavirin treatment, hyphal tip- (Van Alfen et al. 1975).
ping, and subculture (Cai and Hillman 2013), a
technically challenging and time-consuming
120 B. I. Hillman and M. Turina

Fig. 5.3 Virus transmission by hyphal anastomosis of need not be isogenic for anastomosis and transmission to
vegetatively compatible strains, or restriction of transmis- occur. Bottom left, vegetative incompatibility results in
sion by vegetative incompatibility. Bottom right, vegeta- failure of transmission and no change in phenotype of
tively compatible strains of C. parasitica allow for virus-free strain. Cartoons showing hyphal interactions
transmission of the virus CHV2. Note that the strains are above photos

5.3.2.2 Purified Particles Transfected same years also filamentous fungi were shown to
in Protoplasts: be transfected by particles of dsRNA viruses
The first successful attempts at establishing (Victorivirus) (Ghabrial and Mernaugh 1983) and
infections of mycovirus from purified particles Gaeumannomyces graminis var. tritici (Stanway
were considered a major breakthrough. Such was and Buck 1984). Recently such an approach was
the case for dsRNA viruses of Penicillium used to extend the host range of Rosellinia necatrix
stoloniferum (Eurotiales) in the early 1970 (Lhoas victorivirus 1 into C. parasitica wild-type (WT)
1971) and particularly in the killer yeast system, and silencing deficient mutant strains, showing
since, previously yeast viruses were thought to be that Victoriviruses are targeted by the antiviral
transmitted only through cytoplasmic mixing (bud- silencing defense (Chiba et al. 2013). The same
ding, mating, cytoduction, or protoplast fusion). In particle transfection approach was used to test a
this breakthrough work, yeast protoplasts of a sen- mix of 6 mycoviruses purified from a single Peni-
sitive strain were infected by both the K1 and K2 cillium aurantiogriseum (Eurotiales) strain and to
killer viruses though polyethylene glycol (PEG) demonstrate that two of them can replicate in plant
inoculation (Elsherbeini and Bostian 1987). In the cell protoplasts (Nerva et al. 2017). When
5 Viruses that Affect Phenotype and Fitness of Fungi 121

employing this approach, it is important to plate the transcribed in vitro from full-length cDNA clones
purified virus preparation to make sure no fungal to transfect plants (Ahlquist et al. 1984) and then
colonies are born from the original infected isolate. yeast cells (Janda and Ahlquist 1993).
Furthermore, it is important that the transfection Nuss and colleagues used both DNA transfor-
mix is well characterized (possibly by next-genera- mation and RNA transcript approaches success-
tion sequencing, NGS) to exclude unforeseen or fully for the CHV1 mycovirus (Choi and Nuss
unwanted contamination from undetected mixed 1992; Chen et al. 1994). Transfection of
infections. Purified particles from the ssDNA protoplasts was accomplished with viral RNA
mycovirus infecting S. sclerotiorum, were also transcribed in vitro with the T7 RNA polymerase,
shown to be infectious, both for fungi and a possi- with the T7 promoter engineered in front of the 5′
ble dipteran insect vector Lycoriella ingenua end of the viral sequence, and a restriction site for
(Yu et al. 2010, 2013). linearization added after a 20 nt poly A tail (Chen
et al. 1994). In the second approach, the viral
5.3.2.3 Transfection with Nucleic Acid genome was placed in a fungal transformation
Use of native nucleic acid from purified or par- plasmid with a selectable hygromycin
tially purified virus can be used to start mycovirus phosphotransferase resistance gene, between the
replication in a new host (Li et al. 2019). A glyceraldehyde-3-phosphate dehydrogenase
particular application of this approach was used (GAPDH) promoter and terminator from
to demonstrate that dsRNA can be infectious in C. parasitica. In this case, the protoplasts were
the case Aspergillus fumigatus tetramycovirus transformed with the plasmid, but only resistant
1, one of the first polymycovirus characterized. (transgenic) isolates were retrieved and shown to
The researchers treated RNA extracted from a harbor CHV1 mycovirus infection. This approach
purified virion-like colloidal preparation with allows the virus to be maintained indefinitely in
proteinase K and the ssRNA-specific S2 nuclease, the genome, possibly improving the biocontrol
but not the dsRNA-specific RNase III, approach because the virus can be spread by
demonstrating for the first time that dsRNA mating, thus overcoming vegetative compatibility
from a purified virus can be directly infectious barriers and will be present in all the conidial
(Kanhayuwa et al. 2015). progeny without segregation having occurred.
For many years, CHV1 was the only mycovirus
5.3.2.4 Transfection with Infectious infecting filamentous fungi that could be studied
Clones using an infectious clone. Then in 2003, infec-
A major breakthrough in establishing the link tious clones were reported for yeast narnavirus
between a host phenotype (mostly disease) and a 20S (Esteban and Fujimura 2003) and Diaporthe
specific virus, was the ability to start an infection ambigua RNA virus (Moleleki et al. 2003). More
from the cloned DNA or cDNA of the virus. Such recently, in vitro synthesis and transfection
clones, generally harbored by plasmids approach were used for an hypovirus (Sclerotinia
maintained and grown in E. coli, are the basis sclerotiorum Hypovirus 2) from S. sclerotiorum
for reverse genetic approaches in virology. The (Marzano et al. 2015); the first infectious clone of
first infectious cDNA clone for an RNA virus was a member of the Botourmiaviridae family was
assembled for a bacteriophage, a prokaryotic also recently established (Wang et al. 2020).
virus in 1978 (Taniguchi et al. 1978) and for Even more recently, the first infectious clone
Poliovirus in 1981 (Racaniello and Baltimore for a CRESS-like ssDNA mycovirus was assem-
1981). These milestones in the study of RNA bled and verified. Tandem repeat of the circular
viruses were soon replicated for plant viruses genomic components were cloned in pBluescript
with an important modification; rather than II for each of the three genomic components of
transforming cells with the full-length cDNA Fusarium graminearum gemytripvirus
clone as in the first two examples, Ahlquist and 1 (FgGMTV1) and transfected in protoplasts of
colleagues pioneered the use of infectious RNA F. graminearum to establish infection; this
122 B. I. Hillman and M. Turina

approach allowed the identification of replication- 5.4 Specific Selected Systems

required virus components and those that are
symptom-associated. Finally, another mycovirus 5.4.1 Cryphonectria Viruses
infectious transcript allowed the dissection of the
very interesting virus symbiotic lifestyle Cryphonectria parasitica is the most thoroughly
discussed later (see Rosellinia viruses, below). studied fungus–virus system. While viruses have
An infectious clone of the Yado-kari positive- been identified in other Cryphonectria species,
strand virus was verified to be dependent on they are for the most part of greatest interest in
Yado-nushi co-infection to complete its life the context of C. parasitica comparisons. While
cycle (Zhang et al. 2016b). we will provide an overview here, we direct
Still more model cDNA mycovirus infectious readers to many useful reviews on different
clones should be assembled. DsRNA viruses, like aspects of the fungus as a pathogen and as virus
mycoreovirus, could in principle be derived, but host, and of the different viruses that have been
reoviruses have proven to be the most challenging studied in that fungus (Hillman and Suzuki 2004;
systems for such engineering. The first such sys- Macdonald and Fulbright 1991; Milgroom and
tem was not developed until 2007 (see Kanai and Cortesi 2004; Dawe and Nuss 2013; Eusebio-
Kobayashi 2021 for review). Monopartite or mul- Cope et al. 2015; Nuss 2011; Rigling and Pros-
tipartite negative-sense and ambisense RNA pero 2018; Van Alfen et al. 1975; Anagnostakis
viruses are more approachable, and many 1982, 1987; Turina and Rostagno 2007).
examples of negative-strand RNA virus infec- C. parasitica is the ascomycete that caused the
tious clones are described in literature (see pandemic that wiped out the American chestnut,
Neumann et al. 2002 for review). Castanea dentata, as a dominant hardwood spe-
cies in eastern North America, and also virtually
5.3.2.5 Synthetic Biology from eliminated related chinquapin species in the
Metatranscriptomics: southeastern U.S. during the early twentieth cen-
Most of the recently described mycoviruses that tury (Anagnostakis 1987). Its independent inva-
greatly increased our view of mycovirus biodiver- sion into Europe, while impactful, did not
sity and evolution come from metatranscriptomic ultimately result in the equivalent dramatic effect
samples, often complex matrixes, where the on European chestnut forests, owing in part to the
biological sample is no longer available. Never- slightly greater resistance of the tree host,
theless, the principles of synthetic biology can C. sativa, and differences in tree management,
easily be applied to viruses. This approach was as well as the early presence of virulence-
used to demonstrate the host of a ssDNA virus suppressing viruses that spread efficiently in the
(Feng et al. 2021). Infectious clones of a invading populations of the fungus (Rigling and
genomovirus were derived from metagenomic Prospero 2018).
samples by amplifying the genome by PCR and Viruses of C. parasitica have been studied
used to transfect S. sclerotiorum. The virions extensively and one in particular, CHV1, has
recovered were also used to support the virus as been the subject of many hundreds of published
a cause of hypovirulence, but additionally of studies ranging from epidemiology, natural and
infection in B. cinerea and Monilinia fructicola, introduced biocontrol, transmission, virus molec-
and Spodoptera insect cells. Given the ease and ular biology, and the many effects on host biol-
cost-effectiveness of synthetic DNA, we envision ogy. Such exhaustive studies have provided
that more viruses will be revived simply by deeper and broader information on that virus
sequence data available in silico, through the than we have on any other fungal virus. Its
synthesis of infectious clones. study, almost to the exclusion of other
C. parasitica viruses, has had the unintended
consequence that many researchers extrapolate
5 Viruses that Affect Phenotype and Fitness of Fungi 123

results from that singular virus/host system to worked with CHV1 at some point in their
other virus/fungus systems. It is the equivalent training.
of studying smallpox and its causal variola virus Although only a few viruses of C. parasitica
to the exclusion of other human viruses. Never- other than CHV1 have been described outside of
theless, its study has provided great insight into North America (Forgia et al. 2021), several have
fungal viruses, and like other model systems, been found in North America, where the viral
many of the studies performed with CHV1 landscape is diverse and interesting [(Turina and
would have been much more challenging if Rostagno 2007; Hillman and Suzuki 2004) for
attempted with other C. parasitica viruses. reviews]. CHV2, the closest known relative of
The dominance of CHV1 across Europe CHV1 (Hillman et al. 1992, 1994), has been
speaks to its fitness as a virus species, despite found in North America on resprouted American
the fact that it has marked detrimental effects on chestnut only in a small area representing a few
the fungus it infects, greatly reducing its virulence hundred square miles in eastern New Jersey
and fecundity as measured by asexual sporulation (Chung et al. 1994; Peever et al. 1997), close to
and sexual reproduction (Anagnostakis 1987; New York City and close to where the invasion of
Rigling and Prospero 2018). Although few com- C. parasitica into North America is thought to
petition experiments have been published, our have occurred. CHV2 was also isolated from
own anecdotal evidence indicates that infected trees on a single occasion in China
co-cultivation of vegetatively compatible (Peever et al. 1998). CHV3 is only distantly
C. parasitica isolates infected with CHV1 and related to CHV1 and CHV2, and also has been
isolates infected with any of several other viruses, isolated only from a single localized region of the
followed by serial passage, usually results in an Great Lakes region of Michigan, USA, and
isolate infected only with CHV1—the other virus nearby in Canada (Fulbright et al. 1983; Smart
does not survive. Furthermore, unlike many other et al. 1999). CHV4 dominates the landscape of
mycoviruses, CHV1 is virtually never lost from North America (Linder-Basso et al. 2005). In
cultures in storage. This may be an indication of interesting contrast to CHV1, CHV4 causes little
how well adapted it is to its fungal host. measurable phenotypic effect in the infected fun-
A few of the many “firsts” for CHV1 are these: gus (Enebak et al. 1994b), and yet it is present in
(1) it was the first virus associated with and found ~1/4 of C. parasitica isolates throughout North
to be the causal agent of hypovirulence in fungi; America (Peever et al. 1997). CHV4 is relatively
indeed, the term hypovirulence was coined based closely related to CHV3 (Linder-Basso et al.
on CHV1 (Grente 1965); (2) it was the first fungal 2005), but it likely represents an independent
virus for which infectious cDNA clones were entry event into the fungus.
made (Choi and Nuss 1992; Chen et al. 1994), Several other viruses or viral elements have
leading to immense advances in our understand- been isolated from C. parasitica, but few of
ing of mycovirus host/pathogen interactions them have been examined in detail. A mitochon-
[reviewed in (Dawe and Nuss 2013; Nuss 2005, drial element that had little effect on host pheno-
2011)]; (3) it was the first virus released into type was identified (Polashock and Hillman
nature as a transgenic construct for the purpose 1994), leading to the eventual recognition of the
of biological control of a plant disease large viral family Mitoviridae. The two reoviruses
(Anagnostakis et al. 1998); (4) it was the first identified in C. parasitica (Hillman et al. 2004)
virus released in the context of a transgenic fun- resulted in the description of a new virus genus,
gus that had been genetically engineered for uni- Mycoreovirus, and important research on viral
versal vegetative compatibility so as to RNA recombination in fungi (Sun and Suzuki
anastomose and transmit virus to a broader 2008; Aulia et al. 2019; Eusebio-Cope et al.
range of isolates in nature (Stauder et al. 2019). 2010). Interestingly, the C. parasitica mitovirus
(5) Furthermore, many mycovirologists world- and the two reoviruses were isolated only a single
wide who are currently working on other systems time and appear not to have spread in chestnut
124 B. I. Hillman and M. Turina

populations. Recently, the first negative-strand Genomoviridae, to accommodate such viruses

RNA virus was identified in C. parasitica in (Krupovic et al. 2016). Although only one other
isolates from Azerbaijan, along with a novel similar virus has been characterized since then,
ambisense RNA virus (Forgia et al. 2021), further the potential importance of this virus family in the
expanding the known viral diversity in the constellation of mycoviruses is evident as you
fungus. will see below and likely will grow. Indeed,
Marzano and Domier (Marzano and Domier
2016) assembled the genome of soybean leaf-
5.4.2 Sclerotinia Viruses associated gemycircularvirus from soybean
field-derived metatranscriptomes. The virus was
Sclerotinia is the type genus of the ascomycete later renamed soybean leaf-associated
family Sclerotiniaceae. Its most economically genomovirus 1 (SlaGemV1), and an infectious
important species, S. sclerotiorum, an exception- cDNA clone was constructed in the laboratory
ally broad host range soilborne plant pathogen of based on the assembled sequence as noted above
great agronomic importance, is often the major (Feng et al. 2021). Similarly, construction and
pathogen of a particular crop (Xia et al. 2020). deployment of an infectious cDNA clone of the
Although viruses of S. sclerotiorum were not tripartite-genome DNA virus FgGMTV1, the first
among the subjects of early mycovirus study, infectious clone assembled for a DNA mycovirus,
they have gained appreciably in importance over allowed for the investigation of viral sequences
the last two decades (for reviews, see (Jiang et al. critical for virus replication and virulence
2013; Ghabrial et al. 2015; Boland 2004)). (Li et al. 2020).
Coinfections with more than one virus are com- SsHADV1 has been a fascinating virus in sev-
mon in S. sclerotiorum (Ran et al. 2016; Hu et al. eral respects. Besides being the first fungal
2014; Mu et al. 2021), and several viruses have ssDNA virus described, the virus was shown to
been shown to cause hypovirulence (Xie et al. have several other unusual characteristics. The
2006; Hu et al. 2005; Yu et al. 2010; Xu et al. virus was shown to be infectious either as purified
2015; Azhar et al. 2019). particles or as purified viral DNA, first to fungal
Some years after the initial description of protoplasts (Yu et al. 2010), and then to whole
virus-associated hypovirulence of mycelia either in culture or on plant leaves
S. sclerotiorum by Boland (Boland 1992), (Yu et al. 2013). The mechanism of virus uptake
research groups at Huazhong University led by into fungal hyphae with their cell walls intact
Daohong Jiang and then Jiatao Xie began remains unknown, but that property opened the
characterizing the virome of S. sclerotiorum possibility of using SsHADV1 as a biofungicide,
with the objective of finding viruses that might albeit with caveats pertaining to efficacy and
be useful in its control. To date, members of at safety (Yu et al. 2013). Recent studies have
least 15 virus families have been identified in shown not only that SsHADV1 is potentially an
S. sclerotiorum (Qu et al. 2021). Among the effective biocontrol agent in field situations, but
many viruses that have been identified and that it in effect converts the necrotrophic fungus
characterized, several are particularly notable. into a beneficial endophyte (Zhang et al. 2020).
The first single-stranded DNA virus identified Another unexpected finding about SsHADV1
in a filamentous fungus was found in is that it can be vectored from an infected fungus
S. sclerotiorum (Yu et al. 2010) and named to an uninfected fungus by a mycophagous insect,
S. sclerotiorum hypovirulence-associated DNA Lycoriella ingenua, and in fact was able to repli-
virus 1 (SsHADV1). Although few such viruses cate in that insect and was transmitted vertically
have been identified since through classical virol- through insect eggs (Liu et al. 2016). Besides the
ogy approaches, the ubiquitous presence of simi- obvious implications for field spread in agricul-
lar DNA sequences in environmental samples led tural settings, these findings have profound
to the formation of a new virus family, the
5 Viruses that Affect Phenotype and Fitness of Fungi 125

implications for our view of mycovirus ecology Thus, while the function of this situationally dis-
and evolution. pensable segment remains unknown, its universal
presence in isolates retrieved from naturally
infected roots suggests that it is important to
5.4.3 Rosellinia Viruses virus fitness in natural settings. In light of the
examples cited elsewhere for mycovirus replica-
The ascomycete Rosellinia necatrix (Xylariales) tion in plants and insects, it is tempting to specu-
causes white root rot disease on a wide range of late that MyRV3 segment 8 may have a role in
plants, especially perennial woody plants. infection of another host or possible vector
Although the fungus itself is not so well-known (Hillman et al. 2004).
as most of the other exemplar fungi highlighted in Characterization of a bipartite genome virus in
this chapter, it has an important place as a R. necatrix with two large RNA segments
mycovirus host [(Kondo et al. 2013b) for review]. encapsidated in rigid icosahedral particles
This is in large part because of excellent and (Chiba et al. 2009) led to the description of the
thorough descriptions of members of several new virus family Megabirnaviridae (Sato et al.
new virus families and clever experimental 2019). Only one other such virus has been
methods envisioned and planned, especially by characterized thoroughly, a closely related virus
Nobuhiro Suzuki at Okayama University and his in S. sclerotiorum (Wang et al. 2015), but other
colleagues. White root rot and similar root possible members of the family have been
diseases incited by R. necatrix are particularly identified through transcriptome databases (e.g.,
difficult to control because the fungus survives Nerva et al. 2015). The two characterized viruses
well in soil and spreads effectively (ten Hoopen cause reduced virulence and phenotypic changes
and Krauss 2006). Because of the limited effec- to their respective hosts in culture (Chiba et al.
tiveness of fungicides, biological control of 2009; Wang et al. 2015). Another novel virus
R. necatrix with naturally occurring viruses was discovered in R. necatrix that led to description
pursued as a control measure by Matsumoto and of a new virus family was a four-segment dsRNA
colleagues (Ikeda et al. 2004) and later by other virus named Rosellinia necatrix quadrivirus
plant pathologists, especially in Japan. These 1 (RnQV1) (Lin et al. 2012), resulting in the
efforts led to a large collection of fungal isolates establishment of the virus family Quadriviridae
that were then mined for viruses of potential value (Chiba et al. 2018). Like the Megabirnaviridae,
as biocontrol agents. targeted and metagenomic approaches have
Several viruses of importance were discovered identified other probable Quadriviridae family
in R. necatrix, and it continues to be a system of members, but to date, none has been shown to
interest to mycovirologists. The first sequence- affect fungal phenotype.
based evidence for a reovirus in a filamentous The virus mentioned above (Transfection with
fungus came from R. necatrix (Osaki et al. nucleic acid) characterized by a novel mutualistic
2002). The virus, later named Mycoreovirus lifestyle between two unrelated viruses, was first
3-RnW370 (MyRV3-W370) had 12 segments of described in R. necatrix by Suzuki and
dsRNA, in contrast to the two reoviruses found in colleagues. In this instance, one virus yado-kari
C. parasitica, which have 11 segments each. virus 1 (YkV1; yado-kari meaning “borrowing a
Interestingly, one of the 12 dsRNA segments of room”), an ssRNA virus, was hypothesized to use
MyRV3, segment 8, is often lost during labora- the capsid of an unrelated dsRNA virus, yado-
tory subculture. Fungal cultures infected with nushi virus 1 (YnV1; yado-nushi meaning “room
virus strains lacking segment 8 show no owner”), and use it as a base for replication and
discernable phenotype differences compared to genome encapsidation (Zhang et al. 2016b)
their 12-segment counterparts, and they are (reviewed in Hisano et al. 2018). The yado-kari/
hypovirulent based on apple fruit assays yado-nushi hypothesis was supported by later
(Kanematsu et al. 2004; Sasaki et al. 2007). experiments showing that, indeed, YkV1 used
126 B. I. Hillman and M. Turina

its own encoded RdRp enzyme to replicate its without major mycotoxin contamination. Outside
RNA, but in YnV1-encoded capsids (Das et al. these four complexes, there are other important
2021). Recently, this unique model of virus mutu- plant pathogenic Fusariums that produce Type A
alism has been expanded with the discovery in trichothecenes; F. langsethiae, F. poae, and
S. sclerotiorum of a virus related to YkV1 F. sporotrichioides, often referred to as the
partnering in the same manner with an unrelated F. sambucinum species complex that cause Fusar-
virus (Jia et al. 2022). Based on the growing ium head blight (Laraba et al. 2021).
constellation of now more than 10 YkV1-related Given the importance of this genus to agricul-
virus sequences in the proposed family ture and human health, it is not surprising that it
Yadokariviridae (Das et al. 2021), it is reasonable has been the focus of a number of mycovirus
to speculate that this virus lifestyle, perhaps studies (reviewed by Li et al. 2019), so we will
unique to fungi and oomycetes, is relatively com- summarize some of the best studied virus–fungus
mon in these organisms. interactions in this genus, and update the litera-
ture with more recent publications. Looking at the
biodiversity of mycoviruses present in Fusarium
5.4.4 Fusarium Viruses in a recent study, one finds reports of the first
ssDNA virus in this genus with the interesting
The genus Fusarium (Hypocreales) includes a feature of being tripartite; it represents a new
large group of fungal species that are among the genus, distinct from previously described
most important for plant pathology. The group mycovirus genera (Li et al. 2020). Some of the
also includes saprophytes living mostly in the other viruses found in Fusarium are type
soil. Their economic damage is not only directly members of new families, as is the case of the
to the crop, but also, these fungi affect food and dsRNA genome members of the family
feed indirectly because they produce mycotoxins Fusagraviridae, from Fusarium graminearum
that cause major adverse effects on human and virus 3 DK3 (Yu et al. 2009); another mycovirus
livestock health. The taxonomy of the genus has characterized from Fusarium, Fusarium
evolved considerably over the time when it was graminearum virus 1 DK21 (Chu et al. 2002), is
based mostly on morphological features until the the type isolate of the newly established family
end of the 1980, then biological (sexual compati- Fusariviridae that includes a large number of
bility), to current phylogenetic systematics viruses with a ssRNA genome. DK21 is also
(Munkvold 2017). Currently more than 300 spe- one of the five viruses so far described that causes
cies can be distinguished through phylogenetic hypovirulence in its host and has potential for
evidence, but our focus is on four main plant biocontrol including a 60-fold decrease in pro-
pathogenic species complexes based on a RNA duction of trichothecene mycotoxins. Among
polymerase II subunit gene phylogeny: (1) the the other viruses causing hypovirulence, we will
Fusarium fujikuroi species complex that includes mention Fusarium graminearum hypovirus
important diseases of maize, rice, sugarcane, 2 (Li et al. 2015) and Fusarium graminearum
mango, and pine trees (caused by F. circinatum) virus (FgV) strain ch9 (Darissa et al. 2012), a
important for toxic fumonisin contamination; member of the family Chrysoviridae. Fusarium
(2) the Fusarium graminearum species complex oxysporum dianthi virus 1, also a member of the
responsible for B trichothecene and zearalenone Chrysoviridae, interferes with virulence in its
mycotoxin contamination causing, among other host (Lemus-Minor et al. 2018). The most recent
plant diseases, fusarium head blight; (3) Fusarium inclusion of a hypovirulence-causing mycovirus
oxysporum species complex, causing vascular in Fusarium is a megabirnavirus, which not only
wilt and root rot, but not involved in major myco- causes hypovirulence, but also strongly
toxin contamination for humans or livestock; diminishes deoxynivalenol (DON) mycotoxin
(4) the Fusarium solani species complex, mostly production (Li et al. 2022; Xie et al. 2022).
causing foot and root rot of many crops but Some of the above mycoviruses became the
5 Viruses that Affect Phenotype and Fitness of Fungi 127

subject of thorough studies of their interactions T7-derived RNA transcripts, could potentially be
with the fungal host, particularly regarding the engineered to include fragments of essential host
role of RNAi and the mechanism of pathogenesis. genes to increase the virus pathogenicity through
a virus-induced gene silencing method (VIGS)
(Cordoba et al. 2022). Also, a potential tool for
5.4.5 Botrytis Viruses Botrytis manipulation is the infectious clone of
the ssDNA virus from Sclerotinia assembled syn-
Botrytis cinerea (Helotiales) is a necrotrophic thetically from metatranscriptomic sequence, as
fungus endemic worldwide, affecting both the detailed below, since it can infect B. cinerea as
vegetative stage of a number of crops and fruits well as Sclerotinia (Feng et al. 2021).
(such a grapes) during post-harvest storage. Often A partitivirus was also associated with
known as gray mold, Botrytis has the capacity to hypovirulence of B. cinerea after the virus was
survive in the environment by production of resis- transferred through anastomosis to three recipient
tant sclerotia and is considered a model system virulent strains (Kamaruzzaman et al. 2019). The
for the study of plant phytopathogenic fungi isolate that originally harbored the partitivirus
(Williamson et al. 2007), particularly the (QT5–19) was pink and had interesting features
necrotrophic ones. Given the importance of this related to its biocontrol potential through compet-
fungus, it is not surprising that alternatives to itive saprophytic ability (CSA). Volatile organic
fungicides are sought among the mycoviruses compounds (VOCs) specifically emitted by this
that infect it. Over the years, a number of pink B. cinerea strain, seemed to be associated
mycoviruses were shown to cause hypovirulence with this CSA, although the causal link with the
in B. cinerea (Hao et al. 2018a, b; Wu et al. 2010; mycovirus presence still needs to be established
Potgieter et al. 2013; Yu et al. 2015) and reviewed (Kamaruzzaman et al. 2020). The same VOCs
in (Wu et al. 2016). Untargeted approaches have also have an effect on plant growth, stimulated
revealed a complex virome associated with this by the interaction of this strain with the plant
fungus in collections from different regions of the (Kamaruzzaman et al. 2021).
world: the widest virome characterization effort
recently reported a number of interesting new
viruses for this fungus. Among these are a tripar- 5.4.6 Trichoderma Viruses
tite member of the negative-strand RNA
Bunyavirales (Botrytis cinerea bocivirus 1), the Trichoderma (Hypocreales) is a genus of fungi
report of the type member of a subclade of viruses that includes a large number of species that are
with palm domains split on two genomic commonly isolated from soil and rhizosphere
segments (Botrytis cinerea binarnavirus 1, 2, where their saprotrophic lifestyle plays a signifi-
and 3) and a ssDNA virus, named Botrytis cinerea cant role in degradation of plant polysaccharides
ssDNA virus 1 (BcssDV1) (Ruiz-Padilla et al. (Druzhinina et al. 2011). They are particularly
2021). interesting for their beneficial interactions with
Other isolates of the same ssDNA virus were plants, including symbiosis (Harman et al.
also found in China (Hao et al. 2021) and 2004). Trichoderma spp. can also directly attack
Australia (Khalifa and MacDiarmid 2021). The other fungi (mycoparasitism), and for this reason
virus isolate from Australia, tentatively they are of interest as biocontrol agents (Sood
designated Botrytis gemydayirivirus et al. 2020). Mycoparasitism also is a cause of
1 (BGDaV1), was demonstrated to be transfected concern for diseases they cause to edible
to new fungal hosts as purified particles, and the mushrooms.
resulting infected isolate B. cinerea was Given their ecological importance and their
hypovirulent (Khalifa and MacDiarmid 2021). biotechnological relevance, it is surprising that
Recently an infectious clone of Botrytis virus the Trichoderma-associated mycovirome was
F was assembled. The clone, infectious through only recently investigated. Initially only dsRNA
128 B. I. Hillman and M. Turina

segment polymorphism was associated with long term on the presence or absence of
Trichoderma spp. collections (Jom-in and mycoviruses (You et al. 2019).
Akarapisan 2009; Yun et al. 2016). Particularly In the case of ThPV1, inhibition of growth in
interesting is the polymorphism described in co-cultured Pleurotus ostreatus and Rhizoctonia
Trichoderma spp. causing green mold disease in solani increased in ThPV1-containing strains
315 cultivated shiitake isolates from compared with ThPV1-cured isogenic strains,
contaminated logs and sawdust (Yun et al. associated with a significantly higher
2016). A number of dsRNA genome mycoviruses β-1,3-glucanase activity but not of chitinases
were characterized, including partitiviruses: (Chun et al. 2018a); TaPV1 isogenic strains, how-
Trichoderma atroviride partitivirus 1 (TaPV1) ever, did not show significant changes in colony
(Chun et al. 2018b), Trichoderma harzianum morphology. In addition, no changes in the enzy-
partitivirus 1 (ThPV1) (Chun et al. 2018a), and matic activities of β-1,3-glucanase and chitinase
Trichoderma harzianum partitivirus 2 (ThPV2) were observed in virus-cured strains (Chun et al.
(Wang et al. 2022); Fusagraviridae: Trichoderma 2018b). Finally, the bipartite-like ThMV1 on the
atroviride mycovirus 1 (TaMV1) (Lee et al. one hand, increased biomass of the fungal host
2017), Trichoderma asperellum dsRNA Virus and its growth-promoting effect on cucumber
1 (TaRV1) (Zhang et al. 2018); and unclassified plants, but at the same time decreased the biocon-
viruses belonging to the “bipartite” clade: trol potential (Liu et al. 2019a).
Trichoderma harzianum bipartite mycovirus
1 (ThBMV1) (Liu et al. 2019b), and Trichoderma
harzianum mycovirus 1 (ThMV1) (Liu et al. 5.4.7 Agaricus and Other Cultivated
2019a) in the Beauveria-like clade as defined in Mushrooms:
(Nerva et al. 2015). Furthermore, Trichoderma
harzianum hypovirus 1 and 2 and two members Around 200 species of fruitbodies of fungi
of the family Hypoviridae were also described (Agaricales) are consumed across the world.
from Trichoderma (You et al. 2019; Chun et al. Interest in edible mushrooms is continuously
2022). growing because of the nutraceutical properties
Some of these viruses were cured from the associated with both wild harvested and
original strains or transferred to new virus-free cultivated mushrooms (Kalac 2013). World pro-
isolates by anastomosis, and therefore the effect duction from 2010 to 2020 increased steadily
of the virus can be inferred. In particular in the from 24.9 to 42.7 million tons (https://www.fao.
case of ThPV2, the partitivirus has no negative org/faostat/en/#data/QCL); the highest produc-
effect on the qualitative biocontrol performance tion is known from China, Japan, USA,
of the Trichoderma host, but instead shows a Netherland, India, Poland, and Spain. Among
moderate but statistically significant improved those species cultivated, Agaricus bisporus (but-
biocontrol activity in experiments with cucumber ton mushroom, white and brown mushroom,
seeds inoculated with Fusarium oxysporum f. sp. including portobello) is by far the most popular,
cucumerinum (Wang et al. 2022). followed by shiitake (Lentinula edodes), hiratake
In another study, ThHV1 was discovered in or oyster mushroom (Pleurotus ostreatus), and
two forms in two different isolates. In one isolate, Flammulina velutipes (golden needle mushroom,
the virus accumulated with an abundant defective enokitake) (Marcal et al. 2021).
form. Transmission of the two versions of the Cultivated mushroom species harbor many
infectious ThHV1 (with and without defective mycoviruses, and their number has increased
RNA) showed that the one with defective RNA through untargeted HTS of their RNA (Zhang
was highly transmissible and was detrimental to et al. 2022). Nevertheless, their biological role
the biocontrol properties of two Trichoderma has been difficult to ascertain. Although the first
species, raising the concern that successful appli- mycoviruses were indeed described as associated
cation of Trichoderma might depend over the with a mushroom disease (Hollings 1962), proper
5 Viruses that Affect Phenotype and Fitness of Fungi 129

formal establishment of disease etiology through also because of bioactive ingredients shown to be
Koch’s postulates was not achieved due to the effective in treating tumor and infections (Liu
complexity of the infection, and the lack of trac- et al. 2015). A number of symptoms (degenera-
table genetic systems for cultivated mushroom tion of mycelia, abnormal browning of the bag
species. Here we will mention examples of the cultures, and incapability of fruiting body forma-
historically important virus-associated diseases of tion) have been associated with mycovirus pres-
cultivated species, highlighting some of the bio- ence (Sun et al. 2022), but in most cases,
logically relevant associations between virus detection of specific mycoviruses in symptomless
presence and disease. fruiting bodies complicates the evaluation of the
The primary Agaricus bisporus diseases impact of the virus found. In particular, mycelial
associated with mycovirus etiology are La France fragmentation of clonal isolates allowed
disease (originally also known as dieback), obtaining mycovirus-free and mycovirus-positive
described in the mid-1950 in Pennsylvania isogenic lines, and is a good alternative to single
(Sinden and Hauser 1950) and two distinct conidial cultures that are not feasible for edible
syndromes described in the 1990, initially mushrooms (Kim et al. 2015). Here it is worth
named mushroom virus X disease, now named pointing out that three similar viruses (LeV-HKB,
brown cap mushroom disease (BCMD) and Patch LeV-HKA, and LeSV) belonging to the recently
disease (mushroom virus X sensu stricto). proposed family Phlegiviridae (characterized as
La France disease is characterized by slow monopartite, bicistronic with a genome of 11 kb
mycelial growth, distorted or dramatically dsRNA, in one case with associated spherical
delayed fruitbody emergence. A number of particles) (Picarelli et al. 2019), are widely pres-
published works have associated the particulate ent in L. edodes. Certain strains have been
isometric virus Agaricus bisporus virus 1 (AbV1) demonstrated to cause disease in mushroom pro-
with the disease, consisting of 6 RNA and duction (Kim et al. 2015; Sun et al. 2022). A
3 associated RNA viruses (VanDerLende et al. partitivirus (LePV1) was also shown to cause
1994, 1996; Goodin et al. 1992). Currently AbV1 disease in L. edodes (Guo et al. 2017), but no
in the National Center for Biotechnology Infor- biological effect was evident from three negative-
mation (NCBI) is an unassigned member of the stranded viruses (a mymonavirid and two in the
genus Chrysovirus, but it is only very distantly Phenuiviridae family) (Lin et al. 2019).
related to current members of the Chrysoviridae Pleurotus ostreatus is also affected by diseases
family and its taxonomic position should be caused by mycoviruses. In particular, a severe
revised. dieback epidemic was associated with oyster
The complexity of the virome associated with mushroom spherical virus (OMSV) (Yu et al.
Mushroom virus X (MVX) was only recently 2003). OMSV is an isometric virus with a
described, first by elucidation of the dsRNA genome of ~5.7 kb of positive-strand RNA that
segments present (Grogan et al. 2003), and later is related to members of the plant-infecting genus
by their molecular description through HTS Tymovirus. The genome contains 7 open reading
(Deakin et al. 2017). Recently, this multiple frames (ORFs), but only RdRP and coat protein
infection was examined in cultures harboring have been assigned a function. Recent work
9 viruses by statistically analyzing their abun- investigated a strain of OMSV from China for
dance in fruitbodies, distinguishing viruses which isogenic virus-infected and virus-free
upregulated in symptomatic fruiting bodies and lines were obtained, and OMSV-Ch infection
downregulated in asymptomatic fruiting bodies. did inhibit growth and caused malformation of
AbV16 and Orphan 8 often seems to be the host (Hu et al. 2022). Some effects on growth
associated with the disease (Dobbs et al. 2021). also were established for Pleurotus ostreatus
Lentinula edodes is the second most cultivated Virus (PoV); (Korean isolate ASI2792). This iso-
edible mushroom. Shiitake mushroom is widely late was studied using virus-free and virus-
cultivated in East Asia, not only as an edible, but infected isogenic strains obtained through
130 B. I. Hillman and M. Turina

mycelial fragmentation, and its effect on vegeta- 2019), the absence of an obvious phenotype
tive growth was demonstrated. PoV is a bipartite associated with most of those elements has
virus with two segments of dsRNA of circa 2.3 resulted in little follow-up research on them.
and 2.2 kB pairs. An association with extracellu- Recently, small RNA sequencing has been
lar enzyme expression and activity was employed for virus discovery in Phytophthora
established using this system, providing a molec- species (Botella and Jung 2021; Raco et al.
ular mechanism for the diminished capacity of 2022; Poimala et al. 2021; Sutela et al. 2019).
vegetative growth on complex growth substrate Mining of genomes and follow-up expression
(Song et al. 2020). analysis showed that giant DNA virus invasion
Flammulina velutipes was linked to a occurred in the past in Phytophthora (Hannat
partitivirus, Flamminula velutipes browning et al. 2021; Sharma et al. 2014).
virus (FvBV), and was primarily associated with Cai and colleagues undertook investigations of
darker fruitbodies (Magae and Sunagawa 2010) possible RNA viruses infecting Phytophthora
even though in this case Koch’s postulates were infestans with the goal of developing a virus-
not established. induced gene silencing (VIGS) system for molec-
ular manipulation of the pathogen (Cai et al.
2009; Cai and Hillman 2013).
5.4.8 Phytophthora Viruses P. infestans was the organism associated with
the plant pandemic that led to the famine in
Phytophthora spp. (Peronosporales) are Ireland in the mid-nineteenth century, and it
oomycetes that are related to some of the algal continues to be a pathogen of concern on potato
groups having the accessory chlorophyll c, bifla- and tomato varieties. Although the goal of devel-
gellate zoospores, and cellulose cell walls but oping a VIGS system for P. infestans was not
usually lacking chitin. Although oomycetes are ultimately achieved, four unrelated RNA viruses
evolutionarily quite distant from fungi, their were described in a series of studies (Cai et al.
structures, morphologies, and lifestyles are simi- 2009, 2012, 2013, 2019a, b; Cai and Hillman
lar to fungi; furthermore, there are marked 2013).
similarities in genome components (Randall Of the four viruses, only one, PiRV2, was
et al. 2005) and in host defense mechanisms shown to have an effect on phenotype of the
including vegetative incompatibility (Govers host (Cai et al. 2019a). Presence of PiRV2 in
et al. 1997) and RNA silencing systems (Vetukuri P. infestans was shown to cause an increase in
et al. 2011). Likely because of these similarities in asexual sporulation as measured by sporangium
host properties, viruses that infect oomycetes production in culture, and an increase in lesion
appear to be generally similar in their properties size induced by infected P. infestans in detached
to the more thoroughly studied viruses of fungi. potato leaves. Comparative transcriptome analy-
Oomycetes including Phytophthora spp. are sis suggested downregulation of ammonium and
generally more challenging to study in laboratory amino acid intake as a possible mechanism
settings than many fungi. Unlike most filamen- through which PiRV2 resulted in increased spor-
tous fungi that have been the subjects of virus ulation. Interestingly, in a sampling of
studies, Phytophthora and other oomycetes con- 54 P. infestans isolates, PiRV2 was found to be
tain diploid vegetative hyphae that are devoid of especially abundant in the US-8 lineage of
cross walls, in contrast to the generally haploid, P. infestans, one of the dominant lineages of
septate mycelia of true fungi. Oomycetes have P. infestans around the turn of the twenty-first
generally lagged behind true fungi in terms of century, consistent with the hypothesis that
molecular manipulations (Judelson et al. 1991). PiRV2 may have a positive effect on fitness of
Although dsRNAs, presumably of viral origin, its host.
have been identified in many oomycetes (see
reviews by Cai and Hillman 2013; Sutela et al.
5 Viruses that Affect Phenotype and Fitness of Fungi 131

5.4.9 RNA Silencing as an Antivirus electrophoresis, must be disentangled to deter-

Defense System mine whether they represent components of a
single virus or multiple viruses. If multiple, how
RNA silencing is an ancient host defense system many viruses are in the fungal thallus? Making
deployed against viruses by fungi, oomycetes, this determination usually involved methods such
plants, invertebrates, and vertebrates. While as those described above to fulfill Koch’s
RNA silencing appears to be less important to Postulates and obtain singly-infected fungal
vertebrates with more sophisticated antibody- cultures.
based adaptive immune systems, it is critically We are really only beginning to learn the
important for organisms without such antibody- importance of mixed viral infections in fungi in
based systems. In fungi, RNA silencing was first the context of virus effects on fungal ecology.
shown formally to function in antiviral defense of Hillman et al. (2018) categorized virus to virus
C. parasitica against CHV1 by Segers and interactions in coinfections as being: (1) synergis-
colleagues (Segers et al. 2007), after the authors tic, in which one virus enhances replication of
had demonstrated that the p29 protein of CHV1 another during coinfection; (2) mutualistic, in
served as a suppressor of RNA silencing (Segers which both viruses derive benefit from each
et al. 2006). In the same fungus, roles of the two other’s presence in natural settings; (3) antagonis-
principal silencing-associated proteins, dicer-like tic, in which viruses impair each other’s replica-
protein 2 (DCL2) and argonaute-like protein tion or maintenance; or (4) genome
2 (AGL2), in defense against viruses other than rearrangement-inducing viruses. Examples of
CHV1 have been examined (Chiba and Suzuki each have been discussed (Hillman et al. 2018).
2015; Chiba et al. 2016). Antiviral RNA silencing A recent example of a synergistic-mutualistic
and its suppression by viruses has since been virus infection of C. parasitica was initially
investigated in other fungi and virus types thought to involve a single mycoreovirus,
(Mochama et al. 2018). The role of antiviral MyRV2 (Enebak et al. 1994a) but was later
RNA silencing in the ecology of yeast viruses as found to involve a natural coinfection by
situationally dispensable host genes was MyRV2 and the normally symptomless
hypothesized following the observations that hypovirus CHV4 (Aulia et al. 2019). In this
agl2 and dcl2 have been lost and gained repeat- case, CHV4 was necessary for infection of
edly in some fungi, and thus the fitness cost of MyRV2: CHV4 alone was stable in
retaining the genes for antivirus defense is bal- C. parasitica, but MyRV2 was lost in a relatively
anced against the fitness cost of losing the genes short time without CHV4 presence. Furthermore,
and allowing for virus infection (Drinnenberg presence of CHV4 enhanced the vertical trans-
et al. 2011; Nicolás et al. 2013). As noted in the mission of MyRV2 through conidia by more than
section below, there is growing evidence for the two-fold. The mechanism for this trans-
importance of suppression of antiviral RNA stabilization of MyRV2 was suppression of the
silencing as a component of mixed infections of C. parasitica host RNA silencing antiviral
viruses in a single fungal thallus. defense response by CHV4, which has an
identified suppressor of RNA silencing, while
MyRV2 apparently does not (Aulia et al. 2019,
5.4.10 Mixed Infections 2021).
Antagonistic interactions are probably com-
Examples of a single fungal culture infected with mon in natural settings, but likely more difficult
more than one virus are abundant in the literature to discover, because one of the virus partners
(Thapa and Roossinck 2019; Hillman et al. 2018). leads to elimination of the other during the coin-
In many instances, multiple RNA species, usually fection. Therefore, these interactions tend to be
detected as multiple dsRNA bands by gel identified in experimental systems in the labora-
tory. For example, we have found it difficult to
132 B. I. Hillman and M. Turina

maintain cultures coinfected with CHV1 and ecological niches, under the constant threat of
CHV2, although the mechanism for possible multiple abiotic and biotic stressors. Furthermore,
antagonism has not been investigated the effect of a mycovirus might not be obvious
(B. Hillman and B. Halpern, unpublished). directly on the fungal host, but even can be dra-
Chiba and Suzuki found that replication of matic when considering multipartite trophic
Rosellinia necatrix victorivirus 1 (RnVV1) was interactions: this effect on multitrophic
suppressed during coinfection with either the interactions has been suggested for the interaction
mycoreovirus MyRV1 or a mutant of the of the mycovirus Curvularia thermal tolerance
hypovirus CHV1 lacking a RNA silencing sup- virus and its endophytic fungal host Curvularia
pressor (Chiba and Suzuki 2015). In this instance, protuberata (Pleosporales). The effect of the
suppression of RnVV1 likely resulted from mycovirus was recognized only after considering
induction of the host RNA silencing-associated the third partner, a plant host, the grass
genes, dcl2 and agl2, by the antagonistic viruses, Dichanthelium lanuginosum. Only the
and the inability of RnVV1 to combat the mycovirus-infected fungal endophyte provided
antiviral response. the thermal tolerance phenotype to the grass
Some fungal viruses are able to replicate in host necessary to resist high temperature soils
plant cells without adaptation to the new host close to geothermal activity in Yellowstone
(Nerva et al. 2017), and it is reasonable to conjec- Park, (Marquez et al. 2007). Interestingly, the
ture that mixed infection of plant and fungal role of the virus was not discovered until 5 years
viruses in single host cells likely occurs in natural after the original report of high temperature resis-
settings. The demonstrated replication of fungal tant plants. Below we will summarize some of the
viruses in mycophagous insects (Liu et al. 2016) best studied phenotypical changes caused by
and insect cell lines (Feng et al. 2021) further mycoviruses, focusing on those where a molecu-
supports the hypothesis that such heterologous lar mechanism of the perturbation has been
mixed infections may be hotspots of virus recom- studied.
bination and evolution.
5.4.11.1 Virulence
As mentioned above, for many years,
5.4.11 Phenotype Changes mycoviruses have been studied as possible
and Mechanisms biological and biotechnological tools for biocon-
trol of fungal and oomycetes-caused plant
As mentioned briefly above, one of the main diseases (to a lesser extent for human-caused
dogmas still difficult to debunk is the fact that fungal diseases). The approaches to develop bio-
most mycovirus infections are symptomless, control tools from mycoviruses are still mostly
often despite the very high accumulation of the based on natural transmission of hypovirulence
virus, which should somehow come at the through anastomosis. Nevertheless, newer
expense of basal metabolism, deviated toward groundbreaking approaches that rely directly on
viral progeny production. It is now clear that the mycocidal effect of the mycovirus (used as a
indeed in some cases, the effect of a mycoviral proxy of a fungicide molecule) or others that
infection is minimal, but in most cases, it is the include a change in lifestyle of the host that
experimental system that is not developed well makes the fungal host protective of further
enough to measure effects: particularly, we infections have been recently reviewed in detail
mycovirus researchers should keep in mind that (Wagemans et al. 2022). Here we will take a
attempts at measuring effects of a mycovirus deeper dive into the best studied system, CHV1-
under “axenic” conditions in laboratory settings, C. parasitica, and the many studies that tried to
can result in missing the effects of virus infections reveal the precise molecular mechanism of
on “interactions.” It is possible that mycovirus hypovirulence, in order to attempt mimicking it
effects could be unveiled only in natural in other interactions.
5 Viruses that Affect Phenotype and Fitness of Fungi 133

Initial work on the molecular aspects of the Regarding regulation of virulence in other
CHV1–C. parasitica interaction looked at up or mycovirus-host models, it is worth pointing out
downregulated accumulation of proteins and that while hypovirulence is indeed a preferable
mRNA, initially with coarse techniques that outcome from an anthropocentric point of view
later developed into more comprehensive omics for plant pathogens or also human pathogens such
approaches. Two different pathways were at the as Aspergillus (Eurotiales), (see detailed reviews,
center of the investigation: the perturbation Kotta-Loizou and Coutts 2017a; Kotta-Loizou
of signal transduction, and in particular of 2021) in some cases, hypervirulence is of interest,
G-protein related alterations, and to a lesser of for example enhancing the biocontrol potentials
extent of MAP kinases and perturbation of secre- of Trichoderma (see above) or entomopathogenic
tion. A detailed review of the main fungi used for biocontrol, such as mycovirus-
accomplishments of those approaches can be infected Beauveria bassiana (Kotta-Loizou and
found in previous works (Nuss 2005; Turina and Coutts 2017b).
Rostagno 2007). As mentioned earlier, these stud- Two other examples also point to the necessity
ies elucidated very important fungal pathways of looking at mycovirus effects on virulence in
and the effect on virulence when perturbing wider field contexts and not only in pathogenicity
some of those pathways. Nevertheless, the link assays in laboratory settings. A mycovirus in
to virus-caused hypovirulence remained elusive, Leptosphaeria maculans (Pleosporales) induces
and still is after further studies that looked more hypervirulence, but at the same times triggers
globally at virus-caused changes. Overall systemic resistance toward L. maculans: since
hypovirulence caused by CHV1 infection is prob- only the coinfection causes Phoma stem canker
ably the result of a multi-faceted complex rela- (blackleg), it could be argued that pre-inoculation
tionship. Even more recent attempts at describing with the hypervirulent mycovirus-infected isolate
the perturbation of the whole secretome revealed of one fungus decreases the overall severity
a massive hypovirus-caused regulation (one-third caused by the L. biglobosa secondary infection
of the secretome) that, among many others, affect (Shah et al. 2020). In another important biological
known infection-related proteins, including a model, Magnaporthe oryzae chrysovirus 1 strain
long list of plant cell wall-degrading enzymes. A (MoCV1-A) caused hypovirulence in its fungal
At the same time, superoxide dismutase (SOD) host when rice plants were inoculated by punch-
is also downregulated; SOD is an enzyme wounding with virus-infected vs. virus-free
involved in the reduction of reactive oxygen spe- conidia. In contrast, when a battery of susceptible
cies produced by plant to counteract fungal infec- or resistance rice varieties was inoculated by
tion; its downregulation might result in spraying seedlings with virus-infected or virus-
hypovirulent fungal hosts (Wang et al. 2016). free conidial suspensions, mycovirus infection
More targeted recent approaches based on spe- resulted in variable outcomes depending on the
cific gene knock-outs have revealed the role of presence of the combination of resistance gene
both the autophagy pathway with a specific role and avirulence determinant, suggesting that
of ATG-8, likely a susceptibility factor in the mycoviruses could drive physiological diversity,
virus–fungus interaction (Shi et al. 2019), and including pathogenic races (Aihara et al. 2018)
the methylation pathway in maintaining (Note that this virus infects the fungus now
hypovirus infection. In fact, knock out of two known as Pyricularia oryzae (see below, Disrup-
DNA methyltransferases (DNAMTase) strongly tion of known metabolic pathways)).
affected the phenotype of CHV1-infected mutants
resulting in much smaller colony size and higher 5.4.11.2 Common Phenotypic Changes
mycovirus accumulation, pointing to a role of Linked to Mycovirus-Caused
these two DNAMTases in antiviral defense Development Perturbation:
(Ko et al. 2021). Given that most fungi are studied after isolation
from the complex biome they inhabit into a
134 B. I. Hillman and M. Turina

simplified axenic culture rich in substrate, there 5.4.11.3 Sexual Reproduction

are a number of features that are often reported to The effect of mycovirus infection on sexual
be impacted by mycovirus infection. These reproduction was already mentioned for
features are the most immediate to evaluate and C. parasitica (Anagnostakis 1982; Deng et al.
often are used to screen phenotypic variation in 2007a, b); in particular, it was predicted that
populations, a phenomenon often caused by virus-caused downregulation of the mating
mycovirus infection. Among these easily scorable pheromone-encoding genes resulted in limited
phenotypic changes growth rate is likely the sexual reproduction of virus-infected isolates
easiest to notice, but caution must be paid in (Zhang et al. 1993; Turina et al. 2003). Here we
drawing more general conclusions from these will describe a more recent observation that
assays with the tendency to use growth rate as a results from the study of an interesting biological
proxy of hypovirulence. In fact, the two concepts system in which a fungus, Rhizopus microsporus
should be well separated, in view of the fact that (Mucorales) is associated with a virulence factor
growth is a component of fitness, and for some provided by an endobacterium, Mycetohabitans
models of mycovirus-based biocontrol, the fitness (formerly Burkholderia); furthermore, two
of the fungal host should only be minimally narnaviruses are part of the system. While
affected. Another simple character that can be narnaviruses by themselves decrease asexual
judged in vitro is the quantification of conidia reproduction (sporangiospore production), they
and possibly the percentage of mycovirus trans- are strictly required together with the bacterium
mission, both also component of fitness. for sexual reproduction (zygospore formation);
Mycoviruses often affect conidium production this is an example of conditional mutualism,
as is the case of CHV1 infection of where the positive effect on sexual reproduction
C. parasitica. The trade-offs between provided by the narnavirus is conditional on the
hypovirulence, growth, and conidium production presence of the bacteria (Espino-Vazquez et al.
are exemplified by studies that compared different 2020); at the moment the molecular mechanism
isolates of CHV1 that differentially affect these of such interaction remains unknown.
traits and might explain some ineffective attempts
at biocontrol intervention (the EURO-7 CHV1
isolate vs EP713 isolate) (Chen and Nuss 1999; 5.4.12 Disruption of Known Metabolic
Chen et al. 2000). Pathways
Finally, another character that is often judged
and used for visual screens of mycovirus-infected It is noteworthy that, although some mycoviruses
strains is alteration of pigmentation (although the accumulate abundantly in their host (as is the case
biological significance of it is not obvious), as of CHV1-C. parasitica), a considerable energetic
exemplified by lack of orange pigment production expense, very limited studies have examined the
of CHV1-infected C. parasitica (Anagnostakis effect of mycovirus infection on metabolic
1982). In other mycovirus–host interactions, changes, either from a purely physiological
excess pigments are produced. point of view (measuring CO2 and O2 exchanges
These phenotypic changes can be reproduced and substrate utilization) or from a profiling of
by a number of mutants, but here it is worth primary metabolites. Again the model system for
pointing to a specific gene that only affects these this approach is CHV1-C. parasitica. From a
changes when the fungus is infected by CHV1. In general point of view, mycelium on the periphery
a mutant screen, the nam-1 gene, encoding for a of the colony relied heavily on fermentation
Mg2+ transporter, was identified as a specific pathways, while older mycelia shifted to aerobic
modulator of virus-caused symptoms (Faruk carbohydrate metabolism and finally lipid metab-
et al. 2008). olism in the interior, older part of the colony. This
shift left abundant residual glucose in the
5 Viruses that Affect Phenotype and Fitness of Fungi 135

substrate, and mycovirus infection affected the 5.4.13 Mechanisms of Fitness

metabolism in this older part of the plate where Alteration
infected mycelium seemed to be unable to utilize
lipid-related metabolic pathways, which lead to Considerations of fitness alteration caused by
increased glucose depletion (Dawe et al. 2009). mycovirus infection are particularly relevant
This shift related to lipid or carbohydrate metab- when planning natural biocontrol approaches,
olism was confirmed by a more accurate meta- where the hypovirulent strain is released and
bolic profiling, where one third of 164 detected expected to establish naturally. In fact, rarely is
metabolites were either up- or down-regulated reproductive fitness (defined as a quantitative rep-
(Dawe et al. 2009). resentation of reproductive success) measured for
Virus effects on secondary metabolite accu- fungi infected by viruses in natural environments
mulation, including volatile organic compounds and compared to their uninfected counterparts.
(VOCs), represent a major knowledge gap. The This becomes an essential factor when consider-
fact that a comprehensive metabolomic approach ing a successful biocontrol agent. In this sense, an
has not yet been applied to mycovirus infection is interesting recent research paper has applied the
only minimally alleviated by some specific concepts of ecology measuring the life-history
reports, mostly concentrated on the effect on traits in the CHV1–C. parasitica interaction
mycotoxin production. In this regard, the first (Brusini et al. 2017). This work measured how
report was related to the downregulation of afla- hypovirus infection alters different components
toxin production in A. flavus: interestingly, curing of fitness (growth, survival, and reproduction)
of resident virus rendered a nontoxigenic strain and the necessary trade-offs of resource alloca-
toxigenic, and transfection with a Penicillium tion. A number of parameters were measured for
chrysogenum (Eurotiales) virus repristinated the 36 combinations of fungal and virus isolates in
repression of aflatoxin production (Schmidt et al. cross-inoculation experiments. The authors
1986). A number of other studies investigated showed that the classical trade-off between “cur-
mycotoxin production in Aspergillus with differ- rent reproduction” and “somatic growth” (wide-
ent outcomes (Elias and Cotty 1996; Silva et al. spread in nature) is subverted when C. parasitica
2001). In another study, upregulation of is infected by CHV1: in this case, the faster the
ochratoxin production by A. ochraceus was also growth rate, the greater is sporulation; moreover,
linked to the presence of a specific partitivirus the faster the growth rate, the greater vertical
(Nerva et al. 2018). Recently tenuazonic acid transmission of the virus (Brusini et al. 2017).
production was also shown to be induced by a
totivirus infection in Pyricularia oryzae
(Magnaporthales; previous name is 5.4.14 Concluding Thoughts
Magnaporthe oryzae) (Zhang et al. 2016b). The
upregulation of a specific transcription factor The breadth of viruses in the fungal systems that
involved in the teA synthetase gene upregulation have been investigated in some depth, and the
is the proposed molecular mechanism behind the overlap in the types of virus found in those
upregulation (Ninomiya et al. 2020). A different fungi, tells us that the same is true for fungi as
example of a secondary metabolite which is also a with other hosts: when resources available and
virulence factor, AK-toxin, is a host-specific dedicated virologists set out to look for and char-
toxin responsible for the necrotic phenotype: in acterize the viromes of fungi in detail, we find that
this case, a chrysovirus affects growth of the each fungus is host to a wealth of viruses, many of
Alternaria (Pleosporales) host, but at the same which affect host biology and fitness. A few
time it greatly increases AK-toxin production systems have been highlighted here, but there
(Okada et al. 2018). are many other fungal systems that are of long-
term or growing interest, including the genera
136 B. I. Hillman and M. Turina

Saccharomyces (Saccharomycetales), Aspergil- Boland GJ (1992) Hypovirulence and double-stranded-

lus, Colletotrichum (Glomerellales), RNA in Sclerotinia sclerotiorum. Can J Plant Pathol
14:10–17. https://doi.org/10.1080/
Helminthosporium (Pleosporales), Alternaria, 07060669209500900
and Pyricularia, among many others, that could Boland GJ (2004) Fungal viruses, hypovirulence, and
have sections devoted to them. The difficulties of biological control of Sclerotinia species. Can J Plant
assessing phenotypes has also excluded an impor- Pathol 26:6–18
Botella L, Jung T (2021) Multiple viral infections detected
tant group of taxonomically diverse fungi that in Phytophthora condilina by total and small RNA
establish mycorrhizal relationships with plant sequencing. Viruses 13(4). https://doi.org/10.3390/
hosts from our review, but we expect great prog- v13040620
ress in mycovirus–host systems in this field in the Brusini J, Wayne ML, Franc A, Robin C (2017) The
impact of parasitism on resource allocation in a
near future. We are gratified that the task of fungal host: the case of Cryphonectria parasitica and
summarizing the field today is so much more its mycovirus, Cryphonectria hypovirus 1. Ecol Evol 7:
challenging than it was even a few years ago! 5967–5976. https://doi.org/10.1002/ece3.3143
Cai G, Hillman BI (2013) Phytophthora viruses. Adv
Virus Res 86:327–350. https://doi.org/10.1016/B978-
0-12-394315-6.00012-X
References Cai G, Myers K, Hillman BI, Fry WE (2009) A novel virus
of the late blight pathogen, Phytophthora infestans,
Ahlquist PA, French R, Janda MI, Loesch-Fries LS (1984) with two RNA segments and a supergroup
Multicomponent RNA plant virus infection derived 1 RNA-dependent RNA polymerase. Virology
from cloned viral DNA. PNAS 81:7066–7070 392(1):52–61. https://doi.org/10.1016/j.virol.2009.
Aihara M, Urayama S-i, Minh Tuong L et al (2018) 06.040
Infection by Magnaporthe oryzae chrysovirus 1 strain Cai G, Myers K, Fry WE, Hillman BI (2012) A member of
A triggers reduced virulence and pathogenic race con- the virus family Narnaviridae from the plant patho-
version of its host fungus, Magnaporthe oryzae. J Gen genic oomycete Phytophthora infestans. Arch Virol
Plant Pathol 84:92–103. https://doi.org/10.1007/ 157:165–169. https://doi.org/10.1007/s00705-011-
s10327-018-0766-7 1126-5
Anagnostakis SL (1982) Biological control of chestnut Cai G, Krychiw JF, Myers K et al (2013) A new virus from
blight. Science 215(4532):466–471. https://doi.org/ the plant pathogenic oomycete Phytophthora infestans
10.1126/science.215.4532.466 with an 8 kb dsRNA genome: the sixth member of a
Anagnostakis SL (1987) Chestnut blight – the classical proposed new virus genus. Virology 435:341–349.
problem of an introduced pathogen. Mycologia 79:23– https://doi.org/10.1016/j.virol.2012.10.012
37 Cai G, Fry WE, Hillman BI (2019a) PiRV-2 stimulates
Anagnostakis SL, Chen B, Geletka LM, Nuss DL (1998) sporulation in Phytophthora infestans. Virus Res 271:
Hypovirus transmission to ascospore progeny by field- 197674. https://doi.org/10.1016/j.virusres.2019.
released transgenic hypovirulent strains of 197674
Cryphonectria parasitica. Phytopathology 88(7): Cai G, Myers K, Fry WE, Hillman BI (2019b)
598–604. https://doi.org/10.1094/PHYTO.1998.88. Phytophthora infestans RNA virus 2, a novel RNA
7.598 virus from Phytophthora infestans, does not belong to
Aulia A, Andika IB, Kondo H et al (2019) A symptomless any known virus group. Arch Virol 164(2):567–572.
hypovirus, CHV4, facilitates stable infection of the https://doi.org/10.1007/s00705-018-4050-0
chestnut blight fungus by a coinfecting reovirus likely Chen BS, Nuss DL (1999) Infectious cDNA clone of
through suppression of antiviral RNA silencing. Virol- hypovirus CHV1-Euro7: a comparative virology
ogy 533:99–107. https://doi.org/10.1016/j.virol.2019. approach to investigate virus-mediated hypovirulence
05.004 of the chestnut blight fungus Cryphonectria parasitica.
Aulia A, Hyodo K, Hisano S et al (2021) Identification of J Virol 73:985–992
an RNA silencing suppressor encoded by a symptom- Chen BS, Choi GH, Nuss DL (1994) Attenuation of fungal
less fungal hypovirus, Cryphonectria hypovirus virulence by synthetic infectious hypovirus transcripts.
4. Biology 10(2):100. https://doi.org/10.3390/ Science 264:1762–1764
biology10020100 Chen BS, Geletka LM, Nuss DL (2000) Using chimeric
Azhar A, Mu F, Huang H et al (2019) A novel RNA virus hypoviruses to fine-tune the interaction between a
related to sobemoviruses confers hypovirulence on the pathogenic fungus and its plant host. J Virol 74:
phytopathogenic fungus Sclerotinia sclerotiorum. 7562–7567
Viruses-Basel 11(8):759. https://doi.org/10.3390/ Chiapello M, Rodriguez-Romero J, Ayllon MA, Turina M
v11080759 (2020) Analysis of the virome associated to grapevine
downy mildew lesions reveals new mycovirus
5 Viruses that Affect Phenotype and Fitness of Fungi 137

lineages. Virus Evol 6. https://doi.org/10.1093/ve/ 134:181–189. https://doi.org/10.1007/s10658-012-

veaa058 9977-5
Chiba S, Suzuki N (2015) Highly activated RNA silencing Das S, Alam MM, Zhang R et al (2021) Proof of concept
via strong induction of dicer by one virus can interfere of the yadokari nature: a capsidless replicase-encoding
with the replication of an unrelated virus. PNAS but replication-dependent positive-sense single-
112(35):E4911–E4918. https://doi.org/10.1073/pnas. stranded RNA virus hosted by an unrelated double-
1509151112 stranded RNA virus. J Virol 95:e0046721. https://doi.
Chiba S, Salaipeth L, Lin YH et al (2009) A novel bipartite org/10.1128/JVI.00467-21
double-stranded RNA mycovirus from the white root Dawe AL, Nuss DL (2013) Hypovirus molecular biology:
rot fungus Rosellinia necatrix: molecular and from Koch’s postulates to host self-recognition genes
biological characterization, taxonomic considerations, that restrict virus transmission. Adv Virus Res 86:109–
and potential for biological control. J Virol 83:12801– 147. https://doi.org/10.1016/B978-0-12-394315-6.
12812. https://doi.org/10.1128/JVI.01830-09 00005-2
Chiba S, Lin Y-H, Kondo H et al (2013) A novel Dawe AL, Van Voorhies WA, Lau TA et al (2009) Major
Victorivirus from a phytopathogenic fungus, impacts on the primary metabolism of the plant patho-
Rosellinia necatrix, is infectious as particles and gen Cryphonectria parasitica by the virulence-
targeted by RNA silencing. J Virol 87:6727–6738. attenuating virus CHV1-EP713. Microbiol-Sgm 155:
https://doi.org/10.1128/jvi.00557-13 3913–3921. https://doi.org/10.1099/mic.0.029033-0
Chiba S, Lin YH, Kondo H et al (2016) A novel Deakin G, Dobbs E, Bennett JM et al (2017) Multiple viral
betapartitivirus RnPV6 from Rosellinia necatrix infections in Agaricus bisporus - Characterisation of
tolerates host RNA silencing but is interfered by its 18 unique RNA viruses and 8 ORFans identified by
defective RNAs. Virus Res 219:62–72. https://doi.org/ deep sequencing. Sci Rep 7. https://doi.org/10.1038/
10.1016/j.virusres.2015.10.017 s41598-017-01592-9
Chiba S, Caston JR, Ghabrial SA, Suzuki N, ICTV Report Deng F, Allen TD, Hillman BI, Nuss DL (2007a) Com-
C (2018) ICTV virus taxonomy profile: quadriviridae. parative analysis of alterations in host phenotype and
J Gen Virol 99 (11):1480–1481. doi:https://doi.org/10. transcript accumulation following hypovirus and
1099/jgv.0.001152 mycoreovirus infections of the chestnut blight fungus
Choi GH, Nuss DL (1992) Hypovirulence of chestnut Cryphonectria parasitica. Eukaryot Cell 6:1286–1298.
blight fungus conferred by an infectious viral cDNA. https://doi.org/10.1128/EC.00166-07
Science 257:800–803 Deng F, Allen TD, Nuss DL (2007b) Ste12 transcription
Chu YM, Jeon JJ, Yea SJ et al (2002) Double-stranded factor homologue CpST12 is down-regulated by
RNA mycovirus from Fusarium graminearum. Appl hypovirus infection and required for virulence and
Environ Microb 68(5):2529–2534. https://doi.org/10. female fertility of the chestnut blight fungus
1128/aem.68.5.2529-2534.2002 Cryphonectria parasitica. Eukaryot Cell 6:235–244.
Chun J, Yang H-E, Kim D-H (2018a) Identification of a https://doi.org/10.1128/EC.00166-07
novel partitivirus of Trichoderma harzianum Dobbs E, Deakin G, Bennett J et al (2021) Viral
NFCF319 and evidence for the related antifungal activ- interactions and pathogenesis during multiple viral
ity. Front Plant Sci 9. https://doi.org/10.3389/fpls. infections in Agaricus bisporus. MBio 12(1). https://
2018.01699 doi.org/10.1128/mBio.03470-20
Chun J, Yang HE, Kim DH (2018b) Identification and Donaire L, Ayllon MA (2017) Deep sequencing of
molecular characterization of a novel partitivirus from mycovirus-derived small RNAs from Botrytis species.
Trichoderma atroviride NFCF394. Viruses-Basel Mol Plant Pathol 18:1127–1137. https://doi.org/10.
10(11). https://doi.org/10.3390/v10110578 1111/mpp.12466
Chun J, So KK, Ko YH, Kim DH (2022) Molecular Dong K, Xu C, Kotta-Loizou I et al (2023) Novel viroid-
characteristics of a novel hypovirus from Trichoderma like RNAs naturally infect a filamentous fungus. Adv
harzianum. Arch Virol 167(1):233–238. https://doi. Sci 10:202204308
org/10.1007/s00705-021-05253-1 Drinnenberg IA, Fink GR, Bartel DP (2011) Compatibility
Chung P-H, Bedker PJ, Hillman BI (1994) Diversity of with killer explains the rise of RNAi-deficient fungi.
Cryphonectria parasitica hypovirulence-associated Science 333:1592–1592. https://doi.org/10.1126/sci
double-stranded RNAs within a chestnut population ence.1209575
in New Jersey. Phytopathology 84:984–990 Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A et al
Cordoba L, Ruiz-Padilla A, Rodriguez-Romero J, Ayllon (2011) Trichoderma: the genomics of opportunistic
MA (2022) Construction and characterization of a success. Nat Rev Microbiol 9:749–759. https://doi.
Botrytis Virus F infectious clone. J Fungi 8(5). org/10.1038/nrmicro2637
https://doi.org/10.3390/jof8050459 Elias KS, Cotty PJ (1996) Incidence and stability of infec-
Darissa O, Adam G, Schafer W (2012) A dsRNA tion by double-stranded RNA genetic elements in
mycovirus causes hypovirulence of Fusarium Aspergillus section flavi and effects on aflatoxigenicity.
graminearum to wheat and maize. Eur J Plant Pathol Can J Bot 74:716–725. https://doi.org/10.1139/
b96-090
138 B. I. Hillman and M. Turina

Elsherbeini M, Bostian KA (1987) Viruses in fungi - trees in Michigan. Can J Bot 61(12):3164–3171.
infection of yeast with the K1 and K2 killer viruses. https://doi.org/10.1139/b83-354
PNAS 84:4293–4297. https://doi.org/10.1073/pnas.84. Fulbright DW, Paul CP, Garrod SW (1988)
12.4293 Hypovirulence: a natural control of chestnut
Enebak SA, Hillman BI, MacDonald WL (1994a) A blight. In: Mukerji KG, Garg KL (eds) Biocontrol of
hypovirulent isolate of Cryphonectria parasitica with plant diseases. CRC Press, Boca Raton, FL, pp
multiple, genetically unique dsRNA segments. Mol 121–138
Plant Microb Interact 7:590–595 Ghabrial S, Mernaugh R (1983) Biology and transmission
Enebak SA, MacDonald WL, Hillman BI (1994b) Effect of Helminthosporium victoriae mycoviruses, vol 441.
of dsRNA associated with isolates of Cryphonectria Elsevier, Amsterdam, London and New York
parasitica from the central Appalachians and their Ghabrial SA, Suzuki N (2009) Viruses of plant pathogenic
relatedness to other dsRNAs from North America and fungi. Annu Rev Phytopathol 47:353–384. https://doi.
Europe. Phytopathology 84(5):528–534 org/10.1146/annurev-phyto-080508-081932
Espino-Vazquez AN, Bermudez-Barrientos JR, Cabrera- Ghabrial SA, Caston JR, Jiang D et al (2015) 50-plus years
Rangel JF et al (2020) Narnaviruses: novel players in of fungal viruses. Virology 479–480:356–368. https://
fungal-bacterial symbioses. ISME J. https://doi.org/10. doi.org/10.1016/j.virol.2015.02.034
1038/s41396-020-0638-y Goodin MM, Schlagnhaufer B, Romaine CP (1992)
Esteban R, Fujimura T (2003) Launching the yeast 23S Encapsidation of the La France disease-specific dou-
RNA Narnavirus shows 5' and 3' cis-acting signals for ble-stranded RNAs in 36-nm isometric virus-like
replication. PNAS 100:2568–2573. https://doi.org/10. particles. Phytopathology 82:285–290. https://doi.org/
1073/pnas.0530167100 10.1094/Phyto-82-285
Eusebio-Cope A, Sun L, Hillman BI, Suzuki N (2010) Govers F, Drenth A, Pieterse CMJ (1997) The potato late
Mycoreovirus 1 S4-coded protein is dispensable for blight pathogen Phytophthora infestans and other path-
viral replication but necessary for efficient vertical ogenic Oomycota. In: Carroll GC, Tudzynski P (eds)
transmission and normal symptom induction. Virology The Mycota, vol V Part B. Springer, Berlin, pp 17–36
397(2):399–408. https://doi.org/10.1016/j.virol.2009. Grente J (1965) Les formes hypovirulentes d’Endothia
11.035 parasitica et les espoires de lutte contre le chance du
Eusebio-Cope A, Sun L, Tanaka T et al (2015) The chest- chantaingnier. CR Acad Agric France 51:1033–1037
nut blight fungus for studies on virus/host and virus/ Grogan HM, Adie BAT, Gaze RH et al (2003) Double-
virus interactions: from a natural to a model host. stranded RNA elements associated with the MVX dis-
Virology 477:164–175. https://doi.org/10.1016/j.virol. ease of Agaricus bisporus. Mycol Res 107:147–154.
2014.09.024 https://doi.org/10.1017/s0953756203007202
Faruk MI, Eusebio-Cope A, Suzuki N (2008) A host factor Guo MP, Bian YB, Wang JJ et al (2017) Biological and
involved in hypovirus symptom expression in the molecular characteristics of a novel partitivirus
chestnut blight fungus, Cryphonectria parasitica. J infecting the edible fungus Lentinula edodes. Plant
Virol 82:740–754. https://doi.org/10.1128/jvi. Dis 101(5):726–733. https://doi.org/10.1094/pdis-07-
02015-07 16-0951-re
Feng CC, Feng JH, Wang ZY et al (2021) Identification of Hannat S, Pontarotti P, Colson P et al (2021) Diverse
the viral determinant of hypovirulence and host range trajectories drive the expression of a giant virus in the
in Sclerotiniaceae of a Genomovirus reconstructed oomycete plant pathogen Phytophthora parasitica.
from the plant metagenome. J Virol 95(17). https:// Front Microbiol 12:662762. https://doi.org/10.3389/
doi.org/10.1128/jvi.00264-21 fmicb.2021.662762
Forgia M, Isgandarli E, Aghayeva DN et al (2021) Virome Hao F, Wu M, Li G (2018a) Molecular characterization
characterization of Cryphonectria parasitica isolates and geographic distribution of a Mymonavirus in the
from Azerbaijan unveiled a new mymonavirus and a population of Botrytis cinerea. Viruses-Basel 10(8).
putative new RNA virus unrelated to described viral https://doi.org/10.3390/v10080432
sequences. Virology 553:51–61. https://doi.org/10. Hao FM, Ding T, Wu MD et al (2018b) Two novel
1016/j.virol.2020.10.008 hypovirulence-associated mycoviruses in the phyto-
Forgia M, Chiapello M, Daghino S et al (2022) Three new pathogenic fungus Botrytis cinerea: molecular charac-
clades of putative viral RNA-dependent RNA terization and suppression of infection cushion
polymerases with rare or unique catalytic triads discov- formation. Viruses-Basel 10(5). https://doi.org/10.
ered in libraries of ORFans from powdery mildews and 3390/v10050254
the yeast of oenological interest Starmerella bacillaris. Hao FM, Wu MD, Li GQ (2021) Characterization of a
Virus Evol 8(1). https://doi.org/10.1093/ve/veac038 novel genomovirus in the phytopathogenic fungus
Fulbright DW (1984) Effect of eliminating dsRNA in Botrytis cinerea. Virology 553:111–116. https://doi.
hypovirulent Endothia parasitica. Phytopathology org/10.1016/j.virol.2020.11.007
74(6):722–724. https://doi.org/10.1094/Phyto-74-722 Harman GE, Howell CR, Viterbo A et al (2004)
Fulbright DW, Weidlich WH, Haufler KZ et al (1983) Trichoderma species - opportunistic, avirulent plant
Chestnut blight and recovering American chestnut
5 Viruses that Affect Phenotype and Fitness of Fungi 139

symbionts. Nat Rev Microbiol 2:43–56. https://doi. Janda M, Ahlquist P (1993) RNA-dependent replication,
org/10.1038/nrmicro797 transcription, and persistence of brome mosaic-virus
Hillman BI, Suzuki N (2004) Viruses of the chestnut blight RNA replicons in Saccharomyces cerevisiae. Cell 72:
fungus, Cryphonectria parasitica. Adv Virus Res 63: 961–970. https://doi.org/10.1016/0092-8674(93)
423–472. https://doi.org/10.1016/S0065-3527(04) 90584-d
63007-7 Jia JC, Mu F, Fu YP et al (2022) A capsidless virus is
Hillman BI, Tian Y, Bedker PJ, Brown MP (1992) A trans-encapsidated by a bisegmented botybirnavirus. J
North American hypovirulent isolate of the chestnut Virol 96. https://doi.org/10.1128/jvi.00296-22
blight fungus with European isolate-related dsRNA. J Jiang D, Fu Y, Guoqing L, Ghabrial SA (2013) Viruses of
Gen Virol 73:681–686. https://doi.org/10.1099/0022- the plant pathogenic fungus Sclerotinia sclerotiorum.
1317-73-3-681 Adv Virus Res 86:215–248. https://doi.org/10.1016/
Hillman BI, Halpern BT, Brown MP (1994) A viral B978-0-12-394315-6.00008-8
dsRNA element of the chestnut blight fungus with a Jom-in S, Akarapisan A (2009) Characterization of
distinct genetic organization. Virology 201(2): double-stranded RNA in Trichoderma spp. isolates in
241–250. https://doi.org/10.1006/viro.1994.1289 Chiang Mai province. J Agric Technol 5:261–270
Hillman BI, Supyani S, Kondo H, Suzuki N (2004) A Judelson HS, Tyler BM, Michelmore RW (1991) Trans-
reovirus of the fungus Cryphonectria parasitica that formation of the oomycete pathogen, Phytophthora
is infectious as particles and related to the coltivirus infestans. Mol Plant Microb Interact 4:602–607.
genus of animal pathogens. J Virol 78(2):892–898. https://doi.org/10.1094/mpmi-4-602
https://doi.org/10.1128/jvi.78.2.892-898.2004 Kalac P (2013) A review of chemical composition and
Hillman BI, Aulia A, Suzuki N (2018) Viruses of plant- nutritional value of wild-growing and cultivated
interacting fungi. Adv Virus Res 100:99–116. https:// mushrooms. J Sci Food Agr 93:209–218. https://doi.
doi.org/10.1016/bs.aivir.2017.10.003 org/10.1002/jsfa.5960
Hisano S, Zhang R, Faruk MI et al (2018) A neo-virus Kamaruzzaman M, He G, Wu M et al (2019) A novel
lifestyle exhibited by a (+)ssRNA virus hosted in an partitivirus in the hypovirulent isolate QT5-19 of the
unrelated dsRNA virus: taxonomic and evolutionary plant pathogenic fungus Botrytis cinerea. Viruses-
considerations. Virus Res 244:75–83. https://doi.org/ Basel 11(1). https://doi.org/10.3390/v11010024
10.1016/j.virusres.2017.11.006 Kamaruzzaman M, Lyu A, Zhang J et al (2020) Competi-
Hollings M (1962) Viruses associated with a die-back tive saprophytic ability of the hypovirulent isolate
disease of cultivated mushroom. Nature 196:962–965. QT5-19 of Botrytis cinerea and its importance in bio-
https://doi.org/10.1038/196962a0 control of necrotrophic fungal pathogens. Biol Control
Hu X, Roberts DP, Jiang M, Zhang Y (2005) Decreased 142. https://doi.org/10.1016/j.biocontrol.2019.104182
incidence of disease caused by Sclerotinia Kamaruzzaman M, Wang Z, Wu M et al (2021) Promotion
sclerotiorum and improved plant vigor of oilseed rape of tomato growth by the volatiles produced by the
with Bacillus subtilis Tu-100. Appl Microbiol hypovirulent strain QT5-19 of the plant gray mold
Biotechnol 68(6):802–807. https://doi.org/10.1007/ fungus Botrytis cinerea. Microbiol Res 247. https://
s00253-005-1938-x doi.org/10.1016/j.micres.2021.126731
Hu Z, Wu S, Cheng J et al (2014) Molecular characteriza- Kanai Y, Kobayashi T (2021) FAST Proteins: develop-
tion of two positive-strand RNA viruses co-infecting a ment and use of reverse genetics systems for
hypovirulent strain of Sclerotinia sclerotiorum. Virol- Reoviridae viruses. Annu Rev Virol 8:515–536.
ogy 464–465:450–459. https://doi.org/10.1016/j.virol. https://doi.org/10.1146/annurev-virology-
2014.07.007 091919-070225
Hu HJ, Wang JR, Cheng XH et al (2022) Preliminary Kanematsu S, Arakawa M, Oikawa Y et al (2004) a
studies on the effects of oyster mushroom spherical reovirus causes hypovirulence of Rosellinia necatrix.
virus china strain on the mycelial growth and fruiting Phytopathology 94:561–568. https://doi.org/10.1094/
body yield of the edible mushroom Pleurotus PHYTO.2004.94.6.561
ostreatus. Biol-Basel 11(4). https://doi.org/10.3390/ Kanhayuwa L, Kotta-Loizou I, Oezkan S et al (2015) A
biology11040574 novel mycovirus from Aspergillus fumigatus contains
Ikeda K, Nakamura H, Arakawa M, Matsumoto N (2004) four unique dsRNAs as its genome and is infectious as
Diversity and vertical transmission of double-stranded dsRNA. PNAS 112:9100–9105. https://doi.org/10.
RNA elements in root rot pathogens of trees, 1073/pnas.1419225112
Helicobasidium mompa and Rosellinia necatrix. Khalifa ME, MacDiarmid RM (2021) A mechanically
Mycol Res 108:626–634. https://doi.org/10.1017/ transmitted dna mycovirus is targeted by the defence
s0953756204000061 machinery of its host, Botrytis cinerea. Viruses-Basel
Jackson AO, Mitchell DM, Siegel A (1971) Replication of 13(7). https://doi.org/10.3390/v13071315
tobacco mosaic virus.1. Isolation and characterization Kim JM, Song HY, Choi HJ et al (2015) Changes in the
of double-stranded forms of ribonucleic acid. Virology mycovirus (LeV) titer and viral effect on the vegetative
45:182–191. https://doi.org/10.1016/0042-6822(71) growth of the edible mushroom Lentinula edodes.
90125-5
140 B. I. Hillman and M. Turina

Virus Res 197:8–12. https://doi.org/10.1016/j.virusres. alternata. Front Microbiol 10:1076. https://doi.org/

2014.11.016 10.3389/fmicb.2019.01076
Kleinschmidt WJ (1988) Discovery of a mycovirus: an Li P, Wang S, Zhang L et al (2020) A tripartite ssDNA
indirect route. In: Koltin Y, Leibowitz MJ (eds) mycovirus from a plant pathogenic fungus is infectious
Viruses of fungi and simple eukaryotes, vol 7. Marcel as cloned DNA and purified virions. Sci Adv 6:14.
Dekker, New York, pp 1–14 https://doi.org/10.1126/sciadv.aay9634
Ko Y-H, So K-K, Chun J, Kim D-H (2021) Distinct roles Li K, Liu DM, Pan X et al (2022) Deoxynivalenol biosyn-
of two DNA methyltransferases from Cryphonectria thesis in Fusarium pseudograminearum significantly
parasitica in fungal virulence, responses to hypovirus repressed by a megabirnavirus. Toxins 14. https://doi.
infection, and viral clearance. MBio 12(1). https://doi. org/10.3390/toxins14070503
org/10.1128/mBio.02890-20 Lin Y-H, Chiba S, Tani A et al (2012) A novel quadripar-
Kondo H, Chiba S, Toyoda K, Suzuki N (2013a) Evidence tite dsRNA virus isolated from a phytopathogenic fila-
for negative-strand RNA virus infection in fungi. mentous fungus, Rosellinia necatrix. Virology 426:42–
Virology 435(2):201–209. https://doi.org/10.1016/j. 50. https://doi.org/10.1016/j.virol.2012.01.013
viro1.2012.10.002 Lin YH, Fujita M, Chiba S et al (2019) Two novel fungal
Kondo H, Kanematsu S, Suzuki N (2013b) Viruses of the negative-strand RNA viruses related to
white root rot fungus, Rosellinia necatrix. Adv Virus mymonaviruses and phenuiviruses in the shiitake
Res 86:177–214. https://doi.org/10.1016/B978-0-12- mushroom (Lentinula edodes). Virology 533:125–
394315-6.00007-6 136. https://doi.org/10.1016/j.virol.2019.05.008
Kotta-Loizou I (2021) Mycoviruses and their role in fun- Linder-Basso D, Dynek JN, Hillman BI (2005) Genome
gal pathogenesis. Curr Opin Microbiol 63:10–18. analysis of Cryphonectria hypovirus 4, the most com-
https://doi.org/10.1016/j.mib.2021.05.007 mon hypovirus species in North America. Virology
Kotta-Loizou I, Coutts RH (2017a) Mycoviruses in 337:192–203. https://doi.org/10.1016/j.virol.2005.
Aspergilli: a comprehensive review. Front Microbiol 03.038
8:1699 Liu L, Xie J, Cheng J et al (2014) Fungal negative-
Kotta-Loizou I, Coutts RHA (2017b) Studies on the stranded RNA virus that is related to bornaviruses
virome of the entomopathogenic fungus Beauveria and nyaviruses. PNAS 111:12205–12210. https://doi.
bassiana reveal novel dsRNA elements and mild org/10.1073/pnas.1401786111
hypervirulence. PLoS Pathog 13(1). https://doi.org/ Liu J, Wang ZR, Li C et al (2015) Evaluating genetic
10.1371/journal.ppat.1006183 diversity and constructing core collections of Chinese
Krupovic M, Ghabrial SA, Jiang D, Varsani A (2016) Lentinula edodes cultivars using ISSR and SRAP
Genomoviridae: a new family of widespread single- markers. J Basic Microbiol 55:749–760. https://doi.
stranded DNA viruses. Arch Virol 161:2633–2643. org/10.1002/jobm.201400774
https://doi.org/10.1007/s00705-016-2943-3 Liu S, Xie J, Cheng J et al (2016) Fungal DNA virus
Laraba I, McCormick SP, Vaughan MM et al (2021) infects a mycophagous insect and utilizes it as a trans-
Phylogenetic diversity, trichothecene potential, and mission vector. PNAS 113:12803–12808. https://doi.
pathogenicity within Fusarium sambucinum species org/10.1073/pnas.1608013113
complex. PLoS One 16(1):e0245037 Liu C, Li M, Redda ET et al (2019a) A novel double-
Lee SH, Yun S-H, Chun J, Kim D-H (2017) Characteriza- stranded RNA mycovirus isolated from Trichoderma
tion of a novel dsRNA mycovirus of Trichoderma harzianum. Virol J 16(1). https://doi.org/10.1186/
atroviride NFCF028. Arch Virol 162:1073–1077. s12985-019-1213-x
https://doi.org/10.1007/s00705-016-3214-z Liu CC, Li M, Redda ET et al (2019b) Complete nucleo-
Lemus-Minor CG, Canizares MC, Garcia-Pedrajas MD, tide sequence of a novel mycovirus from Trichoderma
Perez-Artes E (2018) Fusarium oxysporum f. sp harzianum in China. Arch Virol 164:1213–1216.
dianthi virus 1 accumulation is correlated with changes https://doi.org/10.1007/s00705-019-04145-9
in virulence and other phenotypic traits of its fungal Macdonald WL, Fulbright DW (1991) Biological-control
host. Phytopathology 108:957–963. https://doi.org/10. of chestnut blight - use and limitations of transmissible
1094/phyto-06-17-0200-r hypovirulence. Plant Dis 75:656–661
Lhoas P (1971) Infection of protoplasts from Penicillium Magae Y, Sunagawa M (2010) Characterization of a
stoloniferum with double-stranded RNA viruses. J Gen mycovirus associated with the brown discoloration of
Virol 13:365–367. https://doi.org/10.1099/0022-1317- edible mushroom, Flammulina velutipes. Virol J
13-2-365 7. https://doi.org/10.1186/1743-422x-7-342
Li PF, Zhang HL, Chen XG et al (2015) Molecular char- Marcal S, Sousa AS, Taofiq O et al (2021) Impact of
acterization of a novel hypovirus from the plant patho- postharvest preservation methods on nutritional value
genic fungus Fusarium graminearum. Virology 481: and bioactive properties of mushrooms. Trends Food
151–160. https://doi.org/10.1016/j.virol.2015.02.047 Sci Technol 110:418–431. https://doi.org/10.1016/j.
Li H, Bian R, Liu Q et al (2019) Identification of a novel tifs.2021.02.007
hypovirulence-inducing hypovirus from Alternaria Marquez LM, Redman RS, Rodriguez RJ, Roossinck MJ
(2007) A virus in a fungus in a plant: three-way
5 Viruses that Affect Phenotype and Fitness of Fungi 141

symbiosis required for thermal tolerance. Science 315: Neumann G, Whitt MA, Kawaoka Y (2002) A decade
513–515. https://doi.org/10.1126/science.1136237 after the generation of a negative-sense RNA virus
Marzano S-YL, Domier LL (2016) Novel mycoviruses from cloned cDNA – what have we learned? J Gen
discovered from metatranscriptomics survey of soy- Virol 83:2635–2662. https://doi.org/10.3389/fmicb.
bean phyllosphere phytobiomes. Virus Res 213:332– 2020.01641
342 Nicolás FE, Torres-Martínez S, Ruiz-Vázquez RM (2013)
Marzano S-YL, Hobbs HA, Nelson BD et al (2015) Trans- Loss and retention of RNA Interference in fungi and
fection of Sclerotinia sclerotiorum with in vitro parasites. PLoS Pathog 9(1):e1003089. https://doi.org/
transcripts of a naturally occurring interspecific recom- 10.1371/journal.ppat.1003089
binant of Sclerotinia sclerotiorum hypovirus 2 signifi- Ninomiya A, Urayama S, Suo R et al (2020) Mycovirus-
cantly reduces virulence of the fungus. J Virol 89: induced tenuazonic acid production in a rice blast
5060–5071. https://doi.org/10.1128/jvi.03199-14 fungus Magnaporthe oryzae. Front Microbiol 11.
Marzano S-YL, Nelson BD, Ajayi-Oyetunde O et al https://doi.org/10.3389/fmicb.2020.01641
(2016) Identification of diverse mycoviruses through Nuss DL (2005) Hypovirulence: mycoviruses at the
metatranscriptomics characterization of the viromes of fungal-plant interface. Nat Rev Microbiol 3(8):
five major fungal plant pathogens. J Virol 90:6846– 632–642. https://doi.org/10.1038/nrmicro1206
6863. https://doi.org/10.1128/jvi.00357-16 Nuss DL (2011) Mycoviruses, RNA silencing, and viral
Milgroom MG, Cortesi P (2004) Biological control of RNA recombination. Adv Virus Res 80:25–48. https://
chestnut blight with hypovirulence: a critical analysis. doi.org/10.1016/B978-0-12-385987-7.00002-6
Annu Rev Phytopathol 42:311–338. https://doi.org/10. Oh CS (1995) Characterization of double-stranded RNA
1146/annurev.phyto.42.040803.140325 viruses and biology of Atkinsonella hypoxylon. PhD,
Mochama P, Jadhav P, Neupane A, Lee Marzano SY Rutgers University, New Brunswick, NJ
(2018) Mycoviruses as triggers and targets of RNA Oh CS, Hillman BI (1995) Genome organization of a
silencing in white mold fungus Sclerotinia partitivirus from the filamentous ascomycete
sclerotiorum. Viruses 10(4). https://doi.org/10.3390/ Atkinsonella hypoxylon. J Gen Virol 76:1461–1470.
v10040214 https://doi.org/10.1099/0022-1317-76-6-1461
Moleleki N, van Heerden SW, Wingfield MJ et al (2003) Okada R, Ichinose S, Takeshita K et al (2018) Molecular
Transfection of Diaporthe perjuncta with Diaporthe characterization of a novel mycovirus in Alternaria
RNA virus. Appl Environ Microbiol 69:3952–3956 alternata manifesting two-sided effects: down-
Morris TJ, Dodds JA (1979) Isolation and analysis of regulation of host growth and up-regulation of host
double-stranded-RNA from virus-infected plant and plant pathogenicity. Virology 519:23–32. https://doi.
fungal tissue. Phytopathology 69:854–858. https:// org/10.1016/j.virol.2018.03.027
doi.org/10.1094/Phyto-69-854 Osaki H, Wei CZ, Arakawa M et al (2002) Nucleotide
Mu F, Li B, Cheng SF et al (2021) Nine viruses from eight sequences of double-stranded RNA segments from a
lineages exhibiting new evolutionary modes that hypovirulent strain of the white root rot fungus
co-infect a hypovirulent phytopathogenic fungus. Rosellinia necatrix: possibility of the first member of
PLoS Pathog 17(8):e1009823. https://doi.org/10. the Reoviridae from fungus. Virus Genes 25:101–107.
1371/journal.ppat.1009823 https://doi.org/10.1023/A:1020182427439
Munkvold GP (2017) Fusarium species and their Pearson MN, Beever RE, Boine B, Arthur K (2009)
associated mycotoxins. Mycotoxigenic Fungi Method Mycoviruses of filamentous fungi and their relevance
Protocols 1542:51–106. https://doi.org/10.1007/978- to plant pathology. Molec Plant Pathol 10:115–128.
1-4939-6707-0_4 https://doi.org/10.1111/j.1364-3703.2008.00503.x
Nerva L, Ciuffo M, Vallino M et al (2015) Multiple Peever TL, Liu YC, Milgroom MG (1997) Diversity of
approaches for the detection and characterization of hypoviruses and other double-stranded RNAs in
viral and plasmid symbionts from a collection of Cryphonectria parasitica in North America. Phytopa-
marine fungi. Virus Res. https://doi.org/10.1016/j. thology 87:1026–1033. https://doi.org/10.1094/
virusres.2015.10.028 PHYTO.1997.87.10.1026
Nerva L, Varese GC, Falk BW, Turina M (2017) Peever TL, Liu YC, Wang K et al (1998) Incidence and
Mycoviruses of an endophytic fungus can replicate in diversity of double-stranded RNAs occurring in the
plant cells: evolutionary implications. Sci Rep 7(1): chestnut blight fungus, Cryphonectria parasitica, in
1908. https://doi.org/10.1038/s41598-017-02017-3 China and Japan. Phytopathology 88:811–817.
Nerva L, Chitarra W, Siciliano I et al (2018) Mycoviruses https://doi.org/10.1094/PHYTO.1998.88.8.811
mediate mycotoxin regulation in Aspergillus Picarelli M, Forgia M, Rivas EB et al (2019) Extreme
ochraceus. Environ Microbiol. https://doi.org/10. diversity of mycoviruses present in isolates of Rhizoc-
1111/1462-2920.14436 tonia solani AG2-2 LP from Zoysia japonica from
Nerva L, Forgia M, Ciuffo M et al (2019) The mycovirome Brazil. Front Cell Infect Microbiol 9. https://doi.org/
of a fungal collection from the sea cucumber 10.3389/fcimb.2019.00244
Holothuria polii. Virus Res 273. https://doi.org/10. Poimala A, Parikka P, Hantula J, Vainio EJ (2021) Viral
1016/j.virusres.2019.197737 diversity in Phytophthora cactorum population
142 B. I. Hillman and M. Turina

infecting strawberry. Environ Microbiol 23:5200– Phytopathology 97:278–286. https://doi.org/10.1094/

5221. https://doi.org/10.1111/1462-2920.15519 PHYTO-97-3-0278
Polashock JJ, Hillman BI (1994) A small mitochondrial Sato Y, Miyazaki N, Kanematsu S et al, ICTV Report
double-stranded (ds) RNA element associated with a Committee (2019) ICTV virus taxonomy profile:
hypovirulent strain of the chestnut blight fungus and megabirnaviridae. J Gen Virol 100:1269–1270. doi:
ancestrally related to yeast cytoplasmic T and W https://doi.org/10.1099/jgv.0.001297
dsRNAs. PNAS 91(18):8680–8684. https://doi.org/ Sato Y, Shamsi W, Jamal A et al (2020) Hadaka virus 1: a
10.1073/pnas.91.18.8680 capsidless eleven-segmented positive-sense single-
Polashock JJ, Bedker PJ, Hillman BI (1997) Movement of stranded RNA virus from a phytopathogenic fungus,
a small mitochondrial double-stranded RNA element Fusarium oxysporum. mBio 11(3). https://doi.org/10.
of Cryphonectria parasitica: ascospore inheritance and 1128/mBio.00450-20
implications for mitochondrial recombination. Mol Schmidt FR, Lemke PA, Esser K (1986) Viral influences
Gen Genet 256:566–571. https://doi.org/10.1007/ on aflatoxin formation by Aspergillus flavus. Appl
s004380050602 Microbiol Biotech 24:248–252
Potgieter CA, Castillo A, Castro M et al (2013) A wild- Segers GC, van Wezel R, Zhang X et al (2006) Hypovirus
type Botrytis cinerea strain co-infected by double- papain-like protease p29 suppresses RNA silencing in
stranded RNA mycoviruses presents hypovirulence- the natural fungal host and in a heterologous plant
associated traits. Virol J 10. https://doi.org/10.1186/ system. Eukaryot Cell 5(6):896–904. https://doi.org/
1743-422x-10-220 10.1128/EC.00373-05
Qu Z, Fu YP, Lin Y et al (2021) Transcriptional responses Segers GC, Zhang X, Deng F et al (2007) Evidence that
of Sclerotinia sclerotiorum to the infection by RNA silencing functions as an antiviral defense mech-
SsHADV-1. J Fungi 7(7):493. https://doi.org/10. anism in fungi. PNAS 104(31):12902–12906. https://
3390/jof7070493 doi.org/10.1073/pnas.0702500104
Racaniello VR, Baltimore D (1981) Cloned poliovirus Shah UA, Kotta-Loizou I, Fitt BDL, Coutts RHA (2020)
complementary-DNA is infectious in mammalian- Mycovirus-induced hypervirulence of Leptosphaeria
cells. Science 214(4523):916–919. https://doi.org/10. biglobosa enhances systemic acquired resistance to
1126/science.6272391 leptosphaeria maculans in Brassica napus. Mol Plant
Raco M, Vainio EJ, Sutela S et al (2022) High diversity of Microbe Interact 33:98–107. https://doi.org/10.1094/
novel viruses in the tree pathogen Phytophthora mpmi-09-19-0254-r
castaneae revealed by high-throughput sequencing of Sharma V, Colson P, Giorgi R et al (2014)
total and small RNA. Front Microbiol 13:911474. DNA-dependent RNA polymerase detects hidden
https://doi.org/10.3389/fmicb.2022.911474 giant viruses in published databanks. Genome Biol
Ran H, Liu L, Li B et al (2016) Co-infection of a Evol 6:1603–1610. https://doi.org/10.1093/gbe/
hypovirulent isolate of Sclerotinia sclerotiorum with evu128
a new botybirnavirus and a strain of a mitovirus. Virol Shi L, Wang J, Quan R et al (2019) CpATG8, a homolog
J 13:92. https://doi.org/10.1186/s12985-016-0550-2 of yeast autophagy protein ATG8, is required for path-
Randall TA, Dwyer RA, Huitema E et al (2005) Large- ogenesis and hypovirus accumulation in the chestnut
scale gene discovery in the oomycete Phytophthora blight fungus. Front Cell Infect Microbiol 9. https://
infestans reveals likely components of doi.org/10.3389/fcimb.2019.00222
phytopathogenicity shared with true fungi. Mol Plant- Silva VN, Durigon EL, Pires MDC et al (2001) Time
Microbe Interact 18:229–243. https://doi.org/10.1094/ course of virus-like particles (VLPS) double-stranded
MPMI-18-0229 RNA accumulation in toxigenic and non-toxigenic
Rigling D, Prospero S (2018) Cryphonectria parasitica, the strains of Aspergillus flavus. Braz J Microbiol 32:56–
causal agent of chestnut blight: invasion history, popu- 60. https://doi.org/10.1590/
lation biology and disease control. Mol Plant Pathol s1517-83822001000100013
19(1):7–20. https://doi.org/10.1111/mpp.12542 Sinden JW, Hauser E (1950) Report of two new mush-
Roossinck MJ (2014) Metagenomics of plant and fungal room diseases. Mushroom Sci 1:96–100
viruses reveals an abundance of persistent lifestyles. Smart CD, Yuan W, Foglia R et al (1999) Cryphonectria
Front Microbiol 5:767. https://doi.org/10.3389/fmicb. hypovirus 3, a virus species in the family hypoviridae
2014.00767 with a single open reading frame. Virology 265:66–73.
Ruiz-Padilla A, Rodriguez-Romero J, Gomez-Cid I et al https://doi.org/10.1006/viro.1999.0039
(2021) Novel mycoviruses discovered in the Song HY, Kim N, Kim DH, Kim JM (2020) The PoV
mycovirome of a necrotrophic fungus. MBio 12:(3). mycovirus affects extracellular enzyme expression and
https://doi.org/10.1128/mBio.03705-20 fruiting body yield in the oyster mushroom, Pleurotus
Sasaki A, Kanematsu S, Onoue M et al (2007) Artificial ostreatus. Sci Rep 10(1). https://doi.org/10.1038/
infection of Rosellinia necatrix with purified viral s41598-020-58016-4
particles of a member of the genus mycoreovirus Sood M, Kapoor D, Kumar V et al (2020) Trichoderma:
reveals its uneven distribution in single colonies. The “secrets” of a multitalented biocontrol agent.
5 Viruses that Affect Phenotype and Fitness of Fungi 143

Plants -Basel 9(6). https://doi.org/ 10.3390/ small RNAs. J Gen Virol 96:714–725. https://doi.org/
plants9060762 10.1099/jgv.0.000003
Stanway CA, Buck KW (1984) Infection of protoplasts of Van Alfen NK, Jaynes RA, Anagnostakis SL, Day PR
the wheat take-all fungus, Gaeumannomyces graminis (1975) Chestnut blight: Biological control by transmis-
var tritici, with double-stranded-RNA viruses. J Gen sible hypovirulence in Endothia parasitica. Science
Virol 65:2061–2065. https://doi.org/10.1099/0022- 189(4206):890–891. https://doi.org/10.1126/science.
1317-65-11-2061 189.4206.890
Stauder CM, Nuss DL, Zhang D-X et al (2019) Enhanced VanDerLende TR, Harmsen MC, Wessels JGH (1994)
hypovirus transmission by engineered super donor Double-stranded RNAs and proteins associated with
strains of the chestnut blight fungus, Cryphonectria the 34 nm virus-particles of the cultivated mushroom
parasitica, into a natural population of strains Agaricus bisporus. J Gen Virol 75:2533–2536. https://
exhibiting diverse vegetative compatibility genotypes. doi.org/10.1099/0022-1317-75-9-2533
Virology 528:1–6. https://doi.org/10.1016/j.virol. VanDerLende TR, Duitman EH, Gunnewijk MGW et al
2018.12.007 (1996) Functional analysis of dsRNAs (L1, L3, L5,
Sun L, Suzuki N (2008) Intragenic rearrangements of a and M2) associated with isometric 34-nm virions of
mycoreovirus induced by the multifunctional protein Agaricus bisporus (white button mushroom). Virology
p29 encoded by the prototypic hypovirus CHV1- 217:88–96. https://doi.org/10.1006/viro.1996.0096
EP713. RNA 14(12):2557–2571. https://doi.org/10. Vetukuri RR, Avrova AO, Grenville-Briggs LJ et al
1261/rna.1125408 (2011) Evidence for involvement of Dicer-like,
Sun Y, Guo M, Wang J et al (2022) Curing two predomi- Argonaute and histone deacetylase proteins in gene
nant viruses occurring in Lentinula edodes by chemo- silencing in Phytophthora infestans. Mol Plant Pathol
therapy and mycelial fragmentation methods. J Virol 12:772–785. https://doi.org/10.1111/j.1364-3703.
Methods 300:114370. https://doi.org/10.1016/j. 2011.00710.x
jviromet.2021.114370 Wagemans J, Holtappels D, Vainio E et al (2022) Going
Sutela S, Poimala A, Vainio EJ (2019) Viruses of fungi viral: virus-based biological control agents for plant
and oomycetes in the soil environment. FEMS protection. Annu Rev Phytopathol. https://doi.org/10.
Microbiol Ecol 95(9). https://doi.org/10.1093/femsec/ 1146/annurev-phyto-021621-114208
fiz119 Wang M, Wang Y, Sun X et al (2015) Characterization of
Sutela S, Forgia M, Vainio EJ et al (2020) The virome a novel Megabirnavirus from Sclerotinia sclerotiorum
from a collection of endomycorrhizal fungi reveals reveals horizontal gene transfer from single-stranded
new viral taxa with unprecedented genome organiza- RNA Virus to double-stranded RNA virus. J Virol 89:
tion. Virus Evol 6(2). https://doi.org/10.1093/ve/ 8567–8579. https://doi.org/10.1128/JVI.00243-15
veaa076 Wang J, Shi L, He X et al (2016) Comparative secretome
Taniguchi T, Palmieri M, Weissmann C (1978) Q-beta analysis reveals perturbation of host secretion
DNA-containing hybrid plasmids giving rise to pathways by a hypovirus. Sci Rep 6. https://doi.org/
Q-beta phage formation in bacterial host. Nature 274: 10.1038/srep34308
223–228. https://doi.org/10.1038/274223a0 Wang Q, Mu F, Xie J et al (2020) A single ssRNA segment
ten Hoopen GM, Krauss U (2006) Biology and control of encoding RdRp is sufficient for replication, infection,
Rosellinia bunodes, Rosellinia necatrix, and Rosellinia and transmission of Ourmia-like virus in fungi. Front
pepo: a review. Crop Prot 25:89–107 Microbiol 11:379. https://doi.org/10.3389/fmicb.2020.
Thapa V, Roossinck MJ (2019) Determinants of coinfec- 00379
tion in the mycoviruses. Front Cell Infect Microbiol Wang R, Liu C, Jiang X et al (2022) The newly identified
9. https://doi.org/10.3389/fcimb.2019.00169 Trichoderma harzianum partitivirus (THPV2) does not
Turina M, Rostagno L (2007) Virus-induced diminish spore production and biocontrol activity of its
hypovirulence in Cryphonectria parasitica: still an host. Viruses-Basel 14(7). https://doi.org/10.3390/
unresolved conundrum. J Plant Pathol 89(2):165–178 v14071532
Turina M, Prodi A, Van Alfen NK (2003) Role of the Wei S, Bian R, Andika IB et al (2019) Symptomatic plant
Mf1-1 pheromone precursor gene of the filamentous viroid infections in phytopathogenic fungi. PNAS
ascomycete Cryphonectria parasitica. Fungal Genet 116(26):13042–13050. https://doi.org/10.1073/pnas.
Biol 40:242–251. https://doi.org/10.1016/S1087-1845 1900762116
(03)00084-7 Williamson B, Tudzynsk B, Tudzynski P, van Kan JAL
Urayama S, Katoh Y, Fukuhara T et al (2015) Rapid (2007) Botrytis cinerea: the cause of grey mould dis-
detection of Magnaporthe oryzae chrysovirus 1-A ease. Mol Plant Pathol 8(5):561–580. https://doi.org/
from fungal colonies on agar plates and lesions of 10.1111/j.1364-3703.2007.00417.x
rice blast. J Gen Plant Pathol 81:97–102. https://doi. Wu M, Zhang L, Li G et al (2010) Genome characteriza-
org/10.1007/s10327-014-0567-6 tion of a debilitation-associated mitovirus infecting the
Vainio EJ, Jurvansuu J, Streng J et al (2015) Diagnosis and phytopathogenic fungus Botrytis cinerea. Virology
discovery of fungal viruses using deep sequencing of 406:117–126. https://doi.org/10.1016/j.virol.2010.
07.010
144 B. I. Hillman and M. Turina

Wu M, Zhang J, Yang L, Li G (2016) RNA mycoviruses Yu X, Li B, Fu Y et al (2013) Extracellular transmission of

and their role in Botrytis biology. In: Botrytis–the a DNA mycovirus and its use as a natural fungicide.
fungus, the pathogen and its management in agricul- PNAS 110(4):1452–1457. https://doi.org/10.1073/
tural systems. Springer, pp 71–90 pnas.1213755110
Xia ST, Xu Y, Hoy R et al (2020) The notorious soilborne Yu L, Sang W, Wu MD et al (2015) Novel hypovirulence-
pathogenic fungus Sclerotinia sclerotiorum: An update associated RNA mycovirus in the plant-pathogenic
on genes studied with mutant analysis. Pathogens 9(1): fungus Botrytis cinerea: molecular and biological char-
27. https://doi.org/10.3390/pathogens9010027 acterization. Appl Environ Microb 81(7):2299–2310.
Xie J, Wei D, Jiang D et al (2006) Characterization of https://doi.org/10.1128/aem.03992-14
debilitation-associated mycovirus infecting the plant- Yun S-H, Lee SH, So K-K et al (2016) Incidence of
pathogenic fungus Sclerotinia sclerotiorum. J Gen diverse dsRNA mycoviruses in Trichoderma spp.
Virol 87:241–249. https://doi.org/10.1099/vir.0. causing green mold disease of shiitake Lentinula
81522-0 edodes. FEMS Microbiol Lett 363(19). https://doi.
Xie Y, Wang ZF, Li K et al (2022) A Megabirnavirus org/10.1093/femsle/fnw220
alleviates the pathogenicity of Fusarium Zhang L, Churchill ACL, Kazmierczak P et al (1993)
pseudograminearum to wheat. Phytopathology 112: Hypovirulence-associated traits induced by a
1175–1184. https://doi.org/10.1094/phyto-03-21- mycovirus of Cryphonectria parasitica are mimicked
0126-r by targeted inactivation of a host gene. Mol Cellular
Xu Z, Wu S, Liu L, Cheng J et al (2015) A mitovirus Biol 13:7782–7792
related to plant mitochondrial gene confers Zhang R, Hisano S, Tani A et al (2016a) A capsidless
hypovirulence on the phytopathogenic fungus ssRNA virus hosted by an unrelated dsRNA virus. Nat
Sclerotinia sclerotiorum. Virus Res 197:127–136. Microbiol 1(1):15001. https://doi.org/10.1038/
https://doi.org/10.1016/j.virusres.2014.12.023 nmicrobiol.2015.1
You JQ, Zhou K, Liu XL et al (2019) Defective RNA of a Zhang N, Luo J, Rossman AY et al (2016b) Generic names
novel mycovirus with high transmissibility detrimental in Magnaporthales. IMA Fungus 7:155–159. https://
to biocontrol properties of Trichoderma spp. doi.org/10.5598/imafungus.2016.07.01.09
Microorganisms 7(11). https://doi.org/10.3390/ Zhang TT, Zeng XX, Cai XY et al (2018) Molecular
microorganisms7110507 characterization of a novel double-stranded RNA
Yu HJ, Lim D, Lee HS (2003) Characterization of a novel mycovirus of Trichoderma asperellum strain
single-stranded RNA mycovirus in Pleurotus JLM45-3. Arch Virol 163:3433–3437. https://doi.org/
ostreatus. Virology 314:9–15. https://doi.org/10. 10.1007/s00705-018-3988-2
1016/s0042-6822(03)00382-9 Zhang H, Xie J, Fu Y et al (2020) A 2-kb mycovirus
Yu J, Kwon SJ, Lee KM et al (2009) Complete nucleotide converts a pathogenic fungus into a beneficial endo-
sequence of double-stranded RNA viruses from Fusar- phyte for Brassica protection and yield enhancement.
ium graminearum strain DK3. Arch Virol 154:1855– Mol Plant 13:1420–1433. https://doi.org/10.1016/j.
1858. https://doi.org/10.1007/s00705-009-0507-5 molp.2020.08.016
Yu X, Li B, Fu Y et al (2010) A geminivirus-related DNA Zhang YJ, Gao J, Li Y (2022) Diversity of mycoviruses in
mycovirus that confers hypovirulence to a plant patho- edible fungi. Virus Genes. https://doi.org/10.1007/
genic fungus. PNAS 107(18):8387–8392. https://doi. s11262-022-01908-6
org/10.1073/pnas.0913535107
 Lichens
6
Martin Grube

Abstract 6.1 Introduction

Lichenized fungi initiate their symbiotic
structures from microscopic stages after recog- Lichens are a unique group of organisms in many
nition of compatible algae. The partnerships ways. They are perhaps the only fungal life forms
ultimately emerge as complex macroscopic that can be recognized when looking down
phenotypes which are unrivaled in the fungal through the windows of a flying airplane. Lichen
kingdom by their resilience and durability. symbioses can develop ground-covering carpets
This chapter presents an overview of lichen in landscapes where plant vegetation is open or
symbioses and covers the morphology and absent. In the boreal tundra, along rocky coasts, in
systematics of the fungal phenotypes, as well alpine habitats, or in coastal deserts they may
as their associations with diverse photobionts. represent the dominating life form (Fig. 6.1).
This is followed by a coarse overview of eco- Although it is impossible to get a precise figure,
physiology and the secondary chemistry. A and only a rough estimate claims 8% coverage of
special focus is given to the diversity of and the Earth’s land surface (Larson 1987), lichens
the interactions with additional contribute substantially to biosphere processes
microorganisms. Finally, a few comments on and food chains (Elbert et al. 2012; Asplund and
the effects of pollution and environmental Wardle 2017).
change point to the usefulness of lichens as In earlier days of systematics, lichens were
bioindicators. classified as a separate taxonomic group:
“Lichenes,” but the etymology of this term is
Keywords not entirely clear. The Greek word λειχην (as it
appears in Dioscorides’ works), possibly derived
Symbioses · Morphology · Mycobiont · from λειχω (Latin lingo), means to lick up
Photobiont · Microbiome or “eating around itself,” whereas the word λιχεin
has the meaning of “poor” or “little.” Whether
Linnaeus encapsulated this association when call-
ing lichens “rustici pauperrimi” (“peasants”) of
vegetation is unclear, but he may have
appreciated lichens more had he known that
each of them represents a pocket-sized
ecosystem.
M. Grube (✉)
Institute of Biology, University of Graz, Graz, Austria
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 145
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_6
146 M. Grube

Fig. 6.1 (a) Alpine landscape tinged by lichens. Switzerland; (b) large thallus (c. 1 m diam.) of Hypogymnia physodes,
Sweden. Photographs: M. Grube

more microbial photosynthetic partners

6.2 What Are Lichen Symbioses?
(photobionts) and an indeterminate number of
other microscopic organisms living in association
The recognition of lichens as a dual association
with the fungus and algae (Hawksworth and
between a fungus and an alga is attributed to
Grube 2020). In most cases, the fungus acts as
Simon Schwendener, who initially announced
the main partner to provide structure. The symbi-
his discovery in a talk for the annual general
otic phenotype results from the interaction with
meeting at the Swiss Natural History Society in
the microbial photosynthetic partner, required for
1867, and later summarized it in his publication
the development of fungal sexual structures. Nev-
(Schwendener 1869). He concluded that lichen-
ertheless, the participating fungus may grow sep-
forming fungi are “parasites, although with the
arately under certain conditions—including
wisdom of statesmen,” with their algal partners as
axenic cultivation (and the same holds true for
“helotes,” the class of slaves in ancient Sparta.
the algal partners). We may call the fungus a
The term symbiosis (as “Symbiotismus”) was
physiologically facultative biotroph, while the
later introduced to biology by Albert Bernhard
resulting symbiotic “lichen” phenotype results
Frank (1876), with lichens as an example. After
from ecologically obligate biotrophy of the
vigorous rejection of the symbiotic hypothesis by
lichen-forming fungus (Honegger 2012). As the
many contemporary lichenologists, it took
classification of lichen-forming fungi is fully
decades until lichens were classified as fungal
integrated into the fungal systematics as a
life forms and arranged within the taxonomic
whole, we consider the lichen symbiosis as a
system of fungi. In textbooks, they are commonly
fungal lifestyle. Therefore, lichens also can be
described as a partnership of one fungus with an
described as fungi that form self-sustained
algal partner (considering also cyanobacteria
ecosystems containing an extracellular arrange-
within the algal lifestyle). While this view has
ment of one or more microbial photosynthetic
not changed much, evidence shows that lichens
partners and an indeterminate number of other
evolved as open systems and can be interpreted as
microscopic organisms. All organisms within
miniature ecosystems, which include a variety of
the symbiosis retain their independent taxonomic
organisms operating at different trophic levels
names, and the collective association itself has no
(Farrar 1976). A lichen can therefore be regarded
separate name.
as a self-sustaining ecosystem formed by the
interaction of a fungus (mycobiont) and one or
6 Lichens 147

Various cases of algal–fungal interactions are associations, is missing. The younger (micro-)
not recognized as lichen symbioses, including fossils of lichens from the Lower Devonian
cases where photoautotrophs are harbored inside Welsh Borderland are approximately 415 Ma
fungal cells. For example, Mortierella elongata old (Honegger et al. 2013). They already show
(Mucoromycota), which takes up algae of leaf-like thalli with characteristic internal stratifi-
Nannochloropsis oceanica (Du et al. 2019) in its cation. This level of differentiation is also found
hyphae or Geosiphon pyriforme in younger fossils of lichens, some of which can
(Glomeromycota) containing Nostoc even be determined to genus level (cited in
cyanobacteria in bladder-like swollen fungal Honegger 2012).
cells (Schüßler and Kluge 2001). Other examples
of fungal–algal associations are harder to distin-
guish from lichens as defined above. Loose 6.3 The Lichen Phenotype
fungal–algal associations have sometimes been
dubbed “primitive” or “borderline lichens” The formation of more or less sealed, hydro-
(Kohlmeyer et al. 2004). In such cases, the fungal elastic layers of vegetative mycelium appears to
partners do not develop a well-differentiated cor- be a key evolutionary step for developing a lichen
tical layer or other fungal structures considered phenotype. Usually, the fungus tightly wraps
characteristic of lichen thalli, but they develop algal colonies, which enforces functional coher-
ascomata and conidiomata following ence and lichen thallus integrity. A tight closure
non-pathogenic growth on certain types of algae of mycelial structures is achieved when fungal
(as in e.g., Collemopsidium spp., Mastodia hyphae glue together by their more or less
tessellata, Trizodia acrobia). Others can live gelatinizing outer cell walls, which creates a
either as saprotrophs on bark or as primitive structure where cells are embedded in a joint
lichens depending on the details of substrate matrix. The gelatinizing outer cell wall material
conditions. This phenomenon has been named has been named variably by different authors
“optional lichenization” by Wedin et al. (2004). “Kittsubstanz” (cementing substance)
Also in these cases, characteristic thallus by Peveling (1970), “gelatinous matrix” by
structures are hardly developed. Comparing the Ahmadjian (1993), “conglutinate zone” by
genomes of such poorly developed lichen-like life Honegger (1991), or “extracellular interaction
forms with fully developed lichen phenotypes matrix” (Spribille et al. 2020). The latter term
could provide insights into the genomic architec- included the potential roles of additional
ture required for forming lichens. microorganisms in building this structure.
It has often been claimed that lichens are very Spribille et al. (2020) provided a comprehensive
old fungal lifestyles. Whether fossils found in summary about the current state of knowledge
c. 600 Ma phosphorites of the Doushan Forma- about polysaccharides in lichens. It still needs to
tion are primitive lichens is unclear (Yuan et al. be determined whether proteins targeting cell wall
2005). It has been suggested that they represent polysaccharides could eventually play a role in
fungal interactions with benthic marine the functioning of the intercellular matrix (e.g., in
cyanobacterial colonies. Using time-calibrated cell wall remodeling or as carbohydrate-binding
phylogenies of ascomycete fungi and algae, proteins). The tissue-like functional integration of
focusing on lineages with lichen symbionts, fungal plectenchyma, by conglutination by
Nelsen et al. (2020) estimated ages of several polysaccharides of the outer cell walls, is other-
interacting clades and found out that fungal wise well known from the excipula of fungal
origins of lichenization must have occurred soon sporocarps. Since such sporocarp structures are
after the emergence of land plants known from groups which share ancestry with
(tracheophytes). Clear fossil evidence for this those that contain complex lichen thalli (Díaz-
step, which may inform hypotheses of how lichen Escandón et al. 2022), it is likely that basic onto-
thalli evolved from unspecific fungal–algal genetic processes evolved before lichenization
148 M. Grube

and progressively adapted to form thalline cortex recognize compatible photosynthetic partners by
structures as well. the shape of their cells (Ahmadjian and Jacobs
The proper formation of the extracellular 1981; “thigmotropism hypothesis”). Joneson and
“glue” to establish lichen thalli requires interac- Lutzoni (2009) found no support for this hypoth-
tion with compatible photobionts. Resynthesis esis using glass beads as algal “dummies.” Based
experiments have therefore been conducted to on these results, Joneson et al. (2011) introduced
better understand the processes of interplay a “signalling hypothesis” after they found that
between algae and fungi. These experiments between 11 and 28% of mycobiont and
showed that associations with other than the orig- photobiont genes, including three fungal lipases,
inal algae lead to only loose or temporary attach- are upregulated in the “pre-contact” and the
ment of fungi (e.g., Ahmadjian et al. 1980; “contact stage” prior to physical contact of the
Schaper and Ott 2003). Resynthesis experiments symbionts. Fungal genes upregulated in
in agar plates must nevertheless be interpreted cocultures of Cladonia grayi with its algal part-
with caution, because apart from the identity of ner Asterochloris glomerata include membrane
the photobiont, medium composition and growth transporters, secreted hydrolases, and small
conditions also play a substantial role in the proteins, as well as a specific ribitol transporter
ontogeny of lichens. Mycobionts tend to develop (Armaleo et al. 2019). The latter is of relevance,
only irregular cell clusters in standard culture as it has been shown that the polyol ribitol
media, and even co-culture with the native enhances both growth of mycobionts and their
photobiont does not generally lead to the devel- mucilage production (Meeßen et al. 2013). Inter-
opment of native thallus structures. As an alterna- estingly, ribitol is not secreted in aposymbiotic
tive to growth on agar media, Stocker-Wörgötter cultures, suggesting more complex upstream sig-
(2001) used sterilized soil to successfully naling processes. Further research of ontogenetic
re-establish the native phenotype of other lichens processes ought to consider the role of
starting with axenically cultured symbionts. fluctuating hydric conditions in the natural habi-
However, combined with appropriate culturing tat, since it has been shown that such fluctuations
conditions (e.g., under nutrient poor or changing contribute to algal cell wall remodeling
light conditions) the formation of layered thallus (González-Hourcade et al. 2020), and possibly
structures is possible in certain species. Recently, alter the transmission of signaling metabolites
Kono et al. (2020) managed to grow the native as well.
phenotype of Usnea hakonensis axenically to Current literature suggests that certain groups
study upregulated sets of fungal and algal genes of molecules (including fungal lectins and algal
in a symbiotic state. They found evidence for cyclic peptides) are correlated with early contact
various processes involved in symbiotic estab- of symbionts, while others (e.g., phytohormones
lishment, including cell wall remodeling, produc- and molecules involved in carbon exchange) are
tion of hydrophobins (which seal an apoplastic important throughout all stages of lichen synthe-
continuum between interacting cells of fungi and sis. In the fully formed thallus, specialized (tradi-
algae) and symbiosis-specific nutrient flow tionally called “secondary”) lichen metabolites
(including polyol transporters). and mineral nutrition (via substrate, microbial,
The required sequential process of or airborne) stabilize the thallus functionality
lichenization involves five distinguishable steps: (Pichler et al. 2023). Poorly water-soluble
(1) the “pre-contact stage,” (2) the “contact specialized metabolites can accumulate as sub-
stage,” (3) “envelopment” of algal cells by the stantial amounts of crystalline extracellular
fungus, (4) their “incorporation” into a deposits on the surface of hyphae, particularly in
pre-thallus, and (5) “differentiation” into a thal- the peripheric cortex where they act as light
lus. It was originally proposed that mycobionts filters, among other functions.
6 Lichens 149

6.4 Morphologies shear forces of swelling and shrinking caused by

and Growth Types changing water content. These forces commonly
induce the development of fissures in the thalli,
Lichens display diverse morphological shapes which give rise to the formation of areoles. These
and growth styles. Here the focus is given to small thallus patches can become physiologically
ascomycetous lichens, with thalli that may be independent (removal of an areole does not influ-
coarsely classified as brush-like, crust-like, or ence the vitality of neighboring areoles). The
leaf-like forms (Grube and Hawksworth 2007). distinct fissures between the areoles seen in dry
This is an arbitrary grouping, since there is much stages usually close tightly when thalli of these
greater diversity when studying the details of types are irrigated. The formation of crustose
lichen growth forms. In the following, I would thalli with areole formation is correlated with
prefer to primarily distinguish lichen growth the development of a specifically composed cor-
types which are surface-attached (including tex layer. This type of fungal layer contains the
those penetrating the substrate surface) and the remnants of algal cell walls in the intercellular
remainder, which develops surface-detached matrix between living or decaying fungal cells
forms of thalli. Basidiomycetous lichens have (phenocortex, e.g. in Rhizocarpon geographicum,
thalli which are constructed in simpler ways and Fig. 6.3a, c, e). Algal cell walls are pushed toward
lack the typical stratification found in many asco- and into the upper cortex from the algal layer,
mycetous lichens (Oberwinkler 2012). Most where new algal cells are produced by autospore
could be assigned to crustose types of thalli and formation.
consist of small clusters of algal cells surrounded The anchoring of crustose and squamulose
by fungal hyphae. In other cases (Dictyonema), thalli in the substrate is accomplished either by
the thallus is apparently derived from the hyphae produced over the whole lower surface
basidiocarps, and thus superficially resembles (such as a rhizohyphal felt, which is produced by
foliose thalli (Dal-Forno et al. 2013). individual hyphae extending into the soil sub-
strate) or by central rhizinae (when hyphae
bound together in a root-like structures).
6.5 Surface-Attached: Crustose
Lichens
6.6 Surface-Detached Thalli:
Crustose thalli (Fig. 6.2 a–e) include both, more Foliose and Fruticose Lichens
or less mycelial forms that develop within the
substrate and those with the main body mass While crustose lichens still contain decaying algal
appearing on top of the substrate, where they cell walls in the cortex, surface-detached lichens
often develop a closed upper fungal layer (upper exclude algal cells from their upper cortex, and
cortex). Species developing within the substrate, the uniform extracellular matrix apparently
such as rocks (mostly calcareous rocks) or bark, represents a mechanical reinforcement allowing
usually lack such an upper cortex. Crustose thalli them to grow in three dimensions away from the
attach directly to the substrate with their entire substrate or as flat branches over the substrate
lower surface and usually develop over or inside surface. Such growth strategies are found in unre-
more or less stable substrates. Microstructure, lated lineages of lichenized fungi and have also
chemical cues, mechanical parameters, and evolved among closely related groups of lichens
water holding capacity could be factors determin- (Grube and Arup 2001). The upper cortex layer in
ing which species can colonize the substrate. surface-detached lichens seems more coherent
Episubstratic crustose lichens develop typical because fragmentation into areoles does not
growth pattern as their symbiotic structures are occur (e.g., in Rusavskia elegans, Fig. 6.3b, d,
prone to poikilohydric conditions and recurrent f). By exclusion form the cortex, the walls of algal
150 M. Grube

Fig. 6.2 Crustose (a–e) and microfilamentous (f) lichens: chrysocephala (Austria), a pin lichen, bar = 250 μm, (e)
(a) Pyrenula nitida (Austria), bar = 500 μm; (b) Sporastatia testudinea (Austria), bar = 1 mm, (f)
Leucocarpia biatorella (Sweden), bar = 250 μm, (c) Cystocoleus ebeneus (Austria), bar = 1 mm. Photographs:
Vezdaea aestivalis (Austria), bar = 1 mm (d) Chaenotheca M. Grube

cells often keep their shape in the algal layer even walls is due to loss or down-regulation of fungal
after the cell content decayed, reminiscent of a carbohydrate metabolizing genes.
sort of cell “ghosts” cells in that layer. It is still With increased stability and flexibility, the
unknown whether the persistence of algal cell eucortex facilitates the functional integrity needed
6 Lichens 151

Fig. 6.3 From surface-attached to surface-detached the entire lower surface and has a phenocortex, whereas
lichens, Rhizocarpon geographicum (a: habit, c: section, R. elegans develops a cortex on the lower side, has an
e: section stained in alkaline Congo red) and Rusavskia eucortex on the upper side, and detaches from the surface
elegans (b: habit, d: section, f: section stained in alkaline with its lobes, a, b: bar = 1 mm, c: bar = 100 μm, d:
Congo red) R. geographicum is attached to the rock with bar = 250 μm, e, f: bar = 50 μm. Photographs: M. Grube

for development of the larger thalli of morpholog- vegetative structures in the kingdom of fungi.
ically diversified foliose (leaf-like) and fruticose Leaf-like (foliose) thalli develop
(shrub-like) macrolichens. Many of these lichens flattened branches of diverse shape (Fig. 6.4a).
also have internal hydrophobic layers to facilitate In most cases, these grow at the tips of the
gas exchange. There are also morphological branches, while older parts tend to cease growth
intermediates between foliose and crustose and sometimes degenerate at their rear ends (e.-
growth, notably lichens that develop placodioid g., Arctoparmelia centrifuga, Brodoa
thalli. Their lower sides partially detach from the intestiniformis, and others which can form con-
substrate at the thallus periphery where they also centric rings). Foliose lichens represent the typi-
develop cortex-like plectenchyma on the lower cal stratified anatomy of lichens illustrated in
side upper cortex of such transitional textbooks with an upper cortex protecting the
morphologies is composed of a mosaic of adja- algal layer below, a loose medulla delimited by
cent portions with either pheno- or eucortex.” a closed lower cortex layer, which connects to the
The foliose and fruticose lichens, often called substrate by rhizines as holdfast structures.
“macrolichens,” develop the most complex Besides rhizines extending from the lower surface
152 M. Grube

Fig. 6.4 Foliose and terete, surface-detached lichens, (a) (California), bar = 5 mm; (c) fruticose-terete lichen Cladia
foliose “lung lichen” Lobaria pulmonaria (Austria), retipora with net-like growth (New Zealand), bar = 5 mm.
bar = 5 cm; (b) foliose-terete “California state lichen” Photographs: M. Grube
Ramalina menziesii with typical net-like expansions

of the thalli, many other structures of taxonomic conglutinated fungal hyphae. Thalli of
value can be formed at the periphery (e.g., cilia Dermatocarpon are often also called umbilicate,
which are thick hairs formed by hyphal strands yet they rather extend laterally from a holdfast
emanating from the thallus margins) or on the and are better called monophyllous. In contrast to
surface of thalli, ranging from hair felts to diverse most other foliose and terete lichens, the growth
structures for vegetative dispersal, such as isidia of the thalli in Umbilicariaceae can be more or
(Fig. 6.5a, b) or soredia (Fig. 6.5c, d). Also, less irregular or “diffuse,” (e.g., Lasallia
special structures for gas exchange, such as pustulata) with its patchy thallus protuberances;
pseudocyphellae (patches on upper or lower growth is, however, inconspicuous at the thallus
surfaces where loose hyphae replace the tight cor- tips, where thalli may break up or form lacunae
tex for improved gas exchange in many genera), and gaps. Sometimes the term intercalary is used
cyphellae (distinct holes on the lower surfaces as in synonymously for this type of growth, but this
Sticta spp.), or holes in the upper surfaces of the implies discrete non-apical growth and does not
thallus (e.g., Hypogymnia spp.) are known. apply here.
Umbilicate forms (Umbilicaria species) repre- Terete (shrubby) growth forms vary from
sent a special case of foliose growth. Umbilicate upright, brush-like forms to pendant beard-like
thalli differ by being connected to the substrate forms (Usnea). They usually lack upper and
with a single central, rather rigid holdfast struc- lower side of the thalli, and their thalli look rather
ture, the umbilicus, which is made of strongly similar from all sides. Typically, developed terete
6 Lichens 153

Fig. 6.5 Vegetative dispersal units, (a) Pseudevernia fuscescens (Austria), soralia on surface of terete thallus,
furfuracea (Austria), foliose thallus surface with isidia, bar = 5 mm; (d) soralium with tiny soredia, bar = 150 μm.
bar = 1 mm; (b) isidia detail, bar =200 μm; (c) Bryoria Photographs: M. Grube

forms are found, e.g., in the genera Alectoria,

6.7 Jelly Lichens
Bryoria, Usnea, etc. In certain genera, the thalli
may also become more leaf-like, and then the
Lichen thalli are rather brittle when dry and flexi-
distinction between terete and foliose growth is
ble when wet. A special case is the jelly lichens,
blurred (e.g., Ramalina). Perhaps the most spec-
which become slippery and increase significantly
tacular thalli in that genus are formed by
in size when irrigated (Fig. 6.6a, b). Most of them
Ramalina menziesii, occurring along the North
(Collemataceae) are associated with
American Pacific coast and perhaps the fastest-
cyanobacteria and most of their gelatinous inter-
growing lichen species (Fig. 6.4b). It has flat
cellular matrix originates from the photobionts.
branches composed of net-like units that develop
Jelly lichens generally do not have hyphae with
by diffuse expansion of perforated tissue pro-
hydrophobic surfaces and lack internal air-filled
duced at the growing branch apex (Sanders and
cavities in the thalli. In contrast to typical green
Ascaso 1995). Spectacular net-like growth
algal lichens, they often lack a distinct functional
patterns are also known from the southern
layering. Leptogium species develop a cortex of a
hemispheric genus Cladia (see Fig. 6.4c). More
single layer of fungal cells, but the thalli of
complex thalli are known from Cladonia species,
Collema lack such a cortex. Although the fungal
which are dimorphic and composed of a scaly
hyphae merely extend between the predominant
leaf-like primary thallus and terete outgrowths
algal polysaccharide matrix, the mycobiont has an
called podetial.
154 M. Grube

Fig. 6.6 Massive swelling of a jelly lichen, Enchylium polycarpon (Austria) with cyanobacterial photobiont (Nostoc
sp.), when wet, (a) dry stage, (b) wet stage, bars = 1 mm. Photographs: M. Grube

influence on the overall thallus shape, which loosely interwoven and hydrophobic hyphae
differs from free-living cyanobacterial colonies. developing around small colonies of green
Apart from these cyanobacterial forms, there are a algae. These lichens are often sterile and are dis-
few more or less jelly-like green algal lichens, but persed by regular fragmentation of the thallus
these form thin crusts on soil, mosses, or other (which usually allows for vegetative dispersal).
lichens (e.g., genera Epigloea, Thrombium). Thalli that generally do not fragment for dispersal
but which have a hydrophobic “fluffy” internal
structure without an upper cortex are called
6.8 Other Growth Types: byssoid. Such thallus types are frequently found
Microfilamentous, in the tropics (e.g., Cryptothecia, Sagenidiopsis,
Microglobose, Leprose, Tania). Occasionally they break apart into irregu-
and Byssoid Lichens lar clusters and are then referred to as floccose
thalli.
Apart from the types presented before, there are Thallus morphology has always played an
other forms which are composites of microscopic important role in lichen classification. Molecular
thallus structures. The shapes of phylogenetic analyses, however, data revealed
microfilamentous lichens (Fig. 6.2f) are deter- many cases of convergent evolution of similar
mined by the photobiont partners which are fila- growth styles, either by emergence of surface-
mentous forms of cyanobacteria (e.g., Scytonema detached foliose or terete forms from different
in Pyrenothrix, Scytonema in Ephebe) or crustose ancestors within closely related lineages
trentepohlioid green algae (Trentepohlia in (Grube and Hawksworth 2007), or as similar
Cystocoleus, Racodium, or many Coenogonium morphologies that evolved in unrelated lineages
species). Microglobose lichens are composed of (Grube and Kantvilas 2006; Muggia et al. 2011).
globose colonies of green algae packed with a
coherent, usually single layer of fungal cells
(Micarea or Vezdaea). Similar surface-attached 6.9 Vegetative Dispersal
forms are also developed by certain basidiomyce-
tous lichens (e.g., Lichenomphalia umbellifera Numerous lichens can propagate by specialized
and Multiclavula mucida). These mostly hydro- vegetative diaspores, which combine fungal and
philic thalli contrast with leprose lichens, which algal symbionts in tiny structures which may eas-
have a woolly or powdery appearance, due to ily separate from the parental thallus. This form of
6 Lichens 155

propagation keeps the partners together and 6.11 Lichen Mycobionts and Their
allows them to start thallus reproduction without Systematics
a risky search for germinated fungal spores of
compatible algal symbionts in the environment. The historical development of lichen systematics
Because vegetative dispersal units are heavier has been led by methodological advances, which
than fungal spores alone, they are predominantly initiated periods of taxonomic rearrangements. In
involved in local dispersal (Walser 2004), where the early days of lichen taxonomy, only bare eyes
successful combinations of symbionts in parental and simple lenses helped to classify lichens by
thalli can easily reproduce in the same habitat. external characters, while compound microscopes
The various types of vegetative propagules are facilitated the observation of anatomical
classified by their morphological details, such as characters—particularly spore characters—in the
isidia (mostly pin-like propagules with terete second half of the nineteenth century. Analyses of
organization), schizidia (scaly propagules with secondary chemistry, by thin layer chromatogra-
dorsoventral organization), soredia or other, phy in particular, were introduced in the second
more special types. Basically, soredia are formed half of nineteenth century, while the analysis of
by undifferentiated hyphae which bind a small molecular sequence data was introduced in the
number of algal cells, whereas many other forms 1990s to achieve an even more coherent phyloge-
have a covering fungal cortex layer and usually a netic framework. After algal-excluding (“fungal”)
larger population of algal or cyanobacterial cells. primers were designed (Gargas and Taylor 1992),
Certain propagules of foliicolous lichens, such as it was also possible to use extracts from entire
the stalked discs of Phyllophiale alba, or the thalli for phylogenetic approaches, avoiding the
shallow discs of Chroodiscus mirificus are unique cumbersome axenic cultivation of mycobionts. At
types of vegetative propagules. all times, technological advancements were
sparked by the hope of better understanding the
relationships of lichens and achieve a taxonomy
6.10 Conidium Formation based on their evolutionary history.
The number of independent origins of lichens
Campylidia, commonly present in Pilocarpaceae in the fungal kingdom was one of the prominent
and Ectolechiaceae (but also found in questions when molecular methods became pop-
Arthoniaceae and Monoblastiaceae), are hood- ular. In early analyses with limited sampling of
shaped conidiophores, which may also release lichenized lineages, Gargas et al. (1995)
algae, but there is no tight connection of the suggested only two lichenization events in the
photobionts with the macroconidia (Sanders Ascomycota and three such events in
2014). Beside the joint dispersal of symbionts Basidiomycota. With a more extended sampling,
there are also purely fungal forms of mitotic Lutzoni et al. (2001) found that even a single
propagation, ranging from simple formation of lichenization event could not be excluded and
conidia by hyphae at the thallus margins (e.g., suggested that important non-lichenized lineages
thallospores, not uncommon in various crust may have had lichenized ancestors (including the
lichens from arid zones) to more specialized molds in Eurotiales).
types. Diverse shapes of highly specialized The total number of lichenized species is given
conidium-producing synnemata are characteristic as 19,387 species, which are classified in
of the Gomphillaceae (Ferraro 2004). While 39 orders of fungi (Lücking et al. 2016). The
many Gomphillaceae also produce ascospores, number of lichenized species in the fungal classes
there are species in various major lineages, is unevenly distributed in the fungi. The largest
which are only known from sterile stages (e.g., classes Lecanoromycetes (15,131 species),
species of Cheiromycina, Dictyocatenulata, Arthoniomycetes (1541), but also the smaller
Reichlingia, Sporodochiolichen). Lichinomycetes (390), Candelariomycetes (76),
156 M. Grube

and Coniocybomycetes (31) contain primarily example, starting with a few genus names (Arup
lichenized species, whereas other classes such as et al. 2013), the family Teloschistaceae now
Eurotiomycetes (1203), Dothideomycetes (812), contains more than 100 genera.
Agaricomycetes (172), or Sordariomycetes Technological advances also influenced the
(1) contain a majority of non-lichenized species way in which species of lichenized fungi are
(Lücking et al. 2016). Recently, the recognized. Early concepts considered variation
Lichinomycetes have been expanded to include in the thallus shapes and colors, as well as
Candelariomycetes and Coniomycetes, as well as anatomical characters of the thallus and fruitbody
some previously unclassified non-lichenized (including spore measurements) for a phenotypic
lineages (Díaz-Escandón et al. 2022). The large species concept. Simple histochemical
number of lichenized species in Lecanoromycetes differences using Lugol’s solution on the thallus
and Arthoniomycetes correlates positively with have also played a role in some lineages (Lugol
the phenotypic complexity of their thallus organi- staining variation in the ascus walls became very
zation, which will be discussed below in greater important for recognizing higher categories in
detail. Although these classes are only distantly Lecanorales by the 1980s), and various chemical
related and possibly evolved the lichenized habit spot tests are still quick indicators for recognizing
independently, there are parallels in the construc- species (e.g., using potassium hydroxide,
tion of the symbiotic phenotype which suggest hypochloride, or p-phenylenediamine). Beside
that certain general functional constraints play a phenotypic characters, which remain important
role in the evolution of the lichen thallus. The to classify lichen species, chemical compounds
similarities involve general anatomical structure were included in species circumscriptions since
and the involvement of the same secondary the 1960s in the framework of chemosystematics.
metabolites in these unrelated lineages. Chemical variation, however, is not trivial to
The remainder of lichens are found in few interpret, as the compounds involved have a vary-
lineages of the primarily non-lichenized ing degree of phylogenetic conservation in
basidiomycetes, in the orders Atheliales, lichens. Compounds present in the upper cortex
Cantharellales, Corticiales, and Agaricales. Spe- are often more conserved than those located
cies diversity in this groups still seems to be below the algal layer. The compounds below the
underestimated. Lücking et al. (2014) provided algal layer are often chemically related. Variation
evidence that Dictyonema glabratum, once in compound presence was often considered as
thought to consist of a single species, is much species-specific, or specific for subspecific taxa.
more diverse than expected. They distinguished However, when chemical patterns were in conflict
at least 126 species and predicted more than 400. with inference from phenotypic or molecular
Molecular data have elucidated the large-scale sequence data, classification remained
evolutionary relationships of lichens, yet controversial.
reconciling phylogenetic insights with taxonomy Soon after first attempts to detect genetic vari-
became challenging, in particular at the level of ation using DNA hybridization (Blum and
genera, which were previously circumscribed by Kashevarov 1986), PCR methodology and DNA
growth form and other externally visible sequencing were applied to study the
characters. Many were shown to be relationships of lichenized fungal species. In the
non-monophyletic, which required segregation early studies, however, the low number of
and renaming of lineages. In addition, analyses individuals sampled with only one marker (usu-
of traditionally species-rich genera resulted in ally ITS) was not sufficient to clearly resolve
substantial taxonomic splitting. Both lead to a species relationships. With the analysis of several
recent, perhaps too ambitious, proliferation of independent loci, a gene genealogical approach
genus names. Large traditional genera, such as helped to recognize species in the morphologi-
Lecanora, Arthonia, Caloplaca, or Graphis, are cally variable species complex of Letharia
now split into dozens of smaller entities. For vulpina (Kroken and Taylor 2001). Another
6 Lichens 157

approach used large numbers of samples from lichenized stages on the phylloplane is multicel-
numerous populations in a population genetic lular and similar to those of nearby free-living
approach to draw attention to the slow genetic forms. The growth type of Trentepohlia remains
drift in lichens (Printzen et al. 2003). The thread-like in members of the lichen genus
resulting ancestral polymorphisms can be prob- Coenogonium found on bark in the understory
lematic for application of sequence-based species of tropical rainforests. In other tropical species,
recognition (e.g., DNA barcoding). For practical the thread-like morphology of Trentepohlia may
reasons, the systematics of species in lichenized be modified by the fungus (e.g., variations
fungi still relies on a combination of phenotypic between thallus and isidia in the foliicolous
and genotypic characters. In a few recent cases, Chroodiscus mirificus, Lücking and Grube
the analysis of whole fungal genomes used to 2002). Multicellularity is otherwise more or less
address the relationships of closely related species absent in the lichenized state in bark- or rock-
has shown substantial variation across genomes inhabiting or in surface-detached lichens with
and evidence for hybridization in lichen fungi Trentepohlia symbionts. In these cases, only few
(Keuler et al. 2020). Hybridization and introgres- concatenated or individual cells remain as
sion may be largely underestimated factors in remnants of multicellularity. Notably, Resl et al.
shaping complexes of closely related lineages of (2022) found more genes for carbohydrate-active
lichen species in which the separation of clear-cut enzymes (CAZymes) in fungal genomes of
species has always been a challenge (Fernandez- lichens with Trentepohlia than in those
Mendoza et al. 2023). associating with unicellular green algae. Could
the larger number of CAZymes be linked to an
ability to gain control over the algal growth
6.12 Lichen Photobionts and Their architecture?
Systematics Compared with their fungal partners, algal
partners are much less diversified morphologi-
About 90% of the lichenized fungi are associated cally patterns. In their review, Sanders and
with eukaryotic algae, and of these, almost all Masumoto (2021) included about 50 genera of
belong in equal numbers in the classes algal and cyanobacterial genera as lichen
Trebouxiophyceae and Ulvophyceae of the partners. Since lichen algae are largely micro-
Chlorophyta. Exceptions are the partners of sev- scopic organisms without distinctive features,
eral lichens known or suspected to be members of phenotypic classification has always been diffi-
Chlorophyceae (Sanders and Masumoto 2021). cult. Moreover, the algae lack sexuality and
Photobionts of the Trebouxiophyceae are usually change their ultrastructure in the lichenized state
unicellular and rarely observed in free-living with variations depending on the fungal partner.
stages; they are relatives of free-living unicellular Phenotypically based classification was therefore
algae, except for Prasiola, a multicellular, leafy impossible without axenic cultures under
photobiont of the borderline lichen Mastodia standardized conditions. Meanwhile, molecular
tessellata. On the other hand, photobionts belong- sequence data have massively improved the
ing to Ulvophyceae generally form thread-like or knowledge about photobiont diversity. Recent
branched thalli in free-living stages. The most work in different algal lineages estimated sub-
prominent genus of Ulvophyceae is Trentepohlia, stantial hidden genetic diversity (e.g., Muggia
but delimitation of species and genera is not clear et al. 2014, 2018, 2020 in Trebouxia, Gustavs
(Nelsen et al. 2011). Some of the photobionts are et al. 2017 in Coccomyxa/Elliptochloris, or
assigned to the closely related genus Printzina Grube et al. 2017, Kosecka et al. 2020, Borgato
(Hametner et al. 2014a, b), those of foliicolous et al. 2022 in Trentepohliaceae). Nevertheless, we
lichens are usually assigned to Cephaleuros and are still far from describing diversity with species
Phycopeltis. The growth form of these algae in names.
158 M. Grube

6.13 Cyanobacteria 6.15 Photobiont Flexibility

Approximately 10% of lichens contain For a long time, the association of a fungus with
cyanobacteria as their primary photobiont an alga was supposed to be stable in a species.
(Rikkinen 2017). Cyanobacteria are primary Molecular studies with both crustose species
symbionts in about 50 genera and 1000 species (e.g., Blaha et al. 2006) and foliose species (e.g.,
of lichens and secondary symbionts in about Fernández-Mendoza et al. 2011; dal Grande et al.
20 genera and 500 species (Rai 2002). Most of 2018; Garrido-Benavent et al. 2020), however,
the cyanobacterial lichens include strains of the have shown that lichens are able to associate
genus Nostoc, Rhizonema, and to lesser extent, with different strains of closely related algae or
lineages of Calothrix, Fischerella (syn. cyanobacteria, respectively, depending on the
Stigonema), Scytonema, or the unicellular niche, elevation, and geographic location. Similar
Gloeocapsa, Chroococcus, Chroococcidiopsis, habitat-adapted patterns of symbiosis have also
and Anacystis, all of which are capable of fixing been observed in basidiomycetous lichens
nitrogen. Like eukaryotic photobionts, the (Gasulla et al. 2020). These results suggest that
cyanobacteria are also modified in the lichenized many widespread lichen species can adapt to
state and seem to be regulated by the fungal local conditions by association with a locally
partner both in growth form and in the frequency ideal partner. Nevertheless, specificity for the
of heterocysts (Hyvärinen et al. 2002). Whether photoautotrophic partner varies among species.
unculturable strains are also unable to grow free- There are also species which have high specificity
living in nature still needs to be studied. for their algal partners, for example Lobaria
pulmonaria, which always is found associated
with Dictyochloropsis reticulata. Whether high
6.14 Cyanobacteria as Secondary specificity coincides with ecological specializa-
Symbionts tion of lichens still needs to be explored.
Škvorová et al. (2022) showed that the promiscu-
When occurring as secondary photobionts in ity of Cladonia mycobionts with their algae is
addition to green algae, in the so-called tripartite limited by climatic factors and soil chemistry,
lichens, cyanobacteria are often contained within since most mycobionts analyzed in their study
specialized structures called cephalodia. Different could switch only between algae with similar
cephalodia of the same thallus may contain the ecological preferences. The variable extent of
same of different strains of related cyanobacteria. photobiont specificity could also influence the
Cephalodiate lichens are an example of division patterning of lichen communities (photobiont-
of labor, since the green algal photobionts pro- mediated guilds; Kaasalainen et al. 2021, Peksa
duce the gross amount of photosynthates whereas et al. 2022).
the cyanobionts in the cephalodia focus on nitro- Although it has been suspected that an individ-
gen fixation (Nash 2008), which agrees with ual thallus is not uniform with respect to the
higher heterocyst frequencies and higher rates of photobiont (e.g., by isolation of several strains
N2 fixation than found in bipartite cyanolichens. from the same lichen, Friedl 1989), the
In many species, the cephalodia are external and co-occurrence of two algae has been shown in
gall-like, and then more or less scattered on the the thallus by Casano et al. (2011) using a com-
upper or lower surface of the thallus. Sometimes bined molecular and microscopic approach. This
they are hidden internally in small packets (e.g., strategy may increase the ecological flexibility of
Lobaria and Sticta), while they form a more or lichen symbioses. The significance of this phe-
less closed layer beneath the green algal layer in nomenon remains under studied, and it is not
Solorina crocea. known if this is a transient process, a passive
phenomenon, or an actively regulated pattern of
6 Lichens 159

co-occurrence. Co-occurrence of algal strains has violaceofusca). These patterns of photobiont flex-
been studied in few species so far but has been ibility raise fundamental questions of how
found to occur with very different photobionts. partnerships and interactions are genetically con-
Henskens et al. (2012) found that cyanolichens trolled in different lineages of lichenized fungi
may also incorporate green algae in their thalli and how flexible the variation might be. It also
occasionally. raises the question of how different photobionts
Certain lichens have the ability to form thalli influence the morphology of the lichen symbioses
with entirely different types of photobionts, the and to what extent this could be taxonomically
so-called photosymbiodemes. relevant. Steinová et al. (2022) demonstrated that
Photosymbiodemes have been long known from the photobiont species involved in closely related
certain large leaf-like lichens in the family Cladonia species was a better marker for species
Peltigeraceae. The photobionts involve phenotype than the molecular marker of the fun-
cyanobacteria (Nostoc) or green algae as primary gal symbiont, distinguishing a species without
photobiont (in the latter case Nostoc colonies may soredia from those that do. Again, the specific
remain present in cephalodia). The thallus shapes photobiont seems to influence the reproductive
of photosymbiodemes may look fairly similar strategy of the fungal partner.
(Pseudocyphellaria rufovirescens—green
algal, vs. P. murrayi—cyanobacterial)
irrespective of the involved photobiont, or 6.16 The Interface of Fungi
completely different. Their representatives were With Algae
even classified in different genera of lichens
(e.g. Lobaria amplissima—green Complex haustorial structures found in certain
algal, vs. Dendriscocaulon umhausense— fungal–plant interactions are missing in lichen
cyanobacterial). The mycobiont switches fungi, and the contact of fungi with their
between different kingdoms of photobiont corre- photobionts is fairly simple; in many cases the
late with ecological preferences of the algal cell wall is not even penetrated. The main
photobionts involved (usually with flux of solutes in the apoplast seems to be driven
cyanobacterial morphs under wetter conditions) by wetting and drying cycles, whereas active
and their ability to cope with light stress. Green uptake of carbohydrates is presumable via the
algal morphs are equipped with a photoprotective immediate mycobiont and photobiont contact
xanthophyll cycle that is missing in the sites (Honegger 2012).
cyanobacterial morph (Demmig-Adams et al. Simpler, finger-shaped, transparietal
1990b). Also, there are generally differences in (intracellular) haustoria are commonly formed in
the sexual strategy of the mycobiont depending surface-attached (crustose) lichens, whereas
on the photobiont type. In phycosymbiodemes surface-detached lichens commonly develop
involving cyanobacteria and coccoid green intraparietal haustoria (Honegger 2012). Three
algae, it is usually the green algal morph which types can be distinguished: type 1 is characterized
develops fungal sporocarps. A fascinating new by appressoria and short infection pegs, which do
type of photosymbiodeme was uncovered by not penetrate the algal cell wall. Type 2 has short
Ertz et al. (2018), which is an example of a infection pegs sheathed by the algal cell wall, and
mycobiont associated with either members of type 3 develops very short infection pegs with
Trebouxiophyceae or Trentepohliaceae, includ- thin fungal cell walls at the contact site within
ing a switch of its reproduction strategy and phe- the outer cell walls of still growing algal cells.
notypic dimorphism. Lecanographa amylacea Honegger (2012) noticed coordinated growth of
(Arthoniomycetes) is sexual and lacks soredia haustoria with the developing algal cells and
with a trentepohlioid photobiont, whereas the suggested this growth pattern shifts algal cells
same fungus can form a soredial photomorph over short distances in the algal layer in
with Trebouxia sp. (formerly known as Buellia Teloschistaceae and Parmeliaceae. Softness and
160 M. Grube

more pronounced hygroscopic swelling of young depends on a finely balanced share of carbon
thallus parts (in contrast with older parts; Grube between the photobiont and mycobiont in excess
unpublished) would fit with some flexibility of of respiration (most of which can be attributed to
algal assortment in growing thalli. Appressoria the fungus). Photosynthates taken up by the fungi
seem more commonly developed in lichens with are called transfer sugars. In lichens with green
an eucortex and hydrophobic medullar layer. The algae, transfer sugars comprise acyclic
interfaces of lichens with trentepohlioid algae polyalcohols such as ribitol, sorbitol, and
have been less well studied, but early reports erythritol, whereas mycobionts in association
suggest both intraparietal and transparietal with cyanobacteria receive glucose from their
haustoria in an age-dependent pattern, suggesting partner. To finely tune this system, lichenized
that early intraparietal stages develop into fungi evolved morphological adaptations to regu-
transparietal haustoria in aging algal cells late access to light and water, and gas exchange
(Matthews et al. 1989). for the algal partner. Widespread species with
Additional interaction structures can be broad ecological preferences may optimize their
observed in lichen interactions of basidiomycetes. water relations via anatomical variations within
Most of the fungi wrap their algal partner in small species (Colesie et al. 2017). Such anatomical
globules, but they do not develop distinctly devel- differences can also lead to different organization
oped algal layers. Basidiomycetes associated with of algal layers, as shown by Vondrak and
the filamentous cyanobacterial genus Rhizonema Kubásek (2013). They observed that in morphs
form haustoria from the so-called mantle hyphae from arid or mountain regions, algal cells are
and may penetrate the cyanobacterial cell chains organized in thick stacks (algal stacks) separated
longitudinally with a central hypha (e.g., by vertical channels of likely light-transmitting
Dictyonema). Basidiomycetes associated with fungal hyphae (fungal stacks).
green algae wrap hyphae around clusters of Light is the energy source for photosynthesis,
algal cells and may eventually develop while water acts as an electron acceptor molecule
appressoria. The rather aggressive mycelia of and carbon dioxide as carbon source. Light
Athelia species, which overgrow and attack a access, as well as protection from excess light, is
wide range of lichens and algal colonies, develop regulated through positioning of the photobiont
haustoria within the parasitized algal cells. beneath a protective fungal cover, and gas
exchange in the layer algal beneath is facilitated
by water-repelling fungal hyphae. Water transfer
6.17 Ecophysiology of Lichens is also supported by various hyphal structures,
such as the distinctive rhizines and veins on the
One the most fascinating aspects of lichen biol- lower surface of Peltigera species. Variations in
ogy is the physiological integration of entirely theses phenotypic structures depend on the eco-
unrelated organisms, which react in a logical circumstances. Unsurprisingly, similar
synchronized manner to environmental morphologies may emerge convergently in unre-
conditions. The algae (and cyanobacteria) are lated groups of lichens. For instance, unrelated
kept under fungal control in lichen thalli to sus- epiphytic and epiphyllous lichens in the under-
tainably produce more photosynthates than the story of lowland tropical rainforests usually limit
algae alone require, and which are sufficient to investment in fungal biomass under the prevailing
sustain the entire symbiotic system. This is aston- conditions of dim light and high temperatures that
ishing as the photobiont contributes a minor frac- challenge the balance of carbon fixation of the
tion to the total lichen biomass, sometimes less algae against respiration of the entire lichen.
than 10%. It appears that lichens are—by evolu- Hence, the thalli of epiphyllous lichens from dif-
tionary optimization—highly efficient photosyn- ferent lineages (e.g., Arthonia and Porina) are
thetic “machines.” Growth of the lichen sometimes confined to a single layer of hyphae
symbiosis, depending on positive carbon gain, running along edges of neighboring algal threads.
6 Lichens 161

Lichens are known for their persistence under Furthermore, proteins need to be protected in the
environmental conditions that are hostile for most dry state to retain sufficient molecular order to
other life forms. Depending on fungal species, revive upon rehydration. Osmotic stress is
lichens are able to survive periodic extremes of ameliorated by the production of osmoprotectants
heat or cold, and notably, desiccation. The pro- acting as osmolytes. Various types of small
nounced desiccation tolerance of many lichens is molecules serve this purpose, including various
a key feature for surviving such extremes sugars, sugar alcohols (similar or the same as the
(Kranner et al. 2008). Likely, genomic traces transfer sugars), and other low-molecular-weight
evolved to correlate with desiccation tolerance. molecules with neutral charge and low or no
For instance, Armaleo and Chiou (2021) found a toxicity at high concentrations, such as certain
fascinating link between growth rate, desiccation amino acids and their derivatives, for example
tolerance, and ribosome biogenesis. Ribosome mycosporines, and betaines (Gostinčar et al.
biogenesis requires splicing of group I introns 2012).
present in the ribosomal RNA genes. Most Lichens can resume full metabolic activity
introns populating lichen rDNA are unable to within minutes, apparently with proteins already
self-splice and their insertion into yeast ribosomal in place and thus not dependent on their complete
RNA genes caused the yeast strains to grow 4.4–6 resynthesis by the ribosomal machinery. Upon
times slower, and made them 40–1700 times rehydration, at least the side groups of carbon
more desiccation tolerant depending on intron backbones must be able to move for enzymatic
position and number. Fast growth rates would activity, as is the case in a “rubbery,” but not yet a
indeed poorly reconcile with the effects of recur- “glassy” state. Transcription, translation, and
rent drying on metabolism. other more complex processes require transition
The primary mechanisms of desiccation toler- to the liquid state with full carbon backbone
ance must, however, act directly at the cellular mobility of proteins (Candotto Carniel et al.
level. Desiccation tolerance may generally be 2021; Farrant and Hilhorst 2021). Expressed as
considered as the ability to restore metabolism a percentage of dry weight, lichen water contents
from the air-dried state, or, in a more precise can range from 2–15% in a dry state to 100–300%
definition, the capability to survive drying at rela- under hydrated conditions, and some
tive humidities below 65% (absolute water con- cyanobacterial jelly lichens may even take up
tent equal to or below 0.1 g H2O g-1 dry mass 2000%. Most of the water is kept in the strongly
and a water potential of ≤-100 MPa; Oliver et al. swollen cell walls (mostly fungal in green algal
2020). The mechanisms conferring desiccation lichens or cyanobacterial in jelly lichens). Suit-
tolerance (Kranner et al. 2008; Pichler et al. able levels of humidity are of profound impor-
2023), although still not fully understood, are tance to run photosynthesis. Between 70 and
believed to comprise molecules that support 150% water relative to dry weight appears to be
“vitrification,” i.e., the formation of a “glassy optimal, although lichens may start with net pho-
state.” For example, non-reducing sugars may tosynthesis at c. 20% water weight. Productivity
substitute for water, maintaining the spacing decreases with higher amounts of water due to the
between and within macromolecules, avoiding low solubility of CO2 leading to gas exchange
molecular crowding and cellular collapse, likely inhibition. To lower this type of inhibition, the
in conjunction with LEA-like proteins (“Late anatomical structure of lichens and particularly
Embryogenesis Abundant” dehydrins). More- the hydrophobicity of certain internal parts of
over, a potent antioxidant machinery appears to stratified lichen thalli play a significant role to
be essential for desiccation-tolerant organisms ensure gas exchange for efficient photosynthesis.
(Kranner et al. 2005), serving to protect from The hydrophobicity of fungal structures relies on
oxidative damage by reactive oxygen species pro- secondary lichen compounds covering the surface
duced as inevitable by-products or disrupting of the fungal cell walls, as well as a hydrophobic
electron transport chains upon desiccation. cell wall surface layer. Being highly significant
162 M. Grube

with regard to translocation of solutes between 6.18 Nitrogen

the partners, this layer covers both mycobiont and
photobiont cells as it creates an apoplastic contin- Growth of lichens depends on the availability of
uum for both symbionts. The sealing is achieved nitrogen as an essential macronutrient for synthe-
by the self-assembly of hydrophobins, a family of sis of proteins and nucleic acids, among other
cysteine-rich surface-active proteins forming a important roles. For the approximately 90% of
continuous rodlet-layer on the symbiont cells. green algal lichen species, it is assumed that
Class 1 hydrophobins have been characterized in fixed nitrogen is provided by various forms of
Xanthoria species (Scherrer et al. 2000; Scherrer deposition from the atmosphere, including
and Honegger 2003) and in Dictyonema aerosols but also in more robust manner by bird
glabratum (dikaryotic hyphae, three different manure. It remains to be studied, whether surface-
hydrophobins; Trembley et al. 2002). attached bacteria potentially contribute—either
Many lichens are able to maintain photosyn- dead or alive—to the nitrogen budget of lichens.
thesis despite wide variation in water content. Ten percent of lichen species live together
Photosystem II (Φ, quantum yield) efficiency with cyanobacteria (mostly Nostoc) as the pri-
often used as a proxy of photosynthetic activity, mary symbiont and about 3–5% have specialized
gives a good approximation, but under certain organs called cephalodia to grow cyanobacteria in
conditions does not directly relate to actual CO2 addition to the primary green algal partner (tripar-
uptake considering alternative electron transport tite lichens). Comparison with primarily
routes (e.g., photorespiration, Mehler reaction; cyanobacterial lichens suggested a separation of
Green et al. 1998). High rates of area-related tasks in tripartite lichens. By assessing the num-
photosynthesis and chlorophyll contents have ber of heterocysts, the cells in which nitrogen
been reported, in the range of those known from fixation takes place, Hyvärinen et al. (2002)
leaves of deciduous trees or evergreen forests found that heterocyst frequency in bipartite
(Green et al. 2008). Nevertheless, there seems to lichens ranges from 2 to 8%, and in tripartite
be substantial variation of rates among or within lichens between 10 and 55%. Several, mostly
thalli of a species and with time. Generally, young soil-inhabiting, green algal lichens with squamu-
thallus parts are much more active than older lose morphology are known to grow either facul-
parts, which agrees well with patterns of micro- tatively or obligately near free-living blue-green
scopically assessed cell vitality and chlorophyll algae, mainly Stigonema. This little studied phe-
fluorescence (Grube, unpublished observations). nomenon is known as cyanotrophy (Poelt and
Excess light can cause photoinhibition, i.e., the Mayrhofer 1988), which represents a form of
decrease in photochemical efficiency experienced ecological facilitation. Some of the species occur
in response to irradiation exceeding the energy only on very poor, acidic rocks on Stigonema,
used for photosynthetic ATP and NADPH pro- whereas they may occur independent of
duction and/or photodestruction, i.e. the light- Stigonema in habitats with high nutrient availabil-
induced damage to the photosynthetic apparatus. ity. Nitrogen fixation by cyanobacteria yields
Sensitivity of lichens to photoinhibition is NH4+, which is metabolized to glutamine, part
species-specific and depends on the present type of which is then transferred to the mycobiont.
of algal or cyanobacterial photobiont (Demmig- The mycobiont catalyzes glutamine to glutamate
Adams et al. 1990a) and their capacity for and other amino acids, which can be directed to
non-photochemical quenching (NPQ), including the green algal photobiont. Supplying the
xanthophyll cycles (Demmig-Adams et al. 1990b; photobiont with higher levels of nitrogen could
García-Plazaola et al. 2012).
6 Lichens 163

potentially increase the carbon gain capacity, regions. Johansson et al. (2011) showed varied
whereas directing more nitrogen into fungal tis- effects of simulated phosphorous deposition in
sue may increase the rate of maintenance respira- dependence of nitrogen deposition, as P supply
tion (Lambers 1985; Reich et al. 1998). Thus, a can both mitigate and intensify the negative
finely tuned balance likely contributed to effects of excess nitrogen depending on the (epi-
adjusting growth control of the photobiont and phytic) lichen species, and apparently allocation
the overinvestment in fungal structures. to the symbionts.
Nitrogen fixation requires higher levels of Transcriptomic analysis of resynthesized
moisture than respiration and photosynthesis, Usnea hakonensis thalli showed upregulation of
and apparently do not start below water contents photobiont genes involved in photosynthate
of 80% dry weight, while maximal fixation rates transport and mycobiont genes involved in nitro-
were found at 200-400% ODW in species with gen and phosphorus transport, when compared to
saturation at 500–800% oven dry weight (ODW) isolated and cultured myco- and photobionts
(Kershaw 1985). (Kono et al. 2020). In this species, genes for
Biological fixation occurs via nitrogenase, acid phosphatase and phosphate transport are
which uses the relatively rare element molybde- upregulated in the mycobiont, but are absent in
num as a cofactor. Alternatively, nitrogenases the photobiont, which instead upregulates genes
also may use vanadium or iron in place of molyb- for ATPase, suggesting dependence of the
denum in the active site, and genes for those photobiont from fungal phosphorous supply.
enzymes have been detected in the Nostoc
symbionts of Peltigera species (Hodkinson et al.
2014) in addition to standard molybdenum- 6.20 Secondary Lichen Products
dependent nitrogenases. Thus, these alternative
possibilities for nitrogen fixation provide flexibil- Lichens make the fungal world more colorful
ity of metabolic processes and play a more with their secondary metabolites. For practical
prominent role for lichens in habitats where reasons, the term secondary metabolites will be
molybdenum is limited. Consistent with this used here, although these compounds fulfill
thought, the acquisition of vanadium is strongly important ecological functions. The compounds,
regulated by the abundance of molybdenum produced by the fungal partner are a metabolic
according to Darnajoux et al. (2017), who also sink for excess carbon but more importantly they
found evidence for the activity of alternative serve in the biology of lichen symbioses as
vanadium-based nitrogenase in the Nostoc photoprotectants against intense radiation, as
cyanobiont of Peltigera aphthosa s.l. defense against feeders, or as factors mediating
metal homeostasis and pollution tolerance of
lichen thalli. For their bioactive properties they
6.19 Phosphorous are also of interest for biotechnology, while they
have long served as taxonomic characters.
The requirements of controlled resource alloca- Secondary metabolites are deposited as micro-
tion in a symbiotic system extends to phospho- scopic crystals in the intercellular spaces or are
rous, which in contrast to nitrogen may be lost by present as insoluble amorphous to melanin-like
sedimentation. Lichens cope with this limitation pigments in hyphal walls or outside of the walls
by recycling phosphorous, as shown in Cladonia (e.g., as pigment caps or as deposits between
podetia by Hyvärinen and Crittenden (2000). fungal cells). The crystallized compounds tend
Similar recycling was also shown for nitrogen in to be water-insoluble and accumulate in the thal-
the reindeer lichen Cladonia portentosa by Ellis lus, where they are unevenly distributed. Some
et al. (2005), who suggested this ability as a are only known as compounds present in the
reason for the ecological success and landscape- upper cortex. Others may be present in layers
tinging presence of Cladonia mats in subarctic below the algal layer, either as medullary
164 M. Grube

compounds or—when a lower cortex is mevalonate pathway (isoprenyls), and shikimic

missing—as compounds possibly shielding the acid pathway (shikimates). The compounds can
lower surface against the environment or chelat- be classified in major classes of lichen secondary
ing ions used during substrate exploitation. The metabolites (summarized in Table 6.1). Some of
gross distribution of compounds within the thalli the compounds can also be dimerized or are able
could be studied merely by localizing spot to form heterodimers such as bisanthraquinones,
reactions, or by fluorescence microscopy (Kauppi dimeric xanthones, or heterodimers of xanthones
and Verseghy-Patay 1990). Progress in mass with chromones (Nguyen et al. 2020; Tuong et al.
spectrometry-based imaging methods now 2020). Otherwise, visually detectable compounds
resolves metabolic patterns at high spatial and present in low amounts (e.g., in ascomata) of
chemical resolution, which makes it possible to many, particularly tropical lichens have poorly
visualize the microscopic distribution of each been explored (Fig. 6.7b, c; Yamamoto et al.
individual compound (Gadea et al. 2020; Garg 2002) and still await their structural discovery.
et al. 2016). Major compounds can be characteristic of
The most widespread lichen substances are lineages at various taxonomic levels, rarely so at
depsides and depsidones, followed by the order level (e.g., most representatives of
representatives of different compound classes, orange colored Teloschistales produce
such as yellow-greenish usnic acids, reddish- anthraquinones), but more commonly at the
orange anthraquinones, and bright yellow genus level (e.g., the ubiquitous yellowish-green
pulvinic acids (Fig. 6.7a). Of the depsides, colored species in Xanthoparmelia or Usnea in
atranorin seems to be the most common com- Parmeliaceae). Chemistry, however, not always
pound turning the lichens grayish-white (present reconciles clearly with phylogeny, as some
in perhaps a third of lichen species). The genomes lineages are chemically more variable than others
of lichen fungi sequenced so far suggest the pres- with respect to their main cortical compounds.
ence of dozens of biosynthetic pathway gene Chemical variation of medullary compounds has
clusters, but only a few compounds are produced been most commonly used in the classification of
in a single thallus; these, however, may accumu- species, either by presence of single compounds
late in massive amounts in the upper cortex or by variation in the patterns of chemically
(sometimes up to 30% of dry weight). Only related substances (chemosyndromatic variation).
rarely, a higher number of compounds is present Because most of the lichen compounds belong
in a single thallus. One notable exception is to the class of polyketides, their corresponding
Lecanora elixii, which contains more than building enzymes, the polyketide synthases
10 compounds, most of which are xanthone (PKS) raised interest during the early exploration
derivatives. In addition to filtering different of lichenized fungal genomes. Schmitt and
wave lengths of light, the upper cortical Lumbsch (2009) found that those genes of class
compounds seem to function as a light switch 1 polyketide synthases (PKS1) in lichens coding
depending on water content: More light can pass for methylsalicylic acids (MSAS) are more
through the cortex to the photosynthetic algal closely related to similar genes from bacteria
layer beneath when the crystals are more widely than to other fungal PKS1 genes. They concluded
spaced in swollen hydrated thalli (Dietz et al. that horizontal gene transfer is responsible for the
2000). The enhanced shielding by light reflection capability of lichenized fungi to produce these
from closely spaced crystals in dry stage may phenolic compounds. Since then, it became a
prevent the detrimental effects of photoinhibition challenge to find out which of the genes are
and radical formation. responsible for the production of the compounds
Different biochemical pathways lead to a vast accumulating in the thalli. In an early attempt,
diversity of more than 1000 known lichen Brunauer et al. (2009) characterized the transcript
compounds. The three most important ones are of a polyketide synthase gene (PKS) from the
the acetyl-polymalonyl pathway (polyketides), cultured mycobiont of Xanthoria elegans
6 Lichens 165

Fig. 6.7 Secondary metabolites in lichens, (a) Fluores- with crimson margin, bar = 1 mm; (c) Crimson crystals
cence of rhizocarpic acid in thalli of Rhizocarpon (quinoid compounds, likely isofuranonaphthoquinones)
geographicum under illumination with UV360-light, on ascomatal margins of C. rubrocinctum, bar = 100 μm.
bar = 2 mm; (b) Coniocarpon rubrocinctum fruitbodies Photographs: M. Grube

(XePKS1) using SMART-rapid amplification of major lichen compounds, such as grayanic acid
cDNA ends (RACE) cDNA synthesis and (Armaleo et al. 2011), usnic acid (Abdel-Hameed
sequencing of cloned cDNA. They chose et al. 2016), atranorin (Kim et al. 2021), or
conditions under which the cultures exclusively gyrophoric acid (Singh et al. 2022). With the
produced anthraquinones to suggest that the present knowledge about the putative usnic acid
detected gene transcript was responsible for the biosynthesis cluster, Pizarro et al. (2020)
synthesis of anthraquinones. Later, comparative conducted a broader analysis of genomes from
analyses revealed gene clusters producing other Parmeliaceae and found that this gene cluster is
166 M. Grube

Table 6.1 Survey of the major classes of specialized metabolites in lichens

Acetyl- Secondary aliphatic acids, esters
polymalonyl and related derivatives
pathway Polyketide-derived aromatic Mononuclear phenolic compounds
compounds Di- and tri-aryl derivatives of Depsides, tridepsides, and
simple phenolic units benzyl esters
Depsidones and diphenyl
ethers
Depsones
Dibenzofurans, usnic
acids and derivatives
Anthraquinones and biogenetically related xanthones
Chromones
Naphthaquinones
Xanthones
Mevalonate pathway Di-, sester-, and triterpenes
Steroids
Shikimic acid pathway Terphenylquinones
Pulvinic acid derivatives

ancestral in Parmeliaceae. All species producing critically reassessed when species are distin-
usnic acid contained this gene cluster, but all guished solely by such characters. However,
species without usnic acid apparently lost genomic variation of biosynthetic genes not
synthesizing genes quickly during evolution. directly reflected by the expressed chemotypes
Genomic comparison indeed shows consider- may raise questions about unknown roles of the
able gene content dynamics even at low taxo- gene products and unstudied correlations with
nomic levels. Singh et al. (2021) detected other characters.
biosynthetic gene cluster variation across Lasallia
pustulata populations from Mediterranean and
cold-temperate climate zones. Although all 6.21 Additional Interactions:
populations produced gyrophoric acid and to Lichenicolous Lichens,
minor extent lecanoric and hiascic acid the Lichen Microbiome,
irrespective of the sampling location, the popula- and the Lichens as Holobionts
tion genomic analyses indicated that U. pustulata
contains three clusters that are highly Being widespread and slow-growing organisms
differentiated between the Mediterranean and with persistent thalli, lichens might be a readily
cold-temperate populations, with one entire clus- available source of nutrition for associated
ter exclusively present and a putatively dysfunc- microorganisms, primarily bacteria and fungi
tional second cluster in cold-temperate (Fig. 6.9a–d). Past studies about lichen-associated
populations. In a third gene cluster, variation is microorganisms have focused on the diversity of
fixed in all cold-temperate populations. The lichen-associated fungi, which have been
authors suggested this pattern is shaped by both characterized by their phenotypes, while bacterial
positive and hitchhiking selection. The pattern of diversity was studied more thoroughly only in the
expressed and detected metabolites seems to be last two decades by molecular methods.
unaffected from genomic variation in this case, In a strict sense, lichenicolous fungi live exclu-
but the results are of considerable interest for sively on lichens, and they may include both,
chemosystematics. If pathway genes are present non-lichenized and lichenized fungi, which are
in genomes within a species, the taxonomic sig- also known as lichenicolous lichens (Fig. 6.8a,
nificance of chemical differences needs to be b). The distinction between these and free-living
6 Lichens 167

Fig. 6.8 Examples of lichenicolous lichens, (a) Rhizocarpon dinothetes on Protoparmelia badia (Switzerland), (b)
Rhizocarpon renneri on Dimelaena oreina (Switzerland), bars = 1 mm. Photographs: M. Grube

lichens can sometimes be difficult when they are “parasymbiosis.” 2319 species of mostly obligate
parasitic on other lichens only in juvenile stages lichenicolous fungi have been named (Diederich
to become independent later (Hafellner 2018) and et al. 2018), but their true diversity is still not
then develop faster than the initial host thallus. known. Species may develop local necroses or
More commonly, lichenicolous lichens grow brain-like outgrowths (galls; Fig. 6.9c) on thalli
slowly and remain dependent on the host thallus. of their specific hosts but most other species
Lichenicolous lichens take advantage of their develop more or less without symptoms. The
host’s algae and may maintain them in the para- architecture of the overall lichen structure in
sitic thallus or, alternatively, switch to another, these cases remains as originally determined by
secondarily acquired photobiont (De Los Ríos the principal lichen mycobiont.
et al. 2002b; Wedin et al. 2016; Moya et al. Since lichens are composite organisms,
2020). The take-over of the already lichenized lichenicolous fungi can have varied affinities to
host algae by another fungus requires physiologi- the partners of lichens. Some of the species are
cal adjustment for symbiosis with another fungus clearly associated with the algal partners of the
and was recently called “translichenization” host, whereas others have a mycoparasitic life-
(Pichler et al. 2023). style, or are preferentially found in the intercellu-
lar gels produced by either the fungal partner of
the host, or by both fungal and algal partners
6.22 Non-lichenized Lichenicolous (Fig. 6.10; De Los Ríos et al. 2002a). Moreover,
Fungi and More: The Fungal the cortical layers of lichens are frequently
Microbiome inhabited by dark-septate fungi, with unknown
affinity to any partner of the host.
Fungi have been recognized as lichen inhabitants In addition to fungi developing diagnostic
long before the symbiotic nature of the biology of spore-producing structures, many asymptomatic
the host has been revealed. Since the fungi can be cultured from crushed lichen thalli
mid-nineteenth century, researchers observed (Petrini et al. 1990). According to DNA-sequence
high specificity of many lichenicolous (=living analyses, these endolichenic fungi largely belong
on/in lichens) fungi for their host lichens to families and genera also known as endophytes
(Berkeley 1844; Nylander 1857). Zopf (1897) of plants (Tripathi and Joshi 2019). Using culti-
had already referred to them as an additional vation techniques, up to 48 different fungi were
part of the lichen symbiosis, forming a recorded from a single lichen species (U’Ren
168 M. Grube

Fig. 6.9 Lichenicolous habit: (a) Melittangium Rhizocarpon geographicum, bar = 2 mm (d)
lichenicola on Xanthoria parietina, bar = 50 μm (b) Sphaerellothecium contextum on Ochrolechia frigida,
Zwackhiomyces coepulonus on Rusavskia elegans, bar = 100 μm. Photographs: M. Grube
bar = 50 μm (c) Cercidospora cephalodiorum on

et al. 2012). Little is known about their role and work revealed they belong to the same species
abundance in their hosts, as they could include (Velmala et al. 2009). Further tests need to con-
trapped resting spores or those with very limited firm whether the presence of yeasts or their chem-
growth, respectively. Microscopic techniques, istry could alter the physical properties of the
which differentially visualize endolichenic fungi cortex or correlate with long pseudocyphellae
to distinguish them from the lichen mycobiont, developing along infected thalli. Such differences
are required to gain more insight into the roles of could indicate modified patterns of gas exchange
these associated fungi. and thus a change in ecological requirements.
The idea of a unitary role of one main fungal Because Spribille et al. (2016) detected
partner in shaping lichen thalli was challenged by representatives of this basidiomycetous yeast
the discovery of basidiomycetous yeasts (genus lineage in a variety of other lichens (particularly
Cyphobasidium, Cystobasidiomycetes, Parmeliaceae) using a sensitive PCR assay with
Pucciniomycotina). Spribille et al. (2016) highly specific primers, they suggested an integral
visualized these yeasts for the first time in the role for these fungi in the upper cortex of lichens.
thallus cortex of Bryoria fremontii, a pendent Their suggestion of a high degree of specificity of
lichen in Parmeliaceae. Those specimens with these fungi was put in perspective by Mark et al.
an abundance of yeast cells had a yellow color, (2020). It appears that these and other yeasts have
due to vulpinic acid in the branches. These a widespread occurrence in lichens (e.g.,
individuals have earlier been classified as a sepa- Fernández-Mendoz et al. 2017; Tuovinen et al.
rate species, Bryoria fremontii, until molecular
6 Lichens 169

Fig. 6.10 Interaction types

of lichenicolous fungi with
their hosts, (a) haustoria
formed in the algal partner
of the host, (b) appressoria
attaching to the algal
partner of the host, (c)
simple haustoria in the host
hyphae, (d) complex
haustoria in the host
hyphae, (e) intrahyphal
hyphae in the host, (f)
exploitation of the
intercellular gels of the
host, (g) aggressive
penetration of host
structures. Drawings by
M. Grube

2021), while their hyphal and fertile states that produce localized infections and symptoms
develop only in certain host species. by species specialized on their hosts.
Surprisingly few representatives of
non-lichenized fungi on lichens are known as
devastating pathogens, which suggests that the 6.23 Bacterial Microbiome
lichen biology is particularly well prepared to
keep the colonizing organisms in balance. The Bacteria associated with lichens have been stud-
poikilohydric lifestyle of many lichens may limit ied more thoroughly during the past two decades,
fast-growing pathogens requiring constant high although their presence was recognized long ago
humidity and instead support osmotolerant and by light microscopy form dotted “sprinkles” of
slow-growing microbiota (while lichens may cells on the surface of sectioned thalli. The much
quickly mold when stored under humid larger bacterial spore-bearing structures (“fruiting
conditions). Nonetheless, even in warmer habitats bodies”) of Melittangium lichenicola (Fig. 6.9a;
with continued levels of high humidity, which are Myxococcales, Deltaproteobacteria; commonly
better suited to promote pathogenic lifestyles, known as myxobacteria), however, led to the
lichens seem to have control over their colonizers, earliest description of a bacterial species on
either by producing bioactive compounds or lichens (Thaxter 1892). Bacteria were later
because of extracellular production of reactive isolated from lichens and cultured (e.g., Cengia
oxygen as a defense mechanism (Beckett et al. Sambo 1926; Henkel and Yuzhakova 1936).
2005). Osmotolerance of lichens could help to Their taxonomic affiliation relied on classic
passively balance commensal associations that microscopic methods including physiological
either do not express symptoms of disease or assays of cultivated strains, while classification
170 M. Grube

Fig. 6.11 Lichen thallus

structure of lung lichen
Lobaria pulmonaria in
cross-section. 3D
reconstruction of FISH
image stacks. Eubacteria
(red) and
Alphaproteobacteria
(yellow) on both, the upper
and the lower cortex,
Betaproteobacteria (pink)
being less abundant and
locally contained. Fungal
hyphae (blue) and algae
(green) visualized by
autofluorescence (from
Grube et al. 2015, with
permission of Springer
Nature)

of isolated bacteria by sequence data was accom- composition of bacterial communities was stud-
plished much later (e.g., González et al. 2005; ied by Cardinale et al. (2012), showing that the
Cardinale et al. 2006; Liba et al. 2006). Counts relative abundance of Alphaproteobacteria
of bacteria prepared by Cardinale et al. (2008) “drops” in older thallus parts.
revealed about 6 × 107 bacteria per gram of lichen Lichen surfaces vary dramatically in their
and showed that lichens are often densely covered hydrophobicity/hydrophily that correlate with
by bacterial colonies in a biofilm-like manner. the numbers of bacteria observed on them:
Using In situ hybridization with specific fluores- Hydrophobic surfaces generally bear fewer bac-
cent probes for ribosomal RNA they also revealed teria than hydrophilic surfaces. Microscopic
that most bacteria belonged to the observations also suggest that samples of wide-
Alphaproteobacteria (Fig. 6.11). The diversity spread lichens from hot and dry habitats have
and specificity of lichen-associated bacterial fewer bacteria than samples of these lichens
communities was subsequently studied in more from cool and humid habitats (Grube,
detail by culture-independent sequencing unpublished).
approaches and more sophisticated-omics With complementary-omics approaches, such
technologies. Such studies demonstrated that as metagenomics and metaproteomics, a better
lichens are furnished with a complex host-specific understanding of bacterial communities and their
bacterial microbiome (Grube et al. 2009; Bates potential functions is possible (Grube et al. 2015).
et al. 2011; Hodkinson et al. 2012; Potential vitamin and hormone production, and
Aschenbrenner et al. 2014; Leiva et al. 2021). various other functions suggest that bacteria can
Further support for the specificity of the lichen be functionally important components of lichen
community was demonstrated in a study of a symbioses. It has been suggested that they poten-
lichenicolous lichen, when the bacterial commu- tially contribute to the fitness of the lichen
nity composition shifted dramatically with the holobiont, similar to the situation of microbiomes
onset of the invasion by the parasitic lichen of plants and animals. In addition, it is suggested
(Wedin et al. 2016). In addition, the effect of that bacterial metabolites could also mediate
thallus age and habitat as parameters driving the defensive functions. Direct evidence for
6 Lichens 171

production of bacterial defensive metabolites in lichens Chrysothrix chlorina (Chrysothrix

the lichen thallus came from imaging mass spec- chrysovirus 1; CcCV1) and Lepraria incana
trometry analyses of Peltigera hymenina by Garg (Lepraria chrysovirus 1; LiCV1) once classified
et al. (2016), who were able to localize the pro- in the genus Alphachrysovirus, rather have a rela-
duction of secondary metabolites in situ. tionship to chrysoviridae known from filamen-
According to metatranscriptomics data, the tous ascomycetous fungi (Petrzik et al. 2019).
various bacterial groups also varied in their tran- Interestingly, CcCV1 was not found in the lichen
scription patterns. Analyses of the mycobiont but in the accompanying endolichenic
metatranscriptome also revealed previously fungus Penicillium citreosulfuratum. The symbi-
unrecognized groups, such as otic partners of lichens may contain several virus
Chthoniobacterales, which contribute biotin and species independently and simultaneously:
folate synthesis in Lobaria pulmonaria (Cernava CaMV as well as the capsid protein gene of
et al. 2017). Moreover, the hundreds of different ApMV was detected in the photobiont of
bacterial strains on a single thallus of this lichen Xanthoria parietina and both, the plant
respond to the poikilohydric conditions. Under cytorhabdovirus and the ApMV were found in
dry conditions, upregulation of a specialized Usnea chaetophora (Petrzik et al. 2014, 2015).
ketone metabolism indicated a switch to lipid- Proteins assigned to rhabdoviruses and
based nutrition (Cernava et al. 2019). With betaflexiviruses were found in the metaproteome
respect to the diversity of bacterial species present of Lobaria pulmonaria (Eymann et al. 2017), and
on lichen thalli, their varied physiological Grube et al. (2015) found bacteriophage
requirements suggest differential reactions under sequences in the metagenome of the same lichen.
varying hygric conditions of their hosts. The presence of bacteriophages in lichens is con-
firmed by occurrences of bacteriophage proteins
assigned to the families Myoviridae and
6.24 Other (Eukaryotic) Colonizers Siphoviridae, which infect Bacteria and Archaea
of Lichens (Eymann et al. 2017). Urayama et al. (2020)
characterized the total dsRNA viral community
Few reports exist on other eukaryotic organisms of a lichen species using dsRNA-seq technology
associated with lichens. Lakatos et al. (2004) and revealed that partitiviruses were dominant
reported on diatoms living in association with and active. Sequences were classified into two
tropical representatives of the genus genera including both plant- and fungi-infecting
Coenogonium. Further microbial organisms partitiviruses. While 17 of 65 OTUs were related
seem to be regularly present in lichens, including to Partitiviridae, viruses related to seven dsRNA
plasmodial myxomycete amoebae, heterotrophic virus families (Amalgaviridae, Botybirnaviridae,
nanoflagellates, and naked and testate amoebae Chrysoviridae, Endornaviridae,
(Anderson 2014; Wilkinson et al. 2015). The Megabirnaviridae, Picobirnaviridae, and
influence of these organisms in the lichen symbi- Totiviridae), three ssRNA virus families
osis is unknown, because they seem to be (Gammaflexviridae, Hypoviridae, and
transient. Narnaviridae), and one unclassified RNA virus
family (Polymycoviridae) were also identified
from a single thallus of lichen (the host lichen
6.25 Lichen-Associated Viruses species was not indicated, but possibly represents
Cladonia pyxidata s.l. according to their Fig. 6.1).
Beside microorganisms, viruses have been found These data suggest a high diversity of viruses
in lichens, including single- (ssRNA) and double- associated with the different partners in lichens
stranded RNA (dsRNA) viruses, apparently algal (i.e., mycobiont, photobiont, associated fungi,
origin due to their similarities with plant viruses and bacteria). When Merges et al. (2021) found
(Petrzik et al. 2014, 2016). DsRNA viruses in the five novel viruses belonging to Caulimoviridae,
172 M. Grube

Myoviridae, Podoviridae, and Siphoviridae in characteristic for nutrient-rich, basic habitats

metagenomic DNA of Umbilicaria phaea, they (Wirth et al. 2013).
suggested that the Caulimovirus is associated Lichens also accumulate pollutants. Particular
with green algal photobionts (Trebouxia) of the focus was given to metal accumulation in the
lichen, as its abundance decreased with increasing past, due to the interest in bioindication of toxic
elevation, reflecting the specific algal lineage metals (“heavy metals”). Accumulation may
hosting this virus. The remaining viruses were occur via particulate entrapment in intercellular
attributed to bacterial hosts, with patterns specific spaces of the thallus, by extracellular binding in
to the population of the host lichen. Functional the cell wall, or by intracellular uptake (Nash
annotation of sequences retrieved from 2008). Various elements can be distributed differ-
cyanolichens indicate numerous viral-encoded ently in the thalli and accumulation also depends
auxiliary metabolic genes (AMGs, involved in on other factors such as morphology, secondary
amino acid, nucleotide, and carbohydrate metab- metabolites, and not least on the species involved
olism, including AMGs for secondary metabo- (Bačkor and Loppi 2009).
lism and fatty acid biosynthesis). These results Lichens are affected by environmental change
raise the question of whether viruses may modu- as well. A recent study revealed that climate
late physiology and interactions within lichens change proceeds faster than the photobionts of
(Ponsero et al. 2021), and thus affect the fitness lichens would be able to adapt (Nelsen et al.
of their hosts as well. 2022). Cryophilic species of tundra habitats
seem to retract from their southernmost
occurrences. However, due to their high depen-
6.26 Lichens as Bioindicators dence on habitat conditions, any alteration in the
of Pollution and Environmental lichen vegetation could result from a combination
Change of accompanying factors as well. Lichens from
exposed rock habitats seem to cope well with
Deposition of toxic elements and molecules warmer temperatures, but more problems are
represents a major constraint for metabolically expected for soil-inhabiting lichens, especially
active lichens because they take up nutrients as these would be easily outcompeted by higher
over their entire thallus surfaces. Hence, lichens plant vegetation. Lichens adapted to cool and
seem generally more affected by gaseous continuously humid sites are sensitive to climate
emissions than many other organisms. Depending change and especially to dry conditions. Many of
on the sensitivity of species, they respond to air these are also found in forests characterized by
pollution by structural changes and bleaching. years of ecological continuity, such as old-growth
Particularly damaging are oxides of sulphur and forests. Unfortunately, these vulnerable forest
nitrogen, which affect basic metabolic processes habitats are threatened by economic interests as
(e.g., decreased electron flow through photosys- well. Lichen decline is not only a problem of
tem II, deactivation of nitrogenase, etc.) and con- climate change but perhaps more importantly a
sequently, sexual and asexual reproduction. problem of habitat destruction and other anthro-
Lichens have, therefore, been frequently used as pogenic impacts. For instance, lichens suffer from
bioindicators of air pollution (Conti 2008). A an excess of nitrogen deposition in areas mas-
plethora of literature exists about this subject, sively influenced by agricultural activities. This
which cannot be discussed in detail here. Amelio- impact seems responsible for the recent
ration from acid emissions since the 1980s led to alterations observed in the lichen flora of both
recolonization of lichen-depleted locations, Alps (Nascimbene et al. 2019) and boreal forests
though not leading to a complete restitution of (Esseen et al. 2022).
the original composition, but favorizing lichens
6 Lichens 173

6.27 Concluding Remark Arup U, Søchting U, Frödén P (2013) A new taxonomy of

the family Teloschistaceae. Nordic J Bot 31:16–83
Aschenbrenner IA, Cardinale M, Berg G, Grube M (2014)
Just as lichens are a prominent example for the Microbial cargo: do bacteria on symbiotic propagules
“living together” of unrelated organisms, the reinforce the microbiome of lichens? Environ
study of lichens has flourished by cooperation Microbiol 16:3743–3752
Asplund J, Wardle DA (2017) How lichens impact on
among researchers with different backgrounds. terrestrial community and ecosystem properties. Biol
To gain new insights in lichen biology, research Rev 92:1720–1738
requires novel methodological approaches and a Bačkor M, Loppi S (2009) Interactions of lichens with
continued interest in interdisciplinarity. As such, heavy metals. Biol Plant 53:214–222
Bates ST, Cropsey GW, Caporaso JG, Knight R, Fierer N
the study of lichens will also move forward as a (2011) Bacterial communities associated with the
symbiotic activity of researchers in the future. lichen symbiosis. Appl Environ Microbiol 77:1309–
1314
Acknowledgments The author thank Fernando Beckett RP, Minibayeva FV, Laufer Z (2005) Extracellu-
Fernandez Mendoza (Graz) for discussions and Ilse lar reactive oxygen species production by lichens.
Kranner (Innsbruck) for comments on the text. Lichenologist 37:397–407
Berkeley MJ (1844) Notices of British fungi. Ann Mag
Nat Hist 13:340–360
Blaha J, Baloch E, Grube M (2006) High photobiont
References diversity associated with the euryoecious lichen-
forming ascomycete Lecanora rupicola (Lecanoraceae,
Abdel-Hameed M, Bertrand RL, Piercey-Normore MD, Ascomycota). Biol J Linn Soc 88:283–293
Sorensen JL (2016) Putative identification of the Blum OB, Kashevarov GP (1986) The DNA homologies
usnic acid biosynthetic gene cluster by de novo as a proof of the legitimacy of the establishment of the
whole-genome sequencing of a lichen-forming fungus. lichen genus Lasallia Merat (Umbilicariaceae).
Fungal Biol 120:306–316 Doklady Akademii Nauk Ukrainskoi SSR Seriya B
Ahmadjian V (1993) The lichen symbiosis. Wiley, 12:61–64
New York Borgato L, Ertz D, Van Rossum F, Verbeken A (2022) The
Ahmadjian V, Jacobs JB (1981) Relationship between diversity of lichenized trentepohlioid algal
fungus and alga in the lichen Cladonia cristatella (Ulvophyceae) communities is driven by fungal taxon-
Tuck. Nature 289:169–172 omy and ecological factors. J Phycol 58(4):582–602
Ahmadjian V, Russel LA, Hildreth KC (1980) Artificial Brunauer G, Muggia L, Stocker-Wörgötter E, Grube M
reestablishment of lichens. I. Morphological (2009) A transcribed polyketide synthase gene from
interactions between the phycobionts of different Xanthoria elegans. Mycol Res 113:82–92
lichens and the mycobionts Cladonia cristatella and Candotto Carniel F, Fernandez-Marín B, Arc E,
Lecanora chrysoleuca. Mycologia 72:73–89 Craighero T, Laza JM, Incerti G, Tretiach M, Kranner
Anderson OR (2014) Microbial communities associated I (2021) How dry is dry? Molecular mobility in relation
with tree bark foliose lichens: a perspective on their to thallus water content in a lichen. J Exp Bot 72:1576–
microecology. J Eukar Microbiol 61:364–370 1588
Armaleo D, Chiou L (2021) Modeling in yeast how rDNA Cardinale M, Puglia AM, Grube M (2006) Molecular
introns slow growth and increase desiccation tolerance analysis of lichen-associated bacterial communities.
in lichens. G3 11:jkab279 FEMS Microbiol Ecol 57:484–495
Armaleo D, Sun X, Culberson C (2011) Insights from the Cardinale M, Vieira de Castro J Jr, Müller H, Berg G,
first putative biosynthetic gene cluster for a lichen Grube M (2008) In situ analysis of the bacterial com-
depside and depsidone. Mycologia 103:741–754 munity associated with the reindeer lichen Cladonia
Armaleo D, Müller O, Lutzoni F, Andrésson ÓS, Blanc G, arbuscula reveals predominance of
Bode HB, Collart FR, Dal Grande F, Dietrich F, Alphaproteobacteria. FEMS Microbiol Ecol 66:63–71
Grigoriev IV, Joneson S, Kuo A, Larsen PE, Logsdon Cardinale M, Steinová J, Rabensteiner J, Berg G, Grube M
JM Jr, Lopez D, Martin F, May SP, McDonald TR, (2012) Age, sun and substrate: triggers of bacterial
Merchant SS, Miao V, Morin E, Oono R, Pellegrini M, communities in lichens. Environ Microbiol Rep 4:23–
Rubinstein N, Sanchez-Puerta MV, Savelkoul E, 28
Schmitt I, Slot JC, Soanes D, Szövényi P, Talbot NJ, Casano LM, del Campo EM, García-Breijo FJ, Reig-
Veneault-Fourrey C, Xavier BB (2019) The lichen Armiñana J, Gasulla F, Del Hoyo A, Guéro A, Barreno
symbiosis re-viewed through the genomes of Cladonia E (2011) Two Trebouxia algae with different physio-
grayi and its algal partner Asterochloris glomerata. logical performances are ever-present in lichen thalli of
BMC Genomics 20:1–33 Ramalina farinacea. Coexistence versus competition?
Environ Microbiol 13:806–818
174 M. Grube

Cengia Sambo M (1926) Ancora della polisimbiosi nei Dietz S, Büdel B, Lange OL, Bilger W (2000) Transmit-
licheni ad alghe cianoficee. 1. Batteri simbionti. Atti tance of light through the cortex of lichens from
Soc Ital Sci Nat Mus Civ Storia Nat Milano 64:191– contrasting habitats. Bibl Lichenol 75:171–182
195 Du ZY, Zienkiewicz K, Vande Pol N, Ostrom NE,
Cernava T, Erlacher A, Aschenbrenner IA, Krug L, Benning C, Bonito GM (2019) Algal-fungal symbiosis
Lassek C, Riedel K, Grube M, Berg G (2017) leads to photosynthetic mycelium. elife 8:e47815
Deciphering functional diversification within the Elbert W, Weber B, Burrows S, Steinkamp J, Büdel B,
lichen microbiota by meta-omics. Microbiome 5:1–13 Andreae MO, Pöschl U (2012) Contribution of crypto-
Cernava T, Aschenbrenner IA, Soh J, Sensen CW, gamic covers to the global cycles of carbon and nitro-
Grube M, Berg G (2019) Plasticity of a holobiont: gen. Nat Geosci 5:459–462
desiccation induces fasting-like metabolism within Ellis CJ, Crittenden PD, Scrimgeour CM, Ashcroft CJ
the lichen microbiota. ISME J 13:547–556 (2005) Translocation of 15N indicates nitrogen
Colesie C, Williams L, Büdel B (2017) Water relations in recycling in the mat-forming lichen Cladonia
the soil crust lichen Psora decipiens are optimized via portentosa. New Phytol 168:423–434
anatomical variability. Lichenologist 49:483–492 Ertz D, Guzow-Krzemińska B, Thor G, Łubek A, Kukwa
Conti ME (2008) Lichens as bioindicators of air pollution. M (2018) Photobiont switching causes changes in the
WIT Transact State-of-the-art Sci Engin 30:111–162 reproduction strategy and phenotypic dimorphism in
Dal Grande F, Rolshausen G, Divakar PK, Crespo A, the Arthoniomycetes. Sci Rep 8:1–14
Otte J, Schleuning M, Schmitt I (2018) Environment Esseen PA, Ekström M, Grafström JBG, Palmquist K,
and host identity structure communities of green algal Westerlund B, Stahl G (2022) Multiple drivers of
symbionts in lichens. New Phytol 217:277–289 large-scale decline in boreal forest canopies. Glob
Dal-Forno M, Lawrey JD, Sikaroodi M, Bhattarai S, Change Biol 28:3293–3309
Gillevet PM, Sulzbacher M, Lücking R (2013) Starting Eymann C, Lassek C, Wegner U, Bernhardt J, Fritsch OA,
from scratch: evolution of the lichen thallus in the Fuch S, Otto A, Albrecht D, Schiefelbein U,
basidiolichen Dictyonema (Agaricales: Cernava T, Aschenbrenner I, Berg G, Grube M, Riedel
Hygrophoraceae). Fungal Biol 117:584–598 K (2017) Symbiotic interplay of fungi, algae, and
Darnajoux R, Zhang X, McRose DL, Miadlikowska J, bacteria within the lung lichen Lobaria pulmonaria
Lutzoni F, Kraepiel AM, Bellenger JP (2017) L. Hoffm. as assessed by state-of-the-art
Biological nitrogen fixation by alternative nitrogenases metaproteomics. J Prot Res 16:2160–2173
in boreal cyanolichens: importance of molybdenum Farrant JM, Hilhorst HW (2021) What is dry? Exploring
availability and implications for current biological metabolism and molecular mobility at extremely low
nitrogen fixation estimates. New Phytol 213:680–689 water contents. J Exp Bot 72:1507–1510
De Los Ríos A, Ascaso C, Grube M (2002a) Infection Farrar JF (1976) The lichen as an ecosystem: observation
mechanisms of lichenicolous fungi studied by various and experiment. In: Brown DH, Hawksworth DL, Bai-
microscopic techniques. Biblioth Lichenol 82:153–161 ley RH (eds) Lichenology: progress and problems.
De Los Ríos A, Ascaso C, Grube M (2002b) An ultrastruc- Academic, London, pp 385–406
tural, anatomical and molecular study of the Fernández-Mendoz F, Fleischhacker A, Kopun T,
lichenicolous lichen Rimularia insularis. Mycol Res Grube M, Muggia L (2017) ITS 1 metabarcoding
106:946–953 highlights low specificity of lichen mycobiomes at a
Demmig-Adams B, Maguas C, Adams WW, Meyer A, local scale. Mol Ecol 26:4811–4830
Kilian E, Lange OL (1990a) Effect of high light on Fernández-Mendoza F, Domaschke S, García MA,
the efficiency of photochemical energy conversion in a Jordan P, Martín MP, Printzen C (2011) Population
variety of lichen species with green and blue-green structure of mycobionts and photobionts of the wide-
phycobionts. Planta 180:400–409 spread lichen Cetraria aculeata. Mol Ecol 20:1208–
Demmig-Adams B, Adams WW, Green TGA, Czygan FC, 1232
Lange OL (1990b) Differences in the susceptibility to Fernandez-Mendoza F, Strasser E, Frolov I, Vondrak J,
light stress in two lichens forming a Muggia L, Mayrhofer H, Gaya E, Grube M (2023)
phycosymbiodeme, one partner possessing and one Introgressive descent and hypersexuality drive the evo-
lacking the xanthophyll cycle. Oecologia 84:451–456 lution of sexual parasitism and morphological reduc-
Díaz-Escandón D, Tagirdzhanova G, Vanderpool D, Allen tion in a fungal species complex. bioRxiv, 2023-01
CCG, Aptroot A, Češka O, Hawksworth DL, Ferraro LI (2004) Morphological diversity in the
Huereca A, Knudsen K, Kocourková J, Lücking R, hyphophores of Gomphillaceae (Ostropales, lichenized
Resl P, Spribille T (2022) Genome-level analyses Ascomycetes). Fungal Div 15:153–169
resolve an ancient lineage of symbiotic ascomycetes. Frank AB (1876) Über die biologischen Verhältnisse des
Curr Biol 32:5209–5218 Thallus einiger Krustenflechten. Cohn, Beitr Biol
Diederich P, Lawrey JD, Ertz D (2018) The 2018 classifi- Pflanzen 2:123–200
cation and checklist of lichenicolous fungi, with 2000 Friedl T (1989) Systematik und Biologie von Trebouxia
non-lichenized, obligately lichenicolous taxa. Bryolo- (Microthamniales, Chlorophyta) als Phycobiont der
gist 121:340–425
6 Lichens 175

Parmeliaceae (lichenisierte Ascomyceten). 218p. Doc- fruit body types in the molecular era. Mycol Res 111:
toral thesis, Universität Bayreuth 1116–1132
Gadea A, Fanuel M, Lamer A-CL, Boustie J, Rogniaux H, Grube M, Kantvilas G (2006) Siphula represents a remark-
Charrier M, Devehat FL-L (2020) Mass spectrometry able case of morphological convergence in sterile
imaging of specialized metabolites for predicting lichens. Lichenologist 38:241–249
lichen fitness and snail foraging. Plan Theory 9:70 Grube M, Cardinale M, de Castro JV, Müller H, Berg G
García-Plazaola JI, Esteban R, Fernández-Marín B, (2009) Species-specific structural and functional diver-
Kranner I, Porcar-Castell A (2012) Thermal energy sity of bacterial communities in lichen symbioses.
dissipation and xanthophyll cycles beyond the ISME J 3:1105–1115
Arabidopsis model. Photosynth Res 113:89–103 Grube M, Cernava T, Soh J, Fuchs S, Aschenbrenner I,
Garg N, Zeng Y, Edlund A, Melnik AV, Sanchez LM, Lassek C, Wegner U, Becher D, Riedel K, Sensen CW,
Mohimani H, Gurevich A, Miao V, Schiffler S, Lim Berg G (2015) Exploring functional contexts of sym-
YW, Luzzatto-Knaan T, Cai S, Rohwer F, Pevzner PA, biotic sustain within lichen-associated bacteria by com-
Cichewicz RH, Alexandrov T, Dorrestein PC (2016) parative omics. ISME J 9:412–424
Spatial molecular architecture of the microbial commu- Grube M, Muggia L, Baloch E, Hametner C, Stocker-
nity of a Peltigera lichen. mSystems 1:e00139–e00116 Wörgötter E (2017) Symbioses of lichen-forming
Gargas A, Taylor JW (1992) Polymerase chain reaction fungi with Trentepohlialean algae. In: Grube M,
(PCR) primers for amplifying and sequencing nuclear Seckbach J, Muggia L (eds) Algal and cyanobacteria
18s rDNA from lichenized fungi. Mycologia 84:589– symbioses. World Scientific, Hackensack, NJ, pp
592 85–110
Gargas A, DePriest PT, Grube M, Tehler A (1995) Multi- Gustavs L, Schiefelbein U, Darienko PT (2017)
ple origins of lichen symbioses in fungi suggested by Symbioses of the green algal genera Coccomyxa and
SSU rDNA phylogeny. Science 268:1492–1495 Elliptochloris (Trebouxiophyceae, Chlorophyta). In:
Garrido-Benavent I, Pérez-Ortega S, de los Ríos A, Grube M, Seckbach J, Muggia L (eds) Algal and
Fernández-Mendoza F (2020) Amphitropical variation cyanobacteria symbioses. World Scientific,
of the algal partners of Pseudephebe (Parmeliaceae, Hackensack, NJ, pp 169–208
lichenized fungi). Symbiosis 82:35–48 Hafellner J (2018) Focus on lichenicolous fungi: Diversity
Gasulla F, Barrasa JM, Casano LM, del Campo EM (2020) and taxonomy under the principle “one fungus – one
Symbiont composition of the basidiolichen name”. In Blanz P (ed) Biodiversity and ecology of
Lichenomphalia meridionalis varies with altitude in fungi, lichens, and mosses, Austrian Academy of
the Iberian Peninsula. Lichenologist 52:17–26 Sciences Biosystematics and Ecology Series. 34.
González I, Ayuso-Sacido A, Anderson A, Genilloud O Austrian Academy of Sciences, Vienna, pp 227–244
(2005) Actinomycetes isolated from lichens: evalua- Hametner C, Stocker-Wörgötter E, Grube M (2014a) New
tion of their diversity and detection of biosynthetic insights into diversity and selectivity of
gene sequences. FEMS Microbiol Ecol 54:401–415 trentepohlialean lichen photobionts from the
González-Hourcade M, Braga MR, Del Campo EM, extratropics. Symbiosis 63:31–40
Ascaso C, Patiño C, Casano LM (2020) Ultrastructural Hametner C, Stocker-Wörgötter E, Rindi F, Grube M
and biochemical analyses reveal cell wall remodelling (2014b) Phylogenetic position and morphology of
in lichen-forming microalgae submitted to cyclic lichenized Trentepohliales (Ulvophyceae,
desiccation–rehydration. Ann Bot 125:459–469 Chlorophyta) from selected species of Graphidaceae.
Gostinčar C, Muggia L, Grube M (2012) Phycol Res 62:170–186
Polyextremotolerant black fungi: oligotrophism, adap- Hawksworth DL, Grube M (2020) Lichens redefined as
tive potential, and a link to lichen symbioses. Front complex ecosystems. New Phytol 227:1281
Microbiol 3:390 Henkel PA, Yuzhakova LA (1936) Azotfiksiruyuschie
Green TGA, Schroeter B, Kappen L, Seppelt RD, Maseyk bakterii v lishaynikah. Izv Biol Inst Permsk Gos Univ
K (1998) An assessment of the relationship between 10:9–10
chlorophyll a fluorescence and CO2 gas exchange from Henskens FL, Green TA, Wilkins A (2012) Cyanolichens
field measurements on a moss and lichen. Planta 206: can have both cyanobacteria and green algae in a
611–618 common layer as major contributors to photosynthesis.
Green TGA, Nash TH, Lange OL (2008) Physiological Ann Bot 110:555–563
ecology of carbon dioxide exchange. In: Nash TH Hodkinson BP, Gottel NR, Schadt CW, Lutzoni F (2012)
(ed) Lichen biology. Cambridge University Press, Photoautotrophic symbiont and geography are major
New York, pp 152–181 factors affecting highly structured and diverse bacterial
Grube M, Arup U (2001) Molecular and morphological communities in the lichen microbiome. Environ
evolution in the Physciaceae (Lecanorales, lichenized Microbiol 14:147–161
Ascomycotina), with special emphasis on the genus Hodkinson BP, Allen JL, Forrest LL, Goffinet B,
Rinodina. Lichenologist 33:63–72 Sérusiaux E, Andrésson ÓS, Miao V, Bellenger JP,
Grube M, Hawksworth DL (2007) Trouble with lichen: the Lutzoni F (2014) Lichen-symbiotic cyanobacteria
re-evaluation and re-interpretation of thallus form and associated with Peltigera have an alternative
176 M. Grube

vanadium-dependent nitrogen fixation system. Eur J show preference to selected mycobiont lineages in
Phycol 49:11–19 lichen symbioses. J Phycol 56:979–993
Honegger R (1991) Functional aspects of the lichen sym- Kranner I, Cram WJ, Zorn M, Wornik S, Yoshimura I,
biosis. Annu Rev Plant Biol 42:553–578 Stabentheiner E, Pfeifhofer H (2005) Antioxidants and
Honegger R (2012) The symbiotic phenotype of lichen- photoprotection in a lichen as compared to its isolated
forming ascomycetes and their endo- and epibionts. In: symbiotic partners. PNAS 102:3141–3146
Hock B (ed) The Mycota IX. Springer, Berlin, pp Kranner I, Beckett R, Hochman A, Nash TH III (2008)
287–339 Desiccation-tolerance in lichens: a review. Bryologist
Honegger R, Edwards D, Axe L (2013) The earliest 111:576–593
records of internally stratified cyanobacterial and Kroken S, Taylor JW (2001) A gene genealogical
algal lichens from the Lower Devonian of the Welsh approach to recognize phylogenetic species boundaries
Borderland. New Phytol 197:264–275 in the lichenized fungus Letharia. Mycologia 93:38–
Hyvärinen M, Crittenden PD (2000) 33P translocation in 53
the thallus of the mat-forming lichen Cladonia Lakatos M, Lange-Bertalot H, Büdel B (2004) Diatoms
portentosa. New Phytol 145:281–288 living inside the thallus of the green algal lichen
Hyvärinen M, Härdling R, Tuomi J (2002) Cyanobacterial Coenogonium linkii in neotropical lowland rain forests.
lichen symbiosis: the fungal partner as an optimal J Phycol 40:70–73
harvester. Oikos 98:498–504 Lambers H (1985) Respiration in intact plants and tissues:
Johansson O, Olofsson J, Giesler R, Palmqvist K (2011) its regulation and dependence on environmental
Lichen responses to nitrogen and phosphorus additions factors, metabolism and invaded organisms. In:
can be explained by the different symbiont responses. Douce R, Day DA (eds) Higher Plant Respiration.
New Phytol 191:795–805 Springer, Berlin, pp 418–465
Joneson S, Lutzoni F (2009) Compatibility and thigmotro- Larson DW (1987) The absorption and release of water by
pism in the lichen symbiosis: a reappraisal. Symbiosis lichens. Bibl Lichenol 25:351–360
47:109–115 Leiva D, Fernández-Mendoza F, Acevedo J, Carú M,
Joneson S, Armaleo D, Lutzoni F (2011) Fungal and algal Grube M, Orlando J (2021) The bacterial community
gene expression in early developmental stages of of the foliose macro-lichen Peltigera frigida is more
lichen-symbiosis. Mycologia 103:291–306 than a mere extension of the microbiota of the subja-
Kaasalainen U, Tuovinen V, Mwachala G, Pellikka P, cent substrate. Microb Ecol 81:965–976
Rikkinen J (2021) Complex interaction networks Liba CM, Ferrara FIDS, Manfio GP, Fantinatti-
among cyanolichens of a tropical biodiversity hotspot. Garboggini F, Albuquerque RC, Pavan C, Ramos PL,
Front Microbiol 12:672333 Moreira-Filho CA, Barbosa HR (2006) Nitrogen-fixing
Kauppi M, Verseghy-Patay K (1990) Determination of the chemo-organotrophic bacteria isolated from
distribution of lichen substances in the thallus by fluo- cyanobacteria-deprived lichens and their ability to sol-
rescence microscopy. Ann Bot Fenn 27:189–202 ubilize phosphate and to release amino acids and
Kershaw KA (1985) Physiological ecology of lichens. phytohormones. J Appl Microbiol 101:1076–1086
Cambridge University Press, Cambridge Lücking L, Grube M (2002) Facultative parasitism and
Keuler R, Garretson A, Saunders T, Erickson RJ, St reproductive strategies in Chroodiscus (Ascomycota,
Andre N, Grewe F, Smith H, Lumbsch HT, Huang Ostropales). Stapfia 80:267–292
J-P, Leavitt SD (2020) Genome-scale data reveal the Lücking R, Dal-Forno M, Sikaroodi M, Gillevet PM,
role of hybridization in lichen-forming fungi. Sci Rep Bungartz F, Moncada B, Yánez-Ayabaca A, Chaves
10:1–14 JL, Coca LF, Lawrey JD (2014) A single macrolichen
Kim W, Liu R, Woo S, Kang KB, Park H, Yu YH, Ha HH, constitutes hundreds of unrecognized species. PNAS
Oh SY, Yang JH, Kim H, Yun SH, Hur JS (2021) 111:11091–11096
Linking a gene cluster to atranorin, a major cortical Lücking R, Hodkinson BP, Leavitt SD (2016) The 2016
substance of lichens, through genetic dereplication and classification of lichenized fungi in the Ascomycota
heterologous expression. MBio 12:e01111–e01121 and Basidiomycota - approaching one thousand
Kohlmeyer J, Hawksworth DL, Volkmann-Kohlmeyer B genera. Bryologist 119:361–416
(2004) Observations on two marine and maritime “bor- Lutzoni F, Pagel M, Reeb V (2001) Major fungal lineages
derline” lichens: Mastodia tessellata and are derived from lichen symbiotic ancestors. Nature
Collemopsidium pelvetiae. Mycol Prog 3:51–56 411:937–940
Kono M, Kon Y, Ohmura Y, Satta Y, Terai Y (2020) In Mark K, Laanisto L, Bueno CG, Niinemets Ü, Keller C,
vitro resynthesis of lichenization reveals the genetic Scheidegger C (2020) Contrasting co-occurrence
background of symbiosis-specific fungal-algal interac- patterns of photobiont and cystobasidiomycete yeast
tion in Usnea hakonensis. BMC Genomics 21:1–16 associated with common epiphytic lichen species. New
Kosecka M, Jabłońska A, Flakus A, Rodriguez-Flakus P, Phytol 227:1362–1375
Kukwa M, Guzow-Krzemińska B (2020) Matthews SW, Tucker SC, Chapman RL (1989) Ultra-
Trentepohlialean algae (Trentepohliales, Ulvophyceae) structural features of mycobionts and
6 Lichens 177

trentepohliaceous phycobionts in selected subtropical structurally diverse xanthone dimers from the Viet-
crustose lichens. Bot Gaz 150:417–438 namese lichen Usnea baileyi. Fitoterapia 141:104449
Meeßen J, Eppenstein S, Ott S (2013) Recognition Nylander W (1857) De fungillis binis lichenicolis
mechanisms during the pre-contact state of lichens: observatio. Bot Not 1857:83–84
II. Influence of algal exudates and ribitol on the Oberwinkler F (2012) Basidiolichens. In: Hock B
response of the mycobiont of Fulgensia bracteata. (ed) Fungal associations. Springer, Berlin, pp 341–362
Symbiosis 59:131–143 Oliver MJ, Farrant JM, Hilhorst HWM, Mundree S,
Merges D, Dal Grande F, Greve C, Otte J, Schmitt I (2021) Williams B, Bewley JD (2020) Desiccation tolerance:
Virus diversity in metagenomes of a lichen symbiosis avoiding cellular damage during drying and rehydra-
(Umbilicaria phaea): complete viral genomes, putative tion. Ann Rev Plant Biol 71:435–460
hosts and elevational distributions. Environ Microbiol Peksa O, Gebouská T, Škvorová Z, Vančurová L, Škaloud
23:6637–6650 P (2022) The guilds in green algal lichens—an insight
Moya P, Molins A, Chiva S, Bastida J, Barreno E (2020) into the life of terrestrial symbiotic communities.
Symbiotic microalgal diversity within lichenicolous FEMS Microbiol Ecol 98(2):fiac008
lichens and crustose hosts on Iberian Peninsula gyp- Petrini O, Hake U, Dreyfuss MM (1990) An analysis of
sum biocrusts. Sci Rep 10:1–14 fungal communities isolated from fruticose lichens.
Muggia L, Nelson P, Wheeler T, Yakovchenko LS, Mycologia 82:444–451
Tønsberg T, Spribille T (2011) Convergent evolution Petrzik K, Vondrák J, Barták M, Peksa O, Kubešová O
of a symbiotic duet: the case of the lichen genus (2014) Lichens, a new source or yet unknown host of
Polychidium (Peltigerales, Ascomycota). Am J Bot herbaceous plant viruses? Eur J Plant Pathol 138:549–
98:1647–1656 559
Muggia L, Pérez-Ortega S, Kopun T, Zellnig G, Grube M Petrzik K, Vondrák J, Kvíderová J, Lukavský J (2015)
(2014) Photobiont selectivity leads to ecological toler- Platinum anniversary: virus and lichen alga together
ance and evolutionary divergence in a polymorphic more than 70 years. PLoS One 10:3
complex of lichenized fungi. Ann Bot 114:463–475 Petrzik K, Koloniuk I, Sarkisová T, Èíhal L (2016) Detec-
Muggia L, Leavitt S, Barreno E (2018) The hidden diver- tion of herbaceous-plant pararetrovirus in lichen her-
sity of lichenised Trebouxiophyceae (Chlorophyta). barium samples. Acta Virol 60:196–200
Phycologia 57:503–524 Petrzik K, Koloniuk I, Sehadová H, Sarkisova T (2019)
Muggia L, Nelsen MP, Kirika PM, Barreno E, Beck A, Chrysoviruses inhabited symbiotic fungi of lichens.
Lindgren, H, Lumbsch HT, Leavitt SD, Trebouxia Viruses 11:12
Working Group (2020) Formally described species Peveling E (1970) Die Darstellung der
woefully underrepresent phylogenetic diversity in the Oberflächenstrukturen von Flechten mit dem Raster-
common lichen photobiont genus Trebouxia Elektronenmikroskop. Deutsche Bot Ges NF 4:89–101
(Trebouxiophyceae, Chlorophyta): an impetus for Pichler G, Muggia L, Candotto Carniel F, Grube M,
developing an integrated taxonomy. Mol Phylogen Kranner I (2023) How to build a lichen: from metabo-
Evol 149:106821 lite release to symbiotic interplay. New Phytol 238:
Nascimbene J, Benesperi R, Giordani P, Grube M, 1362–1378
Marini L, Vallese C, Mayrhofer H (2019) Could hair- Pizarro D, Divakar PK, Grewe F, Crespo A, Dal Grande F,
lichens of high-elevation forests help detect the impact Lumbsch HT (2020) Genome-wide analysis of biosyn-
of global change in the Alps? Diversity 11:45 thetic gene cluster reveals correlated gene loss with
Nash T (2008) Nutrients, elemental accumulation, and absence of usnic acid in lichen-forming fungi. Genome
mineral cycling. In: Nash T (ed) Lichen biology, 2nd Biol Evol 12:1858–1868
edn. Cambridge University Press, Cambridge, pp Poelt J, Mayrhofer H (1988) Über Cyanotrophie bei
234–251 Flechten. Plant Syst Evol 158:265–281
Nelsen MP, Plata ER, Andrew CJ, Lücking R, Lumbsch Ponsero AJ, Hurwitz BL, Magain N, Miadlikowska J,
HT (2011) Phylogenetic diversity of trentepohlialean Lutzoni F, U’Ren JM (2021) Cyanolichen microbiome
algae associated with lichen-forming fungi. J Phycol contains novel viruses that encode genes to promote
47:282–290 microbial metabolism. ISME Commun 1:1–4
Nelsen MP, Lücking R, Boyce CK, Lumbsch HT, Ree RH Printzen C, Ekman S, Tønsberg T (2003) Phylogeography
(2020) No support for the emergence of lichens prior to of Cavernularia hultenii: evidence of slow genetic drift
the evolution of vascular plants. Geobiology 18:3–13 in a widely disjunct lichen. Mol Ecol 12:1473–1486
Nelsen MP, Leavitt SD, Heller K, Muggia L, Lumbsch HT Rai AN (2002) Cyanolichens: nitrogen metabolism. In:
(2022) Contrasting patterns of climatic niche diver- Rai AM, Bergman B, Rasmusson U (eds)
gence in Trebouxia—a clade of lichen-forming algae. Cyanobacteria in symbiosis. Springer, Dordrecht, pp
Front Microbiol 13:791546 97–115
Nguyen VK, Genta-Jouve G, Duong TH, Beniddir MA, Reich PB, Walters MB, Ellsworth DS, Vose JM, Volin JV,
Gallard JF, Ferron S, Boustie J, Mouray E, Grellier P, Gresham C, Bowman WD (1998) Relationships of leaf
Chavasiri W, Le Pogam P (2020) Eumitrins C-E: dark respiration to leaf nitrogen, specific leaf area and
178 M. Grube

leaf life-span: a test across biomes and functional Škvorová Z, Černajová I, Steinová J, Peksa O, Moya P,
groups. Oecologia 114:471–482 Škaloud P (2022) Promiscuity in lichens follows clear
Resl P, Bujold AR, Tagirdzhanova G, Meidl P, Freire rules: partner switching in Cladonia is regulated by
Rallo S, Kono M, Fernández-Brime S, climatic factors and soil chemistry. Front Microbiol
Guðmundsson H, Andrésson ÓS, Muggia L, May- 12:781585
rhofer H, McCutcheon JP, Wedin M, Werth S, Willis Spribille T, Tuovinen V, Resl P, Vanderpool D,
LM, Spribille T (2022) Large differences in carbohy- Wolinski H, Aime MC, Schneider K,
drate degradation and transport potential among lichen Stabentheiner E, Toome-Heller M, Thor G, May-
fungal symbionts. Nat Commun 13:2634 rhofer H, Johannesson H, McCutcheon J (2016) Basid-
Rikkinen J (2017) Cyanobacteria in terrestrial symbiotic iomycete yeasts in the cortex of ascomycete
systems. In: Hallenbeck PC (ed) Modern topics in the macrolichens. Science 353:488–492
phototrophic prokaryotes. Springer, Cham, pp Spribille T, Tagirdzhanova G, Goyette S, Tuovinen V,
243–294 Case R, Zandberg WF (2020) 3D biofilms: in search
Sanders WB (2014) Complete life cycle of the lichen of the polysaccharides holding together lichen
fungus Calopadia puiggarii (Pilocarpaceae, symbioses. FEMS Microbiol Lett 367:5
Ascomycetes) documented in situ: Propagule dis- Steinová J, Holien H, Košuthová A, Škaloud P (2022) An
persal, establishment of symbiosis, thallus develop- exception to the rule? Could photobiont identity be a
ment, and formation of sexual and asexual better predictor of lichen phenotype than mycobiont
reproductive structures. Am J Bot 101:1836–1848 identity? J Fungi 8:275
Sanders WB, Ascaso C (1995) Reiterative production and Stocker-Wörgötter E (2001) Experimental lichenology
deformation of cell walls in expanding thallus nets of and microbiology of lichens: culture experiments, sec-
the lichen Ramalina menziesii (Lecanorales, ondary chemistry of cultured mycobionts, resynthesis,
Ascomycetes). Am J Bot 82:1358–1366 and thallus morphogenesis. Bryologist 104:576–581
Sanders WB, Masumoto H (2021) Lichen algae: the pho- Thaxter R (1892) On the Myxobacteriaceae, a new order
tosynthetic partners in lichen symbioses. Lichenologist of Schizomycetes. Bot Gaz 17:389–406
53:347–393 Trembley ML, Ringli C, Honegger R (2002)
Schaper GM, Ott S (2003) Photobiont selectivity and Hydrophobins DGH1, DGH2, and DGH3 in the
interspecific in interactions in lichen communities. lichen-forming basidiomycete Dictyonema glabratum.
Culture experiments with the mycobiont Fulgensia Fungal Genet Biol 35:247–259
bracteata. Plant Biol 5:441–450 Tripathi M, Joshi Y (2019) Endolichenic fungi: present
Scherrer S, Honegger R (2003) Inter- and intraspecific and future trends. Springer, Singapore
variation of homologous hydrophobin (H1) gene Tuong TL, Do LT, Aree T, Wonganan P, Chavasiri W
sequences among Xanthoria spp. (lichen-forming (2020) Tetrahydroxanthone–chromanone heterodimers
ascomycetes). New Phytol 158:375–389 from lichen Usnea aciculifera and their cytotoxic
Scherrer S, De Vries OMH, Dudler R, Wessels JGH, activity against human cancer cell lines. Fitoterapia
Honegger R (2000) Interfacial self-assembly of fungal 147:104732
hydrophobins of the lichen-forming ascomycetes Tuovinen V, Millanes AM, Freire-Rallo S, Rosling A,
Xanthoria parietina and X. ectaneoides. Fungal Wedin M (2021) Tremella macrobasidiata and
Genet Biol 30:81–93 Tremella variae have abundant and widespread yeast
Schmitt I, Lumbsch HT (2009) Ancient horizontal gene stages in Lecanora lichens. Environ Microbiol 23:
transfer from bacteria enhances biosynthetic 2484–2498
capabilities of fungi. PLoS One 4:e4437 U’Ren JM, Lutzoni F, Miadlikowska J, Laetsch AD,
Schüßler A, Kluge M (2001) Geosiphon pyriforme, an Arnold AE (2012) Host and geographic structure of
endocytosymbiosis between fungus and cyanobacteria, endophytic and endolichenic fungi at a continental
and its meaning as a model system for arbuscular scale. Am J Bot 99:898–914
mycorrhizal research. In: Hock B (ed) The Mycota 9: Urayama SI, Doi N, Kondo F, Chiba Y, Takaki Y,
fungal associations. Springer, Berlin, pp 151–161 Hirai M, Minegshi Y, Hagiwara D, Nunoura T (2020)
Schwendener S (1869) Die Algentypen der Diverged and active partitiviruses in lichen. Front
Flechtengonidien. C. Schultze Microbiol 11:561344
Singh G, Calchera A, Schulz M, Drechsler M, Bode HB, Velmala S, Myllys L, Halonen P, Goward T, Ahti T (2009)
Schmitt I, Dal Grande F (2021) Climate-specific bio- Molecular data show that Bryoria fremontii and
synthetic gene clusters in populations of a lichen- B. tortuosa (Parmeliaceae) are conspecific. Lichenolo-
forming fungus. Environ Microbiol 23:4260–4275 gist 41:231–242
Singh G, Calchera A, Merges D, Valim H, Otte J, Vondrak J, Kubásek J (2013) Algal stacks and fungal
Schmitt I, Dal Grande F (2022) A candidate gene stacks as adaptations to high light in lichens. Lichenol-
cluster for the bioactive natural product gyrophoric ogist 45:115–124
acid in lichen-forming fungi. Microbiol Spectr. Walser JC (2004) Molecular evidence for limited dispersal
https://doi.org/10.1128/spectrum.00109-22 of vegetative propagules in the epiphytic lichen
Lobaria pulmonaria. Am J Bot 91:1273–1276
6 Lichens 179

Wedin M, Döring H, Gilenstam G (2004) Saprotrophy and Wirth V, Hauck M, Schultz M (2013) Die Flechten
lichenization as options for the same fungal species on Deutschlands, vol Band 1. Ulmer, Stuttgart
different substrata: environmental plasticity and fungal Yamamoto Y, Kinoshita Y, Thor G, Hasumi M,
lifestyles in the Stictis–Conotrema complex. New Kinoshita K, Koyama K, Takahashi K, Yoshimura I
Phytol 164:459–465 (2002) Isofuranonaphthoquinone derivatives from
Wedin M, Maier S, Fernandez-Brime S, Cronholm B, cultures of the lichen Arthonia cinnabarina (DC.)
Westberg M, Grube M (2016) Microbiome change by Wallr. Phytochemistry 60:741–745
symbiotic invasion in lichens. Environ Microbiol 18: Yuan X, Xiao S, Taylor TN (2005) Lichen-like symbiosis
1428–1439 600 million years ago. Science 308:1017–1020
Wilkinson DM, Creevy AL, Kalu CL, Schwartzman DW Zopf W (1897) Ueber Nebensymbiose (Parasymbiose).
(2015) Are heterotrophic and silica-rich eukaryotic Ber Deutsch Bot Ges 15:90–92
microbes an important part of the lichen symbiosis?
Mycology 6:4–7
 Umbilicaria muhlenbergii: A Model
for Studying Molecular Mechanisms 7
Regulating Initial Fungal Symbiotic
Interactions with Algal Cells

Yanyan Wang, Xinli Wei, Yuting Hu, and Jin-Rong Xu

Abstract other well-conserved signal transduction

Lichens are known for their importance in pathways that are known to crosstalk and
evolution and ecology but our knowledge of co-regulate fungal–plant interactions in plant
molecular mechanisms regulating symbiont pathogens. The Umbilicaria muhlenbergii
interactions in lichen symbiosis is very lim- genome contains key components of
ited. Recent studies have shown that mitogen-activated protein (MAP) kinase
Umbilicaria muhlenbergii has a relative fast pathways that may regulate fungal–algal
growth rate and is amenable to molecular interactions during lichen symbiosis. Although
genetic studies for efficiently generating there are still challenges, U. muhlenbergii has
transformants and targeted gene disruption the potential to be used as a model to study
mutants. It is the only known dimorphic early stages of symbiotic interactions and sig-
lichen-forming fungus, and pseudohyphal nal transduction pathways involved in the rec-
growth is associated with symbiotic ognition of compatible symbiont cells and
interactions with algal cells of Trebouxia development of highly differentiated masses
jamesii, its photobiont. Pharmacological stud- of fungal–algal cells.
ies and functional characterization of the Gα
subunit UmGpa2 of the trimeric G-proteins Keywords
have shown the involvement of the cAMP- Lichen symbiosis · Signal transduction
PKA (cyclic AMP-protein kinase A) pathway pathways · Fungal–algal interactions ·
in regulating yeast-to-pseudohypha transition Trebouxia jamesii · Early re-lichenization
and fungal–algal interactions. The importance stages
of cAMP-PKA signaling in lichen symbiosis
proved for the first time in U. muhlenbergii
argues for further characterization of this and 7.1 Introduction

Lichen is mainly composed of a lichen-forming

Y. Wang · X. Wei
State Key Laboratory of Mycology, Institute of fungus (the mycobiont, commonly an ascomy-
Microbiology, Chinese Academy of Sciences, Beijing, cete) and a photosynthetic organism (the
China photobiont, often a green alga but can be a cya-
Y. Hu · J.-R. Xu (✉) nobacterium). The fungal–algal association forms
Department of Botany and Plant Pathology, Purdue unique stratified thalli with morphological and
University, West Lafayette, IN, USA physiological features that are different from the
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 181
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_7
182 Y. Wang et al.

mycobiont or photobiont. Besides the primary For the initial symbiotic associations, chemical
algal partner, some lichens have a cyanobacte- and physical signals must be recognized by both
rium as a secondary photobiont in cephalodia. mycobionts and photobionts. Although fungal
Lichens also often harbor endophytic or growth chemotropically toward algal cells may
exophytic bacteria, fungi, and algae. Some of be absent, the pre-contact recognition between
these microorganisms are permanent inhabitants compatible symbionts in proximity through dif-
and may play non-negligible roles in lichen sym- fusible signaling compounds can affect hyphal
biosis (Grube et al. 2009; Spribille et al. 2016). growth, branching frequency, and mucilage for-
The interaction between fungi and mation in the mycobiont (Joneson and Lutzoni
photosynthesizing organisms is an ancient adap- 2009; Meeßen et al. 2013). After the initial con-
tation to terrestrial lifestyle. Independent shifts of tact, compatible photobiont cells are recognized
lichenization and de-lichenization have been via chemical and physical signals and enveloped
observed in different fungal phyla, and many by fungal hyphae and mucilage, which then grow
non-lichenized fungal taxa, including some plant into a mass of undifferentiated fungal and algal
pathogenic fungi, are likely to be derived from cells. For the photobionts, some lectins
lichen ancestors (Lutzoni et al. 2018). Approxi- (carbohydrate-binding glycoproteins) secreted
mately 20,000 species of lichen-forming fungi by fungal cells may function as algal binding
but only fewer than 200 photobiont species have proteins (ABPs) to bind with surface proteins of
been identified. As a result, the diversity of lichen algal cells, such as lectins of Xanthoria parietina
species refers to diversity of lichen-forming fungi and Evernia prunastri binding with algal urease
and many of them share the same photobiont, proteins, which may affect arginine metabolism,
such as the most frequent Trebouxia, intracellular putrescine level, and glucanase
Trentepohlia, and Nostoc species (Friedl and activities in algal cells and symbiotic interactions
Budel 2008). (Nazem-Bokaee et al. 2021; Singh and Walia
For many lichens, the mycobionts and 2014). However, none of these assumed signaling
photobionts can be isolated and cultured on artifi- reactions in algal cells have been characterized by
cial media separately. For the mycobionts, they molecular genetic studies. For the mycobionts,
normally grow extremely slow in axenic cultures different types of sugar alcohols and other
but form typical fungal colonies. Artificial resyn- compounds secreted by the photobionts may be
thesis of lichen biosynthesis with isolated recognized but there is no molecular study on
mycobiont and photobiont cells under controlled photobiont ligands, corresponding fungal
laboratory conditions has been used to study the receptors, and downstream intracellular signaling
re-lichenization processes, which can be divided pathways in the mycobionts. Although some fun-
into four stages: pre-contact (chemical gal lectins have been inappropriately described as
interactions only), contact or post-contact (chem- “receptors,” they are secreted and supposed to
ical and physical interactions), growth of undif- bind with surface proteins of the photobionts
ferentiated mass of symbiont cells, and (Nazem-Bokaee et al. 2021; Singh and Walia
differentiation into to a stratified thallus (Nazem- 2014). More importantly, none of the conserved
Bokaee et al. 2021). Earlier studies have tested signaling pathways and upstream receptors
different conditions, including various substrates identified in the budding yeast and other model
and nutritional or light conditions, for successful fungi (Zhang et al. 2021) have been characterized
reconstruction of lichens such as Acarospora by their roles in fungal–algal interactions during
fuscata and Cladonia cristatella. However, the the establishment of symbiosis.
co-culture process is time-consuming and it usu- In the past three decades, many genes and
ally takes 2–4 weeks to form a tight fungal–algal pathways involved in fungal–plant interactions
contact interface, 3–4 months to develop soredia- have been characterized in plant pathogenic
like structures, and 8–12 months to form a thallus. fungi (Jiang et al. 2018; Zhao et al. 2007). How-
ever, due to their slow growth, there are almost no
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 183

molecular genetic or functional studies in lichen- in spermogonia (asexual fruiting bodies) on

forming fungi. The re-lichenization processes are lichen thalli (in the symbiotic state). However,
drastically under-investigated in lichen-forming the formation of spermatia and spermogonia has
fungi in comparison with plant infection pro- not been observed in cultures, and their biological
cesses in plant pathogenic fungi that are more functions in the life cycle of U. muhlenbergii
amenable to molecular and classical genetic stud- remain to be characterized. Although some
ies, such as the rice blast fungus Magnaporthe Umbilicaria species produce multiseptate
oryzae (Gupta et al. 2021). However, transforma- thalloconidia directly on scleroplectenchymatous
tion is relatively efficient in Umbilicaria tissues or protruding rhizomorphs of the lower
muhlenbergii and transformants can be isolated cortex of lichen thalli, U. muhlenbergii is one of
in 1–2 weeks in Agrobacterium tumefaciens- the Umbilicaria species that does not form
mediated transformation (ATMT) or transforma- thalloconidia (Davydov et al. 2017; Hestmark
tion with PEG-treated protoplasts (Park et al. 1992).
2013; Wang et al. 2020). Furthermore, homolo- The photobiont partner for U. muhlenbergii is
gous recombination frequency is relatively high mainly Trebouxia jamesii (Fig. 7.1), a green algal
in this lichen-forming fungus, and targeted gene species. Species in the genus Trebouxia are the
disruption and knockout mutants have been suc- most common photobionts and they are found in
cessfully identified (Wang et al. 2020). In this approximately 80% of all the known lichens
chapter, we will describe unique features of (Muggia et al. 2020; Nelsen et al. 2021).
U. muhlenbergii and its suitability as a model Although only nonmotile stages with a reduced
for studying the establishment and maintenance chloroplast are observed in lichen thalli,
of symbiotic relationships between the Trebouxia species can develop autospores
mycobionts and photobionts. The emphasis will and zoospores in cultures (Friedl and Budel
be on signal transduction pathways involved in 2008). The features of pyrenoids (micro-
the initial fungal–algal interactions. compartment of chloroplasts of certain algae and
hornworts where the CO2-fixing enzyme
RuBisCO (ribulose-1, 5-bisphosphate carboxyl-
7.2 Unique Features ase/oxygenase) is localized and chloroplast mor-
of U. muhlenbergii phology are used to identify different species of
Trebouxia. Cells of T. jamesii possess impressa-
U. muhlenbergii is a foliose lichen that usually type pyrenoids (Friedl 1989) and shallowly
grows on bare stones at high elevations. It is lobed-type chloroplasts and generally has a diam-
widely distributed in Northern Asia and North eter of 4- to 20-μm (Bordenave et al. 2022).
America (Wei and Jiang 1993). The genus T. jamesii is sensitive to strong light (3000 lux)
Umbilicaria (in Umbilicariaceae, Umbilicariales, and is usually found in lichens that inhabit rocks
Lecanoromycetes) contains over 70 species that with high metal concentrations. Besides
can grow under extreme environmental T. jamesii, the U. muhlenbergii lichen contains
conditions and often produce heavily melanized an endophytic green algal species. Analysis of the
thalli. Commonly known as rock tripe, some of ribosomal DNA (rDNA) sequences showed that it
them are edible and consumed as an emergency is a novel Elliptochloris species closely related to
food. Like other Umbilicaria species, the pre- E. subsphaerica (Wang et al. 2020).
ferred propagules for dispersal in Elliptochloris species have been reported as
U. muhlenbergii are ascospores formed in endolichenic algae present in various lichens
apothecia (sexual fruiting bodies) instead of (Moya et al. 2017) as well as symbionts of sea
reproductive mixtures of vegetative hyphae and anemones (Lewis and Muller-Parker 2004) In
algal cells (Cao et al. 2015). Besides ascospores, comparison with T. jamesii, the endolichenic
the U. muhlenbergii lichen thalli also produce Elliptochloris isolate has a faster growth rate
hyaline, rod-like spermatia on spermatiophores
184 Y. Wang et al.

Fig. 7.1 Thallus structures and symbionts of Umbilicaria P photobiont zone, M medulla, LC lower cortex. (b)
muhlenbergii lichen. (a) A typical cross-section of Colonies and cells of U. muhlenbergii (JL3) and
U. muhlenbergii lichen thallus. UC upper cortex, Trebouxia jamesii (Tre1) in axenic cultures

and requires a higher light intensity for optimal sites in Asia and North America all have been
growth (Wang et al. 2020). observed to undergo dimorphic transition in axe-
Unlike all other cultivated lichen-forming nic cultures in the presence of algal cells of its
ascomycetes that grow only as hyphae, the photobiont T. jamesii but not endolichenic
U. muhlenbergii strains isolated from China and Elliptochloris algal cells (Park et al. 2013; Wang
the US all grow as yeast cells when cultured on et al. 2020). The yeast-to-hypha or pseudohypha
nutritionally rich media (Park et al. 2013; Wang dimorphic transition is well known to be
et al. 2020). The unicellular yeast cells of associated with pathogenesis in a number of
U. muhlenbergii mycobiont grow by budding human and plant pathogenic fungi, including
and form typical yeast-like colonies. When Candida albicans, Talaromyces marneffei,
cultured in liquid or solid potato dextrose medium Blastomyces dermatitidis, Coccidioides immitis,
at room temperature, typical olive-shaped yeast Histoplasma capsulatum, the corn smut fungus
cells of U. muhlenbergii are 3.0–4.5 μm in width Ustilago maydis, and peach leaf curl pathogen
and 5.0–6.5 μm in length (Fig. 7.1). On potato Taphrina deformans (Boyce and Andrianopoulos
dextrose or other common fungal media, it nor- 2015; Svetaz et al. 2017). However, to date,
mally takes only 7–10 days for a single yeast cell U. muhlenbergii is the only lichen-forming fun-
to form a visible colony, which is faster than other gus that has been reported to have dimorphism
cultivated lichen-forming fungi. associated with symbiosis. Nevertheless, it is pos-
Although U. muhlenbergii lives in the yeast sible that other Umbilicaria species or lichen-
form in axenic cultures, it exists in the hyphal or forming fungi also undergo dimorphic transition
pseudohyphal form in the lichen thallus, or switching during lichen symbiosis.
indicating that this lichenized fungus must Besides its association of dimorphism with
undergo the yeast-to-hypha transition during the symbiosis, U. muhlenbergii is amenable for
establishment of symbiosis. The U. muhlenbergii molecular genetic studies because it grows rela-
strains isolated from lichens collected at different tively fast as yeast cells. Transformant colonies
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 185

can be isolated after 10–14 days of incubation. on the Pathogen–Host Interactions (PHI) data-
U. muhlenbergii is highly sensitive to commonly base. Some of these genes, particularly the ones
used antibiotics for selecting fungal encoding secreted proteins, may be involved in
transformants. Hygromycin works efficiently at fungal–algal or fungal–microbial interactions in
20–30 μg/ml in U. muhlenbergii for selecting lichens.
transformants carrying the hygromycin
phosphotransferase (hph) cassette that is com-
monly used in transformation of M. oryzae and 7.3 Regulation of U. muhlenbergii
other ascomycetous plant pathogens (Park et al. Dimorphism by the cAMP-PKA
2013; Wang et al. 2020). Geneticin-resistant Pathway
transformants carrying the neomycin resistance
(neo) gene (Zhou et al. 2011) can be selected at The yeast-to-hypha or pseudohypha transition has
30 μg/ml. More importantly, homologous recom- been observed in various mucoromycetes,
bination frequency in U. muhlenbergii is also ascomycetes, and basidiomycetes but best studied
comparable to that of M. oryzae and many other in the budding yeast Saccharomyces cerevisiae,
plant pathogenic fungi. Targeted gene deletion in which dimorphic switching is important for
mutants can be isolated at the rate of approxi- pseudohyphal growth or filamentation and agar
mately 5% of positive transformants by the gene invasiveness (Gimeno et al. 1992). The well-
replacement approach (Wang et al. 2020). The conserved cAMP-protein kinase A (PKA) path-
Agrobacterium tumefaciens-mediated transfor- way is involved in the regulation of pseudohyphal
mation (ATMT) approach also can be used to growth in yeast and filamentous fungi. In fungal
generate insertional mutants in U. muhlenbergii pathogens, this pathway is known for
(Park et al. 2013). As the first lichen-forming co-regulating dimorphism and pathogenesis. In
fungus with gene deletion and disruption mutants C. albicans, deletion of the key components of
being identified and characterized, Umbilicaria the cAMP-PKA pathway results in defects in
muhlenbergii brings hope to establish a genetic dimorphic switching and reduced virulence
system for studying molecular mechanisms of (Sánchez-Martínez and Pérez-Martín 2002;
lichen symbiosis. Sonneborn et al. 2000). Likely in S. cerevisiae
The published draft genome of and other dimorphic ascomycetes, elevated intra-
U. muhlenbergii isolate KoLRILF000956 has cellular cAMP levels promote hyphal or
297 contigs and contig N50 of 0.21 Mb (Park pseudohyphal growth in C. albicans. In
et al. 2014). The 34.6 Mb assembled genome U. maydis, mutants disrupted in the cAMP-PKA
has a GC content of 47.12%. We have recently pathway also are defective in plant infection and
sequenced the genome of U. muhlenbergii strain morphogenesis. Interestingly, U. maydis grows
JL3 (Wang et al. 2020) by a combination of by budding as haploid yeast cells in cultures but
Illumina PE150 and PacBio sequencing. Strains develops obligate infectious hyphae after mating
KoLRILF000956 and JL3 were collected from and penetration of plant cells. However, the uac1
Mt. Tulaopoding, Ji-Lin Province, China in mutant with reduced intracellular cAMP level
2012 and 2017, respectively (Park et al. 2013; displays a constitutively filamentous growth as
Wang et al. 2020). The 35.4 Mb assembled JL3 hyphae, which can be rescued by exogenous
genome consists of 25 contigs (N50 of 1.58 Mb) cAMP or suppressor mutations in the ubc1 gene
and is predicted to encode 8166 proteins that are (Choi et al. 2015), indicating that low cAMP
over 100 amino acids (Wang, unpublished), levels stimulate hyphal growth in U. maydis, a
including 102 glycoside hydrolases, 1251 basidiomycete pathogen.
proteins with transmembrane domains, and Also interesting, U. muhlenbergii underwent
113 cytochrome P450 proteins. Among the dimorphic switching when cultured on nutrition-
predicted protein-coding genes, 1058 of them ally poor media such as 0.2 × PDA (potato dex-
are related to pathogen–host interactions based trose agar) and BBM (Bold’s Basal Medium) for
186 Y. Wang et al.

extended times (Wang et al. 2020), suggesting G-protein coupled receptors (GPCRs) or other
nutritional starvation may affect dimorphism. sensors such as extracellular mucins. All the key
The cAMP-PKA pathway plays a conserved role components of the cAMP-PKA pathway
in regulating responses to nutritional conditions characterized in S. cerevisiae are conserved in
in fungi. In U. muhlenbergii, treatments with the U. muhlenbergii genome (Fig. 7.2b) although
cAMP or IBMX (3-isobutyl-1-methylxanthine), only functional characterization of the UmGPA3
an inhibitor of cAMP diphosphoesterase, stimu- Gα subunit gene has been published (Wang et al.
late the elongation of yeast cells and 2020). Whereas some lichen-forming fungi such
pseudohyphal growth (Wang et al. 2020). When as Cladonia grayi appear to have an expanded
treated with 10 mM cAMP or 25 nmol of IBMX family of G-alpha subunits, with 5 of them being
for 14 d, colonies of U. muhlenbergii often unique (Armaleo et al. 2019), U. muhlenbergii
become darkly pigmented and have a bumpy has only three that are well conserved in other
(uneven) and hairy appearance associated with ascomycetes. UmGpa3 is homologous to Gpa2 of
pseudohyphal/hyphal growth. Aggregates of C. albicans, Gpa3 of U. maydis, and MagB of
pseudohyphae or hyphae and heavily melanized M. oryzae. The Umgpa3 deletion mutant is nor-
spherical cells (highly differentiated hypha/ mal in growth in the yeast form but it is defective
pseudohyphal compartments) are observed in in switching from yeast cells to pseudohyphal
darkly pigmented bumps. After incubation for growth when incubated on nutritionally poor
1 month, IBMX induces more significant differ- media or under hyperosmotic conditions (Wang
entiation in U. muhlenbergii (Fig. 7.2a). IBMX- et al. 2020). Because the cAMP-PKA pathway
induced bumps have protruding melanized functions downstream Gα subunits (Fig. 7.2b),
hyphae/pseudohyphae on the surface and become these results provide molecular evidence for its
hardened tissue-like structures with a dense epi- involvement in the regulation of dimorphic tran-
dermal layer in which individual compartments or sition in U. muhlenbergii. Recently, we have
cells are no longer distinguishable (Wang et al. identified the Umras2 deletion mutant that
2020). This epidermal layer of highly appears to be defective in pseudohyphal growth
differentiated and melanized hyphae/ based on our preliminary characterization and
pseudohyphae on the bumps of IBMX-treated responses to IBMX treatments (Wang et al.,
colonies is, to certain degree, similar to the epi- unpublished), further confirming the role of
dermal or cortex layer of lichen thalli. Results cAMP signaling in regulating dimorphic
from these pharmacological studies suggest that transition.
the cAMP-PKA pathway is involved in regulating Orthologs of UmGPA3 are important for path-
the yeast-to-pseudohypha transition in ogenesis in a number of fungal pathogens that
U. muhlenbergii. lack dimorphic switching during plant infection,
Like in other eukaryotes, adenylate cyclase such as M. oryzae and Botrytis cinerea. In
(AC) is responsible for the synthesis of intracel- M. oryzae, the initial surface recognition and
lular cAMP, which is degraded by phosphodies- early stages of appressorium formation are
terase (PDE) in fungi. The only known regulated by cAMP signaling and deletion of the
intracellular target of cAMP is the regulatory MAGB Gα subunit gene affects the formation of
subunit of PKA (PKR). Binding of PKR with appressoria that are necessary for plant penetra-
cAMP results in its disassociation from catalytic tion (Fang and Dean 2000). In U. muhlenbergii,
subunits of PKA (CPKAs). Free CPKAs are pseudohyphal growth can be induced by its inter-
enzymatically active and phosphorylate down- action with cells of its compatible photobiont
stream targets such as transcription factors. In T. jamesii, even when they are mixed and
fungi, adenylate cyclase can be activated by co-cultured on nutrient rich medium (Fig. 7.2c).
upstream trimeric G-proteins (Gα/Gβ/Gγ) or Under the same conditions, co-cultivation with
small GTPase (GTP hydrolase) such as RAS in algal cells of endolichenic Elliptochloris species
responses to extracellular stimuli recognized by fails to induce pseudohyphal growth in
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 187

Fig. 7.2 Regulation of yeast-to-hypha dimorphic transi- by AC and degraded by cAMP phosphodiesterase (PDE).
tion by the cAMP-PKA pathway. (a) Colony morphology PKR and CPKA are regulatory and catalytic subunits of
and pseudohyphae of U. muhlenbergii cultures treated PKA. Binding of PKR with cAMP activates the PKA
with IBMX for 2 weeks. (b) A general schematic drawing activities by freeing CPKA from its inhibitory binding
of the well-conserved cAMP-PKA pathway in fungi. In with PKR. Known downstream transcription factors of
the budding yeast Saccharomyces cerevisiae, both Gpr1 PKA include yeast SFL1, FLO11, MSN2, and MSN4. (c)
GPCR functioning upstream from the trimeric G-proteins Algal cells of T. jamesii induce pseudohyphal growth in
(Gα, Gβ, Gγ) and RAS small GTPase proteins can regulate the wild type but not Umgpa3 mutant of U. muhlenbergii
adenylate cyclase (AC). Intracellular cAMP is synthesized in cocultures

U. muhlenbergii, indicating a specific recognition promotes pseudohyphal growth in nutrient rich

of compatible photobiont (Wang et al. 2020). media. However, algal cells in close contact
Longer incubation times lead to the production with pseudohyphae of the UmGPA3G45V and
of darkly pigmented colonies that consist of algal UmGPA3Q208L transformants are often damaged,
cells and melanized pseudohyphae and have suggesting a destructive effect on symbiotic
aerial hyphae/pseudohyphae on the surface interactions between fungal and algal cells.
(Wang et al. 2020). The UmGpa3 mutant is Taken together, these pharmacological and
defective in pseudohyphal growth induced by molecular studies have shown that the
T. jamesii cells. In cocultures of the UmGpa3 cAMP-PKA pathway plays a critical role in the
mutant and T. jamesii cells, melanized colonies dimorphic switching and fungal–algal
with a mixture of pseudohyphae and algal cells interactions during lichen symbiosis in
are not observed (Wang et al. 2020), showing the U. muhlenbergii.
importance of UmGPA3 and cAMP signaling in
T. jamesii-induced differentiation in
U. muhlenbergii. In M. oryzae, dominant 7.4 Map Kinase Signaling
mutations in MAGB lead to appressorium forma- Pathways in U. muhlenbergii
tion on nonconducive conditions but defects in
pathogenesis (Fang and Dean 2000). Similarly, In plant pathogenic fungi, the mitogen-activated
expression of the dominant active UmGPA3G45V protein (MAP) kinase pathways also play impor-
and UmGPA3Q208L alleles in U. muhlenbergii tant roles in regulating responses to various
188 Y. Wang et al.

extracellular cues and different infection pro- do form intracellular haustoria that are
cesses, such as appressorium formation, plant surrounded by thickened cytoplasmic derivatives
cell penetration, and invasive growth (Jiang of algal cells. A few others form intraparietal
et al. 2018; Zhao et al. 2007). A typical MAP haustoria that are short, peg-like protrusions
kinase cascade consists of three protein kinases, compressing but not piercing through algal cell
including a MAP kinase (MAPK), a MAPK wall (Goodenough et al. 2021; Honegger 1986).
kinase (MEK), and a MEK kinase (MEKK) that Although haustoria are abundant in one
are activated sequentially. Like most filamentous Umbilicariaceae species, L. pustulata, haustorium
ascomycetous fungal pathogens, the formation has not been reported in
U. muhlenbergii genome has three MAPK U. muhlenbergii and other Umbilicariaceae spe-
pathways consisting of three non-redundant cies. In haustorium-forming lichenized fungi, the
MAPK, MEK, and MEKK genes (Table 7.1). IG MAPK pathway is involved in the regulation
These three MAPK pathways also are conserved of penetration and haustorium formation.
in other lichen-forming fungi that have been Besides its role in regulating infection
sequenced, including Lasallia pustulata and structures, the IG MAPK pathway is also impor-
C. grayi (Armaleo et al. 2019; Dal Grande et al. tant for infectious growth in plant tissues after
2017). Although these well-conserved MAPK penetration. For lichen-forming fungi, this
pathways likely play important roles in regulating MAPK pathway may be also involved in balanc-
fungal–algal interactions and lichen symbiosis, to ing fungal–algal interactions and later stages of
date, none of them have been functionally the re-lichenization processes. Our preliminary
characterized in any lichen-forming fungi. data showed that U. muhlenbergii and algal cells
In plant pathogenic fungi, the Pmk1 invasive form a biofilm-like complex layer upon initial
growth (IG) pathway has a conserved role in interactions on solid surfaces, which is blocked
regulating the formation of infection structures by deletion of the PMK1 ortholog (Wang, unpub-
(such as appressoria and hyphopodia) and infec- lished). Therefore, this MAPK pathway likely
tious growth in infected plant tissues, often in plays an important role in the initial symbiotic
coordination with the cAMP-PKA pathway associations with algal cells in U. muhlenbergii.
(Jiang et al. 2018; Zhao et al. 2007). Pmk1 is a In human and plant pathogens, this MAPK path-
MAP kinase orthologous to yeast Fus3 and Kss1 way is known to co-regulate various infection
that regulate pheromone response and invasive/ processes with the cAMP signaling pathway. In
pseudohyphal growth in S. cerevisiae. This path- U. muhlenbergii, the IG MAPK and cAMP sig-
way also is known to play a critical role in some naling pathways may co-regulate the initial sym-
human pathogens for virulence and capsule for- biotic interactions as well as further
mation. Although some lichen-forming fungi are differentiation of masses of fungal and algal
known to form appressoria or appressorium-like cells in the re-lichenization process. In C. grayi,
structures such as Lecidea albocaerulescens, orthologs of the MGB1 Gβ and PTH12
Baeomyces rufus, and C. cristatella (Goodenough (MoHOX7) transcription factor genes involved
et al. 2021; Honegger 1986), it is not clear in regulating appressorium formation via the
whether U. muhlenbergii form any specialized cAMP signaling and PMK1 MAPK pathways in
attachment or penetration structures. Neverthe- M. oryzae were identified as differentially
less, it is likely that the invasive growth expressed genes (DEGs) by subtractive
(IG) MAPK pathway regulates fungal responses hybridization (SSH) analysis during fungal–
to chemical and physical signals from algal cells, algal interactions (Joneson et al. 2011).
attachment, and appressorium formation if Studies in a number of plant pathogens have
U. muhlenbergii forms appressoria. Whereas shown that the cell wall integrity (CWI) MAP
many lichen-forming fungi do not form haustoria kinase pathway also has a conserved role in fun-
for nutrient uptakes, some species such as gal pathogenesis. In the budding yeast, the CWI
Lecanora conizaeoides and Lecidella euphoria pathway consisting of the Bck1-Mkk1/Mkk22-
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 189

Table 7.1 Key components of conserved MAPK pathways in U. muhlenbergii

U. muhlenbergiia
Functions S. cerevisiae M. oryzae KoLRILF000956 JL3
Kss1/Pmk1 IG pathway
GPCR Ste2 Ste2 Contig_79: 15259-16434: + A2080a
GPCR Ste3 Ste3 Contig_18: 38439-39570: + A4507
Mucin Msb2 MoMsb2 Contig_235: 25655-26374: + A1409
G-beta Ste4 Mgb1 Contig_33: 163167-164575: - A1076
GTPase Ras2 MoRAS2 Contig_89: 47393-48197: + A0138
PAK Ste20 Mst20 Contig_249: 39860-44611: + A5140
PAK Cla4 Chm1 Contig_17: 5044-7949: - A4476
Adaptor Ste50 Mst50 Contig_143: 62212-63921: - A1025
MEKK Ste11 Mst11 Contig_19: 77033-80129:- A4528
MEK Ste7 Mst7 Contig_264: 43620-45304: + A5885
MAPK Fus3/Kss1 Pmk1 Contig_264: 164503-165751: - A5439
CWI MAPK pathway
Sensor Wsc1 MGG_04325 Contig_266: 184541-185490: + A5663a
Sensor Wsc2 MGG_02754 Contig_92: 152719-155985: + A0313
Sensor Wsc3 MGG_01466 Contig_240: 136301-141700: - A2477
Sensor Wsc4 – Contig_199: 11029-14777: - A6922
Sensor Mid2 MGG_12606 Contig_170: 15231-16130: + A3494a
Sensor Mtl1 –
GTPase Rho1 MGG_07176 Contig_173: 79123-80189: - A3616
PKC Pkc1 MoPkc1 Contig_90: 49578-53475: + A0159
MEKK Bck1 Mck1 Contig_268: 104646-108377: - A5755
MEK Mkk1/Mkk2 Mkk2 Contig_49: 1494-2850: - A0560
MAPK Slt2 Mps1 Contig_264: 279110-280707: + A5473
HOG pathway
Osmosensor Sho1 MoSho1 Contig_163: 400712-401925: - A7020
Histidine kinase Sln1 MoSln1 Contig_288: 84078-87819: - A5981
Histidine kinase – MoHik1 Contig_288: 162918-166419: - A6006
Phosphorelay protein Ypd1 MoYpd1 Contig_109: 67377-68020: - A0844
Phosphorelay protein Ssk1 MoSsk1 Contig_163: 204696-206372: - A7172
MEKK Ste11 Mst11 Contig_19: 77033-80129:- A4528
MEKK Ste11 Mst11 Contig_19: 77033-80129:- A4528
MEKK Ssk1 MoSsk1 Contig_163: 204696-206372: - A7172
MEKK Ssk2/Ssk22 MoSsk2 Contig_203: 346910-351089: - A6737
MEK Pbs2 MoPbs2 Contig_225: 69255-70877: - A2301
MAPK Hog1 Osm1 Contig_70: 173179-174589: + A3836
a
Based on unpublished annotations of predicted genes in strains KoLRILF000956 (Genome accession in NCBI:
GCA_000611775.1) and JL3 (Genome unpublished) of U. muhlenbergii that were isolated from Mt. Tulaopoding,
Ji-Lin Province, China in 2012 and 2017, respectively (Park et al. 2013; Wang et al. 2020)

Slt2 MAPK cascade is activated by Rho1 and roles in regulating various infection and develop-
Pkc1 to regulate gene expression changes via mental processes besides responses to cell wall
transcription factors Rlm1 and Swi6 (Jiménez- stress. In some fungi, it is also involved in nutri-
Gutiérrez et al. 2020) for remodeling of the fungal ent sensing and adaptations to oxidative or other
cell wall during growth and development as well stresses. The key components of this CWI MAPK
as responding to environmental stimuli. In plant pathway are conserved in ascomycetous plant
pathogens, this MAPK pathway plays important pathogens and lichen-forming fungi, including
190 Y. Wang et al.

U. muhlenbergii (Table 7.1). It is possible that the well-conserved role in regulating adaptive
CWI MAPK pathway regulates nutrient sensing responses to hyperosmotic stress. Deletion of the
and symbiotic growth with algal cells. In HOG1 ortholog results in increased sensitivity to
Cochliobolus heterostrophus, the CWI MAPK hyperosmotic stress in all the plant pathogenic
and ChPMK1 co-regulate melanin biosynthesis. fungi studied. In M. oryzae, the osm1 deletion
Old cultures of U. muhlenbergii and its lichen mutant is hypersensitive to osmotic stress and
thalli are heavily melanized. The CWI and IG desiccation (Dixon et al. 1999). The
MAPKs may also co-regulate melanin synthesis U. muhlenbergii genome contains all the key
in U. muhlenbergii. In addition, the Mps1 MAPK components of the HOG pathway, including
regulates the expression of a number of effectors orthologs of yeast Ssk1, Ssk2/22, Pbs2, and
in M. oryzae (Zhang et al. 2017). If fungal–algal Hog1 (Table 7.1). The UmHog1 MAPK has the
interactions also involve effectors, the CWI path- typical TGY dual phosphorylation site and shares
way may play a role in regulating the expression 92% identity with Osm1 of M. oryzae. Therefore,
of U. muhlenbergii effectors to establish and it is likely that the HOG pathway is important for
maintain lichen symbiosis. Furthermore, the survival in nature for U. muhlenbergii and other
CWI MAPK is known to regulate hyphal fusion lichens that have to endure frequent wetting-
and heterokaryon formation in Fusarium drying cycles. In addition, like in other filamen-
graminearum and other ascomycetes (Hou et al. tous fungi (Zhang et al. 2021), U. muhlenbergii
2002). The differentiation of mature lichen thalli may also use the HOG pathway to regulate
likely involves hyphal fusion and cell wall adaptations or responses to oxidative, elevated
modifications, which may be regulated by the temperature, UV radiation, and other environ-
CWI MAPK pathway in U. muhlenbergii. mental stresses that occur frequently in many
Although lack of functional studies, genomic lichens. In the desert lichen fungus Endocarpon
and expression profiling analyses have implicated pusillum, orthologs of the Sho1 osmosensor of
the importance of the CWI pathway in other the HOG pathway (EPUS01068) and BCK1
lichen-forming fungi. For example, in 21-day-old MEK1 of the CWI pathway (EPUS4996) are
cocultures of C. grayi and its algal photobiont, the among the differentially expressed genes under
expression level of a phosphatase gene homolo- PEG-induced drought stress (Wang et al. 2015).
gous to MoYVH1 is up-regulated two-fold For the upstream receptors or sensors of these
(Armaleo et al. 2019). In M. oryzae, MoYvh1 is signaling pathways, the U. muhlenbergii genome
functionally related to both the CWI MAPK and has 73 predicted G-protein coupled receptor
cAMP-PKA pathways (Liu et al. 2016). In (GPCR) genes, including several of them with
Lasallia pustulata, the orthologs of PKC1 and the CFEM (common in several fungal extracellu-
WSC4 that are functionally related to the CWI lar membrane proteins) domain unique to fungal
pathway in S. cerevisiae are among the genes GPCRs, that are known to function upstream
identified by gene–environment correlation anal- from both the cAMP-PKA and IG MAPK
ysis to be related to adaptations for growth at pathways for regulating plant infection processes
different elevations (Dal Grande et al. 2017). in fungal pathogens such as M. oryzae and
Unlike the IG and CWI MAPKs that have the F. graminearum, including (Jiang et al. 2019;
TEY dual phosphorylation site, the third well- Kulkarni et al. 2005). It is possible that some of
conserved MAPK in fungi has the TGY motif, these GPCRs in U. muhlenbergii are involved in
which is similar to p38 stress-activated MAP recognizing chemical and physical signals from
kinases (SAPKs) in animals. In yeast, this algal cells of T. jamesii in early re-lichenization
MAPK of the High-Osmolarity Glycerol (HOG) processes. In C. grayi, 13 of the PTH11-like
pathway mainly regulates responses to GPCR genes are up-regulated over two-fold in
hyperosmotic stress. In plant pathogenic fungi, 21-day-old cocultures with its photobiont cells
although its importance for plant infection varies (Armaleo et al. 2019). The genome sequence of
among different fungi, the HOG pathway has a U. muhlenbergii also has predicted genes
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 191

orthologous to other receptor genes that are genome has two well-conserved DNA
known to function upstream to the MAPK path- methyltransferase (DNMT) genes orthologous to
way in S. cerevisiae and fungal pathogens, such RID and DIM-5 of Neurospora crassa. Besides
as orthologs of SLN1, MSB2, SHO1, WSC1–3, assaying DNA methylation, functional character-
and MID2. Some of them may be functional dur- ization of these two DNMT genes will clarify the
ing fungal–algal interactions by activating their importance of DNA methylation in lichen symbi-
downstream MAPKs and targets. In cocultures of osis. Along the same line, functional characteri-
C. grayi with algal cells, the WSC1 and MSB2 zation of genes involved in histone modifications,
orthologs are up-regulated, although less than such as histone acetyltransferase or deacetylase
two-fold (Armaleo et al. 2019). genes (Aramayo and Selker 2013; Dubey and
Jeon 2017), will determine the importance of
epigenetic controls in lichen symbiosis.
7.5 Conclusions and Perspectives Despite recent advances in genetic studies with
U. muhlenbergii, it is still difficult to pursue
Our knowledge of regulatory mechanisms molecular genetics studies with its photobiont,
controlling symbiont recognition and interactions which poses a problem for advanced studies of
in lichen symbiosis is very limited, likely due to fungal–algal interactions with this lichen system.
the slow growth rate of most lichen-forming fungi Nevertheless, genomics, proteomics, and
and lack of a model system for molecular genetic metabolomics analyses are possible with
studies. With its relative fast growth rate, effi- T. jamesii to study its interactions with
ciency for transformation and gene knockout, U. muhlenbergii. In comparison with fungal
and symbiosis-related yeast-pseudohypha transi- pathogens, another lagging area in lichen-forming
tion, U. muhlenbergii has the potential to be used fungi is advanced cell biology studies with
as a model to study early stages of symbiotic fungal–algal interactions. It will be important to
interactions and signal transduction pathways develop transformants of U. muhlenbergii
involved in the recognition of compatible symbi- expressing fluorescent markers in different cellu-
ont cells and development of highly differentiated lar components and carefully examine by confo-
masses of fungal–algal cells. In addition to the cal microscopy for cellular changes and
cAMP-PKA and MAPK pathways described differentiations associated with initial symbiotic
above, other signaling pathways (such as calcium interactions.
or nitric oxide signaling) may also play roles in
lichen symbiosis. Furthermore, some of these Acknowledgments The authors are grateful to the
conserved signaling pathways are known to regu- National Natural Science Foundation of China for finan-
cial support (grant No. 32170082 and 32070096). The
late fungal–fungal and fungal–bacterial
authors thank Ms. Qiuxia Yang for her assistance in micro-
interactions in ascomycetes (Zhang et al. 2021) manipulation on the lichen thallus.
and they may affect the interactions of
U. muhlenbergii with other microbes present in
lichen thalli. References
Besides genetic regulations, lichen symbiosis
may also involve epigenetic mechanisms. Earlier Aramayo R, Selker EU (2013) Neurospora crassa, a
studies with C. grayi and other lichen-forming model system for epigenetics research. Cold Spring
Harb Perspect Biol 5:a017921. https://doi.org/10.
fungi have shown that DNA methylation is 1101/cshperspect.a017921
increased in the symbiotic condition in compari- Armaleo D, Müller O, Lutzoni F, Andrésson ÓS, Blanc G,
son with axenic cultures, indicating that high Bode HB, Collart FR, Dal Grande F, Dietrich F,
levels of DNA methylation may be associated Grigoriev IV et al (2019) The lichen symbiosis
re-viewed through the genomes of Cladonia grayi
with lichen formation. Although whether
and its algal partner Asterochloris glomerata. BMC
U. muhlenbergii is increased in DNA methylation Genomics 20:605–637. https://doi.org/10.1186/
during symbiotic interactions is not clear, its s12864-019-5629-x
192 Y. Wang et al.

Bordenave CD, Muggia L, Chiva S, Leavitt SD, Cell 68:1077–1090. https://doi.org/10.1016/0092-

Carrasco P, Barreno E (2022) Chloroplast morphology 8674(92)90079-r
and pyrenoid ultrastructural analyses reappraise the Goodenough U, Wagner R, Roth R (2021) Lichen 4. The
diversity of the lichen phycobiont genus Trebouxia algal layer. Algal Res 58:102355. https://doi.org/10.
(Chlorophyta). Algal Res 61:102561. https://doi.org/ 1016/j.algal.2021.102355
10.1016/j.algal.2021.102561 Grube M, Cardinale M, de Castro Jr JV, Muller H, Berg G
Boyce KJ, Andrianopoulos A (2015) Fungal dimorphism: (2009) Species-specific structural and functional diver-
the switch from hyphae to yeast is a specialized mor- sity of bacterial communities in lichen symbioses.
phogenetic adaptation allowing colonization of a host. ISME J 3:1105–1115. https://doi.org/10.1038/ismej.
FEMS Microbiol Rev 39:797–811. https://doi.org/10. 2009.63
1093/femsre/fuv035 Gupta L, Vermani M, Kaur Ahluwalia S, Vijayaraghavan
Cao S, Zhang F, Liu C, Hao Z, Tian Y, Zhu L, Zhou Q P (2021) Molecular virulence determinants of
(2015) Distribution patterns of haplotypes for Magnaporthe oryzae: disease pathogenesis and recent
symbionts from Umbilicaria esculenta and interventions for disease management in rice plant.
U. muehlenbergii reflect the importance of reproduc- Mycology 12:174–187. https://doi.org/10.1080/
tive strategy in shaping population genetic structure. 21501203.2020.1868594
BMC Microbiol 15:212. https://doi.org/10.1186/ Hestmark G (1992) Conidiogenesis in five species of
s12866-015-0527-0 Umbilicaria. Fungal Biol 96:1033–1043. https://doi.
Choi J, Jung WH, Kronstad JW (2015) The cAMP/protein org/10.1016/S0953-7562(09)80113-5
kinase A signaling pathway in pathogenic basidiomy- Honegger R (1986) Ultrastructural studies in lichens:
cete fungi: Connections with iron homeostasis. J I. Haustorial types and their frequencies in a range of
Microbiol 53:579–587. https://doi.org/10.1007/ lichens with trebouxioid photobionts. New Phytol 103:
s12275-015-5247-5 785–795. https://doi.org/10.1111/j.1469-8137.1986.
Dal Grande F, Sharma R, Meiser A, Rolshausen G, tb00853.x
Budel B, Mishra B, Thines M, Otte J, Pfenninger M, Hou ZM, Xue CY, Peng YL, Katan T, Kistler HC, Xu JR
Schmitt I (2017) Adaptive differentiation coincides (2002) A mitogen-activated protein kinase gene
with local bioclimatic conditions along an elevational (MGV1) in Fusarium graminearum is required for
cline in populations of a lichen-forming fungus. BMC female fertility, heterokaryon formation, and plant
Evol Biol 17:93–106. https://doi.org/10.1186/s12862- infection. Mol Plant-Microbe Interact 15:1119–1127.
017-0929-8 https://doi.org/10.1094/Mpmi.2002.15.11.1119
Davydov EA, Peršoh D, Rambold G (2017) Jiang C, Zhang X, Liu H, Xu JR (2018) Mitogen-activated
Umbilicariaceae (lichenized Ascomycota) - Trait evo- protein kinase signaling in plant pathogenic fungi.
lution and a new generic concept. Taxon 66:1282– PLoS Pathog 14:e1006875. https://doi.org/10.1371/
1303. https://doi.org/10.12705/666.2 journal.ppat.1006875
Dixon KP, Xu JR, Smirnoff N, Talbot NJ (1999) Indepen- Jiang C, Cao S, Wang Z, Xu H, Liang J, Liu H, Wang G,
dent signaling pathways regulate cellular turgor during Ding M, Wang Q, Gong C et al (2019) An expanded
hyperosmotic stress and appressorium-mediated plant subfamily of G-protein-coupled receptor genes in
infection by Magnaporthe grisea. Plant Cell 11:2045– Fusarium graminearum required for wheat infection.
2058. https://doi.org/10.1105/tpc.11.10.2045 Nat Microbiol 4:1582–1591. https://doi.org/10.1038/
Dubey A, Jeon J (2017) Epigenetic regulation of develop- s41564-019-0468-8
ment and pathogenesis in fungal plant pathogens. Mol Jiménez-Gutiérrez E, Alegría-Carrasco E, Sellers-Moya Á,
Plant Pathol 18:887–898. https://doi.org/10.1111/mpp. Molina M, Martín H (2020) Not just the wall: the other
12499 ways to turn the yeast CWI pathway on. Int Microbiol
Fang EGC, Dean RA (2000) Site-directed mutagenesis of 23:107–119. http://doi.org/0.1007/s10123-019-
the mag B gene affects growth and development in 00092-2
Magnaporthe grisea. Mol Plant-Microbe Interact 13: Joneson S, Lutzoni F (2009) Compatibility and thigmotro-
1214–1227. https://doi.org/10.1094/Mpmi.2000.13. pism in the lichen symbiosis: a reappraisal. Symbiosis
11.1214 47:109–115. https://doi.org/10.1007/Bf03182294
Friedl T (1989) Comparative ultrastructure of pyrenoids in Joneson S, Armaleo D, Lutzoni F (2011) Fungal and algal
Trebouxia (Microthamniales, Chlorophyta). Plant Syst gene expression in early developmental stages of
Evol 164:145–159. https://doi.org/10.1007/ lichen-symbiosis. Mycologia 103:291–306. https://
BF00940435 doi.org/10.3852/10-064
Friedl T, Budel B (2008) Photobionts. In: Nash TH Kulkarni RD, Thon MR, Pan H, Dean RA (2005) Novel
(ed) Lichen biology. Cambridge University Press, pp G-protein-coupled receptor-like proteins in the plant
21–38. https://doi.org/10.1017/ pathogenic fungus Magnaporthe grisea. Genome Biol
CBO9780511790478.003 6:R24. https://doi.org/10.1186/gb-2005-6-3-r24
Gimeno CJ, Ljungdahl PO, Styles CA, Fink GR (1992) Lewis LA, Muller-Parker G (2004) Phylogenetic place-
Unipolar cell divisions in the yeast S. cerevisiae lead to ment of “Zoochlorellae” (Chlorophyta), algal symbiont
filamentous growth: regulation by starvation and RAS. of the temperate sea anemone Anthopleura
7 Umbilicaria muhlenbergii: A Model for Studying Molecular Mechanisms. . . 193

elegantissima. Biol Bull 207:87–92. https://doi.org/10. manipulation. Genome Announc 2:e00357–e00314.

2307/1543583 https://doi.org/10.1128/genomeA.00357-14
Liu X, Qian B, Gao C, Huang S, Cai Y, Zhang H, Sánchez-Martínez C, Pérez-Martín J (2002) Gpa2, a
Zheng X, Wang P, Zhang Z (2016) The putative pro- G-protein alpha subunit required for hyphal develop-
tein phosphatase MoYvh1 functions upstream of ment in Candida albicans. Eukaryot Cell 1:865–874.
MoPdeH to regulate the development and pathogenic- https://doi.org/10.1128/ec.1.6.865-874.2002
ity in Magnaporthe oryzae. Mol Plant-Microbe Inter- Singh RS, Walia AK (2014) Characteristics of lichen
act 29:496–507. https://doi.org/10.1094/MPMI-11-15- lectins and their role in symbiosis. Symbiosis 62:
0259-R 123–134. https://doi.org/10.1007/s13199-014-0278-y
Lutzoni F, Nowak MD, Alfaro ME, Reeb V, Sonneborn A, Bockmühl DP, Gerads M, Kurpanek K,
Miadlikowska J, Krug M, Arnold AE, Lewis LA, Sanglard D, Ernst JF (2000) Protein kinase A encoded
Swofford DL, Hibbett D et al (2018) Contemporane- by TPK2 regulates dimorphism of Candida albicans.
ous radiations of fungi and plants linked to symbiosis. Mol Microbiol 35:386–396. https://doi.org/10.1046/j.
Nat Commun 9:5451–5461. https://doi.org/10.1038/ 1365-2958.2000.01705.x
s41467-018-07849-9 Spribille T, Tuovinen V, Resl P, Vanderpool D,
Meeßen J, Eppenstein S, Ott S (2013) Recognition Wolinski H, Aime MC, Schneider K,
mechanisms during the pre-contact state of lichens: Stabentheiner E, Toome-Heller M, Thor G et al
II. Influence of algal exudates and ribitol on the (2016) Basidiomycete yeasts in the cortex of ascomy-
response of the mycobiont of Fulgensia bracteata. cete macrolichens. Science 353:488–492. https://doi.
Symbiosis 59:131–143. https://doi.org/10.1007/ org/10.1126/science.aaf8287
s13199-012-0219-6 Svetaz LA, Bustamante CA, Goldy C, Rivero N, Muller
Moya P, Molins A, Martinez-Alberola F, Muggia L, GL, Valentini GH, Fernie AR, Drincovich MF, Lara
Barreno E (2017) Unexpected associated microalgal MV (2017) Unravelling early events in the Taphrina
diversity in the lichen Ramalina farinacea is uncov- deformans-Prunus persica interaction: an insight into
ered by pyrosequencing analyses. PLoS One 12: the differential responses in resistant and susceptible
e0175091. https://doi.org/10.1371/journal.pone. genotypes. Plant Cell Environ 40:1456–1473. https://
0175091 doi.org/10.1111/pce.12942
Muggia L, Nelsen MP, Kirika PM, Barreno E, Beck A, Wang Y, Zhang X, Zhou Q, Zhang X, Wei J (2015)
Lindgren H, Lumbsch HT, Leavitt SD, Fernandez- Comparative transcriptome analysis of the lichen-
Mendoza F, Perez-Ortega S et al (2020) Formally forming fungus Endocarpon pusillum elucidates its
described species woefully underrepresent phyloge- drought adaptation mechanisms. Sci China Life Sci
netic diversity in the common lichen photobiont 58:89–100. https://doi.org/10.1007/s11427-014-
genus Trebouxia (Trebouxiophyceae, Chlorophyta): 4760-9
an impetus for developing an integrated taxonomy. Wang Y, Wei X, Bian Z, Wei J, Xu JR (2020)
Mol Phylogenet Evol 149:106821. https://doi.org/10. Coregulation of dimorphism and symbiosis by cyclic
1016/j.ympev.2020.106821 AMP signaling in the lichenized fungus Umbilicaria
Nazem-Bokaee H, Hom EFY, Warden AC, Mathews S, muhlenbergii. Proc Natl Acad Sci U S A 117:23847–
Gueidan C (2021) Towards a systems biology 23858. https://doi.org/10.1073/pnas.2005109117
approach to understanding the lichen symbiosis: Wei JC, Jiang YM (1993) The Asian Umbilicariaceae
opportunities and challenges of implementing network (Ascomycota). International Academic Publishers,
modelling. Front Microbiol 12:667864. https://doi.org/ Beijing
10.3389/fmicb.2021.667864 Zhang X, Liu W, Li Y, Li G, Xu JR (2017) Expression of
Nelsen MP, Leavitt SD, Heller K, Muggia L, Lumbsch HT HopAI interferes with MAP kinase signalling in
(2021) Macroecological diversification and conver- Magnaporthe oryzae. Environ Microbiol 19:4190–
gence in a clade of keystone symbionts. FEMS 4204. https://doi.org/10.1111/1462-2920.13884
Microbiol Ecol 97:fiab072. https://doi.org/10.1093/ Zhang X, Wang Z, Jiang C, Xu JR (2021) Regulation of
femsec/fiab072 biotic interactions and responses to abiotic stresses by
Park SY, Jeong MH, Wang HY, Kim JA, Yu NH, Kim S, MAP kinase pathways in plant pathogenic fungi. Stress
Cheong YH, Kang S, Lee YH, Hur JS (2013) Biol 1. https://doi.org/10.1007/s44154-021-00004-3
Agrobacterium tumefaciens-mediated transformation Zhao XH, Mehrabi R, Xu JR (2007) Mitogen-activated
of the lichen fungus, Umbilicaria muehlenbergii. protein kinase pathways and fungal pathogenesis.
PLoS One 8:e83896. https://doi.org/10.1371/journal. Eukaryot Cell 6:1701–1714. https://doi.org/10.1128/
pone.0083896 Ec.00216-07
Park SY, Choi J, Lee GW, Jeong MH, Kim JA, Oh SO, Zhou X, Li G, Xu JR (2011) Efficient approaches for
Lee YH, Hur JS (2014) Draft genome sequence of generating GFP fusion and epitope-tagging constructs
Umbilicaria muehlenbergii KoLRILF000956, a in filamentous fungi. In: Xu JR, Bluhm BH (eds)
lichen-forming fungus amenable to genetic Fungal genomics. Springer, pp 199–212. https://doi.
org/10.1007/978-1-61779-040-9_15
 After Air, Light, and Water, the Next
Most Important Thing Is Grass: 8
An Introduction to the Epichloë–Grass
Symbiosis

Daniel A. Bastías, Emma R. Applegate, Pedro E. Gundel,

Linda J. Johnson, Wade J. Mace, Christina D. Moon,
Sushma Prakash, and Stuart D. Card

Abstract fungal diseases and abiotic stresses, such as

Epichloë (Clavicipitaceae) is a genus of fila- those caused by drought and/or nutrient
mentous fungal endophytes that have deficiencies. Strains of mutualistic Epichloë
co-evolved with cool-season grasses with have been developed into highly efficacious
which they form perpetual, symbiotic biocontrol products and are widely utilized
associations. In natural ecosystems Epichloë within the Americas, Australia, and
endophytes have implications for species New Zealand for pasture persistence and wild-
diversity, food web structures, and fundamen- life deterrence.
tal ecological processes. In many managed
pastoral systems, selected Epichloë strains are Keywords
regarded as necessary components of the Alkaloid · Animal toxicosis · Biological
sward as they confer bioprotective traits to control · Endophyte · Fescue · Ryegrass
their host grasses. The most agriculturally
important associations are those between
selected strains of asexual, vertically transmit- 8.1 Background
ted Epichloë spp. and the widely used pasture
species: perennial ryegrass and tall fescue. This review is focussed primarily on the biology
Selected Epichloë strains confer invertebrate, of the Epichloë–grass symbioses, and the science
especially insect, pest deterrence to their plant driving the selection and development of agricul-
hosts from the production of several alkaloidal turally beneficial Epichloë (Clavicipitaceae)
secondary metabolites. Additional Epichloë- strains. While several research groups worldwide
mediated attributes include tolerance against also study Epichloë fungi, this chapter places
emphasis on the farming challenges and research
performed predominantly in New Zealand. Sig-
D. A. Bastías · E. R. Applegate · L. J. Johnson ·
W. J. Mace · C. D. Moon · S. Prakash · S. D. Card (✉) nificant research activity is undertaken by
Resilient Agriculture, AgResearch Limited, Grasslands AgResearch Limited, a Crown Research Institute,
Research Centre, Palmerston North, New Zealand in conjunction with its wholly owned subsidiary,
e-mail: [email protected] Grasslanz Technology Limited, which develops
P. E. Gundel and commercializes innovative plant
Facultad de Agronomía, IFEVA, Universidad de Buenos technologies, providing enormous benefits to pas-
Aires, CONICET, Buenos Aires, Argentina
toral agricultural production systems in
Laboratorio de Biología Vegetal, Instituto de Ciencias New Zealand and worldwide.
Biológicas, Universidad de Talca, Talca, Chile

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 195
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_8
196 D. A. Bastías et al.

Most multicellular life on Earth lives in sym- microorganism that has been identified as also
biosis with microorganisms, and plants are no displaying a mutualistic behaviour, at least for
exception (Rodriguez and Redman 2008). The part of its lifecycle, and in doing so conferring a
term symbiosis is used to describe a close and benefit on its host (for an in-depth discussion,
often long-term interaction between two different read Wennström 1994; Wilson 1995). As
biological species and is derived from the Greek discussed by several authors, the definition of
words simply meaning ‘living together’ and orig- endophyte is subsequently more descriptive and
inally popularized by one of the founding fathers encompasses information regarding the type of
of plant pathology, Heinrich Anton de Bary association between the microorganism and its
(1879). However, there is still some debate on plant host (Carroll 1988; Hardoim et al. 2015;
the exact definition of the term (Martin and Hyde and Soytong 2008; Mercado-Blanco 2015;
Schwab 2012; Schulz et al. 1998). In this chapter, Rodriguez et al. 2009). The added terms then
we have accepted the broader definition of the make this updated definition of the word ‘endo-
term, which incorporates the spectrum of possible phyte’ more informative and excludes dormant
persistent relationships (parasitic, amensalistic, pathogens or saprophytes (read Sinclair and
commensalistic, and mutualistic) between all spe- Cerkauskas 1996 for a detailed discussion). This
cies. These symbioses are complex, ranging from definition would therefore encourage researchers
mutualism through commensalism to parasitism to describe uncharacterized microorganisms
in a continuous manner (Partida-Martinez and isolated from surface-disinfected plant samples
Heil 2011; Redman et al. 2001), although some more appropriately as leaf, root, or seed
authors regard parasitism as an unbalanced sym- ‘associated’ microorganisms (Bent and Chanway
biosis (Kogel et al. 2006). Plants form symbioses 2002; Mercado-Blanco 2015).
throughout their lifecycle with an aggregation of
diverse, interacting microorganisms including
archaea, bacteria, fungi, and protists (Mercado- 8.2 Historical Summary of Epichloë
Blanco 2015; Rodriguez et al. 2009) and these in Agriculture
intimate associations are important ecological
determinants of plant biodiversity (Clay 1988). The historical events that brought Epichloë to the
This microbial community (the plant microbiota) attention of the scientific community are both
coupled with their surrounding environment (the complex and fascinating. Once a relatively
entire habitat = the microbiome) is crucially obscure group of fungi studied by a few botanists,
important for the health and productivity of the these Ascomycetes rose to notoriety in the 1970s
host plant. and early 1980s as being synonymous with live-
For this chapter, we use the definition of the stock toxicity (Bacon et al. 1977; Fletcher and
term endophyte in a stricter sense than originally Harvey 1981; Freeman 1904; Lloyd 1959;
used (also by de Bary). In its original form, endo- McLennan 1920; Neil 1940; Sampson 1933).
phyte simply meant ‘inside the plant’, being However, several events led to this endophyte-
derived from the Greek words endon (=within) derived toxicity in farmed ruminants, originating
and phyton (=plant). It is suggested that in this with the introduction of forage species into sev-
original form, the term is vague and meaningless eral New World countries. Many European grass
and therefore the term should encompass several species were introduced to the USA and
other important behavioural traits (Mercado- New Zealand from the early 1800s as the new
Blanco 2015). Microorganisms defined as settlers experimented with various foods to feed
endophytes should therefore also spend all or their farmed ruminants (Caradus et al. 2021c; Star
part of their lifecycle within the plant, and and Brooking 2006; Stewart 2006). In the USA,
maybe more importantly, should cause no appar- tall fescue [Lolium arundinaceum (Schreb.)
ent disease symptoms. The term ‘mutualistic S.J. Darbyshire = Festuca arundinacea Schreb.]
endophyte’ should be used to define a was the forage that was particularly well adapted,
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 197

Fig. 8.1 A sheep in

summer exhibiting ryegrass
staggers, a neuro-muscular
disorder caused by the
common toxic strain of
endophyte in perennial
ryegrass (Photo courtesy of
Russel Croy, AgResearch
Ltd, New Zealand)

although it was not until the 1950s that the species However, this practice was soon found to be
was widely planted (Hoveland 2009; Sleper and largely undesirable as the fungus was unknow-
West 1996). Perennial ryegrass (Lolium perenne) ingly providing the plant with additional
was the most successful forage species in the bioprotective traits. For example, in most
New Zealand environment and although both jurisdictions of New Zealand, the pastures lacking
these grass species were persistent and productive endophytes soon perished due to unforeseen pres-
in their New World habitats, they started to cause sure from an introduced insect pest, and an alter-
serious toxicity problems in livestock. After much native remedy was required.
investigation of pasture leaves, leaf litter, and soil, The situation in New Zealand was unique,
Epichloë species were identified as the causal whereby a naturalized forage plant and an insect
agents of mycotoxins present in these grazed pas- herbivore arrived from different parts of the world
ture species that manifested as serious livestock and came into direct contact with each other for
toxicoses after ingestion (Bacon et al. 1977; the first time. The first introduction was deliberate
Fletcher and Harvey 1981; Hoveland et al. with the importation of perennial ryegrasses from
1980; Latch and Christensen 1982). The most Europe, mainly England (Daly 1973); but the
potent of these neurotoxic compounds include later introduction of the Argentine stem weevil
the lolitrems (indole diterpenes) that result in (Listronotus bonariensis) in the early part of the
ryegrass staggers (Fig. 8.1) and ergot alkaloids twentieth century, was most likely accidental
responsible for fescue foot, bovine fat necrosis, (Stewart 2006; Williams et al. 1994). This event
and fescue toxicosis (summer slump or syn- illustrates how a species not considered trouble-
drome) (Bacon et al. 1977; Fletcher et al. 1990; some in its native ecosystem can manifest as a
Gallagher et al. 1981; Strickland et al. 2009; serious pest when introduced into a foreign eco-
White et al. 2003). The most obvious remedy system (Goldson et al. 2005).
for this agricultural problem was to remove the A scientific breakthrough came in the
causal fungal agent of these mammalian mid-1980s when peramine, an alkaloid responsi-
syndromes from the fescue and ryegrass hosts ble for insect deterrence, produced by an endo-
(Hoveland 2009; Latch and Christensen 1982). phyte now known as Epichloë festucae var. lolii
198 D. A. Bastías et al.

in its perennial ryegrass host, was discovered compounds known as alkaloids (Diehl 1950;
(Rowan and Gaynor 1986; Rowan et al. 1986). Leuchtmann and Clay 1988; Torres and White
Further secondary metabolites produced by the 2009). The most intensely studied genus within
grass–endophyte association were linked to bio- the Balansiae is Epichloë, which forms perpetual
activity (Bacon 1995; Gallagher et al. 1981; symbioses with more than 100 species of cool-
Lyons et al. 1986; Riedell et al. 1991), and a season grass (subfamily Pooideae), with the
bioprospecting pipeline was developed to identify majority originating from Europe and Asia
Epichloë strains that were less toxic to livestock (Caradus et al. 2021b). As Epichloë species are
while still producing alkaloids that deterred insect ecologically important constituents of many
pests (Johnson et al. 2013a). grassland ecosystems, which cover over 30% of
Since the initial research, scientific the Earth’s land area, they are regarded as key-
programmes were established that centred around stone species (Gibson 2009; Nissinen et al. 2019;
the modification of these bioprospecting pipelines Suttie et al. 2005).
to identify, characterize, and select agriculturally Epichloë–grass associations form a range of
beneficial strains of Epichloë essentially to interactions from mutualistic to antagonistic,
exploit this natural phenomenon for agricultural with the nature of the symbiosis closely linked
gains (Card et al. 2014a; de Bonth et al. 2015; to the reproductive lifecycle of the fungus (Brem
Johnson et al. 2013a). These selected Epichloë and Leuchtmann 1999). Epichloë is a monophy-
strains were then incorporated into elite grass letic genus that includes anamorphic (asexual)
cultivars with the aim to increase pasture persis- and teleomorphic (sexual) species (Fig. 8.2),
tence and productivity, which is of particular with the former previously classified as
importance to farming systems in New Zealand, Neotyphodium (read Leuchtmann et al. 2014 for
Australia, the United States, and parts of South a detailed taxonomic history of the genus). Note
America (Johnson and Caradus 2019). Today, the that species that produce ascospores usually also
genus Epichloë is synonymous with efficacious produce conidia which may serve as spermatia in
biological control of insect pests within applied sexual reproduction (see below). More than
agriculture (Card et al. 2016) and as a fungus that 30 species of Epichloë have been described,
plays a critical role in host plant defences and most exhibiting an asexual lifecycle, being exclu-
grassland ecology (Bastias et al. 2017; Clay sively vertically transmitted via the host’s
1988; Malinowski and Belesky 2000; Omacini infected seeds (Liu et al. 2017; Zhang et al.
et al. 2001; Saikkonen et al. 2006). For further 2017). This plant–endophyte association is essen-
information, please read di Menna et al. (2012) tially a hereditary endosymbiosis as the plant’s
for the New Zealand historical account and Bacon progeny not only inherit their parent’s
(1995) and Ball et al. (2019) for the USA chromosomes and cytoplasm but also their mutu-
perspective. alistic symbiont (Wernegreen 2012).
Asexual Epichloë species are largely mutual-
istic, relying on vertical transmission for their
8.3 Biology of Epichloë Endophytes reproductive success, while sexual species can
transmit horizontally between hosts, and some
Many members of the grass family Poaceae, also species can simultaneously transmit both verti-
known as the true grasses, associate with a pleth- cally and horizontally in pleiotropic symbioses
ora of endophytic microorganisms (Sánchez (Clay 1990). For hosts infected by sexual
Márquez et al. 2012), with the tribe Balansiae Epichloë, vegetative growth is asymptomatic
(Clavicipitaceae) one of the most researched. (as seen also for asexual Epichloë); however, the
Many of the fungi within the Balansiae are endo- sexual state is characterized by the formation of
phytic, systematically colonizing their plant hosts stromata (Fig. 8.3) on the reproductive tillers of
while producing an array of secondary the host, aborting the development of some or all
metabolites, most notably nitrogen-based of the inflorescences in a disease descriptively
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 199

Fig. 8.2 Asexual and sexual lifecycle of Epichloë fungal endophytes (Johnson et al. 2013a reprinted by permission of
Taylor & Francis Ltd)

known as choke (Kirby 1961; White et al. 1991). these flies consume spermatia and therefore act as
The most infamous sexual species is E. typhina, a reservoir of spermatia from the various Epichloë
which is largely antagonistic to several of its grass strains they have fed upon. When the fly
hosts, including the economically important defaecates, a mixture of spermatia from different
orchardgrass (or cocksfoot = Dactylis Epichloë strains is laid along with the flies’ eggs
glomerata), as the fungus inhibits plant seed pro- (Bultman and Leuchtmann 2008), with the
duction, impeding the host’s ability to reproduce stromatal structure forming a cool shelter for
(Chung and Schardl 1997; Leyronas and Raynal Botanophila larval development (Lembicz et al.
2008; Pfender and Alderman 1999; Western and 2021). If these spermatia and stroma are compati-
Cavett 1959). ble, the stroma will thicken allowing formation of
The mating system of sexual Epichloë species numerous perithecia that contain asci, which in
is bipolar heterothallic and is further supported by turn produce ascospores (Chung and Schardl
a tripartite relationship, whereby conidia pro- 1997). The lifecycle is completed when
duced on stromata may be vectored by a ascospores are ejected from the perithecia and
specialized symbiotic insect between opposite dispersed by wind to infect new grass hosts.
mating types of the stromata in a process of Botanophila flies may not be the only vectors of
spermatization found in a number of fungi, anal- Epichloë spermatia. Gastropods, such as slugs,
ogous to plant pollination (Bultman and White have been observed feeding on Epichloë stro-
1988; Kohlmeyer and Kohlmeyer 1974; mata, leading to transmission of viable spermatia
Leuchtmann et al. 2014). Adult flies of the and cross-fertilization (Górzyńska et al. 2011;
genus Botanophila (Anthomyiidae: Diptera) Hoffman and Rao 2013, 2014).
feed on the stromata after being attracted to Our understanding of the molecular
them by volatile compounds produced by the interactions associated with the symbiosis
fungus (Bultman et al. 1995). As feeding ensues, between grass and endophyte has been
200 D. A. Bastías et al.

Fig. 8.3 Stromata

observed on perennial
ryegrass (Photo courtesy of
Alan Stewart, PGG
Wrightson Seeds Ltd,
New Zealand)

significantly enhanced by modern nucleic acid While sexual Epichloë species are haploid, an
sequencing technologies that allow the genomes intriguing feature of this genus is the prevalence
and transcriptomes of both partners to be exam- of interspecific hybrids among the asexual species
ined under a range of conditions and developmen- that have ancestries involving two or three dis-
tal stages. Studies have revealed that Epichloë tinct Epichloë lineages, which contributes to the
species are not silent passengers that evade the genetic diversity of these otherwise clonal species
hosts’ defences within the association as earlier (Leuchtmann et al. 2019; Moon et al. 2004;
thought, but both partners actively interact with Schardl 2010). The presence of hybrids was first
each other. Epichloë can produce a range of small detected in endophytes of tall fescue and peren-
secreted proteins, termed effectors (Hassing et al. nial ryegrass via sequence analyses of specific
2019; Wang et al. 2021b), that modulate host marker genes (Schardl et al. 1994; Tsai et al.
defence responses; the effectors show altered 1994); however, genome-level analyses have
expression in endophyte-infected compared to provided far greater insight into the extent that
endophyte-free grasses (Dinkins et al. 2017; hybridization has had on the genetic diversity of
Dupont et al. 2015; Schmid et al. 2017; Wang Epichloë endophytes (Campbell et al. 2017).
et al. 2019). Moreover, the involvement of a Superinfections of established associations
myriad of processes contributes to symbiotic followed by anastomosis (hyphal fusion) is likely
compatibility, however as this is a broad area of responsible for the generation of interspecific
research, the mechanisms will not be discussed in hybrid endophytes, giving rise to a highly com-
detail here (for reviews on the subject, please read plex evolutionary history (Charlton et al. 2012;
Johnson et al. 2021; Scott et al. 2018). Tsai et al. 1994). These unique symbionts can
also be defined by several further characters,
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 201

including their foliar lifestyle, intercalary hyphal blade is a physical barrier to the advancement of
growth, transmission via seed and host specificity Epichloë hyphae and as grass leaves age, hyphae
(see below for more details). can increase in diameter, and therefore biomass,
but not in frequency (Christensen and Voisey
2006). Critically, Epichloë endophytes colonize
8.3.1 A Foliar Lifestyle meristematic tissues and structures such as axil-
lary buds and inflorescence primordia that allow
Unlike other well-known plant symbionts such as infection of resulting vegetative tillers and repro-
rhizobia and arbuscular mycorrhizal fungi that are ductive tissues for seed transmission (see below).
associated with the rhizosphere, asexual Epichloë
species are only located in the above-ground
tissues of their grass hosts, where they spend 8.3.2 Intercalary Hyphal Growth
their entire lifecycle growing systemically within
the intercellular spaces between plant cells In their vegetative state, the growth of Epichloë is
(Fig. 8.4). Vegetative plants colonized by fully synchronized and highly regulated with that
Epichloë remain completely symptomless and of their hosts, colonizing grass leaves by a unique
cannot be differentiated from plants lacking the mechanism termed intercalary hyphal extension,
symbiosis with the naked eye. Epichloë can, how- and not by the generalized model of hyphal tip
ever, be easily observed in planta, using basic growth (Christensen et al. 2002, 2008; Voisey
microscopy techniques that allow the presence 2010). During intercalary hyphal extension,
of septate, intercellular, infrequently branched Epichloë hyphae attach to the enlarging and
hyphae running longitudinally in leaf sheaths to elongating host grass cells through an extracellu-
be observed (Bacon and White 1994; Latch et al. lar matrix of uncertain origin, and cumulative
1988). Leaf blade colonization patterns can differ growth along the length of the hyphal filament
with host and endophyte genotype, and plant age allows the endophyte to extend at the same rate as
(Christensen et al. 2002). It is speculated that the its host (Christensen et al. 2008).
ligular zone located between the leaf sheath and

Fig. 8.4 Hyphae of

Epichloë coenophiala
growing within the
intercellular spaces of a tall
fescue leaf sheath (Card
et al. 2013 reprinted by
permission of Taylor &
Francis Ltd)
202 D. A. Bastías et al.

8.3.3 Transmission via Seed Epichloë species are found throughout the leaf
sheath, aligned to the leaf axis (Christensen
As plants and their microbial symbionts have et al. 2002). Furthermore, Epichloë associations
co-evolved, the latter have developed sophisti- with tall fescue differ in their distribution of fun-
cated mechanisms of transmission. Seed-borne gal hyphae, with continental-type associations
symbionts (e.g. Alternaria spp.) may be carried exhibiting poor colonization of their leaf blades
in or on the seed as opportunistic, transient compared to Mediterranean associations where
colonizers generally resulting in a low frequency hyphae can be readily observed in leaf blades
of colonized seed. In contrast, seed transmitted and sheaths (Christensen and Voisey 2006;
symbionts (e.g. Epichloë spp.) have evolved Takach et al. 2012). Many of the advantageous
mechanisms that exploit the plants’ reproductive traits conferred by Epichloë to their grass hosts
processes and use this strategy as an exclusive are also beneficial for the development and mar-
mechanism to establish themselves within the keting of efficacious biological control agents for
progeny. These symbionts are therefore totally the benefit of agriculture (Card et al. 2016).
reliant on the success of the plant’s reproductive
strategy for their dissemination and survival
(Barret et al. 2016). Moreover, the frequency of 8.4 Role of Secondary Metabolites
seed transmission among populations and indi- in the Epichloë–Grass
vidual plants can vary widely, and has been Association
shown to be impacted considerably by host genet-
ics (Faeth et al. 2004; Gagic et al. 2018; Leyronas A wide range of agriculturally important traits
and Raynal 2001; Ravel et al. 1997). For a conferred by Epichloë to their grass hosts are
detailed account of vertical transmission by due to their production of a diverse range of
Epichloë spp., read Liu et al. (2017) and Zhang secondary metabolites. Secondary metabolites
et al. (2017). are organic compounds produced by life forms
that are not directly involved in the organism’s
growth, development, or reproduction. These
8.3.4 Host Specificity chemicals are widely distributed throughout all
the biological kingdoms, where they generally
All known species of Epichloë are restricted to a mediate ecological interactions. Secondary
single or several host grass species or closely metabolites produced by fungi generally play an
related grass genera within the cool-season grass important ecological role, enabling these species
subfamily Pooideae due to co-speciation over to colonize diverse habitats and sustain their exis-
many millennia (Schardl 2010; Schardl et al. tence (Bills and Gloer 2017). Fungal-derived sec-
1997; Schardl and Philips 1997). However, the ondary metabolites exhibit a diverse range of
Epichloë lineage may have originally emerged activities, and many play a central role in antago-
from an animal pathogen via an interkingdom nism towards other living organisms (e.g. animal
host jump (Spatafora et al. 2007). and plant toxins, antibiotics, insecticides, and
There is substantial variation between and nematicides), as mineral scavenging agents
within different Epichloë species, particularly (e.g. siderophores), in light absorption
with respect to their in-planta colonization (e.g. pigments), as agents of symbiosis, and as
patterns and chemical diversity (discussed later). enzyme inhibitors and autoregulators (Demain
For example, Epichloë occultans, a symbiont of and Fang 2000; Keller 2019; Vining 1990).
the annual ryegrasses Lolium multiflorum and Most fungal-derived secondary metabolites are
Lolium rigidum, is only found in vegetative plants low molecular weight compounds, categorized
as a dense mycelial mass located at the base of the into three main classes (non-ribosomal peptides,
leaf sheath, while the hyphae of most other polyketides, and terpenes), that are encoded by
contiguous gene assemblies termed biosynthetic
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 203

gene clusters (BGCs). Typically, core enzymes produced by Epichloë spp. exhibit bioactive
within the BGC define the class of secondary properties with several acting as feeding
metabolite produced. Additional enzymes, tran- deterrents or toxins, protecting the host grass
scription factors, and transporters are then from invertebrate and mammalian herbivores
involved in the final modification and delivery (Bush et al. 1997). These bioactive secondary
of the resulting bioactive molecule from the metabolites confer a selective advantage to the
BGC pathway (Keller 2019). The genes within host and are therefore an example of defensive
the BGC are usually co-regulated by global or mutualism between plants and fungi (Clay 1988;
pathway-specific transcription factors which are Saikkonen et al. 2016).
activated in response to an environmental stimu- Epichloë-derived secondary metabolites are
lus. The three common backbone enzymes in produced in variable concentrations within the
BGCs are: polyketide synthases (PKSs), which host plant, with host and endophyte genotype,
are involved in the production of polyketides plant age, plant growth regulators, plant nutrition,
from acetyl CoAs, non-ribosomal peptide and environmental conditions (such as air and soil
synthetases (NRPSs), which can produce temperature, day length, solar radiation, and pre-
non-ribosomal peptides from amino acids, and cipitation) being critically important factors in
terpene synthases/cyclases (TSs and TCs) that their biosynthesis (Agee and Hill 1994; Bastías
generate terpenes from activated isoprene units et al. 2018a, b; Brosi et al. 2011; Bush et al. 1993;
(Cane et al. 1998; Fisch 2013; Keller 2019; Pillay Freitas et al. 2020; Fuchs et al. 2017; Hennessy
et al. 2022; Staunton and Weissman 2001; et al. 2016; Krauss et al. 2007; Rasmussen et al.
Süssmuth and Mainz 2017). However, hybrid 2007; Reinholz and Paul 2000). Epichloë-derived
pathways such as PKS-TC and PKS-NRPS also alkaloids also routinely accumulate in specific
exist, and not all fungal secondary metabolites are locations, e.g., in perennial ryegrass, ergovaline
generated by the three main BGC-type gene accumulates in the pseudostem and basal leaf
clusters. For example, the biosynthesis of the sheath of intermediate aged tillers while in
epichloëcyclins (peptides) are ribosomally meadow fescue, lolines are translocated within
derived (Johnson et al. 2015). the plant and accumulate in shoot and root tissues
There is a wide range of chemotypic diversity (Patchett et al. 2008; Spiering et al. 2002).
among and even within species of Epichloë Peramine, however, does not progressively accu-
underpinned by the resident BGCs, and variation mulate in any plant organ (Koulman et al. 2007).
in the presence of middle- and late-pathway genes Biosynthesis of some secondary metabolites (e.g.,
within alkaloid biosynthesis pathways (Schardl lolines) can also be stimulated after wounding,
et al. 2012, 2013a). Comprehensive genome such as during herbivory by insects (Schardl
sequencing of a range of Epichloë endophytes et al. 2007), indicating that the Epichloë–grass
and close relatives revealed the presence of large symbiosis is able to respond not only to
and pervasive blocks of repeat sequences derived abiotic cues but also biotic ones. Historically,
from retroelements, DNA transposons, and mini- Epichloë-derived secondary metabolites have
ature inverted-repeat transposable elements been challenging to isolate, identify, and
(Schardl et al. 2013b). These sequence repeat classify under standard laboratory conditions,
structures are thought to contribute to genomic although the main pathways are now well defined
instability, including gene duplications and (Caradus et al. 2022; Schardl et al. 2013a). The
losses, resulting in the production of variant alka- most well researched Epichloë-derived secondary
loid compounds (Schardl et al. 2013b). The sec- metabolites are categorized within four main
ondary metabolites derived from Epichloë are chemical classes; the ergot alkaloids
mostly produced in planta by the grass–endo- (e.g. ergovaline), the indole diterpenes
phyte association, although some alkaloids have (e.g. lolitrem B and epoxyjanthitrems),
been detected in fungal cultures at relatively low pyrrolizidines (e.g. lolines), and the
concentrations (Blankenship et al. 2001; Penn pyrrolopyrazines (e.g. peramine) (Fig. 8.5).
et al. 1993). Many secondary metabolites
204 D. A. Bastías et al.

Fig. 8.5 Overview of the key secondary metabolites diterpenes and epoxyjanthitrems from the IDT gene clus-
derived from the three alkaloid biosynthetic gene clusters ter, and (c) lolines from the LOL pathway. Proposed genes
(BGC) in Epichloë: (a) Ergot alkaloids from the ergot that encode the enzymes required for the reactions are
alkaloid synthesis (EAS) gene cluster, (b) indole denoted by arrows

8.4.1 Ergot Alkaloids (Florea et al. 2017; Gerhards et al. 2014).

Although four main groups of ergot alkaloids
Ergot alkaloids are a group of complex, alkaloidal are recognized: clavines, ergopeptines, lysergic
mycotoxins produced by several species of fungi, acid and simple lysergic acid amides, different
including phytopathogens (e.g. Claviceps spp.) ergot alkaloids are produced by several fungal
and mutualistic symbionts (e.g. Epichloë spp.) species within the Ascomycota, with many
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 205

species producing different end products Saikia et al. 2008). The IDT pathway has inter-
(Robinson and Panaccione 2015). In Epichloë, mediate secondary metabolites with invertebrate
this diversity is generated by the presence/ or mammalian bioactivity, with lolitrem B, the
absence of lysergyl peptide synthetases, encoded final product, having the most potent mammalian
by the NRPS EAS gene cluster (Gerhards et al. bioactivity (Fletcher et al. 2017; Gallagher et al.
2014; Schardl et al. 2006, 2012). 1981; Schardl et al. 2013b). Generally, asexual
Dimethylallyltryptophan synthase catalyzes the Epichloë spp. are known to produce higher
first step in ergot alkaloid biosynthesis, and the concentrations of IDTs, including lolitrem B,
deletion of the dmaW gene resulted in the failure compared to their sexual counterparts (Johnson
of Epichloë isolate Lp1 to produce ergot and et al. 2021). In certain Epichloë strains, a NRPS
clavine alkaloids (Wang et al. 2004). The inter- BGC located in the sub-telomeric region of the
mediate secondary metabolites within the ergot chromosome and assembled into 3 clusters is
alkaloid pathway have insect or mammalian bio- responsible for the production of various IDTs
activity although ergovaline, the final product, (Young et al. 2006). A set of four core genes is
has the most potent mammalian bioactivity in required for converting the initial diterpene-
ruminants (Fig. 8.5a; Bacetty et al. 2009; Bacon derived backbone and an indole moiety derived
et al. 1977; Johnson et al. 2021). Ergovaline from tryptophan (or a tryptophan derivative) into
within mammals acts as a potent vasoconstrictor paspaline (Fig. 8.5b) (Schardl et al. 2012, 2013b).
and after consumption, grazing animals exhibit Paspaline is an important and conserved interme-
several clinical symptoms (Dyer 1993). In gen- diate of this gene cluster, which is then acted on
eral, affected animals experience reduced blood by other enzymes in IDT biosynthetic pathways,
flow to the extremities, and therefore are unable to including cytochrome P450 monooxygenases,
dissipate heat in hot environments and lose their flavin adenine dinucleotide (FAD)-dependent
ability to maintain their peripheral body tempera- monooxygenases and prenyl transferases that cat-
ture in cold environments, manifesting as severe alyze various modifications to generate a diver-
animal welfare issues (Klotz and Nicol 2016). sity of IDT products (Schardl et al. 2012, 2013b;
Animal performance is subsequently affected, Young et al. 2009). The common toxic strains of
leading to reduced weight gain, milk production, E. festucae var. lolii, associated with perennial
and reproductive performance (Nicol and Klotz ryegrass, responsible for causing mammalian tox-
2016). Ergovaline is at the centre of fescue toxi- icity in New Zealand, predominantly produce
cosis syndrome in the USA as it is produced by lolitrem B (Johnson et al. 2013a). Ingestion of
the symbiosis between Epichloë coenophiala and lolitrem B can manifest as ryegrass staggers, a
tall fescue, the most common pasture grass in the seasonal neurotoxic disease of grazing livestock,
south-east of the country (Klotz and Nicol 2016). particularly sheep, with symptoms ranging from
However, evidence exists suggesting that other slight muscular tremors through to staggering,
Epichloë-derived secondary metabolites may collapse and even death (di Menna et al. 2012).
contribute to the animal toxicosis often attributed However, compounds other than lolitrem B could
to ergovaline (Gadberry et al. 2003). For an make significant contributions to the incidence of
in-depth review on ergot alkaloids in pastures, ryegrass staggers (di Menna et al. 2012; Finch
read Caradus et al. (2022). et al. 2020; Reed et al. 2016).
Further IDTs, known as epoxyjanthitrems,
were discovered after a survey of many hundreds
8.4.2 Indole Diterpenes of perennial ryegrass accessions for Epichloë
strains incapable of producing toxic levels of
Indole diterpenes (IDTs) are a structurally diverse lolitrem B or ergovaline (Tapper and Lane
group of secondary metabolites produced by 2004). An Epichloë strain, designated AR37,
fungi including species of Aspergillus, Claviceps, was identified with broad-spectrum insect pest
Penicillium, and Epichloë (Ludlow et al. 2019; resistance, excellent animal performance but
206 D. A. Bastías et al.

some ryegrass staggers (Fletcher et al. 2017; involves a Υ-class pyridoxal phosphate enzyme
Fletcher and Sutherland 2009; Hume et al. 2007; encoded by lolC which condenses the precursor
Jensen and Popay 2004; Moate et al. 2012; molecules L-proline and O-acetyl-L-homoserine
Pennell et al. 2005; Popay and Thom 2009; to form N-(3-amino, 3-carboxy) propyl-L-proline.
Popay and Wyatt 1995; Popay and Cox 2016). Another unique feature is the potential involve-
Evidence from a genomic study shows that the ment of host plant acetyltransferases in the con-
biosynthesis of epoxyjanthitrems branches from version of loline to N-acetylloline (Pan et al.
the IDT pathway after terpendole I, due to the 2014; Schardl et al. 2013a). Studies using
activity of idtD and idtO (compared to idtE and isotope-labelled NMR, infrared spectroscopy,
idtJ in the production of lolitrem B) (Fig. 8.3b) and RNA interference spectroscopy have con-
(Miller et al. 2022). Furthermore, the structure firmed the main products of the loline biosyn-
and bioactivity of five epoxyjanthitrems thetic pathway are produced through a sequence
(epoxyjanthitrems I–IV and epoxyjanthitriol) of modifications of the 1-amino group (Faulkner
have been elucidated through their isolation, puri- et al. 2006; Schardl et al. 2012; Spiering et al.
fication, and characterization using nuclear mag- 2008). The most abundant lolines in planta are N-
netic resonance (NMR) spectroscopy, liquid formylloline, N-acetylloline, and N-
chromatography–mass spectrometry, and animal acetylnorloline, which have a broad spectrum of
bioassays (Finch et al. 2020). Epoxyjanthitrem I insecticidal and insect-deterrent activity, and anti-
exhibited a sustained tremor response in mice, nematode activity, with negligible toxicity to
similar to that experienced with lolitrem B but grazing livestock (Bush et al. 1993; Easton et al.
less potent while also reducing weight gain and 2009; Froehlich et al. 2022; Jensen et al. 2009;
feeding in porina (Wiseana cervinata Walker), a Popay et al. 2003, 2009; Wilkinson et al. 2000).
common pasture pest in New Zealand (Finch
et al. 2020). To date, only two species-level taxa
of asexual Epichloë have been reported to pro- 8.4.4 Pyrrolopyrazines
duce epoxyjanthitrems, which are designated
LpTG-3 and LpTG-4 (Hettiarachchige et al. The most researched pyrrolopyrazine is the com-
2015; Kaur et al. 2015). pound peramine, produced by several Epichloë
spp., including strain AR1 that is marketed in
New Zealand for its insect bioactivity and excel-
8.4.3 Pyrrolizidines lent animal performance (Fletcher 1999;
Koulman et al. 2007). Peramine is not toxic to
Hundreds of pyrrolizidine alkaloids have been insects but is a strong feeding deterrent against
identified across thousands of plant species and Argentine stem weevil, where it also affects
most are produced as a defence mechanism development (Pennell et al. 2005; Rowan and
against invertebrate herbivores. The loline Gaynor 1986; Rowan et al. 1986, 1990). A single
alkaloids are a group of saturated pyrrolizidines gene, perA, which codes for a non-ribosomal
with an exo-1-amine and a strained ether bridge peptide synthase was identified by a gene deletion
that joins two aliphatic bridgehead carbon atoms study as being responsible for the production of
(C-2 and C-7) produced by several Epichloë spp., peramine from precursors L-arginine, (S)-
including those that form symbioses with pyrroline-5-carboxylate (precursor to L-proline)
meadow fescue (Festuca pratensis) and tall fes- and a methyl donor (S-adenosylmethionine)
cue grasses (Pan et al. 2014; Schardl et al. 2013a, (Tanaka et al. 2005). Introduction of perA into
2007). The loline BGC (LOL, Fig. 8.5c) consists Epichloë strains that do not produce peramine,
of 11 genes in 3 clusters interspersed with using genome editing techniques, resulted in
transposable elements (Schardl et al. 2012; peramine production in culture and in planta
Spiering et al. 2008). A biologically rare feature (Hettiarachchige et al. 2019). Allelic variations
of the LOL pathway is that the determinant step to perA have been noted in a recent study,
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 207

although their resulting secondary metabolite bio- with enhanced plant growth (Zhong et al. 2022).
activity remains unknown (Berry et al. 2019). This growth promotion is not restricted to abiotic
Peramine appears to be continuously produced stresses as Ueno et al. (2016) have shown that
by Epichloë spp. in planta, however it does not plants symbiotic with Epichloë infested with
progressively accumulate as with other Epichloë- insect herbivores can exhibit increased biomass
derived alkaloids (e.g. ergovaline) (Koulman compared to plants lacking the symbiosis.
et al. 2007). Instead, peramine is translocated Epichloë increased the concentration of
from the endophyte hyphae into the plant’s inter- gibberellins in foliar tissues of F. sinensis with
cellular spaces where it is either metabolized, elevated levels of this hormone correlated to an
mobilized, and/or excreted in guttation fluid increase in plant height and production of tillers
(Koulman et al. 2007). (Wang et al. 2021a). However, potentially not all
Further secondary metabolites have been Epichloë associations boost gibberellin levels.
implicated in the maintenance of the fungal– Tall fescue plants infected with E. coenophiala
grass symbiosis, for example the siderophore exhibited a general downregulation in the expres-
epichloënin A (produced via an NRPS pathway) sion of plant genes involved in the early biosyn-
is required to maintain iron homeostasis between thesis steps of the plant hormone (Dinkins et al.
E. festucae and perennial ryegrass (Johnson et al. 2019). Auxins are the only growth-promoting
2013b). Many novel Epichloë-derived secondary plant hormone found within hyphae of Epichloë
metabolites have also been detected using spp.; however, altered levels of these metabolites
advanced proteomic, metabolomic, and bioinfor- within Epichloë-symbiotic compared to
matic pipelines, however their roles within the endophyte-free plants have yet to be detected
Epichloë–grass symbiotum have yet to be fully (De Battista et al. 1990; Yue et al. 2000).
elucidated (Green et al. 2020; Johnson et al.
2015).
8.6 Interactions Between Epichloë
and Other Microorganisms
8.5 Plant Growth Promotional
Traits Symbiotic microorganisms can alter the pheno-
type of their plant host in ways that may affect
Plants infected with Epichloë spp. can exhibit an interactions with other species as well as the
increase in shoot biomass, number of tillers, and relative fitness of each partner (Clay et al. 2005).
seed yield compared to their endophyte-free Epichloë-derived secondary metabolites can
counterparts (Bastías et al. 2021b; Gundel et al. influence herbivorous invertebrates and mammals
2013; Schardl et al. 2004). This growth promo- and this in turn can affect species diversity, food
tional effect can be linked to an Epichloë- web structures, and fundamental ecological pro-
mediated increment in photosynthetic processes cesses like decomposition (Bultman et al. 2012;
likely brought about by changes to phytohormone Fuchs et al. 2013; Omacini et al. 2001; Popay and
levels and is particularly evident when plants are Bonos 2008). These legacy effects can even per-
exposed to stress (Rozpądek et al. 2015; Xu et al. sist after the death of the Epichloë symbiont and
2021; Zhao et al. 2021). For example, under its grass host (Casas et al. 2016; Zhou et al. 2022).
heavy metal stress (i.e. zinc), Epichloë sinensis Furthermore, Epichloë spp. can also modulate the
infection was associated with increased gibberel- microbiota of their host plants (including the
lin content in plants of Festuca sinensis, which foliar, root, rhizosphere, and seed environments),
correlated with enhanced plant growth (Wang which subsequently has implications for ecologi-
et al. 2021a), while Epichloë gansuensis infection cal succession in natural ecosystems (Bastías
was associated with increased photosynthesis et al. 2021a; Clay et al. 2005; Ju et al. 2020;
levels within plants of Achnatherum inebrians Nissinen et al. 2019; Omacini et al. 2001; Roberts
experiencing drought, which was also correlated and Ferraro 2015; Wakelin et al. 2015). The study
208 D. A. Bastías et al.

of plant microbiomes has received great interest strongly shaped the fungal endophyte
in recent time as molecular approaches have communities of its hosts leaves but not the bacte-
advanced to more readily reveal the composition rial communities (Nissinen et al. 2019).
and dynamics of plant-associated microbial Epichloë spp. can influence their plant host’s
communities, with those associated with microbiota via multiple mechanisms. Although
Epichloë–grass symbioses being no exception no correlation was detected between the antimi-
(Bastías et al. 2021a; Liu et al. 2022a; Mahmud crobial bioactivity expressed by Epichloë spp.
et al. 2021; Nissinen et al. 2019; Tannenbaum and their alkaloid profiles, Roberts and Lindow
et al. 2020). (2014) showed that bacterial communities living
A great deal of knowledge has been obtained on leaf surfaces of Epichloë-infected tall fescue
regarding the bioactivity of Epichloë towards were shaped by the presence of loline alkaloids.
mammalian and invertebrate herbivores but far Lolines promoted bacterial species capable of
less research has been undertaken on the degrading the alkaloid (Roberts and Lindow
interactions between Epichloë and other 2014). Furthermore, several non-alkaloidal sec-
microorganisms (Ren et al. 2009). Of the research ondary metabolites have been implicated in the
that has been conducted, the majority has antagonism exhibited by Epichloë spp. towards
focussed on the biological control of fungal phytopathogens. These compounds include aro-
phytopathogens (Card et al. 2021; Xia et al. matic sterols (Koshino et al. 1989), diacetamides
2018). Despite this applied focus, and the fact (Yue et al. 2000), hydroxyl unsaturated fatty
that several Epichloë-based grass products are acids (Koshino et al. 1987), phenolic glycerides
marketed across several continents for their pest (Koshino et al. 1988), sesquiterpenes (Yoshihara
deterrent properties (Caradus and Johnson 2020), et al. 1985; Yue et al. 2000), indole derivatives
none has been developed or marketed for their (indole 3 acetic acid (IAA) and indole 3 ethanol),
control of plant disease (Card et al. 2021). Fur- and other volatile compounds such as Chokol K
thermore, although a recent meta-analysis has and methyl esters (Bultman and Leuchtmann
shown that many species of phytopathogen 2008). Although Chokol K is known for its insect
(both biotrophic and necrotrophic) are signifi- attractant abilities, particularly Botanophila flies
cantly affected by the presence of Epichloë spp. that are an integral component of the Epichloë
(Pérez et al. 2020), very few published reports sexual cycle, the compound is hypothesized to
describe bioactivity in the field and instead rely have originated as an antimicrobial agent
on data from crudely designed in vitro dual cul- (Schiestl et al. 2006). Perez et al. (2017) describe
ture bioassays and greenhouse experiments (Card an indirect pathway for the protection of
et al. 2021). L. multiflorum from the flower-infecting pathogen
The interactions between Epichloë–grass Claviceps purpurea, the causal agent of ergot
symbioses and other microorganisms are likely disease. The incidence and severity of infection
to be specific to the endophyte and host by C. purpurea was two-fold lower in Epichloë-
genotypes, while also being influenced by envi- symbiotic plants than in non-symbiotic ones, but
ronmental factors. For example, a resident strain when insects were prohibited from visiting the
of E. festucae var. lolii altered the bacterial flowers, this difference disappeared, indicating
communities associated with perennial ryegrass that endophyte-derived volatile compounds may
compared to endophyte-free plants but only when repel insect vectors of C. purpurea and indirectly
infected plants were grown in soil and not sand protect their host plant against this phytopathogen
(Tannenbaum et al. 2020). Furthermore, this (Perez et al. 2017).
Epichloë strain increased the abundance of Bacilli Arbuscular mycorrhizal fungi (AMF) form
and Alphaproteobacteria in shoots but not roots in symbioses with many land plants where they
the second generation of host plants colonize the intracellular spaces of their host’s
(Tannenbaum et al. 2020). However, in tall fes- roots, playing a critical role in the acquisition of
cue, the presence of a strain of E. coenophiala nutrients while increasing the host’s tolerance/
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 209

resistance to abiotic and biotic stresses (Averill grass and endophyte-associated microbiota to
et al. 2019; Branco et al. 2022; Pozo et al. 2015). enhance the fitness of the symbiosis and plant
Epichloë and AMF and can form multi-symbiont persistence is only just being beginning to be
interactions with cool-season grasses, simulta- explored.
neously colonizing the above- and below-ground
tissues of the same host plant, respectively. Novel
grass–Epichloë associations often have a lower 8.7 Factors that Influence
diversity and/or population density of AMF the Frequency
within their roots while wild grasses infected of Endophyte-Infected Plants
with their co-evolved Epichloë symbionts usually in Grass Populations
accommodate a greater abundance of AMF
(Arrieta et al. 2015; Chu-Chou et al. 1992; In pastoral agriculture systems, it is desirable for
Mack and Rudgers 2008; Müller 2003; Novas pastures to contain high infection frequencies of
et al. 2005, 2009; Omacini et al. 2006; Terlizzi bioprotective and animal safe Epichloë
et al. 2022; Vignale et al. 2016, 2018). However, endophytes to support production. However,
the interactions between these fungal symbionts wild (or natural) grass populations have highly
are complex and poorly understood with the variable Epichloë infection frequencies that can
effects of Epichloë on AMF not readily general- range from 0 to 100% (Faeth et al. 2004;
izable across plant-endophyte symbiota (Kalosa- Leyronas and Raynal 2001; Ravel et al. 1997;
Kenyon et al. 2018). Grass host genotype, Saikkonen et al. 1998). One of the most common
Epichloë and AMF genotype, the presence of approaches to studying the ecology of the grass-
Epichloë-derived bioactive compounds endophyte symbiosis is to identify the environ-
(e.g. exudates), environmental stresses mental influences (i.e. abiotic and biotic factors)
(e.g. drought and saline-alkali stress), and that correlate (positively or negatively) with the
resource supply (e.g. nitrogen, phosphate) are incidence of Epichloë symbiosis in grass
listed as important factors that contribute to the populations. To establish causality, the perfor-
interaction (Guo et al. 2019; Liu et al. 2022b; mance of endophyte-infected and endophyte-
Novas et al. 2009; Rasmussen et al. 2007; free plants (ideally collected from different
Slaughter et al. 2018; Vignale et al. 2018; Zhang locations along the environmental gradient) may
et al. 2022; Zhong et al. 2021). be assessed in common garden experiments with
The relationships between Epichloë and the contrasting levels of the same abiotic factor as
microbiota on and within the host grass tissues treatments, or directly in the field through full
are being revealed through detailed culture- cross-transplant experiments. However, in local
independent plant microbiome studies. Members populations, the frequency of symbiotic grasses is
of the phylum Proteobacteria have generally been not only determined by the relative fitness of
observed to dominate grass microbiomes (Bastías endophyte-infected and endophyte-free plants
et al. 2021a), whereby seed transmitted but also by the vertical transmission efficiency
microbiota can colonize and impart beneficial of endophytes in endophyte-infected plants
traits to the establishing seedlings. Epichloë (Gundel et al. 2008, 2011b; Ravel et al. 1997).
occultans infection was associated with an
increase in the seed bacterial community diversity
of L. multiflorum, including Pantoea and Pseudo- 8.7.1 Variation in the Frequency
monas, which contain members that have been of Endophyte-Symbiotic Plants
shown to impart several benefits to their hosts in Natural Populations
(Bastías et al. 2021a). The diversity of microbiota
associated with mature plants is far greater than Certain investigations have found negative
that of seed, and the potential of members of the correlations between symbiosis prevalence and
210 D. A. Bastías et al.

high mean annual precipitation (MAP), or posi- of hosting endophytes was higher in F. rubra
tive ones with areas that experience high summer populations from the north than from the south
temperatures, which have led to the hypothesis (Saikkonen et al. 2000). In a later survey that
that symbiosis with Epichloë endophytes covered a broader geographic scale in northern
improves plant tolerance to drought (Lewis et al. Europe, there was a negative correlation between
1997; Malinowski and Belesky 2006; Żurek et al. endophyte incidence in F. rubra populations (the
2013). This has been experimentally supported in higher the latitude the lower the endophyte inci-
species such as L. perenne, Festuca eskia, and dence) (Dirihan et al. 2016). Interestingly, the
Bromus laevipes where endophyte-infected plants primary productivity (the normalized difference
collected from sites with different MAP showed vegetation index; a dimensionless index that
higher performance under drought treatments describes the difference between visible and
compared to endophyte-free plants (Afkhami near-infrared reflectance of vegetation cover)
et al. 2014; Gibert and Hazard 2013; Gibert was lower with higher latitude; therefore, the
et al. 2012). However, a generalization seems to endophyte incidence in populations of F. rubra
be difficult since the results of studies evaluating was positively associated with primary productiv-
the endophyte-mediated plant tolerance to ity (Dirihan et al. 2016). Primary productivity can
drought are still very variable within and among be used to characterize the quality at the site level
grass species (Decunta et al. 2021; Gundel et al. (where, for example, plant communities in high-
2016). Field surveys carried out in extremely arid quality environments are more productive) and is
environments of Patagonia, which include sites positively associated with MAP and mean annual
with less than 300 mm MAP in the gradients, temperature (Gaston 2000). Therefore, as an inte-
have shown opposite relationships between sym- grative variable of local resources for primary
biosis prevalence and MAP. For example, the producers to grow and sustain higher trophic
prevalence of Epichloë endophytes in populations consumers, variation in primary productivity
of Phleum alpinum, Poa spiciformis, and may underlie the patterns of variation in grass
Hordeum comosum was lower in drier sites, population symbiosis incidence with geographi-
i.e. those with a lower MAP (Casas et al. 2022; cal features like elevation or latitude. Accord-
Victoria et al. 2007). However, no studies have ingly, the global review by Semmartin et al.
assessed the relative performance of endophyte- (2015) showed that symbiosis prevalence in
infected and endophyte-free plants of these grass grass populations was positively correlated with
species under conditions of extremely low water site primary productivity. The initial prediction in
availability. this article was that since herbivory pressure is
The fine fescues that are widely distributed at higher as higher the primary productivity
high latitudes of the Northern Hemisphere have (Oesterheld et al. 1992), the symbiosis would be
been intensively studied with respect to the inci- favourably selected in sites with high productivity
dence of Epichloë fungal endophytes. Field (or MAP) (Semmartin et al. 2015). Alternatively,
surveys, however, have yielded contrasting and motivated by previous works (e.g. Lewis
results when analysed separately or even when et al. 1997; Malinowski and Belesky 2006;
trying to identify the factors driving the endo- Żurek et al. 2013), it was predicted that in the
phyte incidence (Bazely et al. 1997; Santangelo other extreme of the gradient (assuming that low
et al. 2015). For example, while there was a productivity was associated with water deficit),
negative correlation between endophyte inci- the fungal symbiosis incidence would be high
dence and elevation in Festuca rubra occurring because Epichloë can confer tolerance to drought.
in Scandinavian mountains, there was no pattern However, this was not the case, and the probabil-
for the other two fine fescues (Festuca ovina and ity of hosting these endophytes was lower in sites
Festuca vivipara) (Bazely et al. 2007; Granath with low primary productivity (Semmartin et al.
et al. 2007). In west Fennoscandia, the probability 2015). Harsh conditions for growth in very low
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 211

productivity ecosystems hinder the ability of host various North American wild species
plants to maintain the endophyte fungus, thus (Elymus hystrix, Elymus riparius, Elymus
increasing the probability of symbiont loss virginicus, Festuca subverticillata, Poa
(Casas et al. 2022; Gundel et al. 2016). In produc- alsodes, Poa sylvestris, and Sphenopholis
tive environments, endophyte incidence may nitida) at different stages of their lifecycles
respond positively to other factors such as herbi- (Afkhami and Rudgers 2008). Species like
vore pressure (Bazely et al. 1997) and/or plant Festuca arizonica have a variable rate of
competition (Santangelo et al. 2015) in succes- tiller colonization by Epichloë spp.
sional communities. (Schulthess and Faeth 1998), and thus
populations do produce endophyte-free
seeds. Endophyte transmission efficiency
8.7.2 Variation in the Efficiency can respond positively or negatively to envi-
of Endophyte Vertical ronmental factors that affect the host plant.
Transmission For instance, while strong defoliation late in
the growing season of L. multiflorum, an
Epichloë endophytes evolved the ability to syn- annual grass, caused a dramatic depression
chronize their growth to that of their host plants’ in endophyte vertical transmission (Garcia
lifecycle (Christensen et al. 2008), and one of the Parisi et al. 2012), light defoliations
consequences is that most of them are transmitted simulating herbivory by small insects in the
from mother plants to offspring through the seeds leaves of Poa autumnalis promoted the ver-
(i.e. vertical transmission). However, the process tical transmission of the fungus (Gundel et al.
of vertical transmission is not perfect, and the 2020). Interestingly, several field surveys
failures in it can cause endophyte-infected plants found that there was a clear relationship
to produce endophyte-free seeds (Afkhami et al. between rate of endophyte loss or failures in
2014; Gundel et al. 2011b). Thus, as highlighted vertical transmission and the frequency of
previously, transmission efficiency is endophyte-symbiotic plants in extant
important—together with the relative fitness of populations (Afkhami and Rudgers 2008;
symbiotic and non-symbiotic individuals, in Gibert and Hazard 2013; Gundel et al.
determining the endophyte incidence in grass 2009a). By studying endophyte transmission
populations (Gundel et al. 2008; Ravel et al. efficiency of plants collected from different
1997). In relation to the lifecycle of the host, L. perenne field populations in a common
two types of failures have been identified (Gundel garden, transmission was determined to be
et al. 2011b): (i) those that occur during seed an ecotypic characteristic (Gibert and Hazard
production, evaluated as the proportion of 2013). An experiment where L. multiflorum
endophyte-free seeds of the total seed produced plants were crossed with genetically distant
by a plant, and (ii) those that occur due to the pollen donors, concluded that the endophyte
anticipated death of the fungus in the seed, transmission efficiency was mainly affected
evaluated as the proportion of endophyte-free by the host genetic background rather than
seedlings produced by a batch of symbiotic seeds. the level of stress (Gundel et al. 2012).
Experiments with genetically diverse breed-
(i) The fidelity of the vertical transmission pro-
ing populations of L. perenne in association
cess is often imperfect with the fungus being
with Epichloë strain AR37 found that there
lost from plants at many stages of their life
was a strong influence of host genotype on
history, including losses among tillers, spikes
vertical transmission and smaller host
or panicles, and flowers (Afkhami and
genotype-by-environmental interaction,
Rudgers 2008). In that way, symbiotic plants
where the transmission of clonal plants was
can produce endophyte-free seeds. Evidence
examined in two locations of New Zealand
for symbiosis loss has been documented for
212 D. A. Bastías et al.

(Manawatū and Canterbury) (Gagic et al. previously encountered (through maternal

2018). In L. multiflorum, there was a clear effects). For example, the exposure of mother
relationship between the endophyte transmis- plants to ozone increased the rate of endo-
sion efficiency and plant seed production phyte viability loss in L. multiflorum (Gundel
(Gundel et al. 2011a). This latter result et al. 2015; Ueno et al. 2020a). Another pro-
supports the evolutionary theory that predicts cess that can influence the frequency of sym-
interactions between plants and vertically biotic seedlings produced by a seed batch is
transmitted symbiotic microorganisms to be the differential fitness of endophyte-infected
mutually beneficial in part because the fitness and endophyte-free seeds. It has been
of both partners is tightly aligned. observed that under controlled conditions,
(ii) Endophyte-symbiotic seeds can produce the presence of Epichloë spp. depresses the
endophyte-free seedlings presumably due to longevity of the seeds (i.e. the total seed
the death of the fungus (Gundel et al. 2011b). lifespan). This has been observed in
In fact, the high rates of endophyte viability L. multiflorum and F. arundinacea (Gundel
loss in seeds that are still viable are one of the et al. 2009a; Rubin et al. 2022; Ueno et al.
most documented processes across different 2020b). It has been suggested that seed water
host species [e.g. F. arundinacea (Welty content, a parameter that controls seed lon-
et al. 1987; Zhang et al. 2019), L. perenne gevity and depends strongly on environmen-
(Rolston et al. 1986; Tian et al. 2013), tal temperature and humidity, could be
L. multiflorum (Gundel et al. 2009b, 2010), underlying the low endophyte viability in
F. pratensis (Bylin et al. 2016), Bromus host seed and the differential longevity of
auleticus (Stefanoni-Rubio et al. 2022)]. endophyte-infected and endophyte-free
Besides, a given Epichloë strain can show seeds (Gundel et al. 2010; Rolston et al.
different dynamics of viability loss in seeds 1986; Tian et al. 2013; Welty et al. 1987).
of the same host cultivar, thus showing the However, the relationship between endo-
existence of endophyte-by-host genotype phyte presence, seed water content, and
interaction (Hill and Roach 2009; Rubin seed longevity has been elusive (Rubin
et al. 2022; Tian et al. 2013). Despite this et al. 2022; Ueno et al. 2020a). Alternatively,
overall pattern, the ability of fungal Zhang et al. (2019) showed that the presence
endophytes to maintain viability relative to of Epichloë in seed is associated with signifi-
their host seeds can vary with environmental cant alterations of the biochemical composi-
conditions. For example, no premature tion of the seed during ageing through
deaths of endophytes were observed when changes in the relative abundance of different
symbiotic seeds of L. rigidum were buried reserve sugars.
in the field for 3 months (Canals et al. 2008;
Like the relationship between symbiosis prev-
Kirkby et al. 2011). Differences in the results
alence in populations and ecosystem primary pro-
of stored seeds in controlled but continuous
ductivity (Semmartin et al. 2015), the relationship
deteriorative conditions (e.g. accelerated age-
between vertical transmission efficiency and host
ing) versus field conditions could be
plant fitness was found to be positive (Gundel
accounted for by the ability of seeds and
et al. 2011a). Therefore, Epichloë can certainly
endophytes to sense and coordinate with the
improve the fitness of host plants, but the rela-
environmental seasonality, and the possibil-
tionship is highly regulated by the level of
ity of repairing damaged cellular structures
resources available for plant growth. In summary,
between dry and wet periods (McDonald
the evidence suggests that the vertical transmis-
1998). Interestingly, the rate of endophyte
sion process is likely to be just as responsive to
loss in seeds is also affected by the environ-
environmental resources as the relative fitness
mental conditions that the mother plants have
between endophyte-infected and endophyte-free
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 213

plants, and that the ecosystem productivity mammalian toxins are not necessarily discarded
controls the symbiosis prevalence by scaling up and may be developed for their wildlife deterrent
the process from the individual to the population properties (Pennell et al. 2017a). Once potentially
level. mammalian-friendly strains have been identified,
a molecular assay is undertaken with the purpose
of uncovering genetic uniqueness. A full screen
8.8 Commercialization for known secondary metabolites in planta
of Epichloë-Based Products highlights candidate Epichloë strains for inocula-
tion into elite perennial ryegrass grass cultivars.
New Zealand’s economy is export-driven and Parallel discovery programmes were developed
heavily reliant on the productivity of the pastoral for other economically important grass species,
sector to support dairy, beef, sheep, and deer including tall fescue (Johnson et al. 2013a).
farming, supporting the declaration that ‘After One of the most challenging steps in develop-
air, light and water, the next most important ing Epichloë for commerce is the ability to trans-
thing is grass’ (Holford 1933). Perennial ryegrass fer suitable fungal strains from their original wild
is regarded as one of the most important forage host to elite grass cultivars (Easton 2007; Johnson
species in many countries around the world, and Caradus 2019). Although Epichloë had been
including New Zealand. Due to the economic previously isolated from grass tissues and grown
importance of forages the New Zealand govern- in culture (Sampson 1937), Latch and
ment, together with the private sector and other Christensen (1985) were the first to devise a
technology providers, invested substantially in repeatable method of infecting elite grasses with
this area of science from the late 1980s to identify selected strains of Epichloë. This procedure can
a solution to the animal toxicity problems that however lead to incompatibility manifesting as
plagued the agricultural sector in the previous death of inoculated seedlings, stunted tillers with
decades. This significant investment primarily necrosis in the region of the apical meristem, and
financed the establishment of a scientific chlorotic leaf symptoms (Christensen 1995;
programme at AgResearch Limited, a corporat- Christensen et al. 1997). Symptoms of incompat-
ized crown research institute charged with serving ibility are not limited to the host. Endophyte
the agriculture and biotechnology sectors of hyphae can become distorted and collapsed,
New Zealand industry. The programme centred with degenerated cytoplasm (Koga et al. 1993).
around a bioprospecting pipeline to identify, char- Epichloë and their grass hosts have co-evolved,
acterize, and select agriculturally beneficial and it is now generally accepted that endophyte
strains of Epichloë that were less toxic to live- taxonomic groupings are not host specific, but are
stock while maintaining their insect-deterrent confined to forming compatible associations
properties. The pipeline essentially aimed to (i.e. symptomless associations) with related
exploit the natural symbiotic phenomenon grass genera within a tribe (Schardl et al. 1997;
between these fungi and their grass hosts for Schardl and Philips 1997).
agricultural gains (Card et al. 2014a; de Bonth Once compatible associations were developed,
et al. 2015; Johnson et al. 2013a). Seed and the selected endophytes incorporated into the
accessions, either from germplasm holdings or cultivar, grass populations from subsequent
fresh collections, are first entered into a multi- generations were then evaluated through specifi-
phase screening process to identify ryegrass seed cally designed bioactivity, agronomic and toxico-
containing viable Epichloë mycelium (Card et al. logical screens (Fig. 8.6). Under field conditions,
2014b). Following this, Epichloë-infected plants not only were the objectives of insect deterrence
are identified and characterized with respect to and reduced livestock toxicity observed but also
their production of secondary metabolites, partic- additional advantages, including improved grass
ularly mammalian toxins (i.e. IDTs and ergot establishment, higher grass yields, higher tiller
alkaloids). Epichloë strains that produce densities, and reduced weed invasion, leading to
214 D. A. Bastías et al.

an overall agronomic advantage to these grass America, and, to a lesser extent, Europe, where
plants compared to their endophyte-free they play a critical role in pasture productivity,
counterparts (Hume and Sewell 2014; Popay ruminant nutrition, and food production (Caradus
and Hume 2011; Thom et al. 2012). Additional et al. 2021a; Easton and Fletcher 2006; Hill et al.
attributes include tolerance against abiotic 2005).
stresses such as those caused by drought and/or Although delivering a quality seed product
nutrient deficiencies (Caradus and Johnson 2019; containing Epichloë is not free of challenges
Johnson and Caradus 2019). The high rate of (Easton 2007; Rolston and Agee 2006), underpin-
adoption by New Zealand farmers then made ning the development and application of Epichloë
this endophyte-technology a scientific, agricul- technology within the agricultural sector has sig-
tural, and marketing success (Johnson and nificantly hinged upon the biological behaviour
Caradus 2019; Stewart 2006; Woodfield and and characteristics of these endophytic fungi
Judson 2019). Today, over 70% of the ryegrass (Card et al. 2016; Johnson et al. 2021), that can
seed used in New Zealand pastoral farming be separated into five main themes:
contains one of several commercialized, novel
1. Fungal plasticity. Most Epichloë species can
Epichloë strain–host associations. These
be cultured in the laboratory on simple syn-
associations are regarded as important
thetic media that allows scientists to study
components of the microbial communities
them in vitro and develop a reliable method
associated with pasture-based production systems
to transfer selected strains of asexual Epichloë
in Australia, New Zealand, the USA, South
from wild host grasses into elite breeding

Fig. 8.6 A research trial in New Zealand clearly showing lolii (right-hand side plot) compared with an endophyte-
an overall agronomic advantage to perennial ryegrass free plot of perennial ryegrass on the left (Photo courtesy
plants infected with a strain of Epichloë festucae var. of David Hume, AgResearch Ltd, New Zealand)
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 215

material and existing cultivars (Latch and 8.9 Alternative Applications

Christensen 1985). of Epichloë Technology
2. An effective mechanism of action. Although
asexual Epichloë species are generally Epichloë endophytes can also impart beneficial
restricted to the host’s above-ground tissues, attributes to turfgrasses as they enhance the fit-
their bioactivity (via antibiosis) extends fur- ness of the grass when under biotic and abiotic
ther, protecting most of the plant as many stresses (Breen 1994; Funk et al. 1993, 2018;
Epichloë-derived secondary metabolites Stewart et al. 2022). Reports include Epichloë
(e.g. lolines) are mobilized within the plant’s endophytes providing resistance to several impor-
vascular system and translocated to plant tant turfgrass pests [e.g., billbugs (Sphenophorus
organs, such as the roots, where fungal coloni- spp.) and sod webworm (Lepidoptera: Pyralidae)]
zation is absent (Bryant et al. 2010; Popay and and diseases (e.g. red thread and dollar spot
Bonos 2008). caused by Laetisaria fuciformis and Clarireedia
3. Chemical and genetic diversity. A great deal jacksonii, respectively), while some strains can
of chemical and genetic diversity exists within contribute to enhanced tolerance to heavy metals,
Epichloë (Hettiarachchige et al. 2015; Schardl shade, heat, cold, and drought (Bonos et al. 2005;
et al. 2012; van Zijll de Jong et al. 2008). Clarke et al. 2006; Meyer and Funk 1989;
Therefore, once a culture collection is Murphy et al. 1993; Zaurov et al. 2001).
established and characterized, endophyte Researchers in New Zealand have, over many
strains with suitable chemical profiles (little years, accumulated a large collection of geneti-
to no animal toxicity) can be transferred to cally diverse Epichloë strains for research and
elite grass cultivars and evaluated in the field commercial development purposes (Johnson
(Johnson et al. 2013a). This process also has et al. 2013a). Based primarily on their secondary
uncovered new bioactivity from secondary metabolite profiles, many of these strains were
metabolites unknown at the time, deemed unsuitable for pastoral use, specifically
i.e. epoxyjanthitrems from strain AR37 due to their production of ergovaline, a powerful
(Hume et al. 2009; Johnson and Caradus animal toxin. However, this neurotoxin has been
2019). implicated in inducing an avoidance behaviour
4. Propagation via seed. Many other biological observed in several mammals and birds (Coley
control agents (BCA)s are let down by com- et al. 1995; Conover and Messmer 1996;
plicated and expensive formulations to capti- Panaccione et al. 2006). Coupled with their abil-
vate, preserve, and disseminate the product. ity to produce loline alkaloids, potent insecticidal
Asexual Epichloë spp., however, are exclu- and insect-deterrent compounds, these Epichloë
sively disseminated in seed and this lifecycle strains were deemed suitable from a wildlife
adaption has had a profound effect on the deterrent perspective.
marketing, risk assessment, and uptake by Two Epichloë-grass products were developed.
farmers to make the technology a success The first between a turf-type tall fescue cultivar,
(Card et al. 2016; Johnson et al. 2013a). intentionally infected with a strain of
5. Obligate nature. Asexual Epichloë are E. coenophiala, known to produce high levels of
protected from many environmental variables ergovaline and loline alkaloids, which was pri-
due to their obligate lifestyle, including dam- marily developed as an avian deterrent at airports.
aging ultraviolet light, extreme temperatures, The second, a turf-type ryegrass cultivar,
water deficits, and competition for space and intentionally infected with a strain of E. festucae
nutrients from other faster growing, antagonis- var. lolii, known to produce ergovaline,
tic microorganisms (Card et al. 2016). lolitrem B, and peramine, was developed for use
as a wildlife deterrent to discourage herbivores,
including rabbits, granivorous birds, and rodents
216 D. A. Bastías et al.

at sports fields and recreational areas (Finch et al. 2018). Epichloë strains can also closely associate
2016b; Pennell et al. 2016, 2017a, b, c). with bacterial species that have potential to syn-
thesize several secondary metabolites, including
antibiotics and iron chelating compounds (Bastías
8.10 Future Opportunities et al. 2020a). Bacterial symbionts of fungi have
for Epichloë Technology been isolated from many fungal species that form
intimate associations with plants, and these bac-
The Poaceae is arguably the most economically teria can directly affect plant fitness by
important plant family as it not only provides feed modulating plant performance and/or by
for many domesticated animals but also important regulating the performance of their plant-
species suitable for human nutrition [including associated fungi (Bastías et al. 2020b, 2022).
barley (Hordeum vulgare), maize (Zea mays), Symptomless, tripartite associations involving
rice (Oryza spp.), sugarcane (Saccharum spp.), Epichloë spp. are not restricted to bacteria and
and wheat (Triticum aestivum)], building fungi, and Epichloë can also be infected with
materials [such as bamboo (Bambusoideae)], submicroscopic infectious agents
aesthetics for recreational areas such as parks, (i.e. mycoviruses) (Romo et al. 2007;
lawns, and sports grounds [including bluegrass Zabalgogeazcoa et al. 1998). As these tripartite
(Poa spp.), bent grass (Agrostis spp.), Bermuda associations appear to have no detrimental effect
grass (Cynodon dactylon), and red fescue on Epichloë they may offer selective advantages
(F. rubra)] and species suitable for the production to the fungus and/or plant hosts, offering a mutu-
of cellulosic and non-cellulosic biofuels [includ- alistic tripartite symbiosis that could be exploited
ing maize, sweet sorghum (Sorghum bicolor), for agricultural gains.
and sugarcane)]. While Epichloë spp. are not Lolines are agriculturally beneficial alkaloids
naturally found in modern cereal grasses, they produced by Epichloë species that form natural
can be found within their wild relatives, including symbiosis with tall fescue (e.g. E. coenophiala)
Elymus spp. and Hordeum spp. (Card et al. and meadow fescue (e.g. Epichloë uncinatum and
2014b). Selected Epichloë strains isolated from Epichloë siegelii) (Schardl et al. 2007). Lolines
these wild Triticeae grasses have been transferred are not produced from Epichloë species that form
into barley, wheat, and rye (Secale cereale) to natural symbioses with perennial ryegrass.
create artificial symbioses, with the latter associa- Lolines are non-toxic to ruminants and small
tion able to inhibit diseases, caused by Puccinia mammals while exhibiting insect deterring and
recondita and Mycosphaerella recutita (syn. insecticidal activities (Easton et al. 2009; Finch
Cercosporidium graminis), in the field (Hume et al. 2016a, b; Fletcher et al. 2000). An applied
et al. 2018; Li et al. 2021; Simpson et al. 2014). motive therefore existed to develop novel
Further opportunities exist when several agri- symbioses between loline-producing Epichloë
culturally beneficial microorganisms are com- spp. and elite cultivars of perennial ryegrass to
bined. For example, Li et al. (2018) describe the achieve a wider spectrum of invertebrate activity
association of perennial ryegrass with both than achieved with Epichloë strains naturally
E. festucae var. lolii and the AMF, present in perrenial ryegrass (Easton 2007;
Claroideoglomus etunicatum. Although Easton et al. 2009). However, forming compati-
C. etunicatum alone increased the plant’s ability ble associations between these species has been
to uptake phosphorous and increased its dry challenging, with their loline production and ver-
weight, this effect was enhanced by the presence tical transmission frequencies being significantly
of an Epichloë strain. Furthermore, the presence lower than anticipated (Ball and Tapper 1999;
of the AMF and Epichloë exhibited lower disease Croy et al. 2022; Freitas et al. 2020). To maintain
incidence of leaf spot caused by Bipolaris symbiotic compatibilty between elite grass
sorokiniana, compared to plants infected by just cutivars and asexual Epichloë strains that exhibit
one of these mutualistic fungal species (Li et al. a greater number of agriculturally beneficial traits,
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 217

alternative techniques are required. All of the climatic events. For example, Europe experienced
commercial symbiotic associations in cool-season the worst drought in 500 years (Le Page 2022),
grasses developed to date have utilized naturally historic floods submerged more than a third of
occurring Epichloë strains, identified from exotic Pakistan that affected more than 30 million peo-
host germplasm. However, while genetic ple (Sands 2022), and many countries, from
modifications of Epichloë strains using traditional China to the USA, recorded severe heat waves
cloning-based techniques have demonstrated the (Lopez 2022). Climate change is predicted to
roles of key genes in processes such as alkaloid have serious implications on the future of many
biosynthesis, opportunities now exist to edit sec- agricultural systems (Arora 2019). Increasing
ondary metabolite pathways in endophytic fungi temperatures, elevated atmospheric CO2 levels,
using targeted genetic engineering techniques and changing precipitation patterns are likely to
such as CRISPR-Cas9 (Chowdhary et al. 2022). impact agriculturally important insect pests by
For example, Florea et al. (2021) utilized a expanding their geographic range, increasing
CRISPR-based approach to generate their overwintering survival rates and affecting
non-transgenic deletion mutants of their interactions with natural enemies (Skendžić
E. coenophiala, an endophyte that associates et al. 2021). Plant disease outbreaks are expected
with tall fescue, to remove the gene clusters to intensify due to invasions of new
responsible for the biosynthesis of ergovaline. phytopathogens and/or due to increases in the
Additionally, Miller et al. (2022) used CRISPR severity of existing phytopathogens
to demonstrate that new biochemical variation (Kocmánková et al. 2009; Misra et al. 2020).
can be created within the epoxyjanthitrem path- Functional symbiosis is one of the most success-
way of Epichloë strain AR37, a commercialized ful mechanisms by which plants can improve
fungal strain that associates with perennial their ability to tolerate stress (Hereme et al.
ryegrass (Caradus et al. 2021a). 2020; Morales-Quintana et al. 2022). Climate
The manipulation of secondary metabolite change, together with current unsustainable agri-
pathways in Epichloë using CRISPR-Cas9 gene cultural practices that expend valuable resources
inactivation therefore has the potential to deliver while degrading the environment (Pareek et al.
novel Epichloë strains that can be incorporated 2020), highlights the critical importance of
into elite pasture grasses that eliminate, or signifi- integrating knowledge from both agricultural
cantly reduce, animal health and welfare issues and natural ecosystems, from single plants and
while retaining desirable traits such as pest and multispecies plant communities and their
disease protection (Miller et al. 2022). Future microbiota, and from below-ground and above-
opportunities to increase the value of Epichloë ground multitrophic interactions to further
technology will likely be achieved through the improve the resilience and sustainability of
inactivation of secondary metabolite pathways worldwide crop production.
and promoting the expression of the most agricul-
turally beneficial compounds or introducing
Endophytes Everywhere
novel bioactive compounds (Caradus et al.
Endophytic fungi are thought to occur in all
2021a; Caradus and Johnson 2020; Miller et al.
species of land plants as well as brown,
2022). Genome editing approaches are generally
green, and red marine seaweeds. Ergot
viewed as more socially acceptable than tradi-
alkaloid-producing clavicipitaceous
tional cloning-based genetic modifications, and
endophytes with narrow host ranges are
hold promise for future applications in
the subjects of this chapter. These are
New Zealand to help tackle future challenges to
Epichloë and close relatives
food production.
(Clavicipitaceae) that infect certain grasses
The most important challenge currently facing
and sedges. Another group of alkaloid-
mankind is climate change, and in 2022 many
regions around the world experienced severe (continued)
218 D. A. Bastías et al.

References
producing clavicipitaceous fungi infect
some morning glory species Afkhami M, Rudgers J (2008) Symbiosis lost: imperfect
(Convolvulaceae), which have been used vertical transmission of fungal endophytes in grasses.
Am Nat 172:405–416. https://doi.org/10.1086/589893
as entheogens.
Afkhami ME, McIntyre PJ, Strauss SY (2014) Mutualist-
Most endophytic fungi, however, are mediated effects on species’ range limits across large
non-clavicipitaceous (NC) species of geographic scales. Ecol Lett 17:1265–1273. https://
Ascomycota (Pezizomycotina, especially doi.org/10.1111/ele.12332
Agee C, Hill N (1994) Ergovaline variability in
Sordariomycetes and Dothideomycetes)
Acremonium-infected tall fescue due to environment
and a few Basidiomycota and plant genotype. Crop Sci 34:221–226
(Agaricomycotina, Pucciniomycotina, and Arora NK (2019) Impact of climate change on agriculture
Ustilaginomycotina) and Mucoromycota production and its sustainable solutions. Environ Sus-
tain 2:95–96. https://doi.org/10.1007/s42398-019-
with broad host ranges. The
00078-w
NC-endophytes have been placed in three Arrieta AM, Iannone LJ, Scervino JM, Vignale MV,
groups based, not on taxonomy, but largely Novas MV (2015) A foliar endophyte increases the
on life history and ecological traits such as diversity of phosphorus-solubilizing rhizospheric
fungi and mycorrhizal colonization in the wild grass
horizontal or vertical transmission, degree, Bromus auleticus. Fungal Ecol 17:146–154
and site of colonization of the hosts (single Averill C, Bhatnagar JM, Dietze MC, Pearse WD, Kivlin
cells to large areas and not only in leaves SN (2019) Global imprint of mycorrhizal fungi on
and stems, but also roots). They also may whole-plant nutrient economics. Proc Natl Acad Sci
U S A 116:23163–23168. https://doi.org/10.1073/
have saprobic or parasitic lifestyles under pnas.1906655116
some conditions (Rodriguez et al. 2009). Bacetty AA, Snook ME, Glenn AE, Noe JP, Nagabhyru P,
Interactions of plant hosts and Bacon CW (2009) Chemotaxis disruption in
NC-endophytes are not so well studied as Pratylenchus Scribneri by tall fescue root extracts
and alkaloids. J Chem Ecol 35:844–850. https://doi.
clavicipitaceous endophytes, but some are org/10.1007/s10886-009-9657-x
known to convey benefits to the host such Bacon CW (1995) Toxic endophyte-infected tall fescue
as drought tolerance and enhanced growth. and range grasses: historic perspectives. J Anim Sci 73:
A few NC-endophytes benefit their hosts 861–870
Bacon CW, White JF (1994) Stains, media, and
under local conditions of pH and tempera- procedures for analyzing endophytes. Biotechnology
ture stress. Some root-inhabiting species of endophytic fungi of grasses. CRC Press, pp 47–56
are prevalent in non-mycorrhizal plants Bacon C, Porter J, Robbins J, Luttrell E (1977) Epichloë
(i.e. Brassicaceae) (Card et al. 2015). typhina from toxic tall fescue grasses. Appl Environ
Microbiol 34:576–581
NC-endophytes have been important in Ball O-P, Tapper B (1999) The production of loline
bioremediation because the fungi can alkaloids in artificial and natural grass/endophyte
degrade a variety of recalcitrant chemicals associations. Proceedings of the New Zealand plant
(Dubey et al. 2020). Another applied area protection conference, pp 264–269
Ball DM, Lacefield GD, Hoveland CS (2019) The wonder
of research on these fungi is drug discov- grass: the story of tall fescue in the United States.
ery. Endophytic fungi-mediated biosynthe- Oregon Tall Fescue Commission, Salem, OR
sis often results in synthesis of larger Barret M, Guimbaud JF, Darrasse A, Jacques MA (2016)
quantities of chemical compounds in con- Plant microbiota affects seed transmission of phyto-
pathogenic microorganisms. Mol Plant Pathol 17:791
junction with the host plant, a phenomenon Bastias DA, Martínez-Ghersa MA, Ballaré CL, Gundel PE
shown with the cancer drug taxol (Tiwari (2017) Epichloë fungal endophytes and plant defenses:
and Bae 2022; Yan et al. 2018). not just alkaloids. Trends Plant Sci 22:939–948.
https://doi.org/10.1016/j.tplants.2017.08.005
Bastías DA, Alejandra Martínez-Ghersa M, Newman JA,
Card SD, Mace WJ, Gundel PE (2018a) The plant
Acknowledgements The authors thank David Hume and hormone salicylic acid interacts with the mechanism
Anouck de Bonth for critical revision of the manuscript. of anti-herbivory conferred by fungal endophytes in
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 219

grasses. Plant Cell Environ 41:395–405. https://doi. Branco S, Schauster A, Liao HL, Ruytinx J (2022)
org/10.1111/pce.13102 Mechanisms of stress tolerance and their effects on
Bastías DA, Martínez-Ghersa MA, Newman JA, Card SD, the ecology and evolution of mycorrhizal fungi. New
Mace WJ, Gundel PE (2018b) Jasmonic acid regula- Phytol
tion of the anti-herbivory mechanism conferred by Breen J (1994) Acremonium endophyte interactions with
fungal endophytes in grasses. J Ecol 106:2365–2379. enhanced plant resistance to insects. Annu Rev
https://doi.org/10.1111/1365-2745.12990 Entomol 39:401–423
Bastías DA, Jauregui R, Applegate ER, Altermann E, Card Brem D, Leuchtmann A (1999) High prevalence of hori-
SD, Johnson LJ (2020a) Complete genome sequence zontal transmission of the fungal endophyte Epichloë
of Paenibacillus sp. strain E222, a bacterial symbiont sylvatica. Bull Geobot Inst ETH 65:12
of an Epichloë fungal endophyte of ryegrass. Microbiol Brosi GB, McCulley RL, Bush LP, Nelson JA, Classen
Resour Announc 9:e00786–e00720. https://doi.org/10. AT, Norby RJ (2011) Effects of multiple climate
1128/MRA.00786-20 change factors on the tall fescue–fungal endophyte
Bastías DA, Johnson LJ, Card SD (2020b) Symbiotic symbiosis: infection frequency and tissue chemistry.
bacteria of plant-associated fungi: friends or foes? New Phytol 189:797–805
Curr Opin Plant Biol 56:1–8 Bryant R, Cameron N, Edwards G (2010) Response of
Bastías DA, Bustos LB, Jáuregui R, Barrera A, Acuña- black beetle and redheaded pasture cockchafer larvae
Rodríguez IS, Molina-Montenegro MA, Gundel PE to loline alkaloids in meadow fescue roots. N Z Plant
(2021a) Epichloë fungal endophytes influence seed- Prot 63:219–223
associated bacterial communities. Front Microbiol 12: Bultman TL, Leuchtmann A (2008) Biology of the
795354–795354 Epichloë-Botanophila interaction: an intriguing associ-
Bastías DA, Gianoli E, Gundel PE (2021b) Fungal ation between fungi and insects. Fungal Biol Rev 22:
endophytes can eliminate the plant growth–defence 131–138
trade-off. New Phytol 230:2105–2113 Bultman TL, White JF (1988) “Pollination” of a fungus by
Bastías DA, Applegate ER, Johnson LJ, Card SD (2022) a fly. Oecologia 75:317–319
Factors controlling the effects of mutualistic bacteria Bultman TL, White JF Jr, Bowdish TI, Welch AM,
on plants associated with fungi. Ecol Lett 25:1879– Johnston J (1995) Mutualistic transfer of Epichloë
1888. https://doi.org/10.1111/ele.14073 spermatia by Phorbia flies. Mycologia 87:182–189
Bazely DR, Vicari M, Emmerich S, Filip L, Lin D, Inman Bultman TL, Aguilera A, Sullivan TJ (2012) Influence of
A (1997) Interactions between herbivores and fungal isolates infecting tall fescue on multitrophic
endophyte-infected Festuca rubra from the Scottish interactions. Fungal Ecol 5:372–378
islands of St. Kilda, Benbecula and Rum. J Bush LP, Fannin FF, Siegel MR, Dahlman DL, Burton HR
Appl Ecol:847–860 (1993) Chemistry, occurrence and biological effects of
Bazely DR, Ball JP, Vicari M, Tanentzap AJ, Bérenger M, saturated pyrrolizidine alkaloids associated with
Rakocevic T, Koh S (2007) Broad-scale geographic endophyte-grass interactions. Agric Ecosyst Environ
patterns in the distribution of vertically-transmitted, 44:81–102. https://doi.org/10.1016/0167-8809(93)
asexual endophytes in four naturally-occurring grasses 90040-V
in Sweden. Ecography 30:367–374 Bush LP, Wilkinson HH, Schardl CL (1997) Bioprotective
Bent E, Chanway CP (2002) Potential for misidentification alkaloids of grass-fungal endophyte symbioses. Plant
of a spore-forming Paenibacillus polymyxa isolate as Physiol 114:1
an endophyte by using culture-based methods. Appl Bylin A, Card S, Hume D, Lloyd-West C, Huss-Danell K
Environ Microbiol 68:4650–4652 (2016) Endophyte storage and seed germination of
Berry D, Mace W, Grage K, Wesche F, Gore S, Schardl Epichloë-infected meadow fescue. Seed Sci Technol
CL, Young CA, Dijkwel PP, Leuchtmann A, Bode 44:138–155
HB, Scott B (2019) Efficient nonenzymatic cyclization Campbell MA, Tapper BA, Simpson WR, Johnson RD,
and domain shuffling drive pyrrolopyrazine diversity Mace W, Ram A, Lukito Y, Dupont P-Y, Johnson LJ,
from truncated variants of a fungal NRPS. Proc Natl Scott DB (2017) Epichloë hybrida, sp. nov., an
Acad Sci U S A 116:25614–25623 emerging model system for investigating fungal allo-
Bills GF, Gloer JB (2017) Biologically active secondary polyploidy. Mycologia 109:715–729
metabolites from the fungi. In: The fungal kingdom, pp Canals R, San Emeterio L, Oreja A (2008) Chances of loss
1087–1119 of fungal endophytes in agronomic grasses: a case-
Blankenship JD, Spiering MJ, Wilkinson HH, Fannin FF, study for Lolium rigidum. Agric Ecosyst Environ
Bush LP, Schardl CL (2001) Production of loline 127:146–152
alkaloids by the grass endophyte, Neotyphodium Cane DE, Walsh CT, Khosla C (1998) Harnessing the
uncinatum, in defined media. Phytochemistry 58: biosynthetic code: combinations, permutations, and
395–401 mutations. Science 282:63–68
Bonos SA, Wilson MM, Meyer WA, Reed FC (2005) Caradus JR, Johnson LJ (2019) Improved adaptation of
Suppression of red thread in fine fescues through temperate grasses through mutualism with fungal
endophyte-mediated resistance. Appl Turfgrass Sci 2: endophytes. In: Schouten A (ed) Endophyte biotech-
1–7. https://doi.org/10.1094/ATS-2005-0725-01-RS nology: potential for agriculture and pharmacology.
CABI, Wageningen, pp 85–108
220 D. A. Bastías et al.

Caradus JR, Johnson LJ (2020) Epichloë fungal range in a native Patagonian grass—resource
endophytes-from a biological curiosity in wild grasses eco-physiological relations. Funct Ecol 36:583–594
to an essential component of resilient high performing Charlton ND, Craven KD, Mittal S, Hopkins AA, Young
ryegrass and fescue pastures. J Fungi 6:322. https://doi. CA (2012) Epichloë canadensis, a new interspecific
org/10.3390/jof6040322 epichloid hybrid symbiotic with Canada wildrye
Caradus J, Chapman D, Cookson T, Cotching B, (Elymus canadensis). Mycologia 104:1187–1199.
Deighton M, Donnelly L, Ferguson J, Finch S, https://doi.org/10.3852/11-403
Gard S, Hume D (2021a) Epichloë endophytes–new Chowdhary K, Arora H, Sharma S (2022) CRISPR/Cas9-
perspectives on a key ingredient for resilient perennial based genome editing as a way ahead for inducing
grass pastures. NZGA Res Pract Ser 17:347–360 production of bioactive metabolites in endophytes.
Caradus JR, Card SD, Hewitt KG, Hume DE, Johnson LJ Natl Acad Sci Lett 45:275–280. https://doi.org/10.
(2021b) Asexual Epichloë fungi—obligate mutualists. 1007/s40009-022-01107-9
Encyclopedia 1:1084–1100 Christensen MJ (1995) Variation in the ability of
Caradus JR, Goldson SL, Moot DJ, Rowarth JS, Stewart Acremonium endophytes of Lolium perenne, Festuca
AV (2021c) Pastoral agriculture, a significant driver of arundinacea and F. pratensis to form compatible
New Zealand’s economy, based on an introduced associations in the three grasses. Mycol Res 99:466–
grassland ecology and technological advances. J R 470. https://doi.org/10.1016/S0953-7562(09)80647-3
Soc N Z 1-45. https://doi.org/10.1080/03036758. Christensen M, Voisey C (2006) The biology of the endo-
2021.2008985 phyte/grass partnership. In: Popay A, Thom ER (eds)
Caradus JR, Card SD, Finch SC, Hume DE, Johnson LJ, 6th international symposium on fungal endophytes of
Mace WJ, Popay AJ (2022) Ergot alkaloids in grasses. Christchurch, New Zealand Grassland Associ-
New Zealand pastures and their impact. N Z J Agric ation, pp 123–134
Res 65:1–41 Christensen MJ, Ball OJP, Bennett RJ, Schardl CL (1997)
Card SD, Tapper BA, Lloyd-West C, Wright KM (2013) Fungal and host genotype effects on compatibility and
Assessment of fluorescein-based fluorescent dyes for vascular colonization by Epichloë festucae. Mycol Res
tracing Neotyphodium endophytes in planta. 101:493–501. https://doi.org/10.1017/
Mycologia 105:221–229. https://doi.org/10.3852/ S0953756296002833
12-062 Christensen MJ, Bennett RJ, Schmid J (2002) Growth of
Card S, Johnson L, Bonth Ad, Tapper B, Mace W, Epichloë and Neotyphodium and p-endophytes in
Faville M, Pennell C, Caradus J, and Hume leaves of Lolium and Festuca grasses. Mycol Res
D. 2014a. Epichloë endophytes from cool season 106:93–96. https://doi.org/10.1017/
grasses – reaping the rewards from a well-tuned S095375620100510X
bio-prospecting pipeline. The 10th International Myco- Christensen MJ, Bennett RJ, Ansari HA, Koga H, Johnson
logical Congress, Bangkok. RD, Bryan GT, Simpson WR, Koolaard JP, Nickless
Card SD, Faville MJ, Simpson WR, Johnson RD, Voisey EM, Voisey CR (2008) Epichloë endophytes grow by
CR, de Bonth AC, Hume DE (2014b) Mutualistic intercalary hyphal extension in elongating grass leaves.
fungal endophytes in the Triticeae–survey and descrip- Fungal Genet Biol 45:84–93. https://doi.org/10.1016/j.
tion. FEMS Microbiol Ecol 88:94–106 fgb.2007.07.013
Card SD, Hume DE, Roodi D, McGill CR, Millner JP, Chu-Chou M, Guo B, An Z-Q, Hendrix J, Ferriss R,
Johnson RD (2015) Beneficial endophytic Siegel M, Dougherty C, Burrus P (1992) Suppression
microorganisms of Brassica – a review. Biol Control of mycorrhizal fungi in fescue by the Acremonium
90:102–112 coenophialum endophyte. Soil Biol Biochem 24:633–
Card S, Johnson L, Teasdale S, Caradus J (2016) 637
Deciphering endophyte behaviour: the link between Chung KR, Schardl CL (1997) Sexual cycle and horizontal
endophyte biology and efficacious biological control transmission of the grass symbiont, Epichloë typhina.
agents. FEMS Microbiol Ecol 92 Mycol Res 101:295–301. https://doi.org/10.1017/
Card SD, Bastías DA, Caradus JR (2021) Antagonism to S0953756296002602
plant pathogens by Epichloë fungal endophytes—a Clarke BB, White JF, Hurley RH, Torres MS, Sun S, Huff
review. Plan Theory 10:1997 DR (2006) Endophyte-mediated suppression of dollar
Carroll G (1988) Fungal endophytes in stems and leaves: spot disease in fine fescues. Plant Dis 90:994–998.
from latent pathogen to mutualistic symbiont. Ecology https://doi.org/10.1094/pd-90-0994
69:2–9 Clay K (1988) Fungal endophytes of grasses: a defensive
Casas C, Torretta JP, Exeler N, Omacini M (2016) What mutualism between plants and fungi. Ecology 69:10–
happens next? Legacy effects induced by grazing and 16
grass-endophyte symbiosis on thistle plants and their Clay K (1990) Fungal endophytes of grasses. Annu Rev
floral visitors. Plant Soil 405:211–229 Ecol Syst 21:275–297
Casas C, Gundel PE, Deliens E, Iannone LJ, García Clay K, Holah J, Rudgers JA (2005) Herbivores cause a
Martinez G, Vignale MV, Schnyder H (2022) Loss of rapid increase in hereditary symbiosis and alter plant
fungal symbionts at the arid limit of the distribution
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 221

community composition. Proc Natl Acad Sci 102: bioprospecting toward sustainable agriculture. Crit
12465–12470 Rev Biotechnol 40:1210–1231
Coley AB, Fribourg HA, Pelton MR, Gwinn KD (1995) Dupont P-Y, Eaton CJ, Wargent JJ, Fechtner S,
Effects of tall fescue endophyte infestation on relative Solomon P, Schmid J, Day RC, Scott B, Cox MP
abundance of small mammals. Wiley Online Library (2015) Fungal endophyte infection of ryegrass
Conover MR, Messmer TA (1996) Feeding preferences reprograms host metabolism and alters development.
and changes in mass of Canada geese grazing New Phytol 208:1227–1240. https://doi.org/10.1111/
endophyte-infected tall fescue, Condor, pp 859–862 nph.13614
Croy RG, Sutherland BL, Hume DE, Mace WJ, van Dyer DC (1993) Evidence that ergovaline acts on seroto-
Koten C, Finch SC (2022) Animal safety of a tall nin receptors. Life Sci 53:PL223–PL228. https://doi.
fescue endophyte (Epichloë sp.) in a perennial ryegrass org/10.1016/0024-3205(93)90555-H
(Lolium perenne) host. N Z Vet J 70:165–176. https:// Easton H (2007) Grasses and Neotyphodium endophytes:
doi.org/10.1080/00480169.2021.2011795 co-adaptation and adaptive breeding. Euphytica 154:
Daly GT (1973) The grasslands of New Zealand. In: 295–306
Langer RHM (ed) Pastures and pasture plants. Wel- Easton H, Fletcher L (2006) The importance of endophyte
lington, A H and A W Reed Ltd in agricultural systems-changing plant and animal pro-
de Bary A (1879) Die erscheinung der symbiose. Verlag ductivity. NZGA Res Pract Ser 13:11–18
von Karl J. Trübner, Strassburg Easton H, Lyons T, Cooper B, Mace W (2009) Loline
De Battista J, Bacon C, Severson R, Plattner R, Bouton J alkaloids for better protection of pastures from insect
(1990) Indole acetic acid production by the fungal pests. Proc N Z Grassl Assoc:151–154
endophyte of tall fescue. Agron J 82:878–880 Faeth SH, Helander ML, Saikkonen KT (2004) Asexual
de Bonth A, Card S, Briggs L, Faville M, Finch S, Neotyphodium endophytes in a native grass reduce
Hong W, Johnson L, Liu L, Mace W, Pennell C, competitive abilities. Ecol Lett 7:304–313
Popay A, Schmidt J, Sprosen J, Tapper B, and Hume Faulkner JR, Hussaini SR, Blankenship JD, Pal S, Branan
D. 2015. Fungal foray: the pursuit of beneficial endo- BM, Grossman RB, Schardl CL (2006) On the
phyte strains for Australasian pastures. 9th Interna- sequence of bond formation in loline alkaloid biosyn-
tional Symposium on Fungal Endophytes of Grasses thesis. Chembiochem 7:1078–1088
(ISFEG 2015), Melbourne, pp 98–99 Finch S, Munday J, Munday R, Kerby J (2016a) Short-
Decunta FA, Pérez LI, Malinowski DP, Molina- term toxicity studies of loline alkaloids in mice. Food
Montenegro MA, Gundel PE (2021) A systematic Chem Toxicol 94:243–249
review on the effects of Epichloë fungal endophytes Finch SC, Pennell CGL, Kerby JWF, Cave VM (2016b)
on drought tolerance in cool-season grasses. Front Mice find endophyte-infected seed of tall fescue unpal-
Plant Sci 12:644731 atable - implications for the aviation industry. Grass
Demain AL, Fang A (2000) The natural functions of Forage Sci 71:659–666. https://doi.org/10.1111/gfs.
secondary metabolites. In: Fiechter A (ed) History of 12203
modern biotechnology I. Springer, Berlin, pp 1–39 Finch SC, Prinsep MR, Popay AJ, Wilkins AL, Webb NG,
di Menna ME, Finch SC, Popay AJ, Smith BL (2012) A Bhattarai S, Jensen JG, Hawkes AD, Babu JV, Tapper
review of the Neotyphodium lolii/Lolium perenne sym- BA, Lane GA (2020) Identification and structure eluci-
biosis and its associated effects on animal and plant dation of epoxyjanthitrems from Lolium perenne
health, with particular emphasis on ryegrass staggers. infected with the endophytic fungus Epichloë festucae
N Z Vet J 60:315–328 var. lolii and determination of the tremorgenic and anti-
Diehl WW (1950) Balansia and the Balansiae in America: Insect activity of epoxyjanthitrem I. Toxins 12:526
US Department of Agriculture Fisch KM (2013) Biosynthesis of natural products by
Dinkins RD, Nagabhyru P, Graham MA, Boykin D, microbial iterative hybrid PKS–NRPS. RSC Adv 3:
Schardl CL (2017) Transcriptome response of Lolium 18228–18247
arundinaceum to its fungal endophyte Epichloë Fletcher L (1999) “Non-toxic” endophytes in ryegrass and
coenophiala. New Phytol 213:324–337 their effect on livestock health and production. NZGA
Dinkins RD, Nagabhyru P, Young CA, West CP, Schardl Res Pract Ser 7:133–139
CL (2019) Transcriptome analysis and differential Fletcher LR, Harvey IC (1981) An association of a Lolium
expression in tall fescue harboring different endophyte endophyte with ryegrass staggers. N Z Vet J 29:185–
strains in response to water deficit. Plant Genome 12: 186
180071 Fletcher L, Sutherland B (2009) Sheep responses to
Dirihan S, Helander M, Väre H, Gundel PE, Garibaldi LA, grazing ryegrass with AR37 endophyte. Proc N Z
Irisarri JGN, Saloniemi I, Saikkonen K (2016) Geo- Grassl Assoc:127–132
graphic variation in Festuca rubra L. ploidy levels and Fletcher L, Hoglljnd J, Sutherland B (1990) The impact of
systemic fungal endophyte frequencies. PLoS One 11: Acremonium endophytes in New Zealand, past, present
e0166264 and future. Proc N Z Grassl Assoc:227–235
Dubey A, Malla MA, Kumar A, Dayanandan S, Khan ML Fletcher L, Popay AJ, Stewart AV, Tapper B (2000) Herb-
(2020) Plants endophytes: unveiling hidden agenda for age and sheep production from meadow fescue with
222 D. A. Bastías et al.

and without the endophyte Neotyphodium uncinatum. of a fungal Neotyphodium symbiont in an annual grass
Proceedings of the 4th international neotyphodium/ population. Austral Ecol 37:620–628
grass interactions symposium, 27–29 Sep 2000, Gaston KJ (2000) Global patterns in biodiversity. Nature
Soest, pp 447–453 405:220–227
Fletcher L, Finch S, Sutherland B, deNicolo G, Mace W, Gerhards N, Neubauer L, Tudzynski P, Li S-M (2014)
Van Koten C, Hume D (2017) The occurrence of Biosynthetic pathways of ergot alkaloids. Toxins 6:
ryegrass staggers and heat stress in sheep grazing 3281–3295
ryegrass-endophyte associations with diverse alkaloid Gibert A, Hazard L (2013) Genetically based vertical
profiles. N Z Vet J 65:232–241 transmission drives the frequency of the symbiosis
Florea S, Panaccione DG, Schardl CL (2017) Ergot between grasses and systemic fungal endophytes. J
alkaloids of the family Clavicipitaceae. Phytopathol- Ecol 101:743–752
ogy 107:504–518 Gibert A, Volaire F, Barre P, Hazard L (2012) A fungal
Florea S, Jaromczyk J, Schardl CL (2021) Non-transgenic endophyte reinforces population adaptive differentia-
CRISPR-mediated knockout of entire ergot alkaloid tion in its host grass species. New Phytol 194:561–571.
gene clusters in slow-growing asexual polyploid https://doi.org/10.1111/j.1469-8137.2012.04073.x
fungi. Toxins 13:153 Gibson DJ (2009) Grasses and grassland ecology. Oxford
Freeman EM (1904) The seed-fungus of Lolium University Press
temulentum, L., the Darnel. Philos Trans R Soc Lond Goldson SL, Rowarth JS, Caradus JR (2005) The impact
Ser B Containing Papers Biol Char 196:1–27 of invasive invertebrate pests in pastoral agriculture: a
Freitas PP, Hampton JG, Rolston MP, Glare TR, Miller review. N Z J Agric Res 48:401–415
PP, Card SD (2020) A tale of two grass species: tem- Górzyńska K, Lembicz M, Olszanowski Z, Leuchtmann A
perature affects the symbiosis of a mutualistic Epichloë (2011) Botanophila-epichloë interaction in a wild
endophyte in both tall fescue and perennial ryegrass. grass, Puccinellia distans, lacks dependence on the
Front Plant Sci 11. https://doi.org/10.3389/fpls.2020. fly vector. Ann Entomol Soc Am 104:841–846
00530 Granath G, Vicari M, Bazely DR, Ball JP, Puentes A,
Froehlich KA, McAnulty R, Greer A (2022) Loline alka- Rakocevic T (2007) Variation in the abundance of
loid effects on gastrointestinal nematodes. Animals 12: fungal endophytes in fescue grasses along altitudinal
996 and grazing gradients. Ecography 30:422–430
Fuchs B, Krischke M, Mueller MJ, Krauss J (2013) Green KA, Berry D, Feussner K, Eaton CJ, Ram A,
Peramine and lolitrem B from endophyte-grass Mesarich CH, Solomon P, Feussner I, Scott B (2020)
associations cascade up the food chain. J Chem Ecol Lolium perenne apoplast metabolomics for identifica-
39:1385–1389. https://doi.org/10.1007/s10886-013- tion of novel metabolites produced by the symbiotic
0364-2 fungus Epichloë festucae. New Phytol 227:559–571
Fuchs B, Krischke M, Mueller MJ, Krauss J (2017) Plant Gundel PE, Batista WB, Texeira M, Martínez-Ghersa MA,
age and seasonal timing determine endophyte growth Omacini M, Ghersa CM (2008) Neotyphodium endo-
and alkaloid biosynthesis. Fungal Ecol 29:52–58 phyte infection frequency in annual grass populations:
Funk CR, White RH, Breen JP (1993) Importance of relative importance of mutualism and transmission
Acremonium endophytes in turf-grass breeding and efficiency. Proc R Soc B Biol Sci 275:897–905
management. Agric Ecosyst Environ 44:215–232. Gundel PE, Garibaldi LA, Tognetti PM, Aragón R, Ghersa
https://doi.org/10.1016/0167-8809(93)90048-T CM, Omacini M (2009a) Imperfect vertical transmis-
Funk CR, Belanger FC, Murphy JA (2018) Role of sion of the endophyte Neotyphodium in exotic grasses
endophytes in grasses used for turf and soil conserva- in grasslands of the Flooding Pampa. Microb Ecol 57:
tion. Biotechnology of endophytic fungi of grasses. 740–748
CRC Press, pp 201–209 Gundel PE, Martínez-Ghersa MA, Garibaldi LA, Ghersa
Gadberry M, Denard T, Spiers D, Piper E (2003) Effects of CM (2009b) Viability of Neotyphodium endophytic
feeding ergovaline on lamb performance in a heat fungus and endophyte-infected and noninfected
stress environment. J Anim Sci 81:1538–1545 Lolium multiflorum seeds. Botany 87:88–96
Gagic M, Faville MJ, Zhang W, Forester NT, Rolston MP, Gundel PE, Martínez-Ghersa MA, Batista WB, Ghersa
Johnson RD, Ganesh S, Koolaard JP, Easton HS, CM (2010) Dynamics of Neotyphodium endophyte
Hudson D (2018) Seed transmission of Epichloë infection in ageing seed pools: incidence of differential
endophytes in Lolium perenne is heavily influenced viability loss of endophyte, infected seed and
by host genetics. Front Plant Sci 9:1580 non-infected seed. Ann Appl Biol 156:199–209
Gallagher RT, White EP, Mortimer PH (1981) Ryegrass Gundel PE, Garibaldi LA, Martínez-Ghersa MA, Ghersa
staggers: isolation of potent neurotoxins lolitrem a and CM (2011a) Neotyphodium endophyte transmission to
lolitrem B From staggers-producing pastures. N Z Vet Lolium multiflorum seeds depends on the host plant
J 29:189–190. https://doi.org/10.1080/00480169. fitness. Environ Exp Bot 71:359–366
1981.34843 Gundel PE, Rudgers J, Ghersa CM (2011b) Incorporating
Garcia Parisi PA, Casas C, Gundel PE, Omacini M (2012) the process of vertical transmission into understanding
Consequences of grazing on the vertical transmission of host–symbiont dynamics. Oikos 120:1121–1128
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 223

Gundel PE, Martínez-Ghersa MA, Omacini M, Cuyeu R, peramine biosynthesis. Mol Gen Genomics 294:315–
Pagano E, Ríos R, Ghersa CM (2012) Mutualism 328
effectiveness and vertical transmission of symbiotic Hill N, Roach P (2009) Endophyte survival during seed
fungal endophytes in response to host genetic back- storage: endophyte–host interactions and heritability.
ground. Evol Appl 5:838–849 Crop Sci 49:1425–1430
Gundel PE, Pérez LI, Helander M, Saikkonen K (2013) Hill N, Bouton J, Hiatt E, Kittle B (2005) Seed maturity,
Symbiotically modified organisms: nontoxic fungal germination, and endophyte relationships in tall fescue.
endophytes in grasses. Trends Plant Sci 18:420–427 Crop Sci 45:859–863
Gundel P, Sorzoli N, Ueno A, Ghersa C, Seal C, Bastías D, Hoffman GD, Rao S (2013) Association of slugs with the
Martínez-Ghersa M (2015) Impact of ozone on the fungal pathogen Epichloë typhina (Ascomycotina:
viability and antioxidant content of grass seeds is Clavicipitaceae): potential role in stroma fertilisation
affected by a vertically transmitted symbiotic fungus. and disease spread. Ann Appl Biol 162:324–334
Environ Exp Bot 113:40–46 Hoffman GD, Rao S (2014) Fertilization of Epichloë
Gundel P, Irisarri J, Fazio L, Casas C, Pérez L (2016) typhina stromata by mycophagous slugs. Mycologia
Inferring field performance from drought experiments 106:1–7. https://doi.org/10.3852/13-069
can be misleading: the case of symbiosis between Holford G (1933) Grassland work overseas. Proc N Z
grasses and Epichloë fungal endophytes. J Arid Envi- Grassl Assoc:1–4
ron 132:60–62 Hoveland CS (2009) Origin and history. Chapter 1. In:
Gundel PE, Sun P, Charlton ND, Young CA, Miller TE, Fribourg HA, Hannaway DB and West CP, editors.
Rudgers JA (2020) Simulated folivory increases verti- Tall Fescue for the twenty-first century agronomy
cal transmission of fungal endophytes that deter monographs 53. Madison, WI
herbivores and alter tolerance to herbivory in Poa Hoveland CS, Haaland RL, King CC Jr, Anthony WB,
autumnalis. Ann Bot 125:981–991 Clark EM, McGuire JA, Smith LA, Grimes HW,
Guo Y, Gao P, Li F, Duan T (2019) Effects of AM fungi Holliman JL (1980) Association of Epichloë typhina
and grass endophytes on perennial ryegrass Bipolaris fungus and steer performance on tall fescue pasture.
sorokiniana leaf spot disease under limited soil Agron J 72:1064–1065. https://doi.org/10.2134/
nutrients. Eur J Plant Pathol 154:659–671. https://doi. agronj1980.00021962007200060048x
org/10.1007/s10658-019-01689-z Hume D, Sewell J (2014) Agronomic advantages
Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, conferred by endophyte infection of perennial ryegrass
Compant S, Campisano A, Döring M, Sessitsch A (Lolium perenne L.) and tall fescue (Festuca
(2015) The hidden world within plants: ecological arundinacea Schreb.) in Australia. Crop Pasture Sci
and evolutionary considerations for defining function- 65:747–757
ing of microbial endophytes. Microbiol Mol Biol Rev Hume D, Ryan D, Cooper B, Popay A (2007) Agronomic
79:293–320 performance of AR37-infected ryegrass in northern
Hassing B, Winter D, Becker Y, Mesarich CH, Eaton CJ, New Zealand. Proc N Z Grassl Assoc 69:201–205
Scott B (2019) Analysis of Epichloë festucae small Hume D, Cooper B, Panckhurst K (2009) The role of
secreted proteins in the interaction with Lolium endophyte in determining the persistence and produc-
perenne. PLoS One 14:e0209463 tivity of ryegrass, tall fescue and meadow fescue in
Hennessy LM, Popay AJ, Finch SC, Clearwater MJ, Cave Northland. Proc N Z Grassl Assoc 71:145–150
VM (2016) Temperature and plant genotype alter alka- Hume DE, Drummond JB, Rolston MP, Simpson WR,
loid concentrations in ryegrass infected with an Johnson RD (2018) Epichloë endophyte improves
Epichloë endophyte and this affects an insect herbi- agronomic performance and grain yield of rye (Secale
vore. Front Plant Sci 7:1097 cereale). Proceedings of the 10th international sympo-
Hereme R, Morales-Navarro S, Ballesteros G, Barrera A, sium on fungal endophytes of grasses
Ramos P, Gundel PE, Molina-Montenegro MA (2020) Hyde KD, Soytong K (2008) The fungal endophyte
Fungal endophytes exert positive effects on dilemma. Fungal Divers 33:163–173
Colobanthus quitensis under water stress but neutral Jensen J, Popay A (2004) Perennial ryegrass infected with
under a projected climate change scenario in AR37 endophyte reduces survival of porina larvae. N Z
Antarctica. Front Microbiol 11. https://doi.org/10. Plant Prot 57:323–328
3389/fmicb.2020.00264 Jensen J, Popay A, Tapper B (2009) Argentine stem wee-
Hettiarachchige IK, Ekanayake PN, Mann RC, Guthridge vil adults are affected by meadow fescue endophyte
KM, Sawbridge TI, Spangenberg GC, Forster JW and its loline alkaloids. N Z Plant Prot 62:12–18
(2015) Phylogenomics of asexual Epichloë fungal Johnson LJ, Caradus JR (2019) The science required to
endophytes forming associations with perennial deliver Epichloë endophytes to commerce. Endophytes
ryegrass. BMC Evol Biol 15:1–14 Grow World:343
Hettiarachchige IK, Elkins AC, Reddy P, Mann RC, Johnson L, de Bonth A, Briggs L, Caradus J, Finch S,
Guthridge KM, Sawbridge TI, Forster JW, Fleetwood D, Fletcher L, Hume D, Johnson R,
Spangenberg GC (2019) Genetic modification of asex- Popay A, Tapper B, Simpson W, Voisey C, Card S
ual Epichloë endophytes with the perA gene for (2013a) The exploitation of Epichloae endophytes for
224 D. A. Bastías et al.

agricultural benefit. Fungal Divers 60:171–188. https:// Kohlmeyer J, Kohlmeyer E (1974) Distribution of
doi.org/10.1007/s13225-013-0239-4 Epichloë typhina (Ascomycetes) and its parasitic fly.
Johnson LJ, Koulman A, Christensen M, Lane GA, Mycologia 66:77–86. https://doi.org/10.1080/
Fraser K, Forester N, Johnson RD, Bryan GT, 00275514.1974.12019575
Rasmussen S (2013b) An extracellular siderophore is Koshino H, Togiya S, Yoshihara T, Sakamura S,
required to maintain the mutualistic interaction of Shimanuki T, Sato T, Tajimi A (1987) Four fungitoxic
Epichloë festucae with Lolium perenne. PLoS Pathog C-18 hydroxy unsaturated fatty acids from stromata of
9:e1003332 Epichloë typhina. Tetrahedron Lett 28:73–76. https://
Johnson RD, Lane GA, Koulman A, Cao M, Fraser K, doi.org/10.1016/s0040-4039(00)95652-1
Fleetwood DJ, Voisey CR, Dyer JM, Pratt J, Koshino H, Terada S-I, Yoshihara T, Sakamura S,
Christensen M (2015) A novel family of cyclic Shimanuki T, Sato T, Tajimi A (1988) Three phenolic
oligopeptides derived from ribosomal peptide synthe- acid derivatives from stromata of Epichloë typhina on
sis of an in planta-induced gene, gigA, in Epichloë Phleum pratense. Phytochemistry 27:1333–1338.
endophytes of grasses. Fungal Genet Biol 85:14–24 https://doi.org/10.1016/0031-9422(88)80188-2
Johnson LJ, Bastías DA, Caradus JR, Chettri P, Forester Koshino H, Yoshihara T, Sakamura S, Shimanuki T,
NT, Mace WJ, Miller TA, Moon CD, Voisey CR, Sato T, Tajimi A (1989) A ring B aromatic sterol
Zhang W, Card SD (2021) Chapter 6- The dynamic from stromata of Epichloë typhina. Phytochemistry
mechanisms underpinning symbiotic Epichloë–grass 28:771–772. https://doi.org/10.1016/0031-9422(89)
interactions: implications for sustainable and resilient 80112-8
agriculture. In: White J, Kumar A, Droby S (eds) . Koulman A, Lane GA, Christensen MJ, Fraser K, Tapper
Woodhead Publishing, Microbiome stimulants for BA (2007) Peramine and other fungal alkaloids are
crops, pp 73–108 exuded in the guttation fluid of endophyte-infected
Ju Y, Zhong R, Christensen MJ, Zhang X (2020) Effects of grasses. Phytochemistry 68:355–360. https://doi.org/
Epichloë gansuensis endophyte on the root and rhizo- 10.1016/j.phytochem.2006.10.012
sphere soil bacteria of Achnatherum inebrians under Krauss J, Härri SA, Bush L, Husi R, Bigler L, Power SA,
different moisture conditions. Front Microbiol 11:747 Müller CB (2007) Effects of fertilizer, fungal
Kalosa-Kenyon E, Slaughter LC, Rudgers JA, McCulley endophytes and plant cultivar on the performance of
RL (2018) Asexual Epichloë endophytes do not con- insect herbivores and their natural enemies. Funct Ecol
sistently alter arbuscular mycorrhizal fungi coloniza- 21:107–116
tion in three grasses. Am Midl Nat 179(2):157–165. Latch GCM, Christensen MJ (1982) Ryegrass endophyte,
https://doi.org/10.1674/0003-0031-179.2.157 incidence, and control. N Z J Agric Res 25:443–448
Kaur J, Ekanayake PN, Tian P, De Jong EVZ, Latch GCM, Christensen MJ (1985) Artificial infection of
Dobrowolski MP, Rochfort SJ, Mann RC, Smith KF, grasses with endophytes. Ann Appl Biol 107:17–24
Forster JW, Guthridge KM (2015) Discovery and Latch GCM, Christensen MJ, Hickson RE (1988)
characterisation of novel asexual Epichloë endophytes Endophytes of annual and hybrid ryegrasses. N Z J
from perennial ryegrass (Lolium perenne L.). Crop Agric Res 31:57–63. https://doi.org/10.1080/
Pasture Sci 66:1058–1070 00288233.1988.10421364
Keller NP (2019) Fungal secondary metabolism: regula- Le Page M (2022) Worst drought in 500 years? New
tion, function and drug discovery. Nat Rev Microbiol Scientist 255:8. https://doi.org/10.1016/S0262-4079
17:167–180 (22)01459-2
Kirby E (1961) Host-parasite relations in the choke disease Lembicz M, Miszalski Z, Kornaś A, Turnau K (2021)
of grasses. Trans Br Mycol Soc 44:493–503 Cooling effect of fungal stromata in the Dactylis-
Kirkby K, Pratley J, Hume D, An M, Wu H (2011) Viabil- Epichloë-Botanophila symbiosis. Commun Integr
ity of seed and endophyte (Neotyphodium occultans) in Biol 14:151–157. https://doi.org/10.1080/19420889.
annual ryegrass (Lolium rigidum) when buried and in 2021.1938824
long term storage. Seed Sci Technol 39:452–464 Leuchtmann A, Clay K (1988) Atkinsonella Hypoxylon
Klotz JL, Nicol AM (2016) Ergovaline, an endophytic and Balansia Cyperi, epiphytic members of the
alkaloid. 1. Animal physiology and metabolism. Balansiae. Mycologia 80:192–199. https://doi.org/10.
Anim Prod Sci 56:1761–1774. https://doi.org/10. 1080/00275514.1988.12025520
1071/AN14962 Leuchtmann A, Bacon CW, Schardl CL, White JF Jr,
Kocmánková E, Trnka M, Juroch J, Dubrovský M, Tadych M (2014) Nomenclatural realignment of
Semerádová D, Možný M, Žalud Z (2009) Impact of Neotyphodium species with genus Epichloë.
climate change on the occurrence and activity of harm- Mycologia 106:202–215
ful organisms. Plant Protect Sci 45:S48 Leuchtmann A, Young CA, Stewart AV, Simpson WR,
Koga H, Christensen MJ, Bennett RJ (1993) Incompatibil- Hume DE, Scott B (2019) Epichloë novae-zelandiae, a
ity of some grass-Acremonium endophyte associations. new endophyte from the endemic New Zealand grass
Mycol Res 97:1237–1244. https://doi.org/10.1016/ Poa matthewsii. N Z J Bot 57:271–288
S0953-7562(09)81292-6 Lewis G, Ravel C, Naffaa W, Astier C, Charmet G (1997)
Kogel K-H, Franken P, Hückelhoven R (2006) Endophyte Occurrence of Acremonium endophytes in wild
or parasite–what decides? Curr Opin Plant Biol 9:358– populations of Lolium spp. in European countries and
363
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 225

a relationship between level of infection and climate in Martin BD, Schwab E (2012) Symbiosis:“Living together”
France. Ann Appl Biol 130:227–238 in chaos. Stud Hist Biol 4:7–25
Leyronas C, Raynal G (2001) Presence of Neotyphodium- McDonald MB (1998) Seed quality assessment. Seed Sci
like endophytes in European grasses. Ann Appl Biol Res 8:265–276
139:119–127 McLennan E (1920) The endophytic fungus of Lolium.
Leyronas C, Raynal G (2008) Role of fungal ascospores in Proc R Soc Vic (XXXII C N S) 32:252–301
the infection of orchardgrass (Dactylis glomerata) by Mercado-Blanco J (2015) Life of microbes inside the
Epichloë typhina agent of choke disease. J Plant Pathol plant. Principles of plant-microbe interactions.
90:15–21 Springer, pp 25–32
Li F, Guo Y, Christensen MJ, Gao P, Li Y, Duan T (2018) Meyer WA, Funk CR (1989) Progress and benefits to
An arbuscular mycorrhizal fungus and Epichloë humanity from breeding cool-season grasses for turf.
festucae var. lolii reduce Bipolaris sorokiniana disease Contributions from breeding forage and turf grasses,
incidence and improve perennial ryegrass growth. pp 31–48
Mycorrhiza 28:159–169. https://doi.org/10.1007/ Miller TA, Hudson DA, Johnson RD, Singh JS, Mace WJ,
s00572-017-0813-9 Forester NT, Maclean PH, Voisey CR, Johnson LJ
Li C, Wang Z, Chen T, Nan Z (2021) Creation of novel (2022) Dissection of the epoxyjanthitrem pathway in
barley germplasm using an Epichloë endophyte Epichloë sp. LpTG-3 strain AR37 by CRISPR gene
Liu J, Nagabhyru P, Schardl CL (2017) Epichloë festucae editing. Front Fungal Biol 3. https://doi.org/10.3389/
endophytic growth in florets, seeds, and seedlings of ffunb.2022.944234
perennial ryegrass (Lolium perenne). Mycologia 109: Misra A, Yadav S, Mishra S, Tripathi M (2020) Impact of
691–700 meteorological variables and climate change on plant
Liu B, Ju Y, Xia C, Zhong R, Christensen MJ, Zhang X, diseases. Plant pathogens. Apple Academic Press, pp
Nan Z (2022a) The effect of Epichloë endophyte on 313–327
phyllosphere microbes and leaf metabolites in Moate PJ, Williams SRO, Grainger C, Hannah MC,
Achnatherum inebrians. iScience 25:104144. https:// Mapleson D, Auldist MJ, Greenwood JS, Popay AJ,
doi.org/10.1016/j.isci.2022.104144 Hume DE, Mace WJ, Wales WJ (2012) Effects of
Liu H, Tang H, Ni X, Zhang Y, Wang Y (2022b) Effects of wild-type, AR1 and AR37 endophyte-infected peren-
the endophyte Epichloë coenophiala on the root micro- nial ryegrass on dairy production in Victoria, Australia.
bial community and growth performance of tall fescue Anim Prod Sci 52:1117–1130. https://doi.org/10.1071/
in different saline-alkali soils. Fungal Ecol 57-58: AN12126
101159. https://doi.org/10.1016/j.funeco.2022.101159 Moon CD, Craven KD, Leuchtmann A, Clement SL,
Lloyd AB (1959) The endophytic fungus of perennial Schardl CL (2004) Prevalence of interspecific hybrids
ryegrass. N Z J Agric Res 2:1187–1194 amongst asexual fungal endophytes of grasses. Mol
Lopez G (2022) A summer of climate disasters. The Ecol 13:1455–1467
New York Times Morales-Quintana L, Miño R, Mendez-Yañez A, Gundel
Ludlow EJ, Vassiliadis S, Ekanayake PN, Hettiarachchige PE, Ramos P (2022) Do fungal-endosymbionts
IK, Reddy P, Sawbridge TI, Rochfort SJ, Spangenberg improve crop nutritional quality and tolerance to stress
GC, Guthridge KM (2019) Analysis of the indole by boosting flavonoid-mediated responses? Food Res
diterpene gene cluster for biosynthesis of the epoxy- Int 161:111850. https://doi.org/10.1016/j.foodres.
janthitrems in Epichloë endophytes. Microorganisms 2022.111850
7:560 Müller J (2003) Artificial infection by endophytes affects
Lyons PC, Plattner RD, Bacon CW (1986) Occurrence of growth and mycorrhizal colonisation of Lolium
peptide and clavine ergot alkaloids in tall fescue grass. perenne. Funct Plant Biol 30:419–424
Science 232:487–489 Murphy JA, Sun S, Betts LL (1993) Endophyte-enhanced
Mack KM, Rudgers JA (2008) Balancing multiple resistance to billbug (Coleoptera: Curculionidae), sod
mutualists: asymmetric interactions among plants, webworm (Lepidoptera: Pyralidae), and white Grub
arbuscular mycorrhizal fungi, and fungal endophytes. (Coleoptera: Scarabeidae) in tall fescue. Environ
Oikos 117:310–320 Entomol 22:699–703. https://doi.org/10.1093/ee/22.
Mahmud K, Lee K, Hill NS, Mergoum A, Missaoui A 3.699
(2021) Influence of tall fescue Epichloë endophytes on Neil JC (1940) The endophyte of rye-grass (Lolium
rhizosphere soil microbiome. Microorganisms 9:1843 perenne). N Z J Sci Technol A21:280–291
Malinowski DP, Belesky DP (2000) Adaptations of Nicol AM, Klotz JL (2016) Ergovaline, an endophytic
endophyte-infected cool-season grasses to environ- alkaloid. 2. Intake and impact on animal production,
mental stresses: mechanisms of drought and mineral with reference to New Zealand. Anim Prod Sci 56:
stress tolerance. Crop Sci 40:923–940. https://doi.org/ 1775–1786. https://doi.org/10.1071/AN14963
10.2135/cropsci2000.404923x Nissinen R, Helander M, Kumar M, Saikkonen K (2019)
Malinowski DP, Belesky DP (2006) Ecological impor- Heritable Epichloë symbiosis shapes fungal but not
tance of Neotyphodium spp. grass endophytes in bacterial communities of plant leaves. Sci Rep 9:
agroecosystems. Grassl Sci 52:1–14 5253. https://doi.org/10.1038/s41598-019-41603-5
226 D. A. Bastías et al.

Novas MV, Cabral D, Godeas AM (2005) Interaction associations as an avian deterrent. N Z Plant Prot 70:
between grass endophytes and mycorrhizas in Bromus 255–264
setifolius from Patagonia, Argentina. Symbiosis Pennell C, Rolston M, Koten C, Hume D, Card S (2017b)
Novas V, Iannone L, Godeas A, Cabral D (2009) Positive Reducing bird numbers at New Zealand airports using
association between mycorrhiza and foliar endophytes a unique endophyte product. N Z Plant Prot 70:224–
in Poa bonariensis, a native grass. Mycol Prog 8:75– 234
81 Pennell C, Rolston M, Latham A, Mace W, Vlaming J, van
Oesterheld M, Sala O, McNaughton S (1992) Effect of Koten C, Latham M, Brown S, Card S (2017c) Novel
animal husbandry on herbivore-carrying capacity at a grass-endophyte associations reduce the feeding
regional scale. Nature 356:234–236 behaviour of invasive European rabbits (Oryctolagus
Omacini M, Chaneton EJ, Ghersa CM, Müller CB (2001) cuniculus). Wildl Res 43:681–690
Symbiotic fungal endophytes control insect host–para- Perez LI, Gundel PE, Marrero HJ, Arzac AG, Omacini M
site interaction webs. Nature 409:78–81 (2017) Symbiosis with systemic fungal endophytes
Omacini M, Eggers T, Bonkowski M, Gange A, Jones T promotes host escape from vector-borne disease.
(2006) Leaf endophytes affect mycorrhizal status and Oecologia 184:237–245. https://doi.org/10.1007/
growth of co-infected and neighbouring plants. Funct s00442-017-3850-3
Ecol 20:226–232 Pérez LI, Gundel PE, Zabalgogeazcoa I, Omacini M
Pan J, Bhardwaj M, Nagabhyru P, Grossman RB, Schardl (2020) An ecological framework for understanding
CL (2014) Enzymes from fungal and plant origin the roles of Epichloë endophytes on plant defenses
required for chemical diversification of insecticidal against fungal diseases. Fungal Biol Rev 34:115–125
loline alkaloids in grass-Epichloë symbiota. PLoS Pfender WF, Alderman SC (1999) Geographical distribu-
One 9:e115590 tion and incidence of orchardgrass choke, caused by
Panaccione DG, Cipoletti JR, Sedlock AB, Blemings KP, Epichloë typhina, in Oregon. Plant Dis 83:754–758.
Schardl CL, Machado C, Seidel GE (2006) Effects of https://doi.org/10.1094/pdis.1999.83.8.754
ergot alkaloids on food preference and satiety in Pillay LC, Nekati L, Makhwitine PJ, Ndlovu SI (2022)
rabbits, as assessed with gene-knockout endophytes Epigenetic activation of silent biosynthetic gene
in perennial ryegrass (Lolium perenne). J Agric Food clusters in endophytic fungi using small molecular
Chem 54:4582–4587 modifiers. Front Microbiol 13. https://doi.org/10.
Pareek A, Dhankher OP, Foyer CH (2020) Mitigating the 3389/fmicb.2022.815008
impact of climate change on plant productivity and Popay AJ, Bonos SA (2008) Biotic responses in endo-
ecosystem sustainability. Oxford University Press, pp phytic grasses. In: Roberts CA, West CP, Spiers DE
451–456 (eds) Neotyphodium in cool-season grasses, pp
Partida-Martinez LPP, Heil M (2011) The microbe-free 163–185
plant: fact or artifact? Front Plant Sci 2:100 Popay AJ, Cox NR (2016) Aploneura lentisci (Homoptera:
Patchett B, Chapman R, Fletcher L, Gooneratne S (2008) Aphididae) and its interactions with fungal endophytes
Root loline concentration in endophyteinfected in perennial ryegrass (Lolium perenne). Front Plant Sci
meadow fescue (Festuca pratensis) is increased by 7:1395
grass grub (Costelytra zealandica) attack. N Z Plant Popay A, Hume D (2011) Endophytes improve ryegrass
Prot 61:210–214 persistence by controlling insects. NZGA Res Pract Ser
Penn J, Garthwaite I, Christensen M, Johnson C, Towers N 15:149–156
(1993) The importance of paxilline in screening for Popay A, Thom E (2009) Endophyte effects on major
potentially tremorgenic Acremonium isolates. In: insect pests in Waikato dairy pasture. Proc N Z Grassl
Hume D, Latch G, Easton H (eds) Proceedings of the Assoc:121–126
2nd international symposium on acremonium/grass Popay A, Wyatt R (1995) Resistance to Argentine stem
interactions. AgResearch, Palmerston North, pp 88–93 weevil in perennial ryegrass infected with endophytes
Pennell C, Popay A, Ball OJP, Hume D, Baird D (2005) producing different alkaloids. Proceedings of the forty
Occurrence and impact of pasture mealybug eighth New Zealand plant protection conference,
(Balanococcus poae) and root aphid (Aploneura Angus Inn, Hastings, August 8–10, 1995:
lentisci) on ryegrass (Lolium spp.) with and without New Zealand Plant Protection Society, pp 229–236
infection by Neotyphodium fungal endophytes. N Z J Popay A, Townsend R, Fletcher L (2003) The effect of
Agric Res 48:329–337 endophyte (Neotyphodium uncinatum) in meadow fes-
Pennell CGL, Popay AJ, Rolston MP, Townsend RJ, cue on grass grub larvae. N Z Plant Prot 56:123–128
Lloyd-West CM, Card SD (2016) Avanex Unique Popay A, Tapper B, Podmore C (2009) Endophyte
Endophyte Technology – a biological deterrent for infected meadow fescue and loline alkaloids affect
the aviation industries. Environ Entomol 45:101–108. Argentine stem weevil larvae. N Z Plant Prot 62:19–27
https://doi.org/10.1093/ee/nvv145 Pozo MJ, López-Ráez JA, Azcón-Aguilar C, García-
Pennell C, Rolston M, Baird D, Hume D, Mckenzie C, Garrido JM (2015) Phytohormones as integrators of
Card S (2017a) Using novel-grass endophyte environmental signals in the regulation of mycorrhizal
symbioses. New Phytol 205:1431–1436
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 227

Rasmussen S, Parsons AJ, Bassett S, Christensen MJ, Romo M, Leuchtmann A, García B, Zabalgogeazcoa I
Hume DE, Johnson LJ, Johnson RD, Simpson WR, (2007) A totivirus infecting the mutualistic fungal
Stacke C, Voisey CR (2007) High nitrogen supply and endophyte Epichloë festucae. Virus Res 124:38–43
carbohydrate content reduce fungal endophyte and Rowan D, Gaynor D (1986) Isolation of feeding deterrents
alkaloid concentration in Lolium perenne. New Phytol against Argentine stem weevil from ryegrass infected
173:787–797 with the endophyte Acremonium loliae. J Chem Ecol
Ravel C, Michalakis Y, Charmet G (1997) The effect of 12:647–658
imperfect transmission on the frequency of mutualistic Rowan DD, Hunt MB, Gaynor DL (1986) Peramine, a
seed-borne endophytes in natural populations of novel insect feeding deterrent from ryegrass infected
grasses. Oikos, pp 18–24 with the endophyte Acremonium loliae. J Chem Soc
Redman RS, Dunigan DD, Rodriguez RJ (2001) Fungal Chem Commun:935–936
symbiosis from mutualism to parasitism: who controls Rowan DD, Dymock JJ, Brimble MA (1990) Effect of
the outcome, host or invader? New Phytol 151:705– fungal metabolite peramine and analogs on feeding and
716 development of Argentine stem weevil (Listronotus
Reed KFM, Mace WJ, Walker LV, Fletcher LR (2016) bonariensis). J Chem Ecol 16:1683–1695
Endophyte metabolites associated with perennial Rozpądek P, Wężowicz K, Nosek M, Ważny R, Tokarz K,
ryegrass toxicosis. Anim Prod Sci 56:895–907. Lembicz M, Miszalski Z, Turnau K (2015) The fungal
https://doi.org/10.1071/AN14495 endophyte Epichloë typhina improves photosynthesis
Reinholz J, Paul V (2000) Effect of temperature on the efficiency of its host orchard grass (Dactylis
lolitrem B content in Lolium perenne infected by glomerata). Planta 242:1025–1035
Neotyphodium lolii. The 3rd international conference Rubin MAR, Ueno AC, Batlla D, Iannone LJ, Martínez-
on harmful and beneficial microorganisms in grassland Ghersa MA, Gundel PE (2022) Seed functional traits in
pastures and turf cultivars of tall fescue (Schedonorus arundinaceus) are
Ren A-Z, Wang Y-H, Gao Y-B (2009) Difference in affected by the non-toxic fungal endophyte AR584.
antifungal activity of morphotypes of clavicipitaceous Crop Pasture Sci 73(9):1085–1096
endophytes within and between species. Acta Ecol Sin Saikia S, Nicholson MJ, Young C, Parker EJ, Scott B
29:227–231. https://doi.org/10.1016/j.chnaes.2009. (2008) The genetic basis for indole-diterpene chemical
08.005 diversity in filamentous fungi. Mycol Res 112:184–
Riedell W, Kieckhefer R, Petroski R, Powell R (1991) 199. https://doi.org/10.1016/j.mycres.2007.06.015
Naturally-occurring and synthetic loline alkaloid Saikkonen K, Faeth SH, Helander M, Sullivan T (1998)
derivatives: insect feeding behavior modification and Fungal endophytes: a continuum of interactions with
toxicity. J Entomol Sci 26:122–129 host plants. Annu Rev Ecol Syst 29:319–343
Roberts EL, Ferraro A (2015) Rhizosphere microbiome Saikkonen K, Ahlholm J, Helander M, Lehtimäki S,
selection by Epichloë endophytes of Festuca Niemeläinen O (2000) Endophytic fungi in wild and
arundinacea. Plant Soil 396:229–239 cultivated grasses in Finland. Ecography 23:360–366
Roberts E, Lindow S (2014) Loline alkaloid production by Saikkonen K, Lehtonen P, Helander M, Koricheva J, Faeth
fungal endophytes of Fescue species select for particu- SH (2006) Model systems in ecology: dissecting the
lar epiphytic bacterial microflora. ISME J 8:359–368 endophyte–grass literature. Trends Plant Sci 11:428–
Robinson SL, Panaccione DG (2015) Diversification of 433
ergot alkaloids in natural and modified fungi. Toxins 7: Saikkonen K, Young CA, Helander M, Schardl CL (2016)
201–218 Endophytic Epichloë species and their grass hosts:
Rodriguez R, Redman R (2008) More than 400 million from evolution to applications. Plant Mol Biol 90:
years of evolution and some plants still can’t make it on 665–675. https://doi.org/10.1007/s11103-015-0399-6
their own: plant stress tolerance via fungal symbiosis. J Sampson K (1933) The systemic infection of grasses by
Exp Bot 59:1109–1114. https://doi.org/10.1093/jxb/ Epichloë typhina (Pers.) Tul. Trans Br Mycol Soc 18:
erm342 30–47
Rodriguez RJ, White JF Jr, Arnold AE, Redman RS Sampson K (1937) Further observations on the systemic
(2009) Fungal endophytes: diversity and functional infection of Lolium. Trans Br Mycol Soc 21:84–97
roles. New Phytol 182:314–330. https://doi.org/10. Sánchez Márquez S, Bills GF, Herrero N, Zabalgogeazcoa
1111/j.1469-8137.2009.02773.x Í (2012) Non-systemic fungal endophytes of grasses.
Rolston M, Hare M, Moore K, Christensen M (1986) Fungal Ecol 5:289–297. https://doi.org/10.1016/j.
Viability of Lolium endophyte fungus in seed stored funeco.2010.12.001
at different moisture contents and temperatures. N Z J Sands L (2022) Pakistan floods: One third of country is
Exp Agric 14:297–300 under water – minister. BBC News
Rolston M, Agee C (2006) Delivering quality seed to Santangelo JS, Turley NE, Johnson MT (2015) Fungal
specification-the USA and NZ novel endophyte expe- endophytes of Festuca rubra increase in frequency
rience. New Zealand Grasslands Association: Research following long-term exclusion of rabbits. Botany 93:
and Practice Series 13:229–231 233–241
228 D. A. Bastías et al.

Schardl CL (2010) The epichloae, symbionts of the grass Scott B, Green K, Berry D (2018) The fine balance
subfamily poöideae. Ann Mo Bot Gard 97:646–665 between mutualism and antagonism in the Epichloë
Schardl CL, Philips TD (1997) Protective grass festucae–grass symbiotic interaction. Curr Opin Plant
endophytes: where are they from and where are they Biol 44:32–38. https://doi.org/10.1016/j.pbi.2018.
going? Plant Dis 81:430–438 01.010
Schardl CL, Leuchtmann A, Tsai HF, Collett MA, Watt Semmartin M, Omacini M, Gundel PE, Hernández-
DM, Scott DB (1994) Origin of a fungal symbiont of Agramonte IM (2015) Broad-scale variation of
perennial ryegrass by interspecific hybridization of a fungal-endophyte incidence in temperate grasses. J
mutualist with the ryegrass choke pathogen, Epichloë Ecol 103:184–190
typhina. Genetics 136:1307–1317 Simpson WR, Faville MJ, Moraga RA, Williams WM,
Schardl CL, Leuchtmann A, Chung KR, Penny D, Siegel Mcmanus MT, Johnson RD (2014) Epichloë fungal
MR (1997) Coevolution by common descent of fungal endophytes and the formation of synthetic symbioses
symbionts (Epichloë spp.) and grass hosts. Mol Biol in Hordeeae (= Triticeae) grasses. J Syst Evol 52:794–
Evol 14:133–143 806
Schardl CL, Leuchtmann A, Spiering MJ (2004) Sinclair JB, Cerkauskas RF (1996) Latent
Symbioses of grasses with seedborne fungal infection vs. endophytic colonization by fungi. In:
endophytes. Annu Rev Plant Biol 55:315–340. Redlin SC, Carris LM (eds) Endophytic fungi in
https://doi.org/10.1146/annurev.arplant.55.031903. grasses and woody plants systematics, ecology, and
141735 evolution. APS Press, St. Paul, MN
Schardl CL, Panaccione DG, Tudzynski P (2006) Ergot Skendžić S, Zovko M, Živković IP, Lešić V, Lemić D
alkaloids–biology and molecular biology. Alkaloids (2021) The impact of climate change on agricultural
Chem Biol 63:45–86 insect pests. Insects 12:440
Schardl CL, Grossman RB, Nagabhyru P, Faulkner JR, Slaughter LC, Nelson JA, Carlisle E, Bourguignon M,
Mallik UP (2007) Loline alkaloids: currencies of mutu- Dinkins RD, Phillips TD, McCulley RL (2018) Cli-
alism. Phytochemistry 68:980–996 mate change and Epichloë coenophiala association
Schardl CL, Young CA, Faulkner JR, Florea S, Pan J modify belowground fungal symbioses of tall fescue
(2012) Chemotypic diversity of epichloae, fungal host. Fungal Ecol 31:37–46. https://doi.org/10.1016/j.
symbionts of grasses. Fungal Ecol 5:331–344. https:// funeco.2017.10.002
doi.org/10.1016/j.funeco.2011.04.005 Sleper DA, West CP (1996) Tall fescue. Cool-season
Schardl CL, Florea S, Pan J, Nagabhyru P, Bec S, Calie PJ forage grasses, pp 471–502
(2013a) The epichloae: alkaloid diversity and roles in Spatafora J, Sung GH, Sung JM, Hywel-Jones N, White J
symbiosis with grasses. Curr Opin Plant Biol 16:480– Jr (2007) Phylogenetic evidence for an animal patho-
488. https://doi.org/10.1016/j.pbi.2013.06.012 gen origin of ergot and the grass endophytes. Mol Ecol
Schardl CL, Young CA, Hesse U, Amyotte SG, 16:1701–1711
Andreeva K, Calie PJ, Fleetwood DJ, Haws DC, Spiering MJ, Davies E, Tapper BA, Schmid J, Lane GA
Moore N, Oeser B (2013b) Plant-symbiotic fungi as (2002) Simplified extraction of ergovaline and
chemical engineers: multi-genome analysis of the peramine for analysis of tissue distribution in
Clavicipitaceae reveals dynamics of alkaloid loci. endophyte-infected grass tillers. J Agric Food Chem
PLoS Genet 9 50:5856–5862. https://doi.org/10.1021/jf025602b
Schiestl FP, Steinbrunner F, Schulz C, von Reub S, Spiering MJ, Faulkner JR, Zhang D-X, Machado C,
Francke W, Weymuth C, Leuchtmann A (2006) Evo- Grossman RB, Schardl CL (2008) Role of the LolP
lution of ‘pollinator’-attracting signals in fungi. Biol cytochrome P450 monooxygenase in loline alkaloid
Lett 2:401–404 biosynthesis. Fungal Genet Biol 45:1307–1314
Schmid J, Day R, Zhang N, Dupont P-Y, Cox MP, Schardl Star P, Brooking T (2006) Fescue to the rescue: chewings
CL, Minards N, Truglio M, Moore N, Harris DR, Zhou fescue, paspalum, and the application of non-British
Y (2017) Host tissue environment directs activities of experience to pastoral practice in New Zealand,
an Epichloë endophyte, while it induces systemic hor- 1880-1920. Agric Hist 312–335
mone and defense responses in its native perennial Staunton J, Weissman KJ (2001) Polyketide biosynthesis:
ryegrass host. Mol Plant-Microbe Interact 30:138– a millennium review. Nat Prod Rep 18:380–416
149. https://doi.org/10.1094/MPMI-10-16-0215-R Stefanoni-Rubio PJ, Gundel PE, Novas MV, Iannone LJ
Schulthess FM, Faeth SH (1998) Distribution, (2022) Ecotype-specific effects of fungal endophytes
abundances, and associations of the endophytic fungal on germination responses of seeds of the South Ameri-
community of Arizona fescue (Festuca arizonica). can wild forage grass Bromus auleticus. Ann Appl Biol
Mycologia 90:569–578 180:247–258
Schulz B, Guske S, Dammann U, Boyle C (1998) Stewart A (2006) Genetic origins of perennial ryegrass
Endophyte-host interactions. II. Defining symbiosis (Lolium perenne) for New Zealand pastures. NZGA
of the endophyte- host interaction. Symbiosis Res Pract Ser 12:55–61
(Rehovot) 25:213–227 Stewart AV, Barcellos G, Brilman L (2022) Use of endo-
phytic fungi in turfgrasses: difficulties in delivery to
8 After Air, Light, and Water, the Next Most Important Thing Is Grass: An. . . 229

the market. Int Turfgrass Soc Res J 14:1070–1073. Ueno AC, Gundel PE, Ghersa CM, Demkura PV, Card
https://doi.org/10.1002/its2.131 SD, Mace WJ, Martínez-Ghersa MA (2020a) Ontoge-
Strickland J, Aiken G, Spiers D, Fletcher L, Oliver J netic and trans-generational dynamics of a vertically
(2009) Physiological basis of fescue toxicosis. Tall transmitted fungal symbiont in an annual host plant in
Fescue for the Twenty-first Century 53:203–227 ozone-polluted settings. Plant Cell Environ 43:2540–
Süssmuth RD, Mainz A (2017) Nonribosomal peptide 2550
synthesis—principles and prospects. Angew Chem Ueno AC, Gundel PE, Seal CE, Ghersa CM, Martínez-
Int Ed 56:3770–3821 Ghersa MA (2020b) The negative effect of a vertically-
Suttie JM, Reynolds SG, Batello C (2005) Grasslands of transmitted fungal endophyte on seed longevity is
the World. Food & Agriculture Organization of the stronger than that of ozone transgenerational effect.
United Nations, Rome Environ Exp Bot 175:104037
Takach JE, Mittal S, Swoboda GA, Bright SK, Trammell van Zijll de Jong E, Dobrowolski MP, Bannan NR,
MA, Hopkins AA, Young CA (2012) Genotypic and Stewart AV, Smith KF, Spangenberg GC, Forster JW
chemotypic diversity of Neotyphodium endophytes in (2008) Global genetic diversity of the perennial
tall fescue from Greece. Appl Environ Microbiol 78: ryegrass fungal endophyte Neotyphodium lolii. Crop
5501–5510 Sci 48:1487–1501
Tanaka A, Tapper BA, Popay A, Parker EJ, Scott B (2005) Victoria N, Collantes M, Cabral D (2007) Environmental
A symbiosis expressed non-ribosomal peptide synthe- effects on grass-endophyte associations in the harsh
tase from a mutualistic fungal endophyte of perennial conditions of south Patagonia. FEMS Microbiol Ecol
ryegrass confers protection to the symbiotum from 61:164–173
insect herbivory. Mol Microbiol 57:1036–1050 Vignale M, Iannone LJ, Pinget AD, De Battista JP, Novas
Tannenbaum I, Kaur J, Mann R, Sawbridge T, Rodoni B, M (2016) Effect of epichloid endophytes and soil fer-
Spangenberg G (2020) Profiling the Lolium perenne tilization on arbuscular mycorrhizal colonization of a
microbiome: from seed to seed. Phytobiomes J 4:281– wild grass. Plant Soil 405:279–287
289 Vignale M, Iannone LJ, Scervino JM, Novas M (2018)
Tapper B, Lane G (2004) Janthitrems found in a Epichloë exudates promote in vitro and in vivo
Neotyphodium endophyte of perennial ryegrass. arbuscular mycorrhizal fungi development and plant
Proceedings of the 5th international symposium on growth. Plant Soil 422:267–281
neotyphodium/grass interactions, University of Vining LC (1990) Functions of secondary metabolites.
Arkansas Press, Fayetteville, AR Annu Rev Microbiol 44:395–427
Terlizzi NL, Rodríguez MA, Iannone LJ, Lanari E, Novas Voisey CR (2010) Intercalary growth in hyphae of fila-
MV (2022) Epichloë endophyte affects the root colo- mentous fungi. Fungal Biol Rev 24:123–131. https://
nization pattern of belowground symbionts in a wild doi.org/10.1016/j.fbr.2010.12.001
grass. Fungal Ecol 57–58:101143. https://doi.org/10. Wakelin S, Harrison S, Mander C, Dignam B,
1016/j.funeco.2022.101143 Rasmussen S, Monk S, Fraser K, O’Callaghan M
Thom ER, Popay AJ, Hume DE, Fletcher LR (2012) (2015) Impacts of endophyte infection of ryegrass on
Evaluating the performance of endophytes in farm rhizosphere metabolome and microbial community.
systems to improve farmer outcomes–a review. Crop Crop Pasture Sci 66:1049–1057
Pasture Sci 63:927–943 Wang J, Machado C, Panaccione DG, Tsai H-F, Schardl
Tian P, Le T-N, Smith K, Forster J, Guthridge K, CL (2004) The determinant step in ergot alkaloid bio-
Spangenberg G (2013) Stability and viability of novel synthesis by an endophyte of perennial ryegrass. Fun-
perennial ryegrass host–Neotyphodium endophyte gal Genet Biol 41:189–198. https://doi.org/10.1016/j.
associations. Crop Pasture Sci 64:39–50 fgb.2003.10.002
Tiwari P, Bae H (2022) Endophytic fungi: key insights, Wang R, Clarke BB, Belanger FC (2019) Transcriptome
emerging prospects, and challenges in natural product analysis of choke stroma and asymptomatic inflores-
drug discovery. Microorganisms 10:360 cence tissues reveals changes in gene expression in
Torres M, White J (2009) Clavicipitaceae: free-living and both Epichloë festucae and its host plant Festuca
saprotrophs to plant endophytes. Encycl Microbiol 1: rubra subsp. rubra. Microorganisms 7:567
422–430 Wang M, Tian P, Gao M, Li M (2021a) The promotion of
Tsai HF, Liu JS, Staben C, Christensen MJ, Latch GC, Festuca sinensis under heavy metal treatment mediated
Siegel MR, Schardl CL (1994) Evolutionary diversifi- by Epichloë endophyte. Agronomy 11:2049
cation of fungal endophytes of tall fescue grass by Wang R, Luo S, Clarke BB, Belanger FC (2021b) The
hybridization with Epichloë species. Proc Natl Acad Epichloë festucae antifungal protein Efe-AfpA is also a
Sci U S A 91:2542–2546. https://doi.org/10.1073/ possible effector protein required for the interaction of
pnas.91.7.2542 the fungus with its host grass Festuca rubra subsp.
Ueno AC, Gundel PE, Omacini M, Ghersa CM, Bush LP, rubra. Microorganisms 9:140
Martínez-Ghersa MA (2016) Mutualism effectiveness Welty R, Azevedo M, Cooper T (1987) Influence of mois-
of a fungal endophyte in an annual grass is impaired by ture content, temperature, and length of storage on seed
ozone. Funct Ecol 30:226–234 germination and survival of endophytic fungi in seeds
230 D. A. Bastías et al.

of tall fescue and perennial ryegrass. Phytopathology gene cluster for indole-diterpene biosynthesis in the
77:893–900 grass endophyte Neotyphodium lolii. Fungal Genet
Wennström A (1994) Endophyte - the misuse of an old Biol 43:679–693
term. Oikos 71:535–536 Young CA, Tapper BA, May K, Moon CD, Schardl CL,
Wernegreen JJ (2012) Endosymbiosis. Curr Biol 22: Scott B (2009) Indole-diterpene biosynthetic capability
R555–R561. https://doi.org/10.1016/j.cub.2012. of Epichloë endophytes as predicted by ltm gene anal-
06.010 ysis. Appl Environ Microbiol 75:2200–2211
Western J, Cavett J (1959) The choke disease of cocksfoot Yue Q, Miller CJ, White JF, Richardson MD (2000) Isola-
(Dactylis glomerata) caused by Epichloë typhina (Fr.) tion and characterization of fungal inhibitors from
Tul. Trans Br Mycol Soc 42:298–307 Epichloë festucae. J Agric Food Chem 48:4687–4692
White JF, Morrow AC, Morgan JG, Chambless DA (1991) Zabalgogeazcoa I, Benito EP, Ciudad AG, Criado BG,
Endophyte-host associations in forage grasses. XIV. Eslava AP (1998) Double-stranded RNA and virus-
Primary stromata formation and seed transmission in like particles in the grass endophyte Epichloë festucae.
Epichloë typhina: development and regulatory aspects. Mycol Res 102:914–918
Mycologia 83:72–81 Zaurov DE, Bonos S, Murphy JA, Richardson M,
White J, James F, Bacon CW, Hywel-Jones NL, Spatafora Belanger FC (2001) Endophyte infection can contrib-
JW (2003) Historical perspectives: human interactions ute to aluminum tolerance in fine fescues. Crop Sci 41:
with Clavicipitalean fungi. CRC Press, Clavicipitalean 1981–1984. https://doi.org/10.2135/cropsci2001.1981
Fungi, pp 12–25 Zhang W, Card SD, Mace WJ, Christensen MJ, McGill
Wilkinson HH, Siegel MR, Blankenship JD, Mallory AC, CR, Matthew C (2017) Defining the pathways of sym-
Bush LP, Schardl CL (2000) Contribution of fungal biotic Epichloë colonization in grass embryos with
loline alkaloids to protection from aphids in a grass- confocal microscopy. Mycologia 109:153–161.
endophyte mutualism. Mol Plant-Microbe Interact 13: https://doi.org/10.1080/00275514.2016.1277469
1027–1033. https://doi.org/10.1094/mpmi.2000.13.10. Zhang W, Mace WJ, Matthew C, Card SD (2019) The
1027 impact of endophyte infection, seed aging, and imbibi-
Williams CL, Goldson S, Baird D, Bullock D (1994) tion on selected sugar metabolite concentrations in
Geographical origin of an introduced insect pest, seed. J Agric Food Chem 67:6921–6929
Listronotus bonariensis (Kuschel), determined by Zhang J, Deng Y, Ge X, Shi X, Fan X, Dong K, Chen L,
RAPD analysis. Heredity 72:412–419 Zhao N, Gao Y, Ren A (2022) The beneficial effect of
Wilson D (1995) Endophyte: the evolution of a term, and Epichloë endophytes on the growth of host grasses was
clarification of its use and definition. Oikos 73:274– affected by arbuscular mycorrhizal fungi, pathogenic
276 fungi and nitrogen addition. Environ Exp Bot 201:
Woodfield D, Judson H (2019) Balancing pasture produc- 104979. https://doi.org/10.1016/j.envexpbot.2022.
tivity with environmental and animal health 104979
requirements. Improving grassland and pasture man- Zhao Z, Kou M, Zhong R, Xia C, Christensen MJ, Zhang
agement in temperate agriculture. Burleigh Dodds Sci- X (2021) Transcriptome analysis revealed plant hor-
ence Publishing, pp 257–272 mone biosynthesis and response pathway modification
Xia C, Li N, Zhang Y, Li C, Zhang X, Nan Z (2018) Role by Epichloë gansuensis in Achnatherum inebrians
of Epichloë endophytes in defense responses of cool- under different soil moisture availability. J Fungi 7:640
season grasses to pathogens: a review. Plant Dis 102: Zhong R, Xia C, Ju Y, Zhang X, Duan T, Nan Z, Li C
2061–2073 (2021) A foliar Epichloë endophyte and soil moisture
Xu W, Li M, Lin W, Nan Z, Tian P (2021) Effects of modified belowground arbuscular mycorrhizal fungal
Epichloë sinensis endophyte and host ecotype on phys- biodiversity associated with Achnatherum inebrians.
iology of Festuca sinensis under different soil moisture Plant Soil 458:105–122. https://doi.org/10.1007/
conditions. Plan Theory 10:1649 s11104-019-04365-7
Yan L, Zhao H, Zhao X, Xu X, Di Y, Jiang C, Shi J, Zhong R, Bastías DA, Zhang X, Li C, Nan Z (2022)
Shao D, Huang Q, Yang H (2018) Production of Vertically transmitted Epichloë systemic endophyte
bioproducts by endophytic fungi: chemical ecology, enhances drought tolerance of Achnatherum inebrians
biotechnological applications, bottlenecks, and host plants through promoting photosynthesis and bio-
solutions. Appl Microbiol Biotechnol 102:6279–6298 mass accumulation. J Fungi 8:512
Yoshihara T, Togiya S, Koshino H, Sakamura S, Zhou Y, Li X, Gao Y, Ren A (2022) Plant endophytes and
Shimanuki T, Sato T, Tajimi A (1985) Three arbuscular mycorrhizal fungi alter the decomposition
fungitoxic cyclopentanoid sesquiterpenes from stro- of Achnatherum sibiricum litter. Appl Soil Ecol 180.
mata of Epichloë typhina. Tetrahedron Lett 26:5551– https://doi.org/10.1016/j.apsoil.2022.104616
5554. https://doi.org/10.1016/s0040-4039(01)80885-6 Żurek G, Wiewióra B, Gozdowski D (2013) Relations
Young CA, Felitti S, Shields K, Spangenberg G, Johnson between bioclimatic variables and endophyte coloniza-
RD, Bryan GT, Saikia S, Scott B (2006) A complex tion of grasses in Poland. Fungal Ecol 6:554–556
 Signals and Host Cell Remodeling
in Arbuscular Mycorrhizal Symbiosis 9
Andrea Genre, Serena Capitanio, and Paola Bonfante

Abstract Keywords
Mycorrhizas are mutualistic interactions that Symbiosis · Mycorrhiza · Plant nutrition ·
the majority of land plants establish with a Fungal genomics · Signaling · Strigolactone ·
heterogeneous group of soil fungi; their distri- Chitin · Calcium · Cell cycle · Evo-devo
bution and diversity have supported the suc-
cess of plants on the planet. Among all
different types of mycorrhizas, arbuscular 9.1 Introduction
mycorrhiza (AM) is the most ancient and the
most common in host plants of all major crops. Our planet hosts around 390,000 plant species.
The functional core of AM is a finely branched Within their hypogeous and epigeous organs,
fungal structure called the arbuscule. plants store more than 450 Gigatons (Gt) of car-
Arbuscules are hosted inside living root cells, bon, roughly corresponding to 80% of the planet
within a specialized cell compartment that is biomass (Bar-On et al. 2018). Interestingly, the
generated through a precise sequence of belowground biomass is mainly composed of
molecular and cellular events. Over the last plant roots (130 Gt C) and soil-dwelling
10 years, the application of novel microbes. Among them, fungi (with their 12 Gt
technologies, such as genome sequencing, C) are the second most conspicuous soil
high-throughput transcriptomics, and live cell microbes, only outnumbered by bacteria (70 Gt.
imaging, has generated substantial advances in C). Such figures provide solid experimental sup-
our knowledge of such events. Here, we pres- port to the acknowledged claim that
ent a synopsis of the recent literature on the mycorrhizas—symbiotic associations between
interactions between AM fungi and their hosts, around 340,000 plant species and 50,000 species
with an evolutionary-developmental focus on of soil fungi—represent one of the most powerful
the intimate contact that develops between and pervasive ecological processes in terrestrial
plant cells and fungal hyphae, in terms of environments. In these mutually beneficial
molecular signaling, nutrient exchange, and interactions, fungi receive photosynthesis-derived
cell organization. carbon from the plant, while supporting the plant
diet with mineral nutrients, mostly phosphorus
and nitrogen. Present in both natural and
A. Genre (✉) · S. Capitanio · P. Bonfante human-controlled agricultural niches,
Dipartimento di Scienze della Vita e Biologia dei Sistemi, mycorrhizas offer several ecosystem services.
Università di Torino, Torino, Italy Mycorrhizal fungi are in fact well-known drivers
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 231
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_9
232 A. Genre et al.

of carbon sequestration and soil particle aggrega-

tion. They also have a major impact on the com- between epidermal and outer cortical cells
position of microbial and plant communities, as to generate the so-called Hartig net (hn), a
originally demonstrated in a seminal study by dense mesh of hyphae hosted in an
Van der Heijden et al. (1998) where the below- expanded apoplast.
ground diversity of arbuscular mycorrhizal fungi In ericoid mycorrhizas (B), extraradical
emerged as a major factor in the maintenance of hyphae directly penetrate epidermal cell
plant biodiversity. Consensus is growing on the walls without the development of apparent
role of biodiversity in ecosystem functioning, adhesion structures. Convoluted hyphal
and—in this respect—mycorrhizal symbioses coils (c) are then produced inside each epi-
are considered to give a major contribution to dermal cell, enclosed within the symbiotic
the mechanisms that support the whole biosphere interface compartment and the perifungal
and its functioning (Eisenhauer et al. 2022). The membrane (8).
link between mycorrhizas and planet biodiversity In orchids (C), mycorrhizal symbiosis
is quite well defined: 90% of extant vascular and involves both germinating seeds
non-vascular plants can form mycorrhizas and, (protocorms) and seedling roots, but the
among them, angiosperms account for the largest best described colonization process is in
and most diverse group of mycorrhizal species protocorms. Here, epidermal hair cells are
(85–90%). They include annual herbs and peren- the main penetration site for external
nial trees as well as the majority of staple crops hyphae, which then reach the cortical
such as rice, wheat, maize, potato, sweet potato, parenchyma to form large intracellular
tomato, and cassava. The number of fungal hyphal coils, or pelotons (p), within a
mycorrhizal species is more difficult to ascertain membrane-delimited symbiotic interface.
and is currently estimated to account for around Lastly, in arbuscular mycorrhizas (D),
10% of known fungal species (Hyde 2022), presymbiotic hyphae contact the epidermal
belonging to the three major phyla of cells of young lateral roots forming an
Ascomycota, Basidiomycota, and adhesion structure called the hyphopodium
Mucoromycota. (hp). The hyphopodium, often flattened and
branched, adheres strongly to the epidermal
cell walls. A penetrating hypha then
Box 9.1 Main Types of Mycorrhizal
develops in this area, crosses the epidermis
Symbioses
and outer cortical cell layers with both
loose intracellular coils and branched
hyphae, and finally reaches inner cortical
cells, where tree-like arbuscules are formed
(ar). Also in this case, all intracellular
hyphae and arbuscules are accommodated
The figure illustrates four major types of in the symbiotic interface. This interface,
mycorrhizal interactions. In outlined by the periarbuscular membrane,
ectomycorrhizas (A), the epidermal cells is believed to be the main site of nutrient
of lateral roots are targeted by extraradical exchange, thanks to its extensive surface.
hyphae that proliferate on the surface of the These diverse mycorrhizal types involve
whole root tip, engulfing it in a pseudopar- different groups of fungi: Basidiomycota
enchymatous tissue known as the mantle and Ascomycota are the preferential
(m). Inner mantle hyphae further penetrate partners in ectomycorrhizas and ericoid
between epidermal cells, proliferating mycorrhizas, while Basidiomycota taxa
associate with orchids and

(continued)
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 233

AMs are found in the roots of wild plants and

Box 9.1 (continued) of most crops, raising therefore the current inter-
Glomeromycotina develop arbuscular est of scientists, producers and politicians. Under-
mycorrhizas (Genre et al. 2020). standing the mechanisms that regulate plant-
fungal interplay has in fact emerged as a social
responsibility for solving the dilemma of saving
The resulting plant-fungal diversity, supported space for wild nature preservation (Wilson 2016)
by over 400 million years of co-evolution, has or growing plants to feed a growing human
resulted in four main mycorrhizal types, defined population.
according to their major morphological traits In the framework of this complex ecological
(Box 9.1): ectomycorrhizas (ECM), ericoid and ethical scenario, we propose a synopsis of the
mycorrhizas (ERM), orchid mycorrhizas recent literature on the interactions between AM
(ORM), and arbuscular mycorrhizas (AM). fungi and their hosts, with a particular focus on
Their specific cell-to-cell interactions were the intimate contact that develops between plant
initially described by ultrastructural observations cells and fungal hyphae, in terms of molecular
between 1970s and 1990s, and these pioneering signaling, nutrient exchange, and cell organiza-
studies provided the foundation for subsequent tion. Novel technologies, such as genome
investigations on signaling and gene regulation sequencing, high-throughput transcriptomics and
in these symbioses (Bonfante 2018). This corpus proteomics as well as live sub-cellular imaging,
of studies revealed that several cellular and are providing the tools to decipher the genetic,
molecular traits are shared among the four types molecular, and cellular bases of such plant–fungal
of mycorrhizas, even if they originated indepen- interactions.
dently at different times during plant evolution
(Genre et al. 2020) and have an uneven phyloge-
netic distribution (Tedersoo et al. 2020): AMs are 9.2 An Insight in the Biology
the dominant type (involving 72% of plant spe- of Arbuscular
cies), followed by ORM (10%), ECM (2%) and Mycorrhizal Fungi
ERM (1.4%). Nevertheless, this distribution
between plant species does not mirror the real Arbuscular Mycorrhizal (AM) fungi are the most
ecological success of each mycorrhizal type. In relevant representatives of Glomeromycotina, a
fact, ECMs dominate the vast boreal forests, subphylum of the Mucoromycota, which also
despite the low number of gymnosperm and embraces Geosiphon pyriformis, a rare fungus
angiosperm species involved. By contrast, hosting cyanobacteria (Schüßler 2012). Even if
AMs—with their extremely broad host ranges— their ranking as Glomeromycota or
are present across most land ecosystems, from Glomeromycotina is still controversial (Tedersoo
arid sub-arctic environments to sub-tropical et al. 2018; Spatafora et al. 2016), the assignment
rainforests. A reason for the ecological success of AM fungi to Mucoromycota satisfies morpho-
of AMs can be found in their evolutionary his- logical, genomic, and functional parameters. As
tory. AMs are in fact acknowledged as the most in other Mucoromycota, they are aseptate and
ancient form of symbiosis, with the earliest fossil multinucleated fungi which often host
evidence of such plant–fungus associations dat- endobacteria in their cytoplasm (Bonfante and
ing back to 400 million years ago. In fact, the Venice 2020), but also possess unique features:
study of the uniquely preserved ecosystem of the they are described as asexual, obligate symbionts,
Rhynie chert revealed that extinct plants such as which depend on the reduced carbon obtained by
Aglaophyton majus hosted intracellular structures the host plants, while they provide plants with
that are very similar to modern arbuscules (Remy minerals. Glomeromycotina which contain differ-
et al. 1994). ent orders interact with a huge number of plants:
234 A. Genre et al.

liverworts, ferns, gymnosperms (from Ginkgo of AMF as obligate biotrophs. However,

biloba to Cupressus), and angiosperms (from R. irregularis could be grown and reproduced
trees such as poplar to herbs and shrubs). It is axenically on a medium added with fatty acids,
also relevant to remind that AM fungi are proba- among which myristate (Kameoka et al. 2019;
bly not alone to colonize plants producing intra- Sugiura et al. 2020). Some AM fungal genomes
cellular branched hyphae: Mucoromycotina “fine have revealed the presence of pathways leading to
root endophytes,” previously known as Glomus the synthesis of secondary metabolites, as
tenue, are a group of soil fungi that form polyketides (Venice et al. 2020b). The multinu-
endosymbioses with different land plants (Sinanaj cleate status of AM fungi, on the one hand,
et al. 2021), even if significant questions on their complicates transformation procedures and, on
distribution and functions are still open. the other, raises many debates on their level of
Genomics has improved some of our views on genetic variability. Genome sequences and an
AM fungal biology: AMF genomes are among in-depth analysis of MAT-loci, which are related
the largest in the fungal kingdom, ranging from to fungal sexuality events, revealed that while
125 Mbp for the A1 strain of Rhizophagus most of the isolates are homokaryotic, i.e.,
irregularis (Tisserant et al. 2013) to the unex- containing nuclei of one genetically similar
pected sizes of 570 Mbp of Gigaspora rosea nucleotype, a few are dikaryotic, i.e. containing
(Morin et al. 2019) and 770 Mbp of nuclei of two genetically divergent nucleotypes
G. margarita (Venice et al. 2020). By contrast, (Ropars et al. 2016; Kokkoris et al. 2021). Fur-
the gene number is comparable among the AM thermore, the availability of multiple sequenced
sequenced species (around 25,000–26,000 strains from the same species revealed an impres-
genes), even if in the largest genomes, the size sive amount of intra-specific variability (the
was inflated by a huge amount of transposable R. irregularis strains only share around 8000
elements, which occupy around 70% of their conserved genes), such that the concept of
genomes. However, Paraglomus occultum “pangenomes” has been suggested to describe
which belongs to the early diverging order of AM genomes (Mathieu et al. 2018).
Paraglomales has a very reduced genome, In conclusion, current views indicate a long
i.e. 39.6 Mb, fewer genes, and lacks the genes evolutionary story of AM fungi mirroring their
involved in the production of fatty acids and capacity to adapt to their plant hosts and changing
sugars (see below), suggesting that the MRCA environments, and to interact with endobacteria.
of Glomeromycotina was already an obligate However, due to the multinucleate status of AM
plant biotroph (Malar et al. 2022). As for some fungi, their genes cannot be easily tested for their
plant pathogens, AM genomes encode for a large functionality, with the rare exception of the fun-
number of effectors, that might guarantee the gal genes involved in Pi uptake (Xie et al. 2022).
communication between AM fungi and their As a consequence, our current physiological
hosts, and for a limited presence of plant cell knowledge is mostly based on the host plant
wall degrading enzymes. This trait may be side, while many of the striking biological
involved in eluding host defense and maintaining features of AM fungi (i.e., obligate biotrophy,
the viability of host cells. AM fungi are fully multinucleated condition, unknown sexual status)
dependent on plant-derived lipids, since their make them remain a rather intractable experimen-
genomes lack a cytosolic Fatty Acid Synthase tal material (Lanfranco and Bonfante 2023).
complex that is common to fungi (FASI), but
possess a mitochondrial FASII for the synthesis
of lipoic acid (reviewed in Bonfante and Venice 9.3 The Development
2020). This feature, together with the apparent of Arbuscular Mycorrhiza
lack of the pathway for thiamine biosynthesis
and the limited uptake of sugars from soil The establishment of AM symbiosis (Fig. 9.1) has
(Tisserant et al. 2013), contributes to the signature been elucidated in detail and is largely conserved,
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 235

Fig. 9.1 Arbuscular mycorrhiza development starts with (ppa) in the underlying epidermal and cortical cells, which
the germination of resting spores (s) in the rhizosphere. builds the symbiotic interface where intracellular hyphae
This generates a loose explorative mycelium, whose and arbuscules (ar) are hosted. Symbiosis functionality
hyphae eventually reach the host root surface, where they allows the extraradical mycelium to generate new spores
develop adhering hyphopodia (hp). Hyphopodium percep- (ns)
tion triggers the assembly of a prepenetration apparatus

with minor variations depending on the plant and plasma membrane, that contains all intracellular
fungal partners involved (Choi et al. 2018). Fun- hyphae (Bonfante 2001). Once the epidermal
gal spore germination is activated in response to layer is crossed, hyphae can branch and grow
optimal moisture and temperature conditions and both inter- and intracellularly to reach the inner
generates a loose mycelium that grows for a few tissues. The PPA mechanism is replicated in each
days at the expense of spore-stored lipids (Genre colonized cell and reaches the highest complexity
et al. 2020). The resulting hyphae explore the and extension in the deepest cortical cells where
surrounding soil in search of a host root. Follow- hyphae branch repeatedly to generate the core
ing reciprocal plant-fungus recognition through structure of AM symbiosis: arbuscules
an exchange of diffusible molecules, fungal (Luginbuehl and Oldroyd 2017). Arbuscules are
hyphae contacting the root epidermis develop highly branched intracellular hyphae that can
swollen and often branched adhesion structures occupy up to 90% of the host cell volume.
called hyphopodia. In response to both chemical Arbuscules are ephemeral structures that collapse
and physical stimuli produced by the and degenerate within a few days (Gutjahr and
hyphopodium, the contacted epidermal cell Parniske 2013; Kobae and Hata 2010), with the
develops an intracellular accommodation struc- host cell regaining its previous organization.
ture, called the prepenetration apparatus (PPA) While root colonization proceeds, the
(Genre et al. 2005), a columnar cytoplasmic extraradical mycelium also develops extensively
aggregation that concentrates the whole exocytic in the surrounding soil, fed by plant-derived
pathway and anticipates the route of the sugars and lipids. Besides scavenging water
penetrating hypha across the epidermal cell. and mineral nutrients to be delivered to the host
PPA-associated exocytosis drives the biogenesis plant, the extraradical mycelium is also respon-
of the symbiotic interface, a novel cell compart- sible for the production of a new generation of
ment surrounded by an extension of the host spores.
236 A. Genre et al.

9.4 Signaling as a Prerequisite mediated signaling pathway and trigger a cellular

for Symbiosis and molecular prelude to root colonization
(Besserer et al. 2006, 2008; Bonfante and Genre
AM fungi perceive the vicinity of a host via root- 2015).
exuded molecules that induce spore germination AM fungi also release signal molecules
and hyphal branching (Buée et al. 2000; (Fig. 9.2) that trigger plant symbiotic responses
Nagahashi and Douds 2004). The most studied (Bonfante and Requena 2011), including tran-
plant symbiotic signals are carotenoid-derived scriptional regulation, nucleus-associated Ca2+
phytohormones called strigolactones (SL), signals, starch accumulation in roots, and lateral
which have a primary role in plant development root formation (Lanfranco et al. 2016). Repeated
(Al-Babili and Bouwmeester 2015; Ruyter-Spira oscillations in nuclear Ca2+ concentration (spik-
et al. 2013). AM fungi sense SLs in root exudates ing) have been observed in the root epidermal
(Fig. 9.2) at concentrations as low as 10 nM. cells contacted by AM fungal hyphopodia, but
Fungal responses to GR24, a synthetic molecule also when the same cells were treated with
commonly used to study SL actions, include stim- exudates from germinated AM fungal spores
ulation of mitochondrial activity, a rapid increase (Chabaud et al. 2011). Similarly, the expression
in ATP and NADH, and nuclear proliferation of the early symbiotic gene ENOD11 in
(Akiyama et al. 2005; Besserer et al. 2006, M. truncatula is upregulated upon both fungal
2008; Akiyama et al. 2010; Salvioli et al. 2016). contact (Chabaud et al. 2002) and the perception
GR24 exposure also causes a sharp increase in of fungal exudates (Kosuta et al. 2003).
Ca2+ concentration in the fungal cytoplasm Different N-acetylglucosamine
(Moscatiello et al. 2014). Although fungal SL oligosaccharides have been characterized in AM
receptors remain unknown, such observations fungal exudates as bio-active molecules responsi-
suggest that SLs are perceived via a Ca2+- ble for such plant responses. They include tetra-
and penta-chito-oligosaccharides (CO4 and CO5)

Fig. 9.2 Root colonization by AM fungi involves a several transcription factors (RAM1, RAD1, CYCLOPS,
sequence of molecular mechanisms. Firstly, an exchange DELLA, DIP1) control the expression of a number of
of signals ensures reciprocal recognition and triggers sym- genes that are required for fungal accommodation and
biotic responses: root secreted strigolactones (SL) activate arbuscule functioning, such as transporters (PT4, PHT1,
fungal metabolism and branching; Myc factors (LCO and AMT2, STR), lipid biosynthetic enzymes (FATM,
CO) trigger the common symbiotic signaling pathway RAM2), and structural proteins related to the symbiotic
(CSSP), in parallel with CSSP-independent signal trans- interface assembly (VAPYRIN, EXO70)
duction mediated by D14L. Downstream of the CSSP,
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 237

(Genre et al. 2013) as well as lipo-chito- been demonstrated to trigger downstream symbi-
oligosaccharides (LCOs), which are very similar otic responses such as nuclear Ca2+ spiking and
to nodulation (Nod) factors released by nitrogen- ENOD11 expression (Venkateshwaran et al.
fixing rhizobia (Maillet et al. 2011). When 2015).
applied as purified molecules, such Myc factors The remaining known CSSP proteins are
mimic the perception of fungal exudates in the localized to either the nuclear envelope or the
host roots, including nuclear Ca2+ spiking and the nucleoplasm: three nucleoporins—NUP85,
regulation of symbiosis-related genes. Interest- NUP133, and NENA (Kanamori et al. 2006;
ingly, the release of CO4 and CO5 in Saito et al. 2007; Groth et al. 2010)—are likely
R. irregularis exudate is boosted upon GR24 involved in nuclear targeting of downstream
treatment (Genre et al. 2013), suggesting the exis- CSSP actors, such as the nuclear envelope
tence of a positive loop between plant and fungal localized ion channel CASTOR/POLLUX
signal perception and production of these (Charpentier et al. 2008), the Ca2+ channel
oligosaccharides (Lanfranco et al. 2016; Volpe CNGC15 (Charpentier et al. 2016), and the
et al. 2023). SERCA-type ATPase MCA8 (Capoen et al.
Recent investigations are shedding light on the 2011). The nuclear envelope lumen is a major
role of alternative receptor complexes, with cen- Ca2+ store, and indeed Ca2+-mediated signaling
tral roles played by CERK1 and MYR1 LysM is a central hub in the CSSP, with the induction of
receptor-like kinases (Miyata et al. 2014; He et al. characteristic oscillations (spiking) in nuclear
2019) in the plant perception of different fungal- Ca2+ concentration (Genre and Russo 2016).
derived molecules, such as short- and long-chain Ca2+-spiking is interpreted by the
COs (Zhang et al. 2021), leading to the activation nucleoplasm-localized Ca2+-and-calmodulin-
of symbiotic or defense-related plant responses dependent protein kinase CCaMK (Mitra et al.
(Chiu and Paszkowsky 2021). 2004). Its activation regulates gene expression
The study of plant signaling mechanisms through its interacting partner CYCLOPS (Miwa
involved in the perception of AM fungal signals et al. 2006) and the transcription factors NSP1,
has been developed in legumes such as Medicago NSP2, NIN, and RAM1 (Oldroyd 2013; Genre
truncatula, largely following the research on rhi- and Russo 2016).
zobial Nod factor signaling. Such comparative However, this largely characterized pathway is
investigations have revealed the existence of a probably not exclusive (Bonfante and Requena
so-called common symbiotic signaling pathway 2011) and emerging evidence suggests the pres-
(CSSP), which includes several genes that are ence of additional, CSSP-independent signaling.
essential for both symbioses (Oldroyd 2013; On this line, Gutjahr et al. (2015) identified loss
Gobbato 2015). Evidence that the same genes of responsiveness to AM fungi in a rice mutant,
are also involved in diverse symbiotic, patho- which was also mirrored by the absence of physi-
genic, and parasitic plant interactions is cal contact and of characteristic transcriptional
accumulating (Genre and Russo 2016). responses to AM fungal diffusible signals. The
The CSSP (Fig. 9.3) starts on the plant cell responsible gene, DWARF 14 LIKE (D14L),
membrane, with a malectin-like domain (MLD) encodes an alpha/beta-fold hydrolase involved
leucine-rich repeat (LRR) receptor-like kinase in the perception of karrikins and unrelated to
(known as SYMRK, in Lotus japonicus). the recognition of known Myc factors,
SYMRK is believed to be able to form alternative demonstrating the existence of parallel fungal
complexes with either Nod factor or Myc factor recognition strategies in AM host plants.
receptors. In its cytoplasmic domain, SYMRK Despite the remaining unclear aspects, over
interacts with a MAP kinase kinase (Chen et al. 20 years of research have conclusively
2012), and HMGR1, a 3-hydroxy-3- demonstrated that a common genetic basis
methylglutaryl-CoA reductase involved in underpins AM colonization in 72% of land plants,
mevalonate synthesis. Indeed, mevalonate has through the perception of fungal signals and the
238 A. Genre et al.

plasma membrane
SYMRK

HMGR1 MAPKK

MEVALONATE

? ?
nuclear envelope

Ca+2 NUP85
NUP133
CASTOR NENA
CNGC15 MCA8
POLLUX

Ca+2 SPIKING

CCaMK

CYCLOPS NSP1 NSP2 NIN RAM1

GENE EXPRESSION

Fig. 9.3 Scheme of the “common symbiotic signaling NUP133, and NENA—likely involved in nuclear targeting
pathway” (CSSP), mediating the perception of AM fungal of the ion channel CASTOR/POLLUX, the Ca2+ channel
signals. The plasma membrane bound leucine-rich repeat CNGC15, and the SERCA-type ATPase MCA8. In turn,
(LRR) receptor-like kinase SYMRK is a central element these generate Ca2+-mediated signals (spiking) in the
on receptor complexes for Nod factors or Myc factors. In nucleoplasm. Ca2+-spiking is interpreted by the nuclear
its cytoplasmic domain, SYMRK interacts with a MAP Ca2+-and-calmodulin-dependent protein kinase CCaMK,
kinase kinase, and HMGR1, an enzyme involved in its interacting partner CYCLOPS and the transcription
mevalonate biosynthesis. Downstream this membrane- factors NSP1, NSP2, NIN, and RAM1, leading to gene
associated module act three nucleoporins—NUP85, regulation

activation of conserved accommodations Parniske 2013; Balestrini and Bonfante 2014)

strategies. where the fungus is hosted and where most of
the signal and nutrient exchanges are believed to
occur (Luginbuehl and Oldroyd 2017; Choi et al.
9.5 The Symbiotic Interface 2018).
A number of plant genes are required for
The perifungal or periarbuscular membrane arbuscule accommodation and symbiotic inter-
surrounding intracellular hyphae and arbuscules, face development and functioning. Besides cell
respectively, (Harrison 2012) outlines the reorganization-related proteins, such as Vesicle-
so-called symbiotic interface, a novel cell com- Associated Membrane Proteins (Ivanov et al.
partment containing a thin layer of unstructured 2012), EXO70I (Zhang et al. 2015), proteases
cell wall materials (Bonfante 2001; Gutjahr and (Takeda et al. 2009; Rech et al. 2013), Vapyrin
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 239

(Pumplin et al. 2010), arbuscule-associated genes pathogenesis-related transcription factor Ethylene

include lipid biosynthetic enzymes such as Response Factor 19. In this frame, AM fungal
FATM and RAM2 (Bravo et al. 2017) and a genomes code for a large number of putative
specific set of membrane-associated proteins that secreted proteins (Sędzielewska-Toro and Delaux
reside on the periarbuscular membrane through- 2016), and a few of them have been given a
out arbuscule functioning and are directly possible role in the accommodation of fungal
involved in nutrient exchange. These include a structures (Tsuzuki et al. 2016; Fiorilli et al.
proton ATPase (Krajinski et al. 2014; Wang et al. 2016). Even if the mechanisms of action for
2014), ATP-binding cassette (ABC) transporters, these putative secreted proteins have not been
Stunted Arbuscule (STR) and STR2 (Zhang et al. elucidated yet, the functional characterization of
2010), phosphate transporters PT4 (Javot et al. the periarbuscular interface is currently the focus
2007) and PHT1 (Yang et al. 2012), ammonium of AM research, and important advances are
transporters AMT2 (Guether et al. 2009). Inter- expected in the coming years.
estingly, trafficking of some of these proteins,
such as phosphate transporters, to the
periarbuscular membrane requires their genes to 9.6 Evolutionary
be expressed during arbuscule branching and Developmental
(Pumplin et al. 2012), leading to the hypothesis Perspectives on Fungal
that PAM construction is achieved by Accommodation by Plant
synchronizing the massive reorientation of exo- Tissues
cytosis with the transcription of periarbuscular
membrane-resident proteins (Gutjahr and It was recently shown (Russo et al. 2019a, b;
Parniske 2013). In line with this, a few plant Carotenuto et al. 2019a, b) that prepenetration
transcription factors required for AM symbiosis responses culminating in interface construction
and arbuscule development have been identified, include cell cycle reactivation in cortical cells,
such as CYCLOPS (Horváth et al. 2011), the with the onset of both anticlinal cell divisions
gibberellin repressor protein DELLAs (Takeda and recursive endoreduplication events in
et al. 2015), Reduced Arbuscular Mycorrhiza1 advance of arbuscule accommodation (Russo
(RAM1; Gobbato et al. 2012), Required for and Genre 2021).
Arbuscule Development1 (RAD1; Xue et al. On the one hand, the combined use of gene
2015), and DELLA-Interacting Protein1 (DIP1; expression and confocal microscopy has
Yu et al. 2014). Additional evidence suggests a highlighted the appearance of ectopic cell
model where DELLA proteins regulate arbuscule divisions in the inner cortex a few hours after
development through modulation of RAM1 and hyphopodium formation (Fig. 9.4), when
RAD1 that in turn regulate genes required to colonizing hyphae are normally confined to the
support arbuscule branching (Park et al. 2015). root epidermis (Russo et al. 2019a, b). Sparse
Arbuscule formation and functioning is also couples of “split cells” (half the length of the
expected to be under fungal control; however, surrounding parenchymal cells) were consistently
our understanding of such processes is limited. observed in the inner root cortex of diverse AM
Fungal effectors are thought to promote compati- host plants (Russo et al. 2019a). The occurrence
bility or to suppress plant defense responses by of cell division in the fully differentiated tissue
interfering with metabolism or signaling was confirmed by the direct observation of divid-
pathways, in analogy with other plant–microbe ing cells as early as 48 hours post hyphopodium
interactions (Lo Presti et al. 2015). Indeed, formation in Daucus carota expressing a GFP
Secreted Protein 7 (Kloppholz et al. 2011) is fusion with TPLATE (Russo et al. 2019a), an
secreted into the host cell and localizes to the adaptin-related protein that accumulates on the
plant nucleus, where it counteracts the plant cell plate membrane and plasmalemma at the
immune response by interacting with the cortical division zone (Van Damme et al. 2006).
240 A. Genre et al.

Fig. 9.4 Confocal images of DAPI (green, nuclei) and cells is preceded by an increase in cell ploidy, represented
SR2200 (gold, cell walls) stained sections of M. truncatula by the numbers indicating nuclear volume in μm3
(cv “Jemalong” A17) roots colonized by the AM fungus (estimated by image analysis according to Carotenuto
G. margarita (Strain BEG34). The images are representa- et al. 2019b). (c) Recursive events of both cell division
tive of the induction of cell cycle-related processes for and endoreduplication may lead to higher ploidy and
arbuscule accommodation. (a) Cell division (arrowheads) couples of shorter “split cells” (double arrowheads). All
generates “split cells” in the inner cortex during early root four cells in this image host an arbuscule. Bar = 50 μm
colonization. (b) Arbuscule (ar) accommodation in split

On the other hand, flow cytometry, confocal Altogether, these observations outlined a dif-
imaging, and gene expression studies revealed the fuse context of cell cycle reactivation in associa-
diffuse occurrence of endoreduplication (DNA tion with AM fungal accommodation (Russo and
duplication in the absence of cell division) in the Genre 2021). Firstly, cell divisions in the inner
root cortex (Carotenuto et al. 2019b), allowing cortex have been observed when intraradical
the precise localization of inner cortical cells hyphae were limited to epidermal and outer corti-
with different levels of increased ploidy in the cal layers but not in later stages; by contrast,
colonized areas of the root (Carotenuto et al. recursive endoreduplication cycles appear to be
2019a). active for a longer period of time, with
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 241

arbusculated and neighboring cells reaching Besides tissue differentiation,

levels of 128 and even 256C ploidy, endoreduplication is also common in plant
corresponding to up to 6 cycles of interactions with diverse microbes: replicating
endoreduplication. In more detail, DNA produces multiple copies of each gene,
endoreduplication appeared to surge at the front intensifying cell responsiveness to microbial
of fungal expansion and reach the highest peaks signals. Examples are numerous from pathogens
in the central area of infection units, suggesting and parasites (de Almeida Engler and Gheysen
the existence of a proportion between ploidy and 2013; Chandran and Wildermuth 2016;
the abundance (or age) of intraradical fungal Wildermuth et al. 2017) to symbionts (Suzaki
structures. Importantly, Carotenuto et al. et al. 2014; Lace and Ott 2018). Furthermore,
(2019b) also observed that the couples of “split” endoreduplication-related cell enlargement is typ-
cortical cells derived from cell division often ically associated with the accommodation of sev-
displayed different ploidy levels, with higher eral microbes, and specifically with arbuscules in
ploidy in cells that were closer to the fungus or AM (Balestrini and Bonfante 2014; Heck et al.
hosting an older arbuscule. This confirms that cell 2016).
division takes place before endoreduplication, or The requirement of a specific rearrangement in
at least that endoreduplication can proceed after the host cell organization for arbuscule accommo-
cell division. dation is apparent from a simple observation of
Secondly, the observation that both cell divi- the structural and functional complexity of the
sion and ploidy increase at a distance from periarbuscular interface (Luginbuehl and Oldroyd
arbuscules or colonizing hyphae suggests the 2017; Choi et al. 2018; Roth et al. 2019; Ivanov
existence of a yet unidentified signaling process et al. 2019), compared to the tunnel-like interface
reactivate the cell cycle before fungal arrival. hosting linear hyphae in outer root tissues. In fact,
In addition, the concentration of both ectopic while epidermal and outer cortical PPAs are
cell divisions and endocycle events to the inner structured as roughly linear cytoplasmic bridges
cortex envisages a remarkable correlation with across the vacuole, the PPAs that generate
the accommodation of arbuscules, which nor- periarbuscular interfaces are much more complex
mally develop in the same cell layer. Cell prolif- and extensive, appearing as large accumulations
eration, with its limited occurrence, appears to of cytoplasm that extend from the hyphal pene-
have a secondary role, if any, in the generation tration site and occupy most of the host cell cen-
of additional space for arbuscule accommodation. tral volume (Genre et al. 2008). Such a massive,
By contrast, the sparse cell divisions observed in centripetally oriented exocytic event has striking
AM colonized areas might relate to the develop- ultrastructural and molecular similarities with the
mental fate of cortical cells. In the roots of most assembly of the cell plate on the cell equatorial
plants, in fact, cortical cell differentiation is deter- plane at the end of mitosis, and indeed an evolu-
mined by an endocycle that doubles their DNA tionary correlation between symbiotic interface
content from 2C to 4C (Cebolla et al. 1999; Edgar biogenesis and cell plate deposition has been
et al. 2014) with a consequent size increase envisaged in both AM (Russo et al. 2019b) and
(Robinson et al. 2018). In line with that, in situ N-fixing nodulation (Downie 2014). In support of
studies of cell ploidy in uncolonized roots of this hypothesis, in vivo imaging of GFP-TPLATE
M. truncatula (Carotenuto et al. 2019a) revealed fusions revealed a strong accumulation of
that most cortical cells had 4C nuclei, while a few TPLATE at sites of PPA assembly and at sites
of them displayed 8C and 16C ploidy levels. of cell-to-cell hyphal passage, where the
Even if experimentally challenging, it would be perifungal membrane fuses with the plasma-
very interesting to investigate if there is a rela- lemma in striking analogy with cell plate fusion
tionship between initial cell ploidy and the occur- with the cell membrane at the end of mitosis
rence of ectopic cell division in early AM (Russo et al. 2019b).
interaction.
242 A. Genre et al.

If the recruitment of cell division processes to of inner cortical cells enter the endocycle,
assemble the extensive symbiotic interface duplicating their DNA content up to several
surrounding each arbuscule now appears more times, continuously stimulated by the
convincing, developmental restraints could con- approaching fungal symbiont—in fact,
tribute to explain why sparse cell division and endoreduplication also extends to those cells
diffuse endoreduplication are limited to the cor- that had divided earlier (Carotenuto et al.
tex. Dong et al. (2021) have recently highlighted 2019a, b). Such a model implies that host cells
that an SHR-SCR module (known to regulate largely anticipate and direct fungal colonization,
cortex/endodermis initial cell division in the root in line with previous suggestions that the plant
meristem) maintains its activity and is required holds substantial control over symbiosis develop-
for cell cycle reactivation in legume inner cortex ment (Gutjahr and Parniske 2013).
during nodule organogenesis (Suzaki et al. 2014).
Even if analogous studies in rice (which does not
form root nodules, but hosts AM fungi) did not 9.7 Conclusions
confirm SHR-SCR expression in cortical cells, it
is reasonable to speculate that analogous We currently have no information on how the
mechanisms involving meristematic transcription earliest land plants acquired the ability to host a
factors maintain a disposition to reactivate the cell symbiotic fungus inside their cells. One can spec-
cycle in the inner cortical cells. This peculiarity is ulate that initial surface interactions provided an
likely related to the evolution of root branching advantageous exchange of nutrients, pressing
(Xiao et al. 2019), but appears to have later been toward more intimate contacts, such as the pene-
co-opted in several plant interactions, from tration of fungal hyphae between the plant cells
N-fixing symbioses (Dong et al. 2021) to nema- and eventually inside their lumen. In this context,
tode parasitism (de Almeida Engler and Gheysen creating de novo a fully functional symbiotic
2013), where both cell division and interface—as in modern plants—appears unreal-
endoreduplication are required for microbe istic. By contrast, stimulating cell divisions in
accommodation. While such processes involve differentiated organs could have been a more
the formation of new organs (i.e., lateral roots, amenable strategy to generate both crack
N-fixing nodules or nematode-hosting cysts), openings in the surface tissues (an entry route
their occurrence in AM, where organogenesis is that is conserved in many extant plant–microbe
absent, appears puzzling; even more so, if we interactions; Ibáñez et al. 2017) and irregular
consider that AM symbiosis appeared in land intercellular spaces in the inner ones, producing
plants before the evolution of true roots (Strullu- a protected niche for the fungus. The subsequent
Derrien et al. 2014, 2018). re-routing of cell plate formation toward the crea-
By discussing the developmental and evolu- tion of a more efficient symbiotic interface
tionary context where cell cycle processes inter- appears achievable, especially in the light of the
weave with AM symbiosis, a scenario emerges current findings, and the observation of split cells
(Fig. 9.1) in which the perception of AM fungal in some of the earliest fossils of AM hosts
colonization in outer root tissues triggers a so-far indicates that this is indeed an ancient response
unknown intraradical signaling process activating associated with fungal accommodation (Strullu-
cell cycle-related processes ahead of the Derrien et al. 2018).
penetrating intraradical hyphae. Inner cortical Based on the information originated by
cells, due to their peculiar developmental status genome sequencing, it will be interesting to
deploy two downstream responses: a few of them focus future investigation on AM fungi (such as
(possibly depending on their ploidy) complete Paraglomerales) and plants (such as bryophytes)
mitosis, splitting into two smaller cells, as cell belonging to more ancient taxa, in order to char-
elongation is very limited in a mature tissue acterize their root colonization process: are
(Russo et al. 2019a, b); the remaining majority hyphopodia crucial for root penetration or could
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 243

crack-entry play a role in such interactions as it Bonfante P, Venice F (2020) Mucoromycota: going to the
does in other plant–fungus and plant–bacterium roots of plant interacting fungi. Fungal Biol Rev 34:
100–113
interactions? Is the PPA model a common hall- Bravo A, Brands M, Wewer V et al (2017) Arbuscular
mark of fungal accommodation? Is arbuscule mycorrhiza-specific enzymes FatM and RAM2 fine-
morphology conserved, alongside the expression tune lipid biosynthesis to promote development of
of known markers of their functionality? In con- arbuscular mycorrhiza. New Phytol 214:1631–1645
Buée M, Rossignol M, Jauneau A et al (2000) The
clusion, a combination of cellular and molecular pre-symbiotic growth of arbuscular mycorrhizal fungi
biology applied to plant-fungal couples different is induced by a branching factor partially purified from
from the model organisms that have focused plant root exudates. Mol Plant-Microbe Interact 13:
research so far may offer new ideas on how AM 693–698
Capoen W, Sun J, Wysham D, Otegui MS et al (2011)
fungi became the favorite guests for 72% of land Nuclear membranes control symbiotic calcium signal-
plant species. ling of legumes. Proc Natl Acad Sci U S A 108:14348–
14353
Carotenuto G, Volpe V, Russo G et al (2019a) Local
endoreduplication as a feature of intracellular fungal
References accommodation in arbuscular mycorrhizas. New
Phytol 223:430–446
Akiyama K, Matsuzaki K, Hayashi H (2005) Plant Carotenuto G, Sciascia I, Oddi L et al (2019b) Size
sesquiterpenes induce hyphal branching in arbuscular matters: three methods for estimating nuclear size in
mycorrhizal fungi. Nature 435:824–827 mycorrhizal roots of Medicago truncatula by image
Akiyama K, Ogasawara S, Hayashi H (2010) Structural analysis. BMC Plant Biol 19:180
requirement of strigolactones for hyphal branching in Cebolla A, Vinardell JM, Kiss E et al (1999) The mitotic
AM fungi. Plant Cell Physiol 51:1104–1117 inhibitor ccs52 is required for endoreduplication and
Al-Babili S, Bouwmeester HJ (2015) Strigolactones, a ploidy-dependent cell enlargement in plants. EMBO J
novel carotenoid-derived plant hormone. Annu Rev 18:4476–4484
Plant Biol 66:161–118 Chabaud M, Venard C, Defaux-Petras A et al (2002)
Balestrini R, Bonfante P (2014) Cell wall remodeling in Targeted inoculation of Medicago truncatula in vitro
mycorrhizal symbiosis: a way towards biotrophism. root cultures reveals MtENOD11 expression during
Front Plant Sci 5:237 early stages of infection by arbuscular mycorrhizal
Bar-On YM, Phillips R, Milo R (2018) The biomass fungi. New Phytol 156:265–273
distribution on Earth. Proc Natl Acad Sci U S A 115: Chabaud M, Genre A, Sieberer BJ et al (2011) Arbuscular
6506–6511 mycorrhizal hyphopodia and germinated spore
Besserer A, Puech-Pages V, Kiefer P et al (2006) exudates trigger Ca2+ spiking in the legume and non
Strigolactones stimulate arbuscular mycorrhizal fungi legume root epidermis. New Phytol 189:347–355
by activating mitochondria. PLoS Biol 4:1239–1247 Chandran D, Wildermuth MC (2016) Modulation of host
Besserer A, Becard G, Roux C et al (2008) GR24, a endocycle during plant-biotroph interactions. Enzyme
synthetic analogue of strigolactones, stimulates mitosis 40:65–103
and growth of the arbuscular mycorrhizal fungus Charpentier M, Bredemeier R, Wanner G, Takeda N,
Gigaspora rosea by boosting its energetic metabolism. Schleiff E, Parniske M (2008) Lotus japonicus CAS-
Plant Physiol 148:402–413 TOR and POLLUX are ion channels essential for
Bonfante P (2001) At the interface between mycorrhizal perinuclear calcium spiking in legume root endosym-
fungi and plants: the structural organization of cell biosis. Plant Cell 20:3467–3479
wall, plasma membrane and cytoskeleton. In: Hock B Charpentier M, Sun J, Vaz Martins T, Radhakrishnan GV,
(ed) Fungal associations. The mycota, vol 9. Springer, Findlay K, Soumpourou E, Thouin J, Véry AA,
Berlin, pp 45–61 Sanders D, Morris RJ, Oldroyd GE (2016) Nuclear-
Bonfante P (2018) The future has roots in the past: the localized cyclic nucleotide-gated channels mediate
ideas and scientists that shaped mycorrhizal research. symbiotic calcium oscillations. Science 352:1102–
New Phytol 220:982–995 1105
Bonfante P, Genre A (2015) Arbuscular mycorrhizal Chen T, Zhu H, Ke D et al (2012) A MAP kinase kinase
dialogues: do you speak ‘plantish’ or ‘fungish’? Trends interacts with SymRK and regulates nodule organo-
Plant Sci 20:150–154 genesis in Lotus japonicus. Plant Cell 24:823–838
Bonfante P, Requena N (2011) Dating in the dark: how Chiu CH, Paszkowsky U (2021) How membrane receptors
roots respond to fungal signals to establish arbuscular tread the fine balance between symbiosis and immunity
mycorrhizal symbiosis. Curr Opin Plant Biol 14:451– signaling. Proc Natl Acad Sci U S A 118:e2106567118
457
244 A. Genre et al.

Choi J, Summers W, Paszkowski U (2018) Mechanisms lotus japonicus acquires nitrogen released by
underlying establishment of arbuscular mycorrhizal arbuscular mycorrhizal fungi. Plant Physiol 150:73–83
symbioses. Annu Rev Phytopathol 56:135–160 Gutjahr C, Parniske M (2013) Cell and developmental
de Almeida Engler J, Gheysen G (2013) Nematode- biology of arbuscular mycorrhiza symbiosis. Annu
induced endoreduplication in plant host cells: why Rev Cell Develop Biol 29:593–617
and how? Mol Plant-Microbe Interact 26:17–24 Gutjahr C, Gobbato E, Choi J et al (2015) Rice perception
Dong W, Zhu Y, Chang H et al (2021) An SHR-SCR of symbiotic arbuscular mycorrhizal fungi requires the
module specifies legume cortical cell fate to enable karrikin receptor complex. Science 350:1521–1524
nodulation. Nature 589:586–590 Harrison MJ (2012) Cellular programs for arbuscular
Downie JA (2014) Legume nodulation. Curr Biol 24:184– mycorrhizal symbiosis. Curr Opin Plant Biol 15:691–
190 698
Edgar B, Zielke N, Gutierrez C (2014) Endocycles: a He J, Zhang C, Dai H et al (2019) A LysM receptor
recurrent evolutionary innovation for post-mitotic cell heteromer mediates perception of arbuscular mycorrhi-
growth. Nat Rev Mol Cell Biol 15:197–210 zal symbiotic signal in rice. Mol Plant 12:1561–1576
Eisenhauer N, Bonfante P, Buscot F et al (2022) Biotic Heck C, Kuhn H, Heidt S et al (2016) Symbiotic fungi
interactions as mediators of context-dependent biodi- control plant root cortex development through the
versity-ecosystem functioning relationships. Res Ideas novel GRAS transcription factor MIG1. Curr Biol 26:
Outcomes 8:e85873 2770–2778
Fiorilli V, Belmondo S, Khouja HR et al (2016) RiPEIP1, Horváth B, Yeun LH, Domonkos A et al (2011) Medicago
a gene from the arbuscular mycorrhizal fungus truncatula IPD3 is a member of the common symbiotic
Rhizophagus irregularis, is preferentially expressed in signaling pathway required for rhizobial and mycorrhi-
planta and may be involved in root colonization. zal symbioses. Mol Plant-Microbe Interact 24:1345–
Mycorrhiza 26:609–621. https://doi.org/10.1007/ 1358
s00572-016-0697-0 Hyde KD (2022) The numbers of fungi. Fungal Divers
Genre A, Russo G (2016) Does a common pathway trans- 114:1
duce symbiotic signals in plant-microbe interactions. Ibáñez F, Wall L, Fabra A (2017) Starting points in plant-
Front Plant Sci 7:96 bacteria nitrogen- fixing symbioses: intercellular inva-
Genre A, Chabaud M, Timmers T et al (2005) Arbuscular sion of the roots. J Exp Bot 68:1905–1918
mycorrhizal fungi elicit a novel intracellular apparatus Ivanov S, Fedorova EE, Limpens E et al (2012)
in Medicago truncatula root epidermal cells before Rhizobium-legume symbiosis shares an exocytotic
infection. Plant Cell 17:3489–3499 pathway required for arbuscule formation. Proc Natl
Genre A, Chabaud M, Faccio A et al (2008) Prepenetration Acad Sci U S A 109:8316–8321
apparatus assembly precedes and predicts the coloni- Ivanov S, Austin J, Berg RH et al (2019) Extensive mem-
zation patterns of arbuscular mycorrhizal fungi within brane systems at the host-arbuscular mycorrhizal fun-
the root cortex of both Medicago truncatula and gus interface. Nat Plants 5:194–203
Daucus carota. Plant Cell 20:1407–1420 Javot H, Penmetsa RV, Terzaghi N et al (2007) A
Genre A, Chabaud M, Balzergue C et al (2013) Short- Medicago truncatula phosphate transporter indispens-
chain chitin oligomers from arbuscular mycorrhizal able for the arbuscular mycorrhizal symbiosis. Proc
fungi trigger nuclear Ca2+ spiking in Medicago Natl Acad Sci U S A 104:1720–1725
truncatula roots and their production is enhanced by Kameoka H, Tsutsui I, Saito K (2019) Stimulation of
strigolactone. New Phytol 198:190–202 asymbiotic sporulation in arbuscular mycorrhizal
Genre A, Lanfranco L, Perotto S et al (2020) Unique and fungi by fatty acids. Nat Microbiol 4:1654–1660
common traits in mycorrhizal symbioses. Nat Rev Kanamori N, Madsen LH, Radutoiu S et al (2006) A
Microbiol 18:649–660 nucleoporin is required for induction of Ca2+ spiking
Gobbato E (2015) Recent developments in arbuscular in legume nodule development and essential for rhizo-
mycorrhizal signaling. Curr Opin Plant Biol 26:1–7 bial and fungal symbiosis. Proc Natl Acad Sci U S A
Gobbato E, Marsh JF, Vernié T, Wang E, Maillet F, Kim J, 103:359–364
Miller JB, Sun J, Bano SA, Ratet P, Mysore KS, Kloppholz S, Kuhn H, Requena N (2011) A secreted
Dénarié J, Schultze M, Oldroyd GE (2012) A GRAS- fungal effector of Glomus intraradices promotes sym-
type transcription factor with a specific function in biotic biotrophy. Curr Biol 21:1204–1209
mycorrhizal signaling. Curr Biol 22(23):2236–2241 Kobae Y, Hata S (2010) Dynamics of periarbuscular
Groth M, Takeda N, Perry J et al (2010) NENA, a Lotus membranes visualized with a fluorescent phosphate
japonicus homologue of Sec13, is required for transporter in arbuscular mycorrhizal roots of rice.
rhizodermal infection by arbuscular mycorrhizal fungi Plant Cell Physiol 51:341–353
but dispensable for cortical endosymbiotic develop- Kokkoris V, Chagnon PL, Yildirir G et al (2021) Host
ment. Plant Cell 22:2509–2526 identity influences nuclear dynamics in arbuscular
Guether M, Neuhauser B, Balestrini R et al (2009) A mycorrhizal fungi. Curr Biol 31:1531–1538
mycorrhizal-specific ammonium transporter from Kosuta S, Chabaud M, Lougnon G et al (2003) A diffus-
ible factor from arbuscular mycorrhizal fungi induces
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 245

symbiosis-specific MtENOD11 expression in roots of Nagahashi G, Douds DD (2004) Isolated root caps, border
Medicago truncatula. Plant Physiol 131:952–962 cells, and mucilage from host roots stimulate hyphal
Krajinski F, Courty PE, Sieh D et al (2014) The H+- branching of the arbuscular mycorrhizal fungus,
ATPase HA1 of Medicago truncatula is essential for Gigaspora gigantea. Mycol Res 108:1079–1088
phosphate transport and plant growth during arbuscular Oldroyd GED (2013) Speak, friend, and enter: signalling
mycorrhizal symbiosis. Plant Cell 26:1808–1817 systems that promote beneficial symbiotic associations
Lace B, Ott T (2018) Commonalities and differences in in plants. Nat Rev Microbiol 11:252–263
controlling multipartite intracellular infections of Park H-J, Floss DS, Levesque-Tremblay V et al (2015)
legume roots by symbiotic microbes. Plant Cell Hyphal branching during arbuscule development
Physiol 59:666–677 requires Reduced Arbuscular Mycorrhiza1. Plant
Lanfranco L, Bonfante P (2023) Lessons from arbuscular Physiol 69:2774–2788
mycorrhizal fungal genomes. Curr Opin Microbiol Pumplin N, Mondo SJ, Topp S, Starker CG, Gantt JS,
75:102357 Harrison MJ (2010) Medicago truncatula Vapyrin is
Lanfranco L, Bonfante P, Genre A (2016) The mutualistic a novel protein required for arbuscular mycorrhizal
interaction between plants and arbuscular mycorrhizal symbiosis. Plant J 61:482–494
fungi. Microbiol Spectr 4:1–20 Pumplin N, Zhang X, Noar RD et al (2012) Polar localiza-
Lo Presti L, Lanver D, Schweizer G, Shigeyuki T, tion of a symbiosis-specific phosphate transporter is
Liang L, Tollot M, Zuccaro A, Kahmann R (2015) mediated by a transient reorientation of secretion.
Fungal effectors and plant susceptibility. Annu Rev Proc Natl Acad Sci U S A 109:665–672
Phytopathol 66:513–545 Rech SS, Heidt S, Requena N (2013) A tandem Kunitz
Luginbuehl LH, Oldroyd GE (2017) Understanding the protease inhibitor (KPI106)-serine carboxypeptidase
arbuscule at the heart of endomycorrhizal symbioses (SCP1) controls mycorrhiza establishment and
in plants. Curr Biol 27:R952–R963 arbuscule development in Medicago truncatula. Plant
Maillet F, Poinsot V, André O, Puech-Pagès V, Haouy A, J 75:711–725
Gueunier M, Dénarié J (2011) Fungal Remy W, Taylor TN, Hass H et al (1994) Four hundred-
lipochitooligosaccharide symbiotic signals in million-year-old vesicular arbuscular mycorrhizae.
arbuscular mycorrhiza. Nature 469:58–63 Proc Natl Acad Sci U S A 91(25):11841–11843
Malar C, Wang M, Stajich Y et al (2022) Early branching Robinson DO, Coate JE, Singh A et al (2018) Ploidy and
arbuscular mycorrhizal fungus Paraglomus occultum size at multiple scales in the arabidopsis sepal. Plant
carries a small and repeat-poor genome compared to Cell 30:2308–2329
relatives in the Glomeromycotina. Microb Genom 8: Ropars J, Toro KS, Noel J et al (2016) Evidence for the
000810 sexual origin of heterokaryosis in arbuscular mycorrhi-
Mathieu S, Cusant L, Roux C et al (2018) Arbuscular zal fungi. Nat Microbiol 1:16033
mycorrhizal fungi: intraspecific diversity and Roth R, Hillmer S, Funaya C et al (2019) Arbuscular cell
pangenomes. New Phytol 220:1129–1134 invasion coincides with extracellular vesicles and
Mitra RM, Gleason CA, Edwards A et al (2004) A Ca2+/ membrane tubules. Nat Plants 5:204–211
calmodulin-dependent protein kinase required for sym- Russo G, Genre A (2021) Divide and be conquered - cell
biotic nodule development: gene identification by cycle reactivation in arbuscular mycorrhizal symbiosis.
transcript-based cloning. Proc Natl Acad Sci U S A Front Plant Sci 12:753265
101:4701–4705 Russo G, Carotenuto G, Fiorilli V et al (2019a) TPLATE
Miwa H, Sun J, Oldroyd GE, Downie JA (2006) Analysis recruitment reveals endocytic dynamics at sites of sym-
of calcium spiking using a cameleon calcium sensor biotic interface assembly in arbuscular mycorrhizal
reveals that nodulation gene expression is regulated by interactions. Front Plant Sci 10:1628
calcium spike number and the developmental status of Russo G, Carotenuto G, Fiorilli V (2019b) Ectopic activa-
the cell. Plant J 48:883–894 tion of cortical cell division during the accommodation
Miyata K, Kozaki T, Kouzai Y et al (2014) The bifunc- of arbuscular mycorrhizal fungi. New Phytol 221:
tional plant receptor, Os CERK1, regulates both 1036–1048
chitin-triggered immunity and arbuscular mycorrhizal Ruyter-Spira C, Al-Babili S, van der Krol S et al (2013)
symbiosis in rice. Plant Cell Physiol 55:1864–1872 The biology of strigolactones. Trends Plant Sci 18:72–
Morin ES, Miyauchi H, San Clemente ECH et al (2019) 83
Comparative genomics of Rhizophagus irregularis, Saito K, Yoshikawa M, Yano K et al (2007)
R. cerebriforme, R. diaphanus and Gigaspora rosea NUCLEOPORIN85 is required for calcium spiking,
highlights specific genetic features in fungal and bacterial symbioses, and seed production
Glomeromycotina. New Phytol 222:1584–1598 in Lotus japonicus. Plant Cell 19:610–624
Moscatiello R, Sello S, Novero M et al (2014) The intra- Salvioli A, Ghignone S, Novero M et al (2016) Symbiosis
cellular delivery of TAT-aequorin reveals calcium- with an endobacterium increases the fitness of a mycor-
mediated sensing of environmental and symbiotic rhizal fungus, raising its bioenergetic potential. ISME J
signals by the arbuscular mycorrhizal fungus 10:130–144
Gigaspora margarita. New Phytol 203:1012–1020
246 A. Genre et al.

Schüßler A (2012) The Geosiphon–Nostoc endosymbiosis biodiversity, ecosystem variability and productivity.
and its role as a model for arbuscular mycorrhiza Nature 396:69–72
research. In: Hock B (ed) Fungal associations. The Venice F, Ghignone A, Salvioli A et al (2020) At the nexus
mycota, vol 9. Springer, Berlin, pp 77–91 of three kingdoms: the genome of the mycorrhizal
Sędzielewska-Toro KA, Delaux P-M (2016) Mycorrhizal fungus Gigaspora margarita provides insights into
symbioses: today and tomorrow. New Phytol 209:917– plant, endobacterial and fungal interactions. Environ
920 Microbiol 22:122–141
Sinanaj B, Hoysted GA, Pressel S et al (2021) Critical Venice F, Desirò A, Silva G et al (2020b) The mosaic
research challenges facing Mucoromycotina ‘fine root architecture of NRPS-PKS in the arbuscular mycorrhi-
endophytes’. New Phytol 232:1528–1534 zal fungus Gigaspora margarita shows a domain with
Spatafora JW, Chang Y, Benny GL et al (2016) A phylum- bacterial signature. Front Microbiol 11:581313
level phylogenetic classification of zygomycete fungi Venkateshwaran M, Jayaraman D, Chabaud M et al (2015)
based on genome-scale data. Mycologia 108:1028– A role for the mevalonate pathway in early plant sym-
1046 biotic signaling. Proc Natl Acad Sci U S A 112:9781–
Strullu-Derrien C, Kenrick P, Pressel S et al (2014) Fungal 9786
associations in Horneophyton ligneri from the Rhynie Volpe V, Chialva M, Mazzarella T, Crosino A, Capitanio
Chert (c. 407 million year old) closely resemble those S, Costamagna L, Koehler W, Genre A (2023) Long-
in extant lower land plants: novel insights into ances- lasting impact of chitooligosaccharide application on
tral plant-fungus symbioses. New Phytol 203:964–979 strigolactone biosynthesis and fungal accommodation
Strullu-Derrien C, Selosse MA, Kenrick P et al (2018) The promotes arbuscular mycorrhiza in Medicago
origin and evolution of mycorrhizal symbioses: from truncatula. New Phytol 237:2316–2331
palaeomycology to phylogenomics. New Phytol 220: Wang E, Yu N, Bano SA et al (2014) A H+-ATPase that
1012–1030 energizes nutrient uptake during mycorrhizal
Sugiura Y, Akiyama R, Tanaka S et al (2020) Myristate symbioses in rice and Medicago truncatula. Plant Cell
can be used as a carbon and energy source for the 26:1818–1183
asymbiotic growth of arbuscular mycorrhizal fungi. Wildermuth MC, Steinwand MA, McRae AG et al (2017)
Proc Natl Acad Sci U S A 117:25779–25788 Adapted biotroph manipulation of plant cell ploidy.
Suzaki T, Ito M, Yoro E et al (2014) Endoreduplication- Annu Rev Phytopathol 55:537–564
mediated initiation of symbiotic organ development in Wilson EO (2016) We must keep every scrap of nature in
Lotus japonicus. Development 141:2441–2445 and around our cities. Nature holds the key to our
Takeda S, Asamizu E, Tabata S et al (2009) Apoplastic aesthetic, intellectual, cognitive and even spiritual sat-
plant subtilase support arbuscular mycorrhiza develop- isfaction. Landscape 18:26–35
ment in Lotus japonicus. Plant J 58:766–777 Xiao TT, van Velzen R, Kulikova O, Franken C, Bisseling
Takeda N, Handa Y, Tsuzuki S et al (2015) Gibberellins T (2019) Lateral root formation involving cell division
interfere with symbiosis signaling and gene expression in both pericycle, cortex and endodermis is a common
and alter colonization by arbuscular mycorrhizal fungi and ancestral trait in seed plants. Development 146:
in Lotus japonicus. Plant Physiol 167:545–557 dev182592
Tedersoo L, Sánchez-Ramírez S, Kõljalg U et al (2018) Xie X, Lai W, Che X, Wang S et al (2022) A SPX domain-
High-level classification of the Fungi and a tool for containing phosphate transporter from Rhizophagus
evolutionary ecological analyses. Fungal Divers 90: irregularis handles phosphate homeostasis at symbi-
135–159 otic interface of arbuscular mycorrhizas. New Phytol
Tedersoo L, Bahram M, Zob M (2020) How mycorrhizal 234:650–671
associations drive plan population and community. Xue L, Cui H, Buer B, Vijayakumar V, Delaux PM,
Science 367:eaba1223 Junkermann S, Bucher M (2015) Network of GRAS
Tisserant E, Malbreil M, Kuo A et al (2013) Genome of an transcription factors involved in the control of
arbuscular mycorrhizal fungus provides insight into the arbuscule development in Lotus japonicus. Plant
oldest plant symbiosis. Proc Natl Acad Sci U S A 110: Physiol 167:854–871
20117–20122 Yang SY, Grønlund M, Jakobsen I et al (2012)
Tsuzuki S, Handa Y, Takeda N, Kawaguchi M (2016) Non-redundant regulation of rice arbuscular mycorrhi-
Strigolactone-induced putative secreted protein 1 is zal symbiosis by two members of the PHOSPHATE
required for the establishment of symbiosis by the TRANSPORTER1 gene family. Plant Cell 24:4236–
arbuscular mycorrhizal fungus Rhizophagus 4251
irregularis. Mol Plant-Microbe Interact 29:277–286 Yu N, Luo D, Zhang X et al (2014) A DELLA protein
Van Damme D, Coutuer S, De Rycke R et al (2006) complex controls the arbuscular mycorrhizal symbiosis
Somatic cytokinesis and pollen maturation in in plants. Cell Res 24:130–133
Arabidopsis depend on TPLATE, which has domains Zhang Q, Blaylock LA, Harrison MJ (2010) Two
similar to coat proteins. Plant Cell 18:3502–3518 Medicago truncatula half-ABC transporters are essen-
van der Heijden M, Klironomos J, Ursic M et al (1998) tial for arbuscule development in arbuscular mycorrhi-
Mycorrhizal fungal diversity determines plant zal symbiosis. Plant Cell 22:1483–1497
9 Signals and Host Cell Remodeling in Arbuscular Mycorrhizal Symbiosis 247

Zhang X, Pumplin N, Ivanov S, Harrison MJ (2015) Zhang C, He J, Dai H et al (2021) Discriminating symbio-
EXO70I is required for development of a sub-domain sis and immunity signals by receptor competition in
of the periarbuscular membrane during arbuscular rice. Proc Natl Acad Sci U S A 118:e2023738118
mycorrhizal symbiosis. Curr Biol 25:2189–2195
 Masters of Manipulation: How Our
Molecular Understanding of Model 10
Symbiotic Fungi and Their Hosts Is
Changing the Face of “Mutualism”

Jonathan M. Plett, Annegret Kohler, and Francis Martin

Abstract mutualism evolved and how to manage these

Ectomycorrhizal (ECM) fungi are soil-borne symbioses under variable climate scenarios.
organisms found predominantly in forest
environments where they colonize the roots Keywords
of trees and shrubs to gain nutrients that sup- Mycorrhiza · Effector · Symbiosis · Climate ·
port growth. Classed as mutualistic fungi, this Microbiome · Pathogen
group of organisms also supports their host
plants through a variety of mechanisms. How-
ever, to gain a foothold in these competitive 10.1 Mutually Dependent:
environments, ECM fungi have evolved the Plant-Fungal Mutualism
ability to manipulate both their host plants Supports Ecosystem Stability
and other organisms to carve a significant
niche. In this chapter, using well-established One of the fundamental factors that plants must
and developing models, we will consider overcome to adapt/survive is the ability to acquire
the role of ECM fungal signals involved in sufficient nutrients from the soil to complement
the structuring of their environment during carbon fixed through photosynthesis. In temper-
the colonization of soil and plant tissues. We ate forests, approximately 90% of available
will also give an overview of the different nutrients are locked into organic polymers (Wild
signaling network hubs that ECM fungi must 1988), while in high latitude ecosystems this per-
overcome or control to establish a nutrient centage can rise higher than 95% (Chalot and
exchange between both partners. We will con- Brun 1998). As plants favor the use of inorganic
clude with the outlook in this area of research nutrients, a pool that is typically insufficient to
and future directions that are necessary to fur- support the plant life in these environments,
ther our understanding of how fungal another mechanism must exist to supply the nutri-
ent needs in these ecosystems. In general, it is
theorized that most plants rely on symbioses with
J. M. Plett (✉) mycorrhizal fungi, bacteria, and upon the activity
Hawkesbury Institute for the Environment, Western of rhizospheric arthropods and saprotrophic fungi
Sydney University, Richmond, NSW, Australia that recycle nutrients (Fig. 10.1; Ma et al. 2021;
e-mail: [email protected] Yan et al. 2022). A large effort has focused on the
A. Kohler · F. Martin role of mycorrhizal fungi in these environments;
UMR INRA/UHP 1136, Interactions Arbres/Micro- although this group of fungi is typically poor in
organismes, Centre INRA de Nancy, Champenoux, France

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 249
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_10
250 J. M. Plett et al.

the enzymes necessary to scavenge humus-bound following emergence (Taylor et al. 2000; Ruess
nutrients, they do encode genes for secreted et al. 2003; Adams et al. 2006). Physically, sym-
proteins and enzymatic pathways involved in biosis establishment between ECM fungal
habitat engineering and in nutrient acquisition hyphae and a favorable host involves growth of
necessary to meet the needs of the plants through- hyphae around a nascent short root to form a
out their lifecycles (Kohler et al. 2015; Miyauchi sheath (mantle) followed by an inward growth
et al. 2020; Wang et al. 2020). The complexity of in-between root cells into the plant apoplastic
unraveling the interplay between the two partners space (forming the Hartig net; Horan et al. 1988;
has challenged researchers for over a century. In Selosse et al. 2006). The Hartig net can range in
the last decade, the research community has made depth from just around the root epidermal cells
significant advances in understanding how (in hardwood species) to several cell layers deep
mycorrhizal fungi structure their environment to within the root parenchyma (in conifer hosts).
facilitate symbiosis—but are current findings Mechanistically, the formation of these hybrid
commensurate with original hypotheses of how tissues involves hyphal division while concur-
mutualisms were thought to function? Or are we rently loosing septation and apical coherence
seeing a blurring of fungal lifestyle boundaries in while physical connections between plant cells
between pathogenic/saprotrophic/mycorrhizal are lost and plant cell shapes change to more
fungi? ovoid, regular structures (Köttke and
One class of mycorrhizal fungi, typically Oberwinkler 1986).
found in forest environments, are the The underlying mechanisms that physically
ectomycorrhizal (ECM) fungi. ECM fungi are shape the mycorrhizal root and control the flow
not a phylogenetically distinct group, but an of nutrients inherent to the mutualism are based
assemblage of very different fungal species that on an ongoing signaling dialogue between the
have independently developed a mutualistic sym- two partners involving metabolites, nucleic
biotic lifestyle (Van der Heijden et al. 2015; acids, and proteins. A number of these signaling
Lutzoni et al. 2018; Strullu-Derrien et al. 2018). pathways and developmental gene networks are
The hyper-diverse lineages from which these common among ECM fungi, suggesting that,
fungi have evolved, including white- and despite the disparate fungal lineages from which
brown-rot fungi, soil/litter decomposers, and they have evolved, there may be a common mutu-
root endophytes, have led to an incredible rich- alistic “toolbox.” This symbiosis toolbox was
ness of genotypic variation that can be plumbed originally hypothesized to be unique to mutualis-
to understand the molecular foundation upon tic organisms and not bear resemblance to the
which mutualism can evolve (Martin et al. methods used by pathogenic microbes to colonize
2016). Unlike other mycorrhizal fungal types a host. Similarly, methods of nutrient acquisition
(e.g., arbuscular mycorrhiza [AM] or orchid from the soil were hypothesized to be distinct
mycorrhiza), ECM fungi are not obligate from saprotrophic ancestors, thereby increasing
biotrophs and can survive independently on soil reliance of the fungus on the host for carbon
substrates without a host, although they will be (Lindahl and Tunlid 2015). Although our under-
severely limited in their speed of growth and standing of the physiological mechanisms and
reproduction as they are not efficient soil organic signaling pathways at play during mutualistic
matter decomposers and would thus be limited by development and functioning need further refine-
the low concentration of soluble carbohydrates ment, the past decade has brought exciting
available in forest soils. Given the high density discoveries in this area that challenge long-held
of ECM fungal spores, propagules, and hyphae notions of what defines a “mutualist.”
within the soils of forest ecosystems, most of Since the first published genome of an ECM
these fungi are not long without receptive hosts fungus (Martin et al. 2008), the number of ECM
and most lateral roots of trees and shrubs in the genome sequencing programs completed, or in
upper soil profiles are colonized within days progress, is growing rapidly (Kohler et al. 2015;
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 251

Fig. 10.1 The

rhizospheric zoo. Like the
quintessential iceberg
metaphor, often what we
see above ground does not
prepare us for the diversity
of life below ground,
interacting with plant roots.
Organisms ranging from
mycorrhizal fungi (e.g.,
AM and ECM fungi),
bacteria, mites, and
nematodes all affect the
root system and the greater
plant. Graphic Credit:
David Laugesen

Miyauchi et al. 2020; Looney et al. 2022; Tang metabolic programs that lead to the development
et al. 2021; Lebreton et al. 2022; Wu et al. 2022) of symbiosis are driven by both the differential
as are application of the first molecular tools expression of transcription factors (Charvet-
necessary to manipulate these fungi to better Candela et al. 2002; Montanini et al. 2011; Garcia
study the role of individual genes or gene et al. 2015; Law et al. 2022) and transduction
products (e.g., RNA silencing; Kang et al. 2020; pathways conserved with other fungi (i.e., tran-
Chowdhury et al. 2022). Sequencing projects of scription factors also found in saprotrophic
ECM plant hosts such as Populus (Tuskan et al. fungi). In addition, the expression of novel
2006), Picea (Nystedt et al. 2013), Eucalyptus symbiosis-specific gene networks is critical to
(Myburg et al. 2014; Wang et al. 2020), Pinus these early stages (Paparokidou et al. 2021;
(Zimin et al. 2014), Quercus (Plomion et al. Ruytinx et al. 2021; Rivera Pérez et al. 2022;
2018), and Fagus (Mishra et al. 2018, 2022) Tang et al. 2021). Table 10.1 summarizes recent
allow for transcriptomic and proteomic analysis ECM transcriptome studies.
of signaling events in both partners during the Despite the complex factors that regulate the
establishment of symbiosis. Using these establishment and functioning of these
techniques it appears that ontogenic and symbioses, the interaction between ECM fungi
252 J. M. Plett et al.

Table 10.1 Contemporary published works investigating the transcriptional control of the colonization of plant tissues
by ectomycorrhizal fungi
Ectomycorrhizal fungus Host tree Experimental conditions References Year
Cenococcum geophilum Pinus Mature mycorrhizal root tips Peter et al. 2016
sylvestris (2016)
Suillus sp. Pinus sp. Mature mycorrhizal root tips Liao et al. 2016
(2016)
Hebeloma cylindrosporum Pinus pinaster Early ectomycorrhizal development Doré et al. 2017
(2017)
Tuber melanosporum, Quercus robur, Symbiosis-induced genes, comparative Murat et al. 2018
Tuber magnatum Corylus transcriptomics (2018)
avellana
Suillus luteus Pinus Tolerance to aluminium stress Liu et al. 2020
massoniana (2020)
10 ectomycorrhizal fungi diverse host Phylostratigraphy approach to identify Miyauch et al. 2020
plants conserved and species-specific symbiosis- (2020)
induced genes
Tricholoma vaccinum Picea abies Abdulsalam 2021
et al. (2021)
Paxillus involutus Pinus Phosphate availability Paparokidou 2021
sylvestris et al. (2021)
Pisolithus tinctorius Quercus suber Colonization process; transcriptome and Sebastiana 2021
metabolome et al. (2021)
Laccaria bicolor Populus Time course of ectomycorrhizal development Ruytinx et al. 2021
tremula x alba (2021)
Lactarius species Pinus Symbiosis-induced genes, in particular Tang et al. 2021
proteases, comparative transcriptomics (2021)
Pisolithus microcarpus Eucalyptus Intra-species variations Plett et al. 2021
grandis (2021)
Fungal community Picea abies Metatranscriptome of ectomycorrhizal roots Schneider 2021
including Cortinarius and et al. (2021)
other ECM
Diverse ectomycorrhizal Fagus Nitrogen-Uptake, metatranscriptome Rivera Pérez 2022
fungi sylvatica et al. (2022)
Pisolithus microcarpus Eucalyptus Role of abscisic acid during colonization Hill et al. 2022
grandis (2022)
Pisolithus microcarpus Eucalyptus Role of small RNAs Wong- 2022
grandis Bajracharya
et al. (2022)
Tricholoma matsutake Pinus Symbiosis-induced genes, in particular Sakamoto 2022
densiflora MiSSPs et al. (2022)

and their hosts continues to be crucial to the genotypic variation in mycorrhizal individuals
health and sustainability of forest ecosystems and communities will affect the overall outcome
globally. For example, recent work by Anthony of intentional inoculation strategies (Stuart and
and colleagues (Anthony et al. 2022) was key in Plett 2020). The aim of this chapter is to highlight
demonstrating that across Europe, the composi- the most recent work into the understanding of
tion of ECM fungal communities in root systems signaling within the rhizosphere during the estab-
was linked to forest growth. Further, there is now lishment of ECM symbiosis and to illustrate the
renewed attention in the use of ECM fungi during way comparative and functional genomics
restoration forestry (Policelli et al. 2020), informs our understanding about the mechanisms
although the implementation of this worldwide used by mutualistic organisms during the estab-
is hampered by several knowledge gaps in how lishment of a relationship between an ECM
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 253

fungus, its hosts, and the organisms that determine the impact of different chemicals on
surround them. the formation of soil microbiomes (Buckley
et al. 2020, 2022). It is unlikely that the large
majority of signals produced by roots are meant
10.2 Finding a Host: Plant to specifically signal ECM fungi (as they could be
Communication also used by saprotrophic or pathogenic fungi to
to the Teaming Masses recognize the presence of a plant), but rather that
ECM fungi have “taken advantage” of these con-
The soil near plant roots is inhabited by a high centration gradients to find host plants.
diversity of bacteria, fungi, and invertebrate spe- Compounds from two groups found within the
cies (Fig. 10.1; Buée et al. 2009). Within this root exudate, the flavonoids and the
niche, called the rhizosphere, mycorrhizal fungi strigolactones, have received special attention in
must perceive the presence of host plants while this regard due to their effect on the physiology of
filtering the signals from other organisms, both mycorrhizal fungi.
beneficial and detrimental, around them. In this Over 4000 different flavones have been
section, we will address how ECM fungi and their isolated from root exudates, each differently
hosts signal in the “microbial zoo” of the functionalized. It is thought that a number of
rhizosphere. these flavonoids have a role as chemical
Prospecting hyphae originating from spore attractants or repellants to mycorrhizal fungi.
germination, from nascent fungal colonies, or Early work demonstrated that a range of different
from colonized roots sense the presence of a flavonoids induce germination in the spores of
root by perceiving concentration gradients of dif- AM and ECM fungi (Fries et al. 1989; Kikuchi
ferent nutrients and metabolites in soil (Fries et al. et al. 2007). Further, flavonoids from Eucalyptus
1989). Characterizing the diversity of molecules root exudates stimulate the growth and branching
in the complex environment of rhizospheric soil of the ECM fungus Pisolithus microcarpus, mor-
in the stage just prior to fungal/root contact, phological changes which likely facilitate the
termed pre-symbiosis for the purposes of this possibility that a hyphal tip will encounter a root
chapter, is very difficult as soils are complex (Köttke and Oberwinkler 1986; Horan et al. 1988;
matrices and determining the timing of when Lagrange et al. 2001; Martin et al. 2007). More
fungal hyphae come into physical contact with recently, the use of microdialysis in this latter
the plant is nearly impossible. In recent years, interaction has further supported an increase in
however, adaptations of different techniques phenylpropanoids secreted during the first few
from the medical field have led to new under- hours of pre-symbiosis and physical interaction
standing of compounds and proteins secreted between P. microcarpus and E. grandis (Plett
from plant roots and fungi during this stage. et al. 2021). In this case, the authors found that
One of the most interesting techniques is the use there were significant increases in flavylium,
of microdialysis. Used originally to determine the phenylpropanoic acid, coumarin, and tannin
activity of metabolic pathways in animal tissues within the first 48 h of interaction. In addition to
in situ (Ungerstedt 1991; Westerink 1995), phenylpropanoids, there were dozens of other
microdialysis utilizes fine capillary probes that, metabolites produced at higher concentration in
through the process of diffusion, are able to take the rhizosphere, but the majority were novel and
up nutrients, metabolites, and proteins from com- therefore identification of the compound or the
plex tissues or matrices. Microdialysis has now class of compound was impossible. This indicates
seen novel applications in forestry settings to that a richness of plant signals released into the
assess nutrient bioavailability and dissolved rhizosphere remain to be characterized, a fact that
organic compounds in real time with minimal currently limits our understanding of these early
disturbance (Randewig et al. 2019), as well as to stages of the interaction. Interestingly, as opposed
emulate the function of root or hyphal tissues to to the AM symbiosis where colonized roots can
254 J. M. Plett et al.

decrease their exudation of certain flavonoid biosynthesis pathway to directly test the role of
compounds (e.g., genistein; Larose et al. 2002), strigolactones in ECM colonization of host roots,
flavonoids are found enriched in rhizospheric soil recent work suggests that elements of the larger
following the establishment of functional mutual- biochemical pathway (i.e., that of carotenoids) are
istic symbiosis (Wong-Bajracharya et al. 2020). facilitative to the mycorrhization process (Hill
This latter finding was unanticipated as, energeti- et al. 2022). In this latter paper, across a time
cally, we would expect the loss of a course of colonization, one of the main plant
chemoattractant in the exudates of roots already pathways that was induced across the integration
colonized by a mutualistic fungus. This strategy of fungal hyphae into plant roots was the caroten-
would mutually benefit both organisms: it is met- oid biosynthesis pathway. In the early stages of
abolically expensive for the plant to produce colonization, gene expression and metabolic anal-
these secondary metabolites when already ysis suggests that a receptive host prioritizes flux
benefiting from a mutualistic exchange while it away from the strigolactone pathway in favor of
would be counterproductive for free living production of abscisic acid (ABA). Addition of
mycorrhizal mycelium to search out a colonized ABA, meanwhile, facilitates the integration of the
host root system. Further work needs to be fungus within the root as denoted by a Hartig net
undertaken in this area to determine if possible, with a greater surface area. The biochemical reg-
changes in the class of flavonoids produced by ulation of this pathway changes toward the end of
bare roots as opposed to colonized roots differen- colonization when genes encoding the enzymes
tially attract ECM fungi. for strigolactone biosynthesis, CCD7 and CCD8,
It is likely that flavonoids do not act alone, but are slightly induced (Hill et al. 2022). This finding
rather with other root exudate compounds. further reinforces the notion that the root
Strigolactones, a class of sesquiterpene lactones secretome changes between the early stages of
produced by a wide range of mono- and dicotyle- ECM colonization and later, functional stages.
donous plants found in the root exudates (Machin These results would also suggest that the signal-
et al. 2020). In AM fungi, recognition of these ing programs in plants that facilitate one type of
compounds is required for host specificity mycorrhizal lineage may not be utilized by
(Akiyama et al. 2005), and they play important another.
roles in the control of AM hyphal growth and While we can identify secreted signals from
branching in the proximity to the root host plants, our understanding of the mechanisms
(Bouwmeester et al. 2003; Akiyama et al. 2005; by which root exudates are sensed by the ECM
Besserer et al. 2008; Bonfante and Genre 2010). fungus has not appreciably advanced in the last
Recent studies suggest that strigolactones decade. Early work demonstrated that upon
originated as AM symbiosis-inducing rhizo- receipt of different signals from a plant host,
sphere signaling molecules and were later L. bicolor genes involved in transduction
recruited as plant hormones (Kodama et al. pathways were among the most abundant of the
2022). Strigolactone concentrations decrease rap- mRNAs in ectomycorrhizal tissues (Voiblet et al.
idly in the root exudates of plants that are not 2001). The regulated genes correspond to the
receptive to colonization, although strigolactone different sub-units of G-proteins (e.g., a fungal
production seems to be linked more closely to G-protein α subunit), response regulators (e.g.,
nutrient status of the root (Akiyama and Hayashi Ras protein kinases), and calcium binding
2008). The role of these compounds in ECM proteins (e.g., calmodulin). A Ras cDNA has
fungi, however, remains elusive. While also been described in L. bicolor and may be
strigolactones play a role in fungal growth in involved in the control of cell growth and prolif-
AM fungi, this has not been found for two previ- eration of hyphae (Sundaram et al. 2001). Simi-
ously tested ECM fungi (i.e., L. bicolor and larly, the T. melanosporum genome encodes the
P. involutus; Steinkellner et al. 2007). While signaling genes documented in other filamentous
mutants have yet to be created in the strigolactone fungi that are involved in pathways controlling
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 255

stress response, filamentous growth, virulence, step, new proteins are also being discovered
and mating. During the interaction with the host within the fungal tree of life that can modify the
plant most signaling transcripts are strongly chemical properties of plant cell wall surfaces that
expressed, although very few are specific to improve adhesion. Within the Pezizomycotina,
mycorrhizal root tips (Martin et al. 2010). With comparative genomics identified a conserved
ever increasing fungal genomes (Miyauchi et al. sequence motif (C-CXXXC-C-C-C-C-C) within
2020) and the ability to transform ECM fungi surface-active protein that, when tested, changed
with gene reporter constructs (Kemppainen and the hydrophobicity of a surface to promote hyphal
Pardo 2021), this area deserves special attention attachment regardless of fungal lifestyle (Zhao
as understanding the breadth of perception et al. 2021). In the endophytic fungus Serendipita
systems in ECM fungi will enable us to narrow indica (also known as Piriformospora indica;
our focus on what plant-based compounds are Sebacinales), secretion of the protein Fungal
critical to the early stages of this interaction. Glycan-Binding 1 alters cell wall composition to
facilitate host colonization (Wawra et al. 2016).
Within mutualistic fungi, a range of secreted
10.3 Deconstruction of a Root: hydrophobins also may support attachment to
Formation of the Symbiotic host surfaces during colonization due to their
Interface amphipathic structure; they accumulate at the sur-
face of hyphae with their hydrophobic domains
Upon physical contact between ECM hyphae and directed outward, aiding aggregation of hyphae or
a host root, the fungus must attach to the plant adhesion to hydrophobic host surfaces
tissue and form an interface with the host that will (Casarrubia et al. 2018). These proteins also
protect hyphae from plant-based defenses but also may enable the fungus to avoid plant defenses
allow for the continued exchange of signaling by coating hyphal surfaces to mask the antigenic-
molecules as well as nutrients. To some degree, ity of fungal β-1,3-D glucans and chitin, known
ECM fungi are at a “disadvantage” compared to elicitors of the plant defense system (Sharp et al.
other lifestyles of fungi at this stage of symbiosis 1984a, b; Roby et al. 1987; Templeton et al.
because convergent evolution within this lifestyle 1994). Due to their incredible stability,
has led to the loss or reduction of several plant hydrophobin layers have also been proposed to
cell wall active enzyme families (Kohler et al. protect the fungal hyphae against plant proteases
2015; Miyauchi et al. 2020). For instance, the and reactive oxygen species (Wösten and Wessels
genome of L. bicolor only encodes one 1997).
endoglucanase GH5 with a fungal cellulose-bind- While our current models concerning the
ing module while there are no cellulases from activity of hydrophobins have been largely
families GH6 and GH7. Instead, ECM fungi are informed by their role in pathogenic fungi (e.g.,
reliant on a range of proteins and compounds that Moonjely et al. 2018; Yu et al. 2020), recent
attach to, and then gently restructure (i.e., not publications on insect mutualists (Lovett et al.
degrade), the plant cell wall to form a more cohe- 2022), ericoid mycorrhizal fungi (Casarrubia
sive structure known as the symbiotic interface. et al. 2018), saprotrophs (Cai et al. 2020), as
Over the last decade, we have seen a range of well as ECM fungi are beginning to broaden our
proteins and enzymes involved in this step understanding of this class. To date, the
characterized for the first time. hydrophobins encoded by ECM fungi have been
As the formation of mycorrhizal root tips studied in a restricted set of lineages. In Pisolithus
never occurs in a static environment as roots are microcarpus (originally identified as P. tinctorius
constantly growing, ECM fungal hyphae must prior to phylogenetic reconstruction of the genus;
excrete a mucilage of different compounds to Martin et al. 2002), early work demonstrated that
attach firmly to the root. While a range of the hydrophobins hydPt-1 and hydPt-2 were
adhesins or oligosaccharides are involved in this induced during the early stages of fungal
256 J. M. Plett et al.

aggregation on roots (Tagu et al. 1996; Voiblet hypothesis, however, was the finding that the
et al. 2001; Duplessis et al. 2005). Similarly, majority of L. bicolor hydrophobins are under
increased accumulation of hydrophobin purifying selection suggesting that maintenance
transcripts was observed in Paxillus involutus/ of a high number of hydrophobins in ECM fungi
Betula pendula ectomycorrhiza (Le Quéré et al. is not evolutionarily advantageous (Plett et al.
2006). The genome of L. bicolor S238N-H82 2012a). While it has been found that hydrophobin
encodes 12 hydrophobin genes (Martin et al. expression correlates to the pathogenesis of ani-
2008), some of which exhibit interesting mal pathogens, in this same link it has not been
differences when compared to classical found as a causal phenotype in other plant-
hydrophobin genes (Plett et al. 2012a). A first colonizing fungi (Parta et al. 1994; Thau et al.
defining characteristic of hydrophobins encoded 1994; Brasier et al. 1995; Bowden et al. 1996).
by L. bicolor is the level of hydrophobicity within Therefore, further work is needed to understand
the core of the consensus sequence: the role of these compounds during host coloni-
hydrophobicity between cysteine doublets is zation and in possibly being a key determinant in
much greater when compared to other class I host specificity.
(e.g., SC3 from Schizophyllum commune) or Other proteins may also support adhesion of
even to class II hydrophobins (e.g., HFBI from fungal hyphae to the root. One class that may play
Trichoderma reesei) (Kim et al. 2005; Jensen a role are the Symbiosis-Regulated Acidic
et al. 2010). A second characteristic observed in Mannoproteins (SRAPs; Martin et al. 1999,
a subset of the hydrophobins encoded by 2008). While a large number of different SRAP
L. bicolor (LbH3, LbH11, and LbH14) is an classes have been identified in fungi (Hilbert et al.
altered frequency of cysteine residues (Jensen 1991; Burgess et al. 1995), one class in particular,
et al. 2010; Plett et al. 2012a), suggesting that the 32 kDa class of SRAPs has received attention
the mature protein has an altered function as in their role as potential adhesins based on timing
seen in other fungi that encode non-canonical of expression, an encoded cell adhesion RGD
hydrophobins (Kershaw and Talbot 1998). motif, and its location on the outer hyphal cell
There are also interesting differences in the wall. In the model of P. microcarpus (previously
panel of hydrophobins expressed during root col- mis-identified as P. tinctorius), expression of
onization whereby the variety of hydrophobins PmSRAP32-1 is especially high at 3 days post
expressed depend upon the plant host being contact between fungal hyphae and Eucalyptus
colonized. In the L. bicolor model, if the fungus globus roots, a time period when hyphae are
is able to colonize a plant species at a high level aggregating around plant roots. The PmSRAP32
(used as a proxy for degree of host receptivity), class is composed of at least 6 isoforms (Kohler
far fewer hydrophobins are expressed as opposed et al. 2015) that range in pI from 4.5 to 5.5 and are
to a plant species with much lower levels of secreted to the plant/hyphal interface during col-
colonization (i.e., poorly receptive host; Plett onization of the plant root (De Carvalho 1994).
et al. 2012a). As hydrophobins are regulated by RGD motifs such as those found in SRAPs are
changes in the external environment (e.g., during thought to be involved in adhesion as RGD
host colonization; Wessels 1996), are produced to containing proteins from the fungus Lentinus
hide antigenic hyphal compounds from the plant edodes induce flocculation in yeast (Kondoh
immune system (Aimanianda et al. 2009), or act et al. 1995) and aggregation of hyphae in
as a base for enzymes that loosen connections Schizophyllum commune (Yasuda and Shishido
between plant tissues (Corvis et al. 2005, 2006, 1997). However, the role of SRAP32 proteins in
2007; Qin et al. 2007; Zhao et al. 2007; Wang cell adhesion has yet to be proven and more
et al. 2010), it is possible that the large variety of recent work would suggest that RGD motifs
hydrophobins encoded by L. bicolor are a neces- could have alternative functions. In plant
sary adaptation to aid in the colonization of a pathogens, the rust fungus Uromyces vignae
wide range of host species. Counter to this uses RGD containing proteins (RGDS and
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 257

RGDSP) to control and repress the defense reac- L. bicolor GH5, meanwhile, was secreted during
tion by the plant host by disrupting the connection the stages of fungal ingrowth into the root and
between the plasma membrane and the plant cell found to localize to the periphery of the hyphae,
wall, a connection necessary for the proper main- and RNAi knockdown mutants of L. bicolor for
tenance of plant cell wall-associated defenses this gene led to a reduction in the development of
(Hostetter 2000). Similarly, in mammalian mycorrhizal root tips. While this activity was key
systems, these proteins have been found to bind to L. bicolor, its broader applicability to other
extracellular proteins to facilitate cell entry ECM lineages is questionable as impairment of
(Chernyavska et al. 2019; Mészáros et al. 2021). this activity in Tricholoma matsutake did not
Another class of secreted fungal proteins that affect colonization of the host: in fact, lack of
could affect fungal aggregation on the root sur- the β-1,4-endoglucanase activity in T. matsutake
face are the Mycorrhiza-induced Small Secreted led to improved host growth rate (Murata et al.
Proteins (MiSSPs). The ECM fungus L. bicolor 2021). These differences highlight a key point
encodes a small secreted protein of 8 kDa that needs refining in our understanding of ECM
(MiSSP8) that undergoes post-translational mod- evolution: while there are many instances of con-
ification and is involved in the formation of the vergent evolution in the function of ECM
fungal mantle during host colonization (Pellegrin mutualisms, there will be distinct differences
et al. 2019). The mechanism by which it supports between different genera due to the number of
this mantle formation is currently unknown, but it times that this lifestyle has independently evolved
may be associated with a repetitive motif found in in the fungal tree of life.
the protein that is common across fungi of multi- The activity of LbGH5-CBM1 was found to
ple lifestyles. Therefore, further work needs to be operate against a variety of different substrates
done to elucidate the full function of these including cellulose, mannans, and
proteins during symbiosis, but these studies galactomannans. This activity was commensurate
show a high degree of similarity among mutualis- with a β-1,4 endoglucanase as the hydrolysis of
tic fungi and saprotrophic and pathogenic these substrates led to release of cellobiose and
microbes in adhesion to their substrates/hosts. cellotriose. Conversely, this enzyme was inactive
Following adhesion, ingrowth of ECM fungal against crystalline cellulose, xyloglucan, xylan,
hyphae into the root apoplastic space to form the arabinoxylan, arabinan, and pectin (Zhang et al.
Hartig net requires alterations to plant cell wall 2018a). As these latter components in the cell
chemistry and the connections that hold cells wall, especially pectin, are necessary for cell
together in a cohesive mass. Early work in this integrity and cell–cell connections, other
area demonstrated that, while plant cell wall mechanisms must be encoded by ECM fungi to
active enzymes were regulated during host colo- enable full apoplastic invasion. L. bicolor
nization in two lineages of ECM fungi (i.e., encodes two possible mechanisms to alter pectin:
L. bicolor and T. melanosporum), only two clas- a GH28 and a series of pectin methyltransferases
ses were in common: the GH5-CBM1 glucanase (PMEs) that may contribute to loosening cell–cell
and a GH28 pectinase (Martin et al. 2008, 2010). connections. Similar to LbGH5-CBM1, the
This specific expression suggested at the time that L. bicolor enzyme LbGH28 production was nec-
these two proteins may play a key role during the essary for elaboration of a full Hartig net, and it
establishment of symbiosis (Martin et al. 2010). was localized both to the hyphal cells and to the
The GH5-CBM1 protein of L. bicolor has since surface of plant cells during symbiosis (Zhang
been characterized (Zhang et al. 2018a) as well as et al. 2022). As predicted based on sequence
a candidate GH5 protein from Tricholoma matsu- analysis, this protein was active against pectins
take (Onuma et al. 2019). The latter protein was as well as polygalacturonic acid but was unable to
characterized as being active during use rhamnogalacturonan, galactan, or pectic
non-symbiotic growth in the fungus and to hydro- galactan as substrates. In a concurrent publica-
lyze β-glucan, lichenan, and CMC-Na. The tion, it was shown that the gene encoding
258 J. M. Plett et al.

LbPME1 exhibited higher expression coinciding alterations are orchestrated has lagged. With the
with PME activity during formation of the mycor- production of genomic resources for a range of
rhizal root tip (Chowdhury et al. 2022). Repres- mycorrhizal fungi and their hosts, however, we
sion of this fungal protein led to a reduction in are now seeing significant advances in this area.
Hartig net formation while over-expression of In this section, we will focus on our advances in
LbPME1 led to a better developed Hartig net as understanding how host hormonal and immunity
well as altered esterification of pectin in plant cell pathways are altered.
walls. Therefore, it was concluded that PMEs One of the earliest studied plant hormones and
form a necessary component of ECM fungal cell its role in mycorrhizal establishment was auxin
wall active enzymes during colonization. Beyond (Rupp and Mudge 1985; Gay et al. 1994; Gea
these two examples, ECM fungi likely use a range et al. 1994; Kaska et al. 1999). In a post-genomic
of other peptidases and proteases to manipulate era, host roots in contact with L. bicolor were
host cell walls. Lactarius deliciosus, for example, found to exhibit altered expression of genes
encodes nearly 13× the average copy number of involved in the polar transport of auxin (PtaPIN
fungalysin proteases, many of which are and PtaAUX genes), auxin conjugation
regulated during the interaction between (PtaGH3), and auxin signaling (PtaIAA; Felten
L. deliciosus and Pinus taeda (Tang et al. 2021; et al. 2009; Vayssières et al. 2015). Similarly, the
Lebreton et al. 2022), proteases that could be interaction between Tricholoma vaccinum and
involved during the integration step. ECM fungi spruce was shown to alter auxin signaling and
may also utilize non-enzymatic routes to gain a movement and that this occurred only in a com-
foothold in the apoplastic space (e.g., through the patible host/fungal interaction and not in non-host
release of auxin which may alter proton levels; plant roots (Krause et al. 2015). Mirroring this,
Gay et al. 1994). Future work in this area com- changes to root architecture induced by the pres-
paring the mechanisms in different ECM evolu- ence of the ECM fungus Tuber borchii required
tionary lineages will provide valuable an intact auxin signaling network (Splivallo et al.
information concerning the diversity of enzymes 2009). We now have a better understanding of
utilized during this stage of colonization. how other hormones are affected during this pro-
cess. The hormones ethylene (ET) and jasmonic
acid (JA) have been found to be inhibitory to the
10.4 Manipulating Host Hub colonization process of L. bicolor on its host
Signaling Pathways: ECM Populus through signal transduction proteins
Colonization Alters Root including ETHYLENE RESPONSE FACTOR 1
Function Directly (Plett et al. 2014). These responses, meanwhile,
and Indirectly are opposed by salicylic acid (SA). Further work
in this model system has shown that most plant
While the growth and development of ECM fungi hormones inhibit mycorrhizal colonization of the
are physically disruptive to the host plant, ECM host including gibberellic acid (GA; Basso et al.
fungi also wield a level of control over host phys- 2020). GA treatment was not found to alter the
iological processes. During colonization, the ability of the fungus to penetrate into the root but
plant cell displays a range of different signaling did decrease the overall number of lateral roots
and biosynthetic pathways that are differentially colonized in the L. bicolor:Populus model. The
regulated. These include altered hormone profiles authors hypothesized, based on transcriptomic
and sensitivity, attenuated immune process, data, that GA may inhibit L. bicolor hyphae
altered secondary metabolism, and induction of from attaching to the root surface due to down-
nutrient pathways and movement. While regulation of glycoprotein synthesis. Basso et al.
identifying which pathways are changed during (2020) also showed that the colonization process
colonization has been studied for many decades, altered not only biosynthesis for a range of plant
the mechanistic understanding of how these hormones, but also sensitivity to the hormones
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 259

produced. Within the first 2 weeks of coloniza- have a wide range of roles and not be a specific
tion, root sensitivity to JA was found to decrease signal of mutualism.
while sensitivity to GA, SA, and ET was ECM fungi may also alter hormonal pathways
increased based on the expression of marker indirectly through the production of Mycorrhiza-
genes (Basso et al. 2020). The means by which Induced Small Secreted Proteins (MiSSPs). Upon
hormone sensitivity is altered still require further the release of the genome of L. bicolor, it was
investigation. Early clues suggest that one such found that over a dozen small secreted proteins
mechanism may be endogenous to the plant: work (SSPs) with no known homology were
with P. microcarpus colonizing the roots of up-regulated during the late stages of colonization
E. grandis found that the host protein methylation (Martin et al. 2008). The size of these proteins,
enzyme EgPRMT10 plays a key role in the regu- their predicted secretion signal, and their expres-
lation of hormone signaling related to ET, JA, and sion in symbiotic tissues were a surprise as they
auxin (Plett et al. 2019). Therefore, one mecha- were reminiscent of “effector” proteins utilized
nism by which hormone sensitivity is altered in by pathogenic organisms to control host cell func-
host plants during ECM colonization may be tion. It was questioned at the time whether or not
through post-secondary modification of proteins. the MiSSPs up-regulated during mutualistic
Evidence also suggests that hormonal sensitiv- exchanges between L. bicolor and its host may
ity may be altered directly due to fungal activity. act in a similar manner to pathogenic effector
The most direct manner is through production of proteins or if they would have unique functions
certain biochemical mimics/agonists. For exam- to mutualism. The first of these MiSSPs to be
ple, one lineage of ECM fungi is able to produce characterized, LbMiSSP7, was induced by root
abundant indolic compounds that, while they are exudates (both host and non-host) before physical
not auxin or auxin-like, affect plant-based auxin contact between the two organisms and continues
signaling. This molecule—hypaphorine—is a at a high level through to a mature mycorrhizal
tryptophan betaine excreted by Pisolithus hyphae root tip (Plett and Martin 2011). LbMiSSP7 was
and is accumulated abundantly in hyphal cells in determined to be one of the essential signals for
contact with host roots. Hypaphorine is able to controlling the in-growth of fungal hyphae into
stimulate the expression of auxin-regulated genes the apoplastic space of the root as RNAi mutant
in Eucalyptus, and it was hypothesized that lines of L. bicolor with lowered production of
hypaphorine might act as an auxin-like com- LbMiSSP7 were not able to establish a functional
pound (Nehls et al. 1998). Work following this mycorrhizal root tip (Plett et al. 2011). The site of
initial publication demonstrated that hypaphorine action of LbMiSSP7 is in the nucleus of the plant
is likely not an auxin mimic but, more surpris- cell where it interacts with multiple JA
ingly, counteracts auxin action on tap root elon- co-receptors leading to the down-regulation of
gation or root hair elongation (Ditengou and certain JA signaling pathways (Plett et al. 2014;
Lapeyrie 2000; Ditengou et al. 2000). It is proba- Fig. 10.2). This understanding was further refined
ble therefore that hypaphorine acts in the plant by Daguerre et al. (2020), who found that PtJAZ6
auxin pathway by competing with auxins for interacts with two poplar transcription factor
auxin-binding proteins and receptors (Kawano families (PtMYC2 and PtJAM1). In the absence
et al. 2001). In the early publications, of JA, these two transcription factors repress tran-
hypaphorine was thought to be exclusive to scription of JA-regulated genes. When
Pisolithus. Since that time, however, it has been LbMiSSP7 localizes to the host nucleus, how-
found to be produced by a wide range of fungi ever, it strengthens the binding of PtJAZ6 to
that are not classified as mutualists as well as in PtMYC2.1 and inhibits the dimerization of
marine organisms and in human milk (Vit et al. PtJAZ6 with other JAZ proteins. DNA Affinity
2015) indicating that it is a metabolite that has Purification sequencing (DAP-seq) analysis has
been co-opted through the evolutionary process to identified that genes directly activated by
MYC2.1 and MYC2.2 contain domains that
260 J. M. Plett et al.

Fig. 10.2 Graphical representation of a host cell and a characterized. Of those that have been studied, processes
selection of pathways impacted by ECM MiSSPs. To date, in the host nucleus, cytoplasm, and cell wall have all been
effector-like MiSSPs have been found in all ECM lineages described
although only a handful have been functionally

would suggest roles as additional transcription transcription factors belongs to the Myb/SANT-
factors, receptor-like kinases, and many defense- like DNA-binding domain family and, as such,
related proteins, including terpene synthases could alter the signaling of a number of hormonal
(TPS; Marques-Galvez et al. 2022). pathways. Similarly, SECRETED PROTEIN7
Monoterpenes produced by some of these TPSs (SP7) from the AM fungus Glomus intraradices
repress ECM formation, suggesting that in addi- also appears to enter the plant cell and localize to
tion to JA inhibition LbMiSSP7 improves ECM the plant nucleus where it interacts with Ethylene
symbiosis through repression of plant terpene Response Factor19 (ERF19) to repress plant
biosynthesis. Another characterized MiSSP from defense signaling (Kloppholz et al. 2011). The
L. bicolor, LbMiSSP7.6, was also found to enter occurrence small secreted proteins in mycorrhizal
into host cells and localize to the nucleus (Kang fungi that target plant hormone signaling
et al. 2020). This protein that interacts with pathways to promote colonization demonstrates
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 261

that mutualists use another mechanism found in not functionally demonstrated. miRNAs, mean-
other lifestyles of fungi to target and manipulate while, are short strands of RNA that can transfer
conserved “hub” pathways during the coloniza- between plant-associated fungi and their hosts
tion of plant tissues (Plett and Martin 2018; Plett through vesicles (Peres da Silva et al. 2015;
and Plett 2022). Wang and Dean 2020) where they directly target
Beyond hormone-controlled pathways messenger RNA (mRNA) in the host cell (Wang
associated with plant defense, the researchers et al. 2017). The formation of a hybrid double-
have asked if ECM fungi are able to control stranded RNA complex between the miRNA and
other aspects of plant immunity. Of the MiSSPs the full-length host mRNA is then identified and
characterized to date that affect non-hormonal degraded stopping translation of the mRNA
pathways, PaMiSSP10b of Pisolithus albus was (Weiberg et al. 2015). Initially characterized
found to interact with the polyamine pathway of only in pathogenic microbes, P. microcarpus
E. grandis (Plett et al. 2020; Fig. 10.2). was the first ECM fungus found to encode
Polyamines are a general class of plant miRNA. Of the 11 identified miRNAs, between
metabolites that function in plant defense through five and six were induced by the colonization
reinforcement of the cell wall, induction of the process (Wong-Bajracharya et al. 2022;
hypersensitive response through, or in addition to, Fig. 10.2). Of these, Pmic_miR-8 was
the production of hydrogen peroxide (Jiménez- characterized and found to enter into host cells
Bremont et al. 2014). Due to their role in defense where it targeted the transcription of several
against pathogens, disease-causing organisms NB-ARC genes. While this family of plant
have evolved small, secreted effector proteins genes have many function, one of their primary
that target polyamine biosynthesis in the plant roles is associated with the perception of invading
(Hewezi et al. 2010; Stes et al. 2011). Simple organisms. Once a foreign tissue is detected, this
polyamines are produced through the activity of gene family is then responsible for the signaling
S-adenosyl methionine decarboxylase that leads to tissue and cellular resistance (van der
(AdoMetDC) proteins, which utilize adenosyl Biezen and Jones 1998). Wong-Bajracharya et al.
methionine from the Yang cycle. PaMiSSP10b (2022) found that Pmic_miR-8 was necessary for
was found to interact with EgAdoMetDC, but the formation of mycorrhizal root tips and the
when compared to pathogenic effectors that inhibition of this signal led to increased
impact the polyamine pathway, PaMiSSP10b NB-ARC transcription and enabled the root to
was found to increase the activity of the enzyme grow away from the fungus. Therefore, we are
(Plett et al. 2020). This activity led to the synthe- beginning to see a range of previously identified
sis of more polyamines and a reduction in reactive (i.e., MiSSP) and novel (e.g., PCP, miRNA) fun-
oxygen species in the host root and that, as gal signals used to alter the signaling or sensitiv-
opposed to pathogenic systems, but similar to ity of plant immune pathways, a view that is
AM model systems (Sannazzaro et al. 2007), currently still limited by the few numbers of
was positive for ECM colonization (Plett et al. systems in which functional characterization has
2020). Two other classes of fungal signaling been undertaken.
compounds beyond the aforementioned MiSSPs
have also been found that support host coloniza-
tion: Proteolytic Cleavage Products (PCPs) and 10.5 Mycorrhizal Fungi Influence
micro-RNA. PCPs may play a similar role during the Developmental Programs
colonization as MiSSPs: these small peptides of of Soil Bacteria
<100 amino acids are formed due to the activity
of cleaved larger precursor proteins. Villalobos Like their effect on plants, mycorrhizal fungi
Solis et al. (2020) suggest that a number of excrete different metabolites, hormones, and
L. bicolor PCPs have putative functions in repres- proteins into the soil that act as rhizospheric
sion of reactive oxygen species, although this was signals (Toljander et al. 2007; Gorka et al. 2019;
262 J. M. Plett et al.

Wong-Bajracharya et al. 2020). It would appear, fungal “host” which produced enzymes largely
based on current research, that these signals may for the organic phosphorus mineralization. In
play a role in structuring rhizospheric microbial Tuber indicum, bacterial communities shifted
populations on and around the hyphae of ECM around fungal hyphae to perform different roles
fungi. Research concerning bacterial populations in amino acid metabolism and nutrient transport
in this zone has been gaining popularity since while functions associated with carbohydrate
Garbaye (1994) described these “mycorrhizal metabolism were decreased, all changes expected
helper bacteria” such that we now have a more to promote ECM host colonization (Li et al.
in-depth understanding of fungal–bacterial 2018).
interactions, interactions that are more wide- As the genera of bacteria associated with the
spread than originally expected (Frey-Klett et al. ECM fungi appear to be fungal lineage-specific,
2007). Interestingly, the bacterial communities how do the fungi cultivate these specific bacterial
that associate with a certain ECM fungi or ECM populations? One possible mechanism of control
colonized root tips tend to be similar, suggesting could be through the production of proteins that
that the ECM fungus may play a role in disrupt or modify quorum sensing between cer-
“cultivating” or attracting specific genera tain bacterial species or that act as antibiotics.
(Nurmiaho-Lassila et al. 1997; Frey-Klett et al. Quorum sensing is a mechanism by which bacte-
2005; Stuart et al. 2022). This is similar to results ria can sense their population density and thereby
from AM fungi (Emmett et al. 2021) and their growth rate as a population. First identified
saprotrophic fungi (Liu et al. 2018). Using micro- in Vibrio fischeri, an aquatic bacterium, quorum
scopic techniques, purification, and isolation as sensing is often mediated by small molecules
well as large-scale sequencing, these bacterial called autoinducers (AIs) that act to transduce
populations are being described, with the domi- messages between bacteria (Fuqua et al. 1994).
nant genera being the Pseudomonas, While there are a number of different molecules
Pedomicrobium, Rhodococcus, Streptomycetes, that act as AIs (e.g., diketopiperazine, furanosyl
Burkholderia, Variibacter, and Bacillus (Abdel- borate diester, or γ-butyrolactone), the most com-
Fattah and Mohamedin 2000; Ochsenreiter et al. mon class of molecules that act as AIs are N-acyl
2003; Schrey et al. 2005; Bomberg and Timonen homoserine lactones (AHLs). AHLs are typically
2007; Schrey and Tarkka 2008; Li et al. 2018). synthesized at a basal level by each bacterium
Bacteria from the listed genera promote the for- which, upon reaching a certain critical level in
mation of a mutualistic relationship between the the medium surrounding the bacteria, induce the
fungus and plant roots by complementing the expression of different suites of genes which
functions of mycorrhizal root tips including nutri- restrict growth of the colony or serve a number
ent acquisition and biological control of host of other purposes (Walker et al. 2003). Quorum
plants (Artursson et al. 2006; Frey-Klett et al. sensing molecules, however, can be made by
2007; Tarkka and Frey-Klett 2008). For example, more organisms than just bacteria. For example,
L. bicolor interacts with Pseudomonas ECM fungi produce compounds that interfere
fluorescens, certain strains of which improve the with bacterial quorum sensing systems (Uroz
growth and survival of L. bicolor hyphae (Brulé and Heinonsalo 2008). These compounds
et al. 2001; Deveau et al. 2007) and feed the exhibited AHL lactonase activity, enzymes that
fungus with thiamine, an essential cofactor of convert AHLs to the inactive N-acyl homoserine
several central enzymes of carbon metabolism (AHS) derivative. The discovery of a mechanism
(Tarkka et al. 2009). In the research by Stuart by which ECM fungi can disrupt AHL signaling
et al. (2022), Pisolithus albus hyphae modified through inactivation of AHLs to AHSs, or the
the soil bacterial community such that phosphate inhibition of their perception, could well be a
solubilization through glucose dehydrogenases strategy developed by the fungi to interfere with
was favored, but not bacterial phosphatases. the growth of populations of bacteria with delete-
This solubilization profile complemented the rious functions. Through this mechanism the
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 263

fungus could control the surrounding bacterial above demonstrates how the last decade has
community and may be a pathway that would seen a sustained increased effort to establish
partially explain the reason why only certain bac- genomic and molecular resources so that we can
teria are present in or on mycorrhizal root tips better understand this complex biological system.
(Nurmiaho-Lassila et al. 1997; Frey-Klett et al. Consistently, it is becoming clear that nearly all
2005). Another mechanism could be through the mechanisms that evolved in pathogenic systems
production of antimicrobial compounds. In work to communicate with hosts are also found in
using six ECM fungal species that colonize mutualistic systems, especially in the case of
Pinus, secretions were found to consistently conserved “hubs” within protein networks of the
inhibit Gram-positive bacteria while they did not host plant and rhizospheric community upon
affect Gram-negative bacterial genera (Shirakawa which fungal proteins/metabolites/nucleic acids
et al. 2019). This was found to correlate with may act to achieve host colonization, while
microbiome studies of colonized roots avoiding/manipulating the plant immune system.
demonstrating that bacterial communities As we have covered in this chapter, we would
associated with Pinus ECM root tips were summarize past research to suggest that ECM
predominated by Gram-negatives. Beyond these fungi likely first target the plant structure by
two mechanisms there are likely others at play. using a variety of methods to manipulate the
Within AM symbioses, for example, fructose physical characteristics of the host tissue to
secreted from fungal hyphae activates phosphate adhere and gain access to the tissues. The
solubilizing bacteria (Zhang et al. 2018b), while mechanisms used here mirror both those found
in pathogenic systems and saprotrophic models, in pathogenic fungi and those used by
small, secreted proteins directly alter microbial saprotrophic fungi to colonize the surface of a
assembly in the hyphosphere (Contreras et al. substrate. ECM fungal proteins then attack the
2018; Kombrink et al. 2019; Snelders et al. second hub which we would suggest is that of
2021, 2022). Therefore, it will be interesting in defense-related hormone signaling (e.g., ethyl-
the coming years if similar pathways to manipu- ene, salicylic acid) to create localized weakening
late ECM-associated microbial communities will of the plant immune system which would then
be uncovered. allow for the beginning of hyphal penetration into
the root apoplastic space. Concurrently with these
actions, research groups around the world have
10.6 Master Manipulators: Using found that ECM fungi also appear to cultivate
Our Understanding of ECM “helper” bacteria in the rhizosphere that aid the
Fungal Communication fungus by providing extra nutrients (e.g., thia-
Strategies to, in Turn, mine), by fending off pathogenic fungi and bacte-
Manipulate Them ria, etc. In this step ECM fungi play upon
for Sustainable Outcomes bacterial sensory interaction hubs such as quorum
sensing or directly through providing nutrition or
It has been a decade since we last updated this by killing using antimicrobial compounds. These
research discussion (Plett et al. 2012b), and our processes are similar to those of pathogenic fungi
views on the genetic and molecular which also manipulate the microbiome
underpinnings that form a “mutualistic” fungus surrounding them to hinder plant immunity and
continue to evolve. Based on the work covered to clear a niche in which the pathogen can estab-
above, we are finding how very similar ECM lish; saprotrophic fungi also produce host distinct
fungi are to pathogens in their methods of com- hyphospheric microbiomes. Therefore, increas-
municating with, and orchestrating colonization ingly, we can see that the molecular definition of
of, a host plant. While ECM fungi continue to fungal lifestyles continues to blur along a
receive far less research attention in comparison saprotrophism-symbiosis-pathogenesis
with pathogenic systems, the work we cover continuum.
264 J. M. Plett et al.

Fig. 10.3 Climate change

impacts symbiotic triangle.
The establishment of
mutualistic mycorrhizal
symbiosis is a factor of
receptive root physiological
status, active plant:microbe
signaling and the
interaction between
microbes. These processes,
in turn, are impacted by
both biotic and abiotic
factors. This delicate
balance is threatened by
climate extremes with
likely outcomes including
disrupted nutrient flow,
increased disease, and
eventual plant and
microbial species loss

The precision and tuning of this process in disruption of these ECM–plant interactions
keeping the plant host healthy and alive is likely could cause untold damage to the sustainability
the means by which ECM fungi can still be clas- of forest habitats and the ecosystems they support
sified as “beneficial.” However, as the mutualistic (Fig. 10.3), continued and sustained effort should
interaction requires a finely tuned, and likely pre- be dedicated to identifying common and diver-
carious, dialogue between two partners in order to gent molecular “toolboxes” related to the ECM
function properly (Fig. 10.3), interruption at any lifestyle. Within these toolboxes we should delve
one of the signaling hubs described above is deeper into how to foster healthy communication
likely to greatly change the nature of the interac- between ECM fungi and their hosts and the soil/
tion between the organisms—if not completely hyphospheric microbial community. We must
abolish the mutualism. Interruption of these also continue to broaden our complement of
signals could come about through a variety of model study systems and look at the impact of
mechanisms including alterations due to abiotic new climate parameters upon small-scale
conditions from global climate change and ecosystems and, hopefully, on population dynam-
increasing anthropomorphic infringement on nat- ics within whole ecosystems. We have access to
ural environments (Fig. 10.3). Similarly, an increasingly large array of genomic and func-
differences in biotic environments as increasingly tional resources that are providing us with candi-
globalized movement of humans, plants, and date biomarkers that could be used under natural
animals have led to changes in many natural settings to evaluate ECM functioning in situ. By
environments that could impact these studying these systems, and their adaptive plas-
relationships. We would hypothesize that ticity under different parameters, we will know
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 265

better how ECM fungi will fare under new cli- salicylate, gibberellin, and ethylene in host roots. Plant
matic conditions and know how to better manage Cell Environ 43:1047–1068
Besserer A, Becard G, Jauneau A, Roux C, Sejalon-
the microbiome of forests. Delmas N (2008) GR24, a synthetic analog of
strigolactones, stimulates the mitosis and growth of
Acknowledgments Research in Jonathan Plett’s lab is the arbuscular mycorrhizal fungus Gigaspora rosea
sponsored by the Australian Research Council, Forestry by boosting its energy metabolism. Plant Physiol 148:
& Wood Products Australia and by the National Institute 402–413
for Forest Products Innovation. Research in Francis Bomberg M, Timonen S (2007) Distribution of Cren- and
Martin’s and Annegret Kohler’s labs is sponsored by the Euryarchaeota in Scots pine mycorrhizospheres and
US Department of Energy, Office of Science, Biological boreal forest humus. Microb Ecol 54:406–416
and Environmental Research as part of the Plant–Microbe Bonfante P, Genre A (2010) Mechanisms underlying ben-
Interfaces Scientific Focus Area (https://pmi.ornl.gov) and eficial plant-fungus interactions in mycorrhizal symbi-
the Laboratory of Excellence ARBRE (grant no. ANR-11- osis. Nat Commun 1:48
LABX-0002_ARBRE). Bouwmeester HJ, Matusova R, Zhongkui S, Beale MH
(2003) Secondary metabolite signalling in host-
parasitic plant interactions. Curr Opin Plant Biol 6:
358–364
References Bowden CG, Smalley E, Guries RP, Hubbes M, Temple B,
Horgen PA (1996) Lack of association between cerato-
Abdel-Fattah GM, Mohamedin AH (2000) Interactions ulmin production and virulence in Ophiostoma novo-
between a vesicular–arbuscular mycorrhizal fungus ulmi. MPMI 9:556–564
(Glomus intraradices) and Streptomyces coelicolor Brasier CM, Kirk SA, Tegli S (1995) Naturally occurring
and their effects on sorghum plants grown in soil noncerato-ulmin producing mutants of Ophiostoma
amended with chitin of brawn scales. Biol Fertil Soils novo-ulmi are pathogenic but lack aerial mycelium.
32:401–409 Mycol Res 99:436–440
Abdulsalam O, Wagner K, Wirth S, Kunert M, David A, Brulé C, Frey-Klett P, Pierrat JC, Courrier S, Gérard F,
Kallenbach M, Boland W, Kothe E, Krause K (2021) Lemoine MC, Rousselet JL, Sommer G, Garbaye J
Phytohormones and volatile organic compounds, like (2001) Survival in the soil of the ectomycorrhizal fun-
geosmin, in the ectomycorrhiza of Tricholoma gus Laccaria bicolor and the effects of a mycorrhiza
vaccinum and Norway spruce (Picea abies). Mycor- helper Pseudomonas fluorescens. Soil Biol Biochem
rhiza 31(2):173–188 33:1683–1694
Adams F, Reddell P, Webb MJ, Shipton WA (2006) Buckley S, Brackin R, Jämtgård S et al (2020)
Arbuscular mycorrhizas and ectomycorrhias on Euca- Microdialysis in soil environments: Current practice
lyptus grandis (Myrtaceae) trees and seedlings in and future perspectives. Soil Biol Biochem 143:
native forests of tropical north-eastern Australia. Aust 107743
J Bot 54:271–281 Buckley S, Brackin R, Näsholm T et al (2022) The influ-
Aimanianda V, Bayry J, Bozza S, Kniemeyer O, ence of sucrose on soil nitrogen availability–a root
Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage exudate simulation using microdialysis. Geoderma
AA, Kaveri SV, Romani L, Latge J-P (2009) Surface 409:115645
hydrophobin prevents immune recognition of airborne Buée M, De Boer W, Martin F, van Overbeek L,
fungal spores. Nature 460:1117–1121 Jurkevitch E (2009) The rhizosphere zoo: an overview
Akiyama K, Hayashi H (2008) Plastid-derived of plant-associated communities of microorganisms,
strigolactones show the way to roots for symbionts including phages, bacteria, archaea and fungi, and of
and parasites. New Phytol 178:695–698 some of their structuring factors. Plant Soil 321:189–
Akiyama K, Matsuzaki K, Hayashi H (2005) Plant 212
sesquiterpenes induce hyphal branching in arbuscular Burgess T, Laurent P, Dell B, Malajczuk N, Martin F
mycorrhizal fungi. Nature 435:824–827 (1995) Effect of fungal-isolate aggressivity on the bio-
Anthony MA, Crowther TW, Van Der Linde S et al (2022) synthesis of symbiosis-related polypeptides in
Forest tree growth is linked to mycorrhizal fungal differentiating eucalypt ectomycorrhiza. Planta 195:
composition and function across Europe. ISME J 16: 408–417
1327–1336 Cai F, Gao R, Zhao Z et al (2020) Evolutionary
Artursson V, Finlay RD, Jansson JK (2006) Interactions compromises in fungal fitness: hydrophobins can hin-
between arbuscular mycorrhizal fungi and bacteria and der the adverse dispersal of conidiospores and chal-
their potential for stimulating plant growth. Environ lenge their survival. ISME J 14:2610–2624
Microbiol 8:1–10 Casarrubia S, Daghino S, Kohler A et al (2018) The
Basso V, Kohler A, Miyauchi S et al (2020) An hydrophobin-like OmSSP1 may be an effector in the
ectomycorrhizal fungus alters sensitivity to jasmonate, ericoid mycorrhizal symbiosis Front. Plant Sci 9:546
266 J. M. Plett et al.

Chalot M, Brun A (1998) Physiology of organic nitrogen Ditengou FA, Béguiristain T, Lapeyrie F (2000) Root hair
acquisition by ectomycorrhizal fungi and elongation is inhibited by hypaphorine, the indole alka-
ectomycorrhizas. FEMS Microbiol Ref 22:21–44 loid from the ectomycorrhizal fungus Pisolithus
Charvet-Candela V, Hitchin S, Ernst D et al (2002) Char- tinctorius, and restored by IAA. Planta 211:722–728
acterization of an Aux/IAA cDNA upregulated in Doré J, Kohler A, Dubost A, Hundley H, Singan V,
Pinus pinaster roots in response to colonization by Peng Y, Kuo A, Grigoriev IV, Martin F,
the ectomycorrhizal fungus Hebeloma Marmeisse R, Gay G (2017) The ectomycorrhizal
cylindrosporum. New Phytol 154:769–777 basidiomycete Hebeloma cylindrosporum undergoes
Chernyavska M, Schmid M, Freitag PC et al (2019) early waves of transcriptional reprogramming prior to
Unravelling receptor and RGD motif dependence of symbiotic structures differentiation. Environ Microbiol
retargeted adenoviral vectors using advanced tumor 19(3):1338–1354
model systems. Sci Rep 9:1–13 Duplessis S, Courty PE, Tagu D, Martin F (2005) Tran-
Chowdhury J, Kemppainen M, Delhomme N et al (2022) script patterns associated with ectomycorrhiza devel-
Laccaria bicolor pectin methylesterases are involved opment in Eucalyptus globulus and Pisolithus
in ectomycorrhiza development with Populus microcarpus. New Phytol 165:599–611
tremula× Populus tremuloides. New Phytol. https:// Emmett BD, Lévesque-Tremblay V, Harrison MJ (2021)
doi.org/10.1111/nph.18358 Conserved and reproducible bacterial communities
Contreras G, Braun MS, Schäfer H et al (2018) Recombi- associate with extraradical hyphae of arbuscular
nant Afusin C, an anionic fungal CSαβ defensin from mycorrhizal fungi. ISME J 15:2276–2288
Aspergillus fumigatus, exhibits antimicrobial activity Felten J, Kohler A, Morin E, Bhalerao RP, Palme K,
against gram-positive bacteria. PLoS One 13: Martin F, Ditengou FA, Legue V (2009) The
pe0205509 ectomycorrhizal fungus Laccaria bicolor stimulates
Corvis Y, Walcarius A, Rink R, Mrabet NT, Rogalska E lateral root formation in poplar and Arabidopsis
(2005) Preparing catalytic surfaces for sensing through auxin transport and signaling. Plant Physiol
applications by immobilizing enzymes via 151:1991–2005
hydrophobin layers. Anal Chem 77:1622–1630 Frey-Klett P, Chavatte M, Clausse M-L, Courrier S, Le
Corvis Y, Brezesinski G, Rink R, Walcarius A, Van der Roux C, Raaijmakers J, Martinotti MG, Pierrat J-C,
Heyden A, Mutelet F, Rogalska E (2006) Analytical Garbaye J (2005) Ectomycorrhizal symbiosis affects
investigation of the interactions between SC3 functional diversity of rhizosphere fluorescent
hydrophobin and lipid layers: elaborating of pseudomonads. New Phytol 165:317–328
nanostructured matrixes for immobilizing redox Frey-Klett P, Garbaye J, Tarkka MT (2007) The mycor-
systems. Anal Chem 78:4850–4864 rhiza helper bacteria revisited. New Phytol 176:22–36
Corvis Y, Trzcinska K, Rink R, Bilkova P, Gorecka E, Fries N, Serck-Hanssen K, Dimberg LH, Theander O
Bilewicz R, Rogalska E (2007) Electron donor- (1989) In vitro enhancement of spore germination
acceptor fullerene derivative retained on electrodes and early hyphal growth of a vesicular-arbuscular
using SC3 hydrophobin. J Phys Chem C 111:1176– mycorrhizal fungus by host root exudates and plant
1179 flavonoids. Symbiosis 7:243–255
Daguerre Y, Basso V, Wittulski S, Schellenberger R, Fuqua WC, Winans SC, Greenberg EP (1994) Quorum
Meyer L, Bailly J, Kohler A, Plett JM, Martin F, sensing in bacteria: the LuxR-LuxI family of cell
Veneault-Fourrey C (2020) The mutualism effector density-responsive transcriptional regulators. J
MiSSP7 of Laccaria bicolor alters the interactions Bacteriol 176(2):269–275
between the poplar JAZ6 protein and its associated Garbaye J (1994) Helper bacteria: a new dimension to the
proteins. Sci Rep 10:20362 mycorrhizal symbiosis. New Phytol 128:197–210
De Carvalho D (1994) Contribution à l’étude des protéines Garcia K, Delaux PM, Cope KR et al (2015) Molecular
régulées par la symbiose ectomycorhizienne. Ph.D. signals required for the establishment and maintenance
thesis. Ecole Nationale de Génie Rurale et Forestier, of ectomycorrhizal symbioses. New Phytol 208:79–87
Nancy Gay G, Normand L, Marmeisse R, Sotta B, Debaud JC
Deveau A, Palin B, Delaruelle C, Peter M, Kohler A, (1994) Auxin overproducer mutants of Hebeloma
Pierrat JC, Sarniguet A, Garbaye J, Martin F, Frey- cylindrosporum Romagnési have increased mycorrhi-
Klett P (2007) The mycorrhiza helper Pseudomonas zal activity. New Phytol 128:645
Fluorescens BBc6R8 has a specific priming effect on Gea L, Normand L, Vian B, Gay G (1994) Structural
the growth, morphology and gene expression of the aspects of ectomycorrhiza of Pinus pinaster (Ait.)
ectomycorrhizal fungus Laccaria bicolor S238N. New Sol. formed by an IAA-overproducer mutant of
Phytol 175:743–755 Hebeloma cylindrosporum Romagnési. New Phytol
Ditengou FA, Lapeyrie F (2000) Hypaphorine from the 128:659
ectomycorrhizal fungus Pisolithus tinctorius Gorka S, Dietrich M, Mayerhofer W et al (2019) Rapid
counteracts activities of indole-3-acetic acid and ethyl- transfer of plant photosynthates to soil bacteria via
ene but not synthetic auxins in eucalypt seedlings. Mol ectomycorrhizal hyphae and its interaction with nitro-
Plant-Microbe Interact 13:151–158 gen availability. Front Microbiol 10:168
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 267

Hewezi T, Howe PJ, Maier TR et al (2010) Arabidopsis Kodama K, Rich MK, Yoda A et al (2022) An ancestral
spermidine synthase is targeted by an effector protein function of strigolactones as symbiotic rhizosphere
of the cyst nematode Heterodera schachtii. Plant signals. Nat Commun 13:3974
Physiol 152:968–984 Kohler A, Kuo A, Nagy LG, Morin E, Barry KW,
Hilbert JL, Costa G, Martin F (1991) Ectomycorrhizal Buscot F, Canbäck B, Choi C, Cichocki N, Clum A,
synthesis and polypeptide changes during the early Colpaert J (2015) Convergent losses of decay
stage of eucalypt mycorrhiza development. Plant mechanisms and rapid turnover of symbiosis genes in
Physiol 97:977–984 mycorrhizal mutualists. Nat Genet 47:410–415
Hill RA, Wong-Bajracharya J, Anwar S et al (2022) Kombrink A, Tayyrov A, Essig A, Stöckli M, Micheller S,
Abscisic acid supports colonization of Eucalyptus Hintze J, Van Heuvel Y, Dürig N, Lin CW, Kallio PT,
grandis roots by the mutualistic ectomycorrhizal fun- Aebi M (2019) Induction of antibacterial proteins and
gus Pisolithus microcarpus. New Phytol 233:966–982 peptides in the coprophilous mushroom Coprinopsis
Horan DP, Chilvers GA, Lapeyrie F (1988) Time sequence cinerea in response to bacteria. ISME J 13(3):588–602
of the infection process in eucalypt ectomycorrhizas. Kondoh O, Muto A, Kajiwara S, Takagi J, Saito Y,
New Phytol 109:451–458 Shishido K (1995) Fruiting body-specific cDNA,
Hostetter MK (2000) RGD-mediated adhesion in fungal mfbAc, from the mushroom Lentinus edodes encodes
pathogens of humans, plants and insects. Curr Opin a high-molecular-weight cell-adhesion protein
Microbiol 3(4):344–348 containing an Arg-Gly-Asp motif. Gene 154:31–37
Jensen BG, Andersen MR, Pedersen MH, Frisvad JC, Köttke I, Oberwinkler F (1986) Root-Fungus interactions
Sondergaard I (2010) Hydrophobins from Aspergillus observed on initial stages of mantle formation and
species cannot be clearly divided into two classes. Hartig net establishment in mycorrhizas of Amanita
BMC Res Notes 3:344 muscaria on Picea abies in pure culture. Can J Bot
Jiménez-Bremont JF, Marina M, Guerrero-Gonzalez 64:2348–2354
MDLL et al (2014) Physiological and molecular Krause K, Henke C, Asiimwe T, Ulbricht A, Klemmer S,
implications of plant polyamine metabolism during Schachtschabel D, Boland W, Kothe E (2015) Biosyn-
biotic interactions. Front Plant Sci 5:95 thesis and secretion of indole-3-acetic acid and its
Kang H, Chen X, Kemppainen M, Pardo AG, Veneault- morphological effects on Tricholoma vaccinum-spruce
Fourrey C, Kohler A, Martin FM (2020) The small ectomycorrhiza. Appl Environ Microbiol 81(20):
secreted effector protein MiSSP7.6 of Laccaria bicolor 7003–7011
is required for the establishment of ectomycorrhizal Lagrange H, Jay-Allemand C, Lapeyrie F (2001) Rutin,
symbiosis. Environ Microbiol 22:1435–1446 the phenolglycoside from eucalyptus root exudates,
Kaska DD, Myllylä R, Cooper JB (1999) Auxin transport stimulates Pisolithus hyphal growth at picomolar
inhibitors act through ethylene to regulate dichotomous concentrations. New Phytol 149:349–355
branching of lateral root meristems in pine. New Larose G, Chênevert R, Moutoglis P, Gagné S, Piché Y,
Phytol 142:49 Vierheilig H (2002) Flavonoid levels in roots of
Kawano T, Kawano N, Hosoya H, Lapeyrie F (2001) Medicago sativa are modulated by the developmental
Fungal auxin antagonist hypaphorine competitively stage of the symbiosis and the root colonizing
inhibits indole-3-acetic acid-dependent superoxide arbuscular mycorrhizal fungus. J Plant Physiol
generation by horseradish peroxidase. Biochem 159(12):1329–1339
Biophys Res Commun 288:546–551 Law SR, Serrano AR, Daguerre Y, Sundh J, Schneider
Kemppainen M, Pardo A (2021) Nucleus-directed fluores- AN, Stangl ZR, Castro D, Grabherr M, Näsholm T,
cent reporter system for promoter studies in the Street NR, Hurry V (2022) Metatranscriptomics
ectomycorrhizal fungus Laccaria bicolor. J Microbiol captures dynamic shifts in mycorrhizal coordination
Meth 190:p106341 in boreal forests. Proc Natl Acad Sci U S A 119(26):
Kershaw MJ, Talbot NJ (1998) Hydrophobins and e2118852119
repellents: proteins with fundamental roles in fungal Le Quéré A, Astrup Eriksen K, Rajashekar B,
morphogenesis. Fungal Genet Biol 23:18–33 Schützendübel A, Canbäck B, Johansson T, Tunlid A
Kikuchi K, Matsushita N, Suzuki K, Hogetsu T (2007) (2006) Screening for rapidly evolving genes in the
Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus Paxillus involutus using
ectomycorrhizal fungus Suillus bovinus. Mycorrhiza cDNA microarrays. Mol Ecol 15(2):535–550
17:563–570 Lebreton A, Tang N, Kuo A, LaButti K, Andreopoulos W,
Kim S, Ahn I-P, Rho H-S, Lee Y-H (2005) MPH1, a Drula E, Miyauchi S, Barry K, Clum A, Lipzen A,
Magnaporthe grisea hydrophobin gene, is required Mousain D (2022) Comparative genomics reveals a
for fungal development and plant colonization. Mol dynamic genome evolution in the ectomycorrhizal
Microbiol 57:1224–1237 milk-cap (Lactarius) mushrooms. New Phytol 235:
Kloppholz S, Kuhn H, Requena N (2011) A secreted 306–319
fungal effector of Glomus intraradices promotes sym- Li Q, Yan L, Ye L, Zhou J, Zhang B, Peng W, Zhang X, Li
biotic biotrophy. Curr Biol 21:1204–1209 X (2018) Chinese black truffle (Tuber indicum) alters
the ectomycorrhizosphere and endoectomycosphere
268 J. M. Plett et al.

microbiome and metabolic profiles of the host tree Martin F, Perotto S, Bonfante P (2007) Mycorrhizal fungi:
Quercus aliena. Front Microbiol 9:2202 a fungal community at the interface between soil and
Liao HL, Chen Y, Vilgalys R (2016) Metatranscriptomic roots. In: Pinton R, Varanini Z, Nannipieri P (eds) The
study of common and host-specific patterns of gene rhizosphere. Biochemistry and organic substances at
expression between pines and their symbiotic the soil-plant interface. Marcel Dekker, New York, pp
ectomycorrhizal fungi in the genus Suillus. PLoS 263–296
Genet 12(10):e1006348 Martin F, Aerts A, Ahrén D, Brun A, Danchin EGJ et al
Lindahl BD, Tunlid A (2015) Ectomycorrhizal fungi– (2008) The genome of Laccaria bicolor provides
potential organic matter decomposers, yet not insights into mycorrhizal symbiosis. Nature 452:88–92
saprotrophs. New Phytol 205(4):1443–1447 Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM
Liu Y, Sun Q, Li J, Lian B (2018) Bacterial diversity et al (2010) Périgord black truffle genome uncovers
among the fruit bodies of ectomycorrhizal and sapro- evolutionary origins and mechanisms of symbiosis.
phytic fungi and their corresponding hyphosphere Nature 464:1033–1038
soils. Sci Rep 8(1):1–10 Martin F, Kohler A, Murat C, Veneault-Fourrey C, Hibbett
Liu H, Chen H, Ding G, Li K, Ren Q (2020) Identification DS (2016) Unearthing the roots of ectomycorrhizal
of candidate genes conferring tolerance to aluminum symbioses. Nat Rev Microbiol 14(12):760–773
stress in Pinus massoniana inoculated with Mészáros B, Sámano-Sánchez H, Alvarado-Valverde J,
ectomycorrhizal fungus. BMC Plant Biol 20(1):1–3 Čalyševa J, Martínez-Pérez E, Alves R, Shields DC,
Looney B, Miyauchi S, Morin E, Drula E, Courty PE, Kumar M, Rippmann F, Chemes LB, Gibson TJ (2021)
Kohler A, Kuo A, LaButti K, Pangilinan J, Lipzen A, Short linear motif candidates in the cell entry system
Riley R (2022) Evolutionary transition to the used by SARS-CoV-2 and their potential therapeutic
ectomycorrhizal habit in the genomes of a implications. Science. Signals 14(665):peabd0334
hyperdiverse lineage of mushroom-forming fungi. Mishra B, Gupta DK, Pfenninger M, Hickler T, Langer E,
New Phytol 233(5):2294–2309 Nam B, Paule J, Sharma R, Ulaszewski B, Warmbier J,
Lovett B, Kasson MT, Gandier JA (2022) Ecology drives Burczyk J (2018) A reference genome of the European
the observed spectrum of hydrophobin protein diver- beech (Fagus sylvatica L.). Gigascience 7(6):giy063
sity across Kingdom Fungi. bioRxiv. https://doi.org/ Mishra B, Ulaszewski B, Meger J, Aury JM, Bodénès C,
10.1101/2022.08.19.504535 Lesur-Kupin I, Pfenninger M, Da Silva C, Gupta DK,
Lutzoni F, Nowak MD, Alfaro ME, Reeb V, Guichoux E, Heer K (2022) A chromosome-level
Miadlikowska J, Krug M, Arnold AE, Lewis LA, genome assembly of the European beech (Fagus
Swofford DL, Hibbett D, Hilu K (2018) Contempora- sylvatica) reveals anomalies for organelle DNA inte-
neous radiations of fungi and plants linked to symbio- gration, repeat content and distribution of SNPs. Front
sis. Nat Commun 9(1):1–11 Genet 12:691058
Ma X, Zhu B, Nie Y, Liu Y, Kuzyakov Y (2021) Root and Miyauchi S, Kiss E, Kuo A, Drula E, Kohler A, Sánchez-
mycorrhizal strategies for nutrient acquisition in forests García M, Morin E, Andreopoulos B, Barry KW,
under nitrogen deposition: a meta-analysis. Soil Biol Bonito G, Buée M (2020) Large-scale genome
Biochem 163:108418 sequencing of mycorrhizal fungi provides insights
Machin DC, Hamon-Josse M, Bennett T (2020) Fellow- into the early evolution of symbiotic traits. Nat
ship of the rings: a saga of strigolactones and other Commun 11(1):1–17
small signals. New Phytol 225(2):621–636 Montanini B, Levati E, Bolchi A, Kohler A, Morin E,
Marques-Galvez JE, Basso V, Kohler A, Barry K, Tisserant E, Martin F, Ottonello S (2011) Genome-
Keymanesh K, Johnson J, Singan V, Grigoriev I, wide search and functional identification of transcrip-
Vilgalys R, Martin F, Veneault-Fourrey C (2022) The tion factors in the mycorrhizal fungus Tuber
establishment of Populus x Laccaria bicolor melanosporum. New Phytol 189:736–750
ectomycorrhiza requires the inactivation of MYC2 Moonjely S, Keyhani NO, Bidochka MJ (2018)
coordinated defense response with a key role for root Hydrophobins contribute to root colonization and
terpene synthases. bioRxiv. https://doi.org/10.1101/ stress responses in the rhizosphere-competent insect
2022.09.06.505662 pathogenic fungus Beauveria bassiana. Microbiology
Martin F, Laurent P, de Carvalho D, Voiblet C, 164(4):517–528
Balestrini R, Bonfante P, Tagu D (1999) Cell wall Murat C, Payen T, Noel B, Kuo A, Morin E, Chen J,
proteins of the ectomycorrhizal basidiomycete Kohler A, Krizsán K, Balestrini R, Da Silva C,
Pisolithus tinctorius: identification, function and Montanini B et al (2018) Pezizomycetes genomes
expression in symbiosis. Fungal Genet Biol 27:161– reveal the molecular basis of ectomycorrhizal truffle
174 lifestyle. Nat Ecol Evol 2(12):1956–1965
Martin F, Díez J, Dell B, Delaruelle C (2002) Murata H, Nakano S, Yamanaka T, Shimokawa T, Abe T,
Phylogeography of the ectomycorrhizal Pisolithus spe- Ichida H, Hayashi Y, Tahara K (2021) Argon-ion beam
cies as inferred from nuclear ribosomal DNA ITS induced mutants of the ectomycorrhizal agaricomycete
sequences. New Phytol 153(2):345–357 Tricholoma matsutake defective in β-1,
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 269

4-endoglucanase activity promote the seedling growth Plett JM, Martin F (2011) Blurred boundaries: lifestyle
of Pinus densiflora in vitro. Botany 99(3):139–149 lessons from fungal ectomycorrhizal genomes. Trends
Myburg AA, Grattapaglia D, Tuskan GA, Hellsten U, Genet 27:14–22
Hayes RD, Grimwood J, Jenkins J, Lindquist E, Plett JM, Martin FM (2018) Know your enemy, embrace
Tice H, Bauer D, Goodstein DM (2014) The genome your friend: using omics to understand how plants
of Eucalyptus grandis. Nature 510(7505):356–362 respond differently to pathogenic and mutualistic
Nehls U, Béguiristain T, Ditengou F, Lapeyrie F, Martin F microorganisms. Plant J 93(4):729–746
(1998) The expression of a symbiosis-regulated gene Plett JM, Plett KL (2022) Leveraging genomics to under-
in eucalypt roots is regulated by auxins and stand the broader role of fungal small secreted proteins
hypaphorine, the tryptophan betaine of the in niche colonization and nutrition. ISME Comm 2:49
ectomycorrhizal basidiomycete Pisolithus tinctorius. Plett JM, Kemppainen M, Kale SD, Kohler A, Legue V,
Planta 207:296–302 Brun A, Tyler B, Pardo A, Martin F (2011) A secreted
Nurmiaho-Lassila EL, Timonen S, Haahtela K, Sen R effector protein of Laccaria bicolor is required for
(1997) Bacterial colonization patterns of intact Pinus symbiosis development. Curr Biol 21:1197–1203
sylvestris mycorrhizospheres in dry pine forest soil: an Plett JM, Gibon J, Kohler A, Duffy K, Hoegger PJ,
electron microscopy study. Can J Microbiol 43:1017– Velagapudi R, Han J, Kües U, Grigoriev IV, Martin
1035 F (2012a) Phylogenetic, genomic organization and
Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin YC, expression analysis of hydrophobin genes in the
Scofield DG, Vezzi F, Delhomme N, Giacomello S, ectomycorrhizal basidiomycete Laccaria bicolor. Fun-
Alexeyenko A, Vicedomini R (2013) The Norway gal Genet Biol 49(3):199–209
spruce genome sequence and conifer genome evolu- Plett JM, Kohler A, Martin F (2012b) De-constructing a
tion. Nature 497(7451):579–584 mutualist: how the molecular blueprints of model sym-
Ochsenreiter T, Selezi D, Quaiser A, Bonch- biotic fungi are changing our understanding of
Osmolovskaya L, Schleper C (2003) Diversity and mutualism. In: Hock B (ed) Fungal Associations. The
abundance of Crenarchaeota in terrestrial habitats stud- Mycota, vol 9. Springer, Berlin, pp 93–117
ied by 16S RNA surveys and real time PCR. Environ Plett JM, Khachane A, Ouassou M, Sundberg B, Kohler A,
Microbiol 5:787–797 Martin F (2014) Ethylene and jasmonic acid act as
Onuma H, Hara K, Sugita K, Kano A, Fukuta Y, Shirasaka negative modulators during mutualistic symbiosis
N (2019) Purification and characterization of a glyco- between Laccaria bicolor and Populus roots. New
side hydrolase family 5 endoglucanase from Phytol 202(1):270–286
Tricholoma matsutake grown on barley based solid- Plett KL, Raposo AE, Anderson IC, Piller SC, Plett JM
state medium. J Biosci Bioeng 128(6):669–676 (2019) Protein arginine methyltransferase expression
Paparokidou C, Leake JR, Beerling DJ, Rolfe SA (2021) affects ectomycorrhizal symbiosis and the regulation of
Phosphate availability and ectomycorrhizal symbiosis hormone signalling pathways. MPMI 32(10):
with Pinus sylvestris have independent effects on the 1291–1302
Paxillus involutus transcriptome. Mycorrhiza 31(1): Plett JM, Plett KL, Wong-Bajracharya J, de Freitas PM,
69–83 Dutra-Costa M, Kohler A, Martin F, Anderson IC
Parta M, Chang Y, Rulong S, Pinto-DaSilva P, Kwon- (2020) Mycorrhizal effector PaMiSSP10b alters poly-
Chung KJ (1994) HYP1, a hydrophobin gene from amine biosynthesis in Eucalyptus root cells and
Aspergilus fuigatus, complements the rodletless phe- promotes root colonization. New Phytol 228(2):
notype in Aspergillus nidulans. Infect Immun 62: 712–727
4389–4395 Plett KL, Buckley S, Plett JM, Anderson IC, Lundberg-
Pellegrin C, Daguerre Y, Ruytinx J, Guinet F, Felten J, Jämtgård S (2021, 1817) Novel microdialysis
Kemppainen M, Frey NFD, Puech-Pagès V, technique reveals a dramatic shift in metabolite secre-
Hecker A, Pardo AG, Martin FM, Veneault-Fourrey tion during the early stages of the interaction between
C (2019) Laccaria bicolor MiSSP8 is a small-secreted the ectomycorrhizal fungus Pisolithus microcarpus
protein decisive for the establishment of the and its host Eucalyptus grandis. Microorganisms 9(9)
ectomycorrhizal symbiosis. Environ Microbiol Plomion C, Aury JM, Amselem J, Leroy T, Murat F,
21(10):3765–3779 Duplessis S, Faye S, Francillonne N, Labadie K, Le
Peres da Silva R, Puccia R, Rodrigues ML, Oliveira DL, Provost G, Lesur I et al (2018) Oak genome reveals
Joffe LS, César GV, Nimrichter L, Goldenberg S, facets of long lifespan. Nat Plants 4(7):440–452
Alves LR (2015) Extracellular vesicle-mediated export Policelli N, Horton TR, Hudon AT, Patterson TR,
of fungal RNA. Sci Rep 5(1):1–12 Bhatnagar JM (2020) Back to roots: the role of
Peter M, Kohler A, Ohm RA, Kuo A, Krützmann J, ectomycorrhizal fungi in boreal and temperate forest
Morin E, Arend M, Barry KW, Binder M, Choi C, restoration. Front For Glob Change 3:97
Clum A (2016) Ectomycorrhizal ecology is imprinted Qin M, Wang LK, Feng XZ, Yang YL, Wang R, Wang C,
in the genome of the dominant symbiotic fungus Yu L, Shao B. and Qiao MQ (2007) Bioactive surface
Cenococcum geophilum. Nat Commun 7(1):1–5 modification of mica and poly (dimethylsiloxane) with
270 J. M. Plett et al.

hydrophobins for protein immobilization. Langmuir, Sharp JA, McNeil M, Albersheim P (1984a) The primary
23(8):4465–4471 structure of one elicitor active and seven elicitor-
Randewig D, Marshall JD, Näsholm T, Jämtgård S (2019) inactive hexa (ß-D-glucopyranolsyl)-D-glucitols
Combining microdialysis with metabolomics to char- isolated from the mycelia walls of Phytophthora
acterize the in situ composition of dissolved organic megasperma f.sp. glycinea. J Biol Chem 25:11321–
compounds in boreal forest soil. Soil Biol Biochem 11326
136:107530 Sharp JA, Valent B, Albersheim P (1984b) Purification
Rivera Pérez CA, Janz D, Schneider D, Daniel R, Polle A and partial characterization of a ß-glucan fragment that
(2022) Transcriptional landscape of ectomycorrhizal elicits phytoalexin accumulation in soybean. J Biol
fungi and their host provides insight into N uptake Chem 25:11312–11320
from forest soil. Msystems 7(1):e00957–e00921 Shirakawa M, Uehara I, Tanaka M (2019)
Roby D, Gadelle A, Toppan A (1987) Chitin Mycorrhizosphere bacterial communities and their sen-
oligosaccharides as elicitors of chitinase activity in sitivity to antibacterial activity of ectomycorrhizal
melon plants. Biochem Biophys Res Commun 143: fungi. Microb Environ pME18146
885–892 Snelders NC, Petti GC, van den Berg GC, Seidl MF,
Ruess RW, Hendrick RL, Burton AJ, Pregitzer KS, Thomma BP (2021) An ancient antimicrobial protein
Sveinbjornsson B, Allen ME, Maurer GE (2003) Cou- co-opted by a fungal plant pathogen for in planta
pling fine root dynamics with ecosystem carbon mycobiome manipulation. Proc Natl Acad Sci 118:49
cycling in black spruce forests of interior Alaska. Snelders NC, Boshoven JC, Song Y, Schmitz N, Fiorin
Ecol Monogr 73:643–662 GL, Rovenich H, van den Berg GC, Torres DE, Petti
Rupp LA, Mudge KW (1985) Ethephon and auxin induce GC, Prockl Z, Faino L et al (2022) A highly polymor-
mycorrhiza-like changes in the morphology of root phic effector protein promotes fungal virulence
organ cultures of Mugo pine. Physiol Plant 64:316 through suppression of plant-associated
Ruytinx J, Miyauchi S, Hartmann-Wittulsky S, de Freitas Actinobacteria. New Phytol. https://doi.org/10.1111/
PM, Guinet F, Churin JL, Put C, Le Tacon F, Veneault- nph.18576
Fourrey C, Martin F, Kohler A (2021) A transcriptomic Splivallo R, Fischer U, Gobel C, Feussner I, Karlovsky P
atlas of the ectomycorrhizal fungus Laccaria bicolor. (2009) Truffles regulate plant root morphogenesis via
Microorganisms 9(12):2612 the production of auxin and ethylene. Plant Phys 150:
Sakamoto Y, Sato S, Takizawa M, Narimatsu M (2022) 2018–2029
Identification of upregulated genes in Tricholoma mat- Steinkellner S, Lendzemo V, Langer I, Schweiger P,
sutake mycorrhiza. FEMS Microbiol Letters 369(1): Khaosaad T, Toussaint JP, Vierheilig H (2007)
fnac085 Flavonoids and strigolactones in root exudates as
Sannazzaro AI, Echiverria M, Alberto EO, Ruiz OA, signals in symbiotic and pathogenic plant-fungus
Menendez AB (2007) Modulation of polyamine bal- interactions. Molecules 12(7):1290–1306
ance in Lotus glaber by salinity and arbuscular mycor- Stes E, Biondi S, Holsters M, Vereecke D (2011) Bacterial
rhiza. Plant Physiol Biochem 45:39–46 and plant signal integration via D3-type cyclins
Schneider AN, Sundh J, Sundström G, Richau K, enhances symptom development in the Arabidopsis-
Delhomme N, Grabherr M, Hurry V, Street NR Rhodococcus fascians interactions. Plant Physiol 156:
(2021) Comparative fungal community analyses 712–725
using metatranscriptomics and internal transcribed Strullu-Derrien C, Selosse MA, Kenrick P, Martin FM
spacer amplicon sequencing from Norway spruce. (2018) The origin and evolution of mycorrhizal
Msystems 6(1):e00884–e00820 symbioses: from palaeomycology to phylogenomics.
Schrey SD, Tarkka MT (2008) Friends and foes: New Phytol 220(4):1012–1030
streptomycetes as modulators of plant disease and Stuart EK, Plett KL (2020) Digging deeper: in search of
symbiosis. Antonie Leeuwenhoek 94:11–19 the mechanisms of carbon and nitrogen exchange in
Schrey SD, Schellhammer M, Ecke M, Hampp R, Tarkka ectomycorrhizal symbioses. Front Plant Sci 10:1658
MT (2005) Mycorrhiza helper bacterium Streptomyces Stuart EK, Castañeda-Gómez L, Macdonald CA, Wong-
AcH 505 induces differential gene expression in the Bajracharya J, Anderson IC, Carrillo Y, Plett JM, Plett
ectomycorrhizal fungus Amanita muscaria. New KL (2022) Species-level identity of Pisolithus
Phytol 168:205–216 influences soil phosphorus availability for host plants
Sebastiana M, Gargallo-Garriga A, Sardans J, Pérez- and is moderated by nitrogen status, but not CO2. Soil
Trujillo M, Monteiro F, Figueiredo A, Maia M, Biol Biochem 165:108520
Nascimento R, Silva MS, Ferreira AN, Cordeiro C Sundaram S, KimSJ SH, McQuattie CJ, Hiremath ST,
(2021) Metabolomics and transcriptomics to decipher Podila GK (2001) Isolation and characterization of a
molecular mechanisms underlying ectomycorrhizal symbiosis-regulated ras from the ectomycorrhizal fun-
root colonization of an oak tree. Sci Rep 11(1):1–6 gus Laccaria bicolor. Mol Plant-Microbe Interact 14:
Selosse MA, Richard F, He X, Simard SW (2006) Mycor- 618–628
rhizal networks: des liaisons dangereuses? Trends Ecol Tagu D, Nasse B, Martin F (1996) Cloning and character-
Evol 21:621–628 ization of hydrophobin-encoding cDNAs from the
10 Masters of Manipulation: How Our Molecular Understanding of Model. . . 271

ectomycorrhizal basidiomycete Pisolithus tinctorius. reveals the structural basis of aromatic amino acid
Gene 168:93–97 betaine biosynthesis. Chem Biochem 16:119–125
Tang N, Lebreton A, Xu W, Dai Y, Yu F, Martin FM Voiblet C, Duplessis S, Encelot N, Martin F (2001) Iden-
(2021) Transcriptome profiling reveals differential tification of symbiosis-regulated genes in Eucalyptus
gene expression of secreted proteases and highly spe- globulus-Pisolithus tinctorius ectomycorrhiza by dif-
cific gene repertoires involved in Lactarius–Pinus ferential hybridization of arrayed cDNAs. Plant J 25:
symbioses. Front Plant Sci 2021:1775 181–192
Tarkka MT, Frey-Klett P (2008) Mycorrhiza helper Walker TS, Bais HP, Grotewold E, Vivanco JM (2003)
bacteria. In: Varma A (ed) Mycorrhiza, 3rd edn. Root exudation and rhizosphere biology. Plant Physiol
Springer, Berlin, pp 113–132 132:44–51
Tarkka MT, Sarniguet A, Frey-Klett P (2009) Inter- Wang M, Dean RA (2020) Movement of small RNAs in
kingdom encounters: recent advances in molecular and between plants and fungi. Mol Plant Pathol 21(4):
bacterium–fungus interactions. Curr Genet 55:233– 589–601
243 Wang X, Wang H, Huang Y, Zhao Z, Qin X, Wang Y,
Taylor AFS, Martin F, Read DJ (2000) Fungal diversity in Miao Z, Chen Q, Qiao M (2010) Noncovalently
ectomycorrhizal communities of Norway spruce functionalized multi-wall carbon nanotubes in aqueous
{Picea abies (L.) Karst.} and beech (Fagus sylvatica solution using the hydrophobin HFBI and their elec-
L.) along north-south transects in Europe. Ecol Stud troanalytical application. Biosens Bioelectron 26:
142:343–365 1104–1108
Templeton MD, Rikkerink EHA, Beever RE (1994) Small, Wang M, Weiberg A, Dellota E Jr, Yamane D, Jin H
cysteine-rich proteins and recognition in fungal-plant (2017) Botrytis small RNA Bc-siR37 suppresses plant
interactions. Mol Plant-Microbe Interact 7:320–325 defense genes by cross-kingdom RNAi. RNA Biol
Thau N, Monod M, Cretani B, Rolland C, Tronchin G et al 14(4):421–428
(1994) Rodletless mutants of Aspergillus fumigatus. Wang W, Das A, Kainer D, Schalamun M, Morales-
Infect Immun 62:4380–4388 Suarez A, Schwessinger B, Lanfear R (2020) The
Toljander JF, Lindahl BD, Paul LR, Elfstrand M, Finlay draft nuclear genome assembly of Eucalyptus
RD (2007) Influence of arbuscular mycorrhizal myce- pauciflora: a pipeline for comparing de novo
lial exudates on soil bacterial growth and community assemblies. Gigasci 9(1):pgiz160
structure. FEMS Microbiol Ecol 61(2):295–304 Wang T, Persson P, Tunlid A (2020) A widespread mech-
Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I anism in ectomycorrhizal fungi to access nitrogen from
et al (2006) The genome of black cottonwood, Populus mineral-associated proteins. Environ Microbiol
trichocarpa. Science 313:1596–1604 23(10):5837–5849
Ungerstedt U (1991) Microdialysis—principles and Wawra S, Fesel P, Widmer H, Timm M, Seibel J, Leson L,
applications for studies in animals and man. J Int Kesseler L, Nostadt R, Hilbert M, Langen G, Zuccaro
Med 230(4):365–373 A (2016) The fungal-specific β-glucan-binding lectin
Uroz S, Heinonsalo J (2008) Inactivation of N-acyl FGB1 alters cell-wall composition and suppresses
homoserine lactone quorum sensing molecules by for- glucan-triggered immunity in plants. Nat Commun
est soil fungi. FEMS Microbiol Ecol 65:271–278 7(1):1–11
van der Biezen EA, Jones JD (1998) The NB-ARC Weiberg A, Bellinger M, Jin H (2015) Conversations
domain: a novel signalling motif shared by plant resis- between kingdoms: small RNAs. Curr Opin
tance gene products and regulators of cell death in Biotechnol 32:207–215
animals. Curr Biol 8:R226–R228 Wessels JGH (1996) Hydrophobins: proteins that change
van der Heijden MG, Martin FM, Selosse MA, Sanders IR the nature of the fungal surface. Adv Microb Physiol
(2015) Mycorrhizal ecology and evolution: the past, 38:1–45
the present, and the future. New Phytol 205(4): Westerink BH (1995) Brain microdialysis and its applica-
1406–1423 tion for the study of animal behaviour. Behav Brain
Vayssières A, Pěnčík A, Felten J, Kohler A, Ljung K, Res 70(2):103–124
Martin F, Legué V (2015) Development of the pop- Wild A (1988) Plant nutrients in soil. In: Wild A
lar-Laccaria bicolor ectomycorrhiza modifies root (ed) Russel’s soil conditions and plant growth, 11th
auxin metabolism, signaling, and response. Plant edn. Longman Scientific and Technical, Harlow, pp
Physiol 169(1):890–902 695–743
Villalobos Solis MI, Poudel S, Bonnot C, Shrestha HK, Wong-Bajracharya J, Castaneda-Gomez L, Plett KL,
Hettich RL, Veneault-Fourrey C, Martin F, Abraham Anderson IC, Carrillo Y, Plett JM (2020) Untangling
PE (2020) A viable new strategy for the discovery of the effect of roots and mutualistic ectomycorrhizal
peptide proteolytic cleavage products in plant-microbe fungi on soil metabolite profiles under ambient and
interactions. MPMI 33(10):1177–1188 elevated carbon dioxide. Soil Biol Biochem 151:
Vit A, Misson L, Blankenfeldt W, Seebeck FP (2015) 108021
Ergothioneine biosynthetic methyltransferase EgtD Wong-Bajracharya J, Singan VR, Monti R, Plett KL,
Ng V, Grigoriev IV, Martin FM, Anderson IC, Plett
272 J. M. Plett et al.

JM (2022) The ectomycorrhizal fungus Pisolithus releases a secreted β-1,4 endoglucanase that plays a
microcarpus encodes a microRNA involved in cross- key role in symbiosis development. New Phytol
kingdom gene silencing during symbiosis. Proc Natl 220(4):1309–1321
Acad Sci U S A 119(3):e2103527119 Zhang L, Feng G, Declerck S (2018b) Signal beyond
Wösten HAB, Wessels JGH (1997) Hydrophorins, from nutrient, fructose, exuded by an arbuscular mycorrhizal
molecular structure to multiple functions in fungal fungus triggers phytate mineralization by a phosphate
development. Mycoscience 38:363–374 solubilizing bacterium. ISME J 12(10):2339–2351
Wu G, Miyauchi S, Morin E, Kuo A, Drula E, Varga T, Zhang F, Labourel A, Haon M, Kemppainen M, Da Silva
Kohler A, Feng B, Cao Y, Lipzen A, Daum C (2022) ME, Brouilly N, Veneault-Fourrey C, Kohler A, Rosso
Evolutionary innovations through gain and loss of MN, Pardo A, Henrissat B (2022) The ectomycorrhizal
genes in the ectomycorrhizal Boletales. New Phytol basidiomycete Laccaria bicolor releases a GH28
233(3):1383–1400 polygalacturonase that plays a key role in symbiosis
Yan H, Freschet GT, Wang H, Hogan JA, Li S, Valverde- establishment. New Phytol 233(6):2534–2547
Barrantes OJ, Fu X, Wang R, Dai X, Jiang L, Meng S Zhao Z-X, Qiao M-Q, Yin F, Shao B, Wu B-Y, Wang
(2022) Mycorrhizal symbiosis pathway and edaphic Y-Y, Wang X-S, Qin X, Li S, Yu L, Chen Q (2007)
fertility frame root economics space among tree spe- Amperometric glucose biosensor based on self-
cies. New Phytol 234(5):1639–1653 assembly hydrophobin with high efficiency of enzyme
Yasuda T, Shishido K (1997) Aggregation of yeast cells utilization. Biosens Bioelectron 22:3021–3027
induced by the Arg-Gly-Asp motif-containing frag- Zhao Z, Cai F, Gao R, Ding M, Jiang S, Chen P et al
ment of high-molecular mass cell-adhesion protein (2021) At least three families of hyphosphere small
MFBA, derived from the basidiomycetous mushroom secreted cysteine-rich proteins can optimize surface
Lentinus edodes. FEMS Microbiol Lett 154:195–200 properties to a moderately hydrophilic state suitable
Yu C, Dou K, Wang S, Wu Q, Ni M, Zhang T, Lu Z, for fungal attachment. Environ Microbiol 23(10):
Tang J, Chen J (2020) Elicitor hydrophobin Hyd1 5750–5768
interacts with Ubiquilin1-like to induce maize systemic Zimin A, Stevens KA, Crepeau MW, Holtz-Morris A,
resistance. J Integr Plant Biol 62(4):509–526 Koriabine M, Marçais G, Puiu D, Roberts M, Wegrzyn
Zhang F, Anasontzis GE, Labourel A, Champion C, JL, de Jong PJ, Neale DB (2014) Sequencing and
Haon M, Kemppainen M, Commun C, Deveau A, assembly of the 22-Gb loblolly pine genome. Genetics
Pardo A, Veneault-Fourrey C, Kohler A (2018a) The 196(3):875–890
ectomycorrhizal basidiomycete Laccaria bicolor
 Nematode-Trapping Fungi
and Caenorhabditis elegans as a Model 11
System for Predator–Prey Interactions

Guillermo Vidal-Diez de Ulzurrun, Sheng-Chian Juan,

Tzu-Hsiang Lin, and Yen-Ping Hsueh

Abstract prey interactions between nematode-trapping

All habitats on earth host various sympatric fungi and C. elegans, focusing on the preda-
organisms that interact in different ways. tion process and the molecular mechanisms
Predator–prey dynamics represent one such underlying it. We envision that, given recent
mode of interaction, which are shaped by advances in experimental techniques devel-
coevolution of the interacting organisms, oped for nematode-trapping fungi, scientists
resulting in complex signal exchanges and will continue to unravel how these carnivorous
eliciting a diversity of hunting and escape fungi have coevolved with their
strategies. A predatory lifestyle has evolved nematode prey.
independently in major phyla of the fungal
kingdom. These carnivorous fungi use diverse Keywords
strategies to hunt fast-moving nematodes, and Nematode-trapping fungi · Arthrobotrys
they are of particular interest due to their oligospora · Caenorhabditis elegans ·
potential as biocontrol agents against parasitic Predator–prey interactions · Co-evolution
nematodes. The fascinating biology of carniv-
orous fungi has interested scientists for
decades. Over the past few years, significant
progress has been made in understanding the The Meiosporic State of Arthrobotrys
molecular mechanisms underlying predation For many years only the conidial state of
by nematode-trapping fungi. Given the exten- Arthrobotrys species was known until
sive research tools and resources that are avail- Donald H. Pfister neglected a Petri dish on
able for the model nematode Caenorhabditis the back of a lab bench for several months.
elegans, the microscopic fungal–nematode Pfister, a mycologist who studies
predator–prey system represents an excep- discomycetes (ascomycetes with apothecial
tional model for exploring coevolution and ascomata), was culturing a species of
predator–prey dynamics. Herein, we summa- Orbilia from ascospores derived from tiny
rize the current knowledge of the predator– cup-like apothecia collected on dung. A
serendipitous discovery came when he
eventually examined the culture plate and
G. Vidal-Diez de Ulzurrun · S.-C. Juan · T.-H. Lin · found he had grown an Arthrobotrys spe-
Y.-P. Hsueh (✉)
cies, the conidial state of a fungus well
Institute of Molecular Biology, Academia Sinica, Taipei,
Taiwan known for its nematode-trapping ability.
e-mail: [email protected]
(continued)
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 273
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_11
274 G. Vidal-Diez de Ulzurrun et al.

prey. Complex communication systems involving

Pfister developed the discovery, linking various kinds of signals between predator and
Orbilia and Arthrobotrys, which he com- prey play critical roles in these interactions.
pared to the mythical twins Castor and Pol- Thus, predation poses a challenging biological
lux. He presented his findings in his question, and several aspects of this core behavior
presidential address to the Mycological remain to be answered.
Society of America (Pfister 1997). Fungi and nematodes are found in most known
Molecular methods have allowed for the ecosystems and display all types of interactions,
in-depth study of these ascosporic fungi ranging from mutualism to predation (Yang et al.
and their diverse conidial states. We now 2020). Their interactions are complex and change
know that Orbilia is distinct from other in response to different biotic and abiotic factors.
discomycetes, and species of Orbilia and Both groups of organisms may be harmful to
related genera are placed in a separate humans, animals, and plants, so understanding
class, the Orbiliomycetes. Several lineages and controlling their dynamics is crucial.
found within Orbiliomycetes correspond to Among the interactions between nematodes and
morphologically, ecologically, and physio- fungi, we find those whereby sessile fungi hunt
logically diverse conidial states. In addition fast-moving nematodes particularly interesting.
to nematode-trapping states such as The reason for this resides in the potential of
Arthrobotrys, Dactylellina, and nematophagous fungi to act as biocontrol agents
Drechslerella, some non-nematode- against parasitic nematodes, such as those causing
trapping fungi are also members of the weight loss in livestock and disease in plants
Orbiliomycetes such as species of the (Lopez-Llorca et al. 2007). These fungi represent
aeroaquatic fungus genus Helicoon. an environmentally friendly alternative to classi-
Conidia of this genus and others disperse cal pesticides, but unfortunately they fail to estab-
in water, buoyed by air bubbles trapped in lish in most agricultural soils (Jaffee 2003). Thus,
the developing conidia. to harness the nematocidal potential of fungi in
complex environments, we need to learn more
about their predatory behavior and the molecular
mechanisms underlying it.
Nematophagous fungi have evolved complex
11.1 Introduction
predatory strategies, such as producing nematoci-
dal toxins, developing conidia that attach to their
Various organisms coinhabit many kinds of
prey, and forming traps that lure and capture
environments, creating biodiverse ecosystems in
nematodes (Li et al. 2015). The distinctive
which they all interact in many ways. Some
morphologies of the fungal traps and the com-
biological interactions result in benefits for the
plexity of the trapping processes have made
interacting species, whereas others result in posi-
nematode-trapping fungi (NTF) fascinating for
tive outcomes for only one of the organisms.
many scientists. Trap morphology varies among
Indeed, some interactions may even be detrimen-
different fungal species, which either use
tal, such as in predation–prey interactions when
adhesives or mechanical force to capture
one species consumes another. Although
nematodes (Nordbring-Hertz et al. 2011). The
predators are often assumed to be fast-moving
trapping process involves various signals
and aggressive, some predators rely on very sub-
between predator and prey in several steps.
tle techniques to lure and capture their prey. For
NTF, such as the ascomycete Arthrobotrys
instance, some make traps, produce attractive
oligospora (Orbiliales), use signals to attract
odors to lure their prey, or use toxins and venoms
nematode to their mycelia. Upon sensing the
to paralyze and kill, all representing strategies that
prey, they begin making adhesive traps. Traps
are the result of years of coevolution with their
capture the nematodes, which are then paralyzed,
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 275

before being pierced by the fungal hyphae that 11.2 Carnivorism in the Fungal
infect and consume them from the inside. Kingdom
Although several genes and pathways have been
described that play roles in prey sensing and trap Fungi are found in most known ecosystems,
formation, the molecular mechanisms underlying where they coexist with a wide variety of
fungal predation remain largely unknown. The organisms, sometimes in synergistic relationships
plasticity of the predatory behavior observed in with their neighbors. Some fungi, however,
wild isolates of the NTF A. oligospora only actively prey on other organisms to obtain addi-
corroborates the complexity and diversity of tional nutrients, and these are carnivorous fungi.
factors involved in this trait (Yang et al. 2020). Carnivorous fungi include amoebophagous fungi,
Additionally, the responses of nematode prey to such as Acaulopage dichotoma (phylum
the predatory behavior of NTF are still poorly Zoopagomycota) which traps and feeds on free-
understood, hindering a complete understanding living amoebas (Corsaro et al. 2018), fungi that
of this predator–prey system. prey on insects, such as Laccaria bicolor (phylum
Nevertheless, the scale at which nematodes Basidiomycota) that paralyzes and eats springtails
and fungi interact means their dynamics can be (Klironomos and Hart 2001), and nematophagous
reproduced under laboratory conditions, with full fungi in several different phyla that prey on
predator–prey interactions occurring in a Petri nematodes (Li et al. 2015).
dish. Both organisms can reproduce easily and The most studied group within the carnivorous
rapidly in laboratories. Thus, several generations fungi is the nematophagous fungi due to the
can be raised within a limited timeframe while diverse strategies that they employ to capture
tracking their genetic background, which their prey, the wide range of habitats in which
facilitates coevolutionary analyses. The genomes these interactions take place, and their potential in
of several nematophagous fungi have been agricultural control measures. More than 700 spe-
sequenced recently, further informing genomic cies of nematophagous fungi have been described
and genetic studies (de Ulzurrun et al. 2020; Lee in various fungal lineages, namely the
et al. 2021). Additionally, the model nematode Ascomycota, Basidiomycota, Zoopagomycota,
Caenorhabditis elegans is a common prey item of and Chytridiomycota (Li et al. 2015) (Fig. 11.1).
various NTF, itself bringing a wide range of tools Although these fungi share common prey, their
and resources to studies of these predator–prey strategies for luring and capturing nematodes dif-
dynamics at the molecular level. fer greatly. For instance, endoparasitic fungi in
In this chapter, we summarize the current the Zoopagomycota use conidia that attach to the
knowledge of the interactions between nematodes nematode cuticle and pierce it to infect their prey
and fungi. We outline the ecology of both from the inside (Lopez-Llorca et al. 2007). Other
organisms, including their habitats, diversity, fungi, such as the ascomycete Metapochonia
and the types of interactions they form in natural rubescens (Clavicipitaceae), attack nematode
ecosystems. This chapter pays special attention to eggs, breaking the eggshell using a special struc-
nematophagous fungi and particularly to NTF. ture called an appressorium (Lopez-Llorca et al.
Therefore, we thoroughly describe the trapping 2007). Another method employed by these fungi
process, as well as the known molecular to capture nematodes is the production of nema-
mechanisms underlying each of its stages. In par- tocidal toxins, such as those produced by hyphae
allel, we review the strategies adopted by of the edible mushroom Pleurotus ostreatus
C. elegans to avoid predation and fungal infec- (Barron 2003). This fungus usually extracts
tion. We hope that this chapter sheds some light nutrients from wood, but under starvation
on the widely unappreciated predator–prey conditions it produces toxins that can paralyze
interactions between fungi and nematodes and and kill nematodes (Lee et al. 2021). Upon
that it will encourage scientists to explore this contact between P. ostreatus and prey,
amazing inter-kingdom system.
276 G. Vidal-Diez de Ulzurrun et al.

Fig. 11.1 Nematophagous fungi rely on different ostreatus release nematocidal toxins that paralyze and
strategies to kill and consume their nematode prey. Endo- kill nematodes. Nematode-trapping fungi in phylum
parasitic fungi in phylum Zoopagomycota use spores and Ascomycota lure nematodes to sophisticated trapping
conidia that attach to the nematode cuticle to pierce it and devices to capture and consume them
infect their prey. Basidiomycetes such as Pleurotus

fungal-derived toxins enter the nematode through more than 200 species of NTF have been
its sensory cilia leading to hypercontraction and described, and they are broadly distributed from
calcium influx of pharyngeal and body wall tropical to polar regions (Nordbring-Hertz et al.
muscles, ultimately causing necrosis of the neu- 2011). For example, NTF within the Orbiliales
romuscular system (Lee et al. 2020). Another evolved from a common ancestor that first preyed
group of nematophagous fungi, i.e., the NTF, on nematodes to fulfill its nitrogen needs in
develop complex and distinctive physiological nitrogen-poor environments (Barron 2003;
structures aimed at trapping nematodes Ahrén and Tunlid 2003; Li et al. 2005). Traps
(Nordbring-Hertz et al. 2011). come in a variety of shapes and they can be
Arthrobotrys oligospora was first described in classified into two main kinds: mechanical traps
1888 (Zopf 1888). Under nutrient-deprived [constricting rings such as those produced by
conditions and in the presence of nematodes, Drechslerella stenobrocha (Orbiliales) (Liu
this fungus produces adhesive, three-dimensional et al. 2012)] and adhesive traps (adhesive nets,
networks known as “traps” to catch nematodes hyphae, knobs and columns, and
(Fig. 11.2). This finding inspired scientists to non-constraining rings) (Nordbring-Hertz et al.
identify and study NTF worldwide. To date, 2011).
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 277

Fig. 11.2 Summary of the trapping process of adhesive adhesive traps (adhesion) and, subsequently, their cuticles
net-forming nematode-trapping fungi. First (from left), are penetrated by the fungus (penetration). Once inside,
nematodes are attracted to the fungal mycelium (attrac- the fungus starts an infection process that culminates with
tion). The fungus senses signals emanating from the digestion of the nematode (digestion). In the end, the
nematodes, which triggers trap formation (sensing). The fungus starts to form conidiophores and conidia
nematodes are then captured and paralyzed by the (conidiation)

potent inducers of trap formation by NTF (Hsueh

11.3 The Trapping Process
et al. 2013). Several chemically synthesized
of Nematode-Trapping Fungi
ascarosides have been shown to induce trap mor-
phogenesis in A. oligospora (Hsueh et al. 2013),
In most natural ecosystems, nematodes and NTF
suggesting that these molecules may be the key
cohabit without harming each other. However, a
signal emanating from nematodes that is
combination of specific biotic and abiotic factors
recognized by NTF. In an effort to uncover the
may alter those dynamics and convert them into a
pathways by which nematode and environmental
predator–prey system (Nordbring-Hertz et al.
signals are sensed, recent studies have revealed
2011). Some abiotic factors that trigger the car-
that conserved signal transduction pathways—
nivorous behavior of NTF are starvation, carbon
including the G-protein signaling pathway,
dioxide levels (Bartnicki-Garcia et al. 1964), and
MAPK cascade, cAMP-PKA pathway, and
the availability of carbon and nitrogen sources
AMPK pathway—are at least partly involved
(Scholler and Rubner 1994). In addition to envi-
(Yang et al. 2020; Kuo et al. 2020; Chen et al.
ronmental factors, biotic stimuli are required to
2021, 2022; Wang et al. 2022) (Fig. 11.3).
induce trap formation, with direct contact
G-protein signaling is critical for sensing of
between living nematodes and fungal hyphae
environments in the fungal kingdom (Alvaro
being the most effective inducer (Åhman et al.
and Thorner 2016). In A. oligospora, deletion of
2002; Su et al. 2017; Xie et al. 2010). Early
GPB1, a homolog of G-protein β subunit STE4
studies showed that nematode extracts and culture
involved in the pheromone signaling pathway in
filtrates such as nemin can induce trap morpho-
the ascomycete Saccharomyces cerevisiae,
genesis in NTF (Lawton 1957; Pramer and Stoll
abolished trap formation (Yang et al. 2020).
1959). Additionally, factors non-specific to
G-protein β subunit STE4 is upstream of the
nematodes, such as dipeptides like
Fus3/Kss1 cascade in S. cerevisiae. Studies have
phenylalanine-valine (Nordbring-Hertz 1973;
shown that components in the Fus3/Kss1 cascade
Friman et al. 1985) or urea and diketopiperazines
of A. oligospora include MAPK Fus3, its
secreted by bacteria (Li et al. 2011, 2016; Wang
upstream kinase MAPKK Ste7, and the down-
et al. 2014), also resulted in trap formation. A
stream transcription factor Ste12 are also required
group of small molecules widely conserved
for nematode-sensing and trap formation (Chen
among nematodes and that function as sex
et al. 2021) (Fig. 11.3). Deletion of any of these
pheromones in C. elegans, i.e., ascarosides, are
278 G. Vidal-Diez de Ulzurrun et al.

Fig. 11.3 Overall signaling pathways involved in trap morphogenesis, vegetative growth, and conidiation processes in
Arthrobotrys oligospora

components resulted in defects in colony growth functions of HOG1 orthologs differ among fungal
and trap development. Additionally, mutants species. For example, MAPK HOG1 controls the
lacking STE7 and FUS3 displayed conidiation osmoadaptive response in S. cerevisiae (Reed
defects, supporting that there might be another et al. 1987), whereas its ortholog in Neurospora
downstream target of Fus3 responsible for crassa is involved in nutrient sensing (Huberman
conidiation. Another MAPK pathway involved et al. 2017). Deletion of HOG1 and MSB2
in nematode sensing is the high osmolarity glyc- (a sensor responsible for osmolarity (Tatebayashi
erol (HOG) pathway, which is involved in the et al. 2007)) orthologs in A. oligospora resulted in
yeast response to altered osmolarity in the envi- diminished trap formation ability (Kuo et al.
ronment (Rodríguez-Peña et al. 2010). The 2020). Moreover, a hog1 deletion mutant also
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 279

exhibited defective conidiation, indicating that sporulation, lipid synthetic regulation, and patho-
there might be downstream targets for HOG1 genicity (Wang et al. 2022) (Fig. 11.3).
that regulate signaling transduction for Contact between a predatory fungus and nem-
conidiation (Fig. 11.3). Aside from the MAPK atode prey is crucial to triggering predation and
pathways, a non-classical pathway may be trap morphogenesis. Furthermore, traps only
involved in prey sensing. This pathway has been work upon direct contact with living and motile
identified in several fungi and it is known to nematodes, since contact with dead or immobile
regulate meiosis, vegetative growth, sporulation, nematodes does not trigger trap development
and environmental adaptation (Irniger 2011). In (Tunlid et al. 1992). Consequently, sessile fungi
A. oligospora, AoIme2 was identified and need to induce nematode contact by attracting
characterized as playing roles in multiple devel- them. Nematodes are attracted to the mycelia of
opmental stages as a meiosis regulator. Mutant Arthrobotrys and Monacrosporium species
lines in which the AoIme2 gene has been deleted (Balan et al. 1974; Field and Webster 1977;
exhibit defects in meiosis and display a lower Saxena et al. 1987; Warcup 1957), and mycelia
number of nuclei in hyphae and conidia, as well hosting traps have been shown to lure even more
as fewer traps with lower trapping ability (Xie prospective prey (Jansson 1982). One of the most
et al. 2020) (Fig. 11.3). In the cAMP-PKA path- attractive compounds used by A. oligospora to
way, deletion of the PKA catalytic subunit gene attract nematodes is 3-methyl-2-butenoate
TPK2 resulted in complete abolition of trap for- (MMB), which mimics the sex and food cue of
mation as well as conidiation defects (Chen et al. nematodes (Hsueh et al. 2017). Aside from
2022). Addition of cAMP restored the trap- MMB, a methylated salicylic acid 6-MSA
forming ability of a gpa2 mutant but not a gpb1 deployed at hyphal tips by another NTF
mutant, indicating that GPA2 regulates the A. flagrans attracts C. elegans (Yu et al. 2021).
cAMP-PKA pathway whereas GPB1 is involved Interestingly, 6-MSA is an upstream product of
in the MAPK pathway. Taken together, these the biosynthetic pathway for arthrosporol, an
results indicate that the cAMP-PKA pathway extract from A. oligospora that negatively
plays a crucial role in sensing nematodes and regulates trap formation. Ascarosides inhibit
trap morphogenesis among NTF and that polyketide synthases involved in the biosynthesis
G-protein coupled receptors (GPCR) operating of arthrosporol (ArtA to ArtD), thereby reducing
upstream of the MAPK and cAMP-PKA signal- its production and triggering trap formation.
ing pathways might act as the receptors for sens- Moreover, expression of artA-cluster genes
ing prey (Chen et al. 2022) (Fig. 11.3). increases after nematodes have been trapped,
In addition to sensing prey, detection of other thus avoiding unnecessary trap formation
energy sources is also crucial for NTF to deter- (Yu et al. 2021).
mine if it is necessary to initiate trap morphogen- When an NTF switches to predatory behavior,
esis. AMP-activated protein kinase (AMPK) is trap formation is initiated at various sites along its
conserved among eukaryotes and plays a role as vegetative hyphae (Nordbring-Hertz et al. 1989).
an energy sensor, especially for sensing carbon The adhesive nets formed by A. oligospora are
sources (Mihaylova and Shaw 2011; Hedbacker built via several steps involving hyphal fusion
and Carlson 2008). In S. cerevisiae, the AMPK events. The initial cells of fungal traps (trap
complex comprises three subunits: catalytic initials) are distinguishable at an early stage due
α-subunit SNF1; regulatory β-subunits GAL83, to their possession of numerous dense bodies
SIP1, and SIP2; and γ-subunit SNF4. A recent (Veenhuis et al. 1985a). These cytosolic
study identified their homologs in A. oligospora, organelles are absent in other fungal cells and
i.e., the α-subunit AoSNF1, β-subunit AoGAL83, bud from special regions of the endoplasmic retic-
and γ-subunit AoSNF4 and demonstrated that this ulum. The role of these dense bodies remains
pathway plays a role in carbon source utilization, unknown, but they are thought to be involved in
prey penetration since their number significantly
280 G. Vidal-Diez de Ulzurrun et al.

declines upon a trap piercing the nematode cuticle 2021). Seven putative RGSs were identified and
(Veenhuis et al. 1989). Traps initially grow per- their deletions were found to reduce trap forma-
pendicular to the parental hypha (Fig. 11.4a) and tion. Among them, the AoflbA mutant line not
then, as they elongate, they arch either backward only exhibited the strongest phenotype but also
or forward until they fuse with another vegetative lacked conidiation ability.
hyphal cell (Nordbring-Hertz 2004) (Fig. 11.4b). Ultimately, the adhesive nets contain numer-
As the trap initial cells elongate and curve, they ous dense bodies that increase in number as the
subdivide into three cells to give rise to a loop trap matures (Nordbring-Hertz 2004; Veenhuis
(Fig. 11.4b), which is formed in about 1–2 h and et al. 1985b). Apart from dense bodies, trap cells
has a diameter of about 25μm. Each cell in a loop differ from the rest of the mycelium in that they
can act as a new trap initial (Fig. 11.4c), subse- are surrounded by an extracellular adhesive layer,
quently resulting in a new loop that anastomoses formed by polymer fibrils composed of proteins
to either other vegetative hyphae or other existing and carbohydrates (Tunlid et al. 1992; Veenhuis
loops (Fig. 11.4d). Since these loops are ran- et al. 1985b; Tunlid and Jansson 1991). When a
domly formed in all possible directions, the final trap contacts a nematode, the structure of this
traps of A. oligospora resemble intricate three- adhesive layer changes; the polymer fibrils that
dimensional networks (Fig. 11.4e). A recent are initially loosely packed and randomly
study revealed a potential gene involved in the distributed become densely packed and reorga-
cell recognition and fusion steps of trap forma- nize to align perpendicularly to the nematode
tion. This gene, sofT, is an ortholog of SO surface (Veenhuis et al. 1985a; Tunlid and
(responsible for cell fusion in N. crassa (Fleißner Jansson 1991). These structural changes result in
et al. 2005)) and it is required for anastomosis in a bridging structure connecting the nematode’s
the NTF A. flagrans (Youssar et al. 2019). Adhe- cuticle to the cell wall of the fungal trap. Since
sive networks developed by A. flagrans mutants this process occurs at multiple sites along the
lacking sofT fail to recognize and fuse to basal or space between the two organisms, the trap cell
neighboring cells, resulting in incomplete loops. wall becomes effectively anchored to the nema-
The striatin-interacting phosphatase and kinase tode cuticle, thereby immobilizing the prey. The
(STRIPAK) complex, a conserved signaling hub adhesion between fungal adhesive traps and the
in eukaryotes (Hwang and Pallas 2014; Kück nematode cuticle was long assumed to be
et al. 2019) that regulates multicellular develop- mediated by lectins and, more specifically, by an
ment, cell fusion, septation, and pathogenicity in N-acetylgalactosamine-specific lectin (AOL)
filamentous fungi (Kück et al. 2016), is also present in the traps of A. oligospora (Tunlid
involved in trap formation. In A. flagrans, one et al. 1992; Nordbring-Hertz and Mattiasson
of its components named SipC is involved in 1979). However, further analyses have shown
growth, trap morphogenesis, and conidiation that the predatory ability of AOL-deficient
(Wernet et al. 2022). Sipc deficiency results in mutants was similar to that observed for the wild
the formation of unusual but still functional traps. type (Balogh et al. 2003). More recent studies on
One phenotype displayed by sipc deletion the polymers of the adhesive layer have indicated
mutants results in trap initials growing back into that adhesion is mediated by proteins or protein-
vegetative hyphae due to delayed actin formation, containing substances (Wang et al. 2015). The
which accumulates at the septa separating the trap entomopathogenic fungus Metarhizium
cells. That study also demonstrated that long anisopliae (sometimes as M. robertsii)
microtubules lie on the inner side of the trap, (Clavicipitaceae) produces proteins called
with disruption of these microtubules resulting MAD1 and MAD2 adhesins to enable it to stick
in unusual trap morphogenesis. Additionally, a to the surface of insects. A homolog of MAD1 in
recent work studied regulators of G-protein sig- A. oligospora, AoMad1, has been identified as a
naling (RGSs) that negatively regulate G-protein factor contributing to its pathogenicity (Liang
signaling pathways in A. oligospora (Ma et al. et al. 2015). AoMad1 is located at the cell wall,
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 281

Fig. 11.4 Trap

morphogenesis by
Arthrobotrys oligospora. A
trap grows perpendicular to
a vegetative hypha (a),
forming an initial three-cell
loop by fusing back into the
parent hypha (b). Cells of
the first loop act as trap
initials (c), giving rise to
new loops (d). The final
trap configuration
comprises several loops that
have grown in different
directions, resulting in
three-dimensional adhesive
nets (e). Fungal cell walls in
fig. (a) to (d) were stained
by SCRI Renaissance 2200
(SR2200) and were
photographed by MD
ImagXpress Micro
L. Fungal cell wall in fig.
(e) was stained by
Calcofluor White (CFW)
and was photographed by
ZEISS LSM 980 inverted
confocal microscope. Scale
bars, 25μm

and mutants in which the respective gene has degrading the cuticle include collagenases
been deleted lack the extracellular layer; as such (Lopez-Llorca et al. 2007), hydrolytic enzymes
they exhibit a smooth trap surface. However, the such as those identified in Arthrobotrys species
traps of those mutants were still functional, and, (Schenck et al. 1975; Tosi et al. 2002), and serine
in fact, the number of traps increased in Aomad1 proteases (Yang et al. 2007). Indeed, serine pro-
deletion mutants. These results suggest that key tease inhibitors suppress the ability of fungi to kill
additional components contributing to the adhe- nematodes (Wang et al. 2014), and the nematoci-
siveness of trap cells of A. oligospora remain to dal activity of these proteases varies among
be defined. nematodes, indicating they exhibit specific host
The immobilized nematode is still protected by affinity (Tunlid et al. 1994a; Wang et al. 2006).
its cuticle so, in order to feed on their prey, NTF Interestingly, the genome of A. oligospora
first have to break through this barrier. The nem- contains lots of protease genes (Yang et al.
atode cuticle consists of three layers of proteins 2011) similar to those involved in host infection
and nematode-specific collagens, and its structure possessed by other pathogenic fungi (Monod
varies at different life-stages and among species et al. 2002). NTF also secrete collagenase and
(Cox et al. 1981; Page and Johnstone 2007). NTF subtilisin-like extracellular serine proteases (PII)
deploy both enzymatic degradation and mechani- to degrade the cuticle (Tosi et al. 2002; Tunlid
cal pressure to penetrate this barrier (Nordbring- et al. 1994a; Minglian et al. 2004). The protease
Hertz et al. 2011; Yang et al. 2007). Enzymes for inhibitor phenylmethylsulfonyl fluoride (PMSF)
282 G. Vidal-Diez de Ulzurrun et al.

was found to significantly reduce the ability of NTF conidium formation may also be affected
NTF to immobilize nematodes (Tunlid and by interactions with their prey. For instance,
Jansson 1991; Tunlid et al. 1994b). However, under nutrient-limited conditions, A. oligospora
PII gene expression was not observed during the forms very few nodes of conidia on
cuticle penetration stage, but during colonization conidiophores on the plate surface and tend to
and digestion, implying that PII may be used to produce the majority of conidiophores on aerial
decompose the cuticle from the inside. Serine hyphae. In contrast, after consuming nematodes,
proteases are highly expressed in trophic hyphae it produces multiple nodal conidia on the plate
growing inside infected nematodes, indicating surface rather than in the air. Though several
that these enzymes might play a role in digesting works exploring nematode-sensing signaling
nematode prey (Åhman et al. 2002). Aside from pathways have shown that mutants lacking
serine proteases, a fungal lectin, AOL is also Fus3, Ste7, Slt2, AoFlbA, and AoSnf1 displayed
accumulated in trophic hyphae (Rosen et al. impaired conidiation (Chen et al. 2021; Wang
1997). However, further functional studies on et al. 2022; Ma et al. 2021; Zhen et al. 2018),
these highly expressed enzymes will be required very little is known about how NTF regulate the
to identify critical players for nematode conidiation process in response to the presence of
digestions. In addition, several genes are known nematodes. A recent work showed that the velvet
to be involved in serine protease production in proteins VelB and VosA regulate the conidation
A. oligospora, including protein kinases such as process in NTF (Park and Yu 2012). In
non-classical MAPK Ime2 and AMPK Snf1 A. oligospora, deletion of AoVelB and AoVosA
(Wang et al. 2022; Xie et al. 2020), the RGSs resulted in no difference in growth rate or
AoRgs and AoFlbA (Ma et al. 2021), and velvet conidiation, but the AoVelB deletion mutants
protein AoVelB (Zhang et al. 2019). completely lacked conidiation ability even
Penetration of the cuticle is mediated by the though they could still form normal
formation of a penetration tube, a structure that conidiophores (Zhang et al. 2019). Significantly,
emerges from the trap cells which decompose the AoVelB is also involved in trap formation and
nematode cuticle and pierce into the worm body. serine protease production. Ime2 that is involved
The newly-forming cell wall can pass through the in trap morphogenesis and serine protease pro-
original trap cell wall due to partial hydrolysis, duction also affects conidium formation (Xie
resulting in a small penetration tube (Veenhuis et al. 2020). Aoime2 deletion mutants produce
et al. 1985b). After penetration of the nematodes, fewer and abnormal conidia with a reduced num-
an enlarged hyphal structure can be observed. ber of nuclei, and sporulation-related genes are
This enlarged hyphal structure was named the down-regulated, revealing a conserved role for
infection bulb, which was first observed and Ime2 in conidium regulation. These studies sug-
described in A. oligospora in 1955 (Shepherd gest that there might be an intriguing link between
1955). To investigate the detailed invasion pro- conidium and trap formation.
cess further, we constructed a cytosolic-GFP The plasticity of the predatory activities
labeled A. oligospora strain to conduct live- observed among wild isolates of A. oligospora
imaging. We observed that the penetration tube hints at a strong genetic factor driving this diver-
became anchored into the worm body within sity (Yang et al. 2020). Possibilities include sig-
10 minutes after contacting C. elegans nificant differences in trap morphology and
(Fig. 11.5a). Then, the invasion hypha started to efficiency, as well as in the response to trap-
swell and developed into large spherical infection triggering stimuli among strains. The complexity
bulb within 20 minutes (Fig. 11.5b) As the infec- of nematode and NTF interactions has been fur-
tion bulb matured, trophic hyphae developed and ther observed in natural ecosystems. In nature,
grew rapidly inside the nematodes. (Fig. 11.5d traps are rarely observed, unlike the propensity
and e). for trapping behavior exhibited by NTF in the
laboratory, which ultimately hinders their use as
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 283

Fig. 11.5 Invasion process of cytosolic-GFP labeled consume nematode (d and e). Scale bars, 30μm. The
Arthrobotrys oligospora growing into Caenorhabditis invasion process was recorded by Andor Revolution WD
elegans. Penetration tube grew into the nematode (a) and with Lambda 20X water lens. Images were analyzed with
the infection bulb developed well within 20 min (b and c), MetaMorph
which eventually formed trophic hyphae to colonize and

biocontrol agents against nematodes. More gen- unravel further the mysteries behind the
eral biological questions such as when and why interactions between NTF and nematodes.
NTF evolved to develop traps remain to be
addressed. In order to answer some of these
questions, whole genomes of several 11.4 Caenorhabditis elegans
nematophagous fungi have been sequenced, as a Model to Study Fungal–
including the network-making NTF Nematode Interactions
A. oligospora strain TWF154 and ATCC24927
(Yang et al. 2011, 2020) and A. flagrans strain Phylum Nematoda is the most abundant on the
CIM1 (Zhang et al. 2020a), the knob-making planet, occurring all over the world and
NTF Dactylellina haptotyla strain CBS 200.50 cohabiting with a diversity of organisms. It has
(as Monacrosporium haptotylum) (Meerupati been estimated that over a million nematode spe-
et al. 2013), the constricting ring-producing cies inhabit the Earth, and yet only 30,000
NTF D. stenobrocha strain CGMCC 3.6768 nematodes have been described to date (Kiontke
(Liu et al. 2014), the endoparasitic and Fitch 2013; Lorenzen 1994). A large propor-
nematophagous fungus Esteya vermicola strain tion of nematodes are parasitic, causing severe
CBS115803 (Ophiostomataceae) (Wang et al. diseases in humans, animals, and plants (Bethony
2018), and the toxin-producing P. ostreatus strain et al. 2006; Jasmer et al. 2003) with those affect-
PC9 (Lee et al. 2021). We believe these genomic ing plants resulting in significant agricultural
resources, combined with additional molecular losses (Jones et al. 2013; Trudgill and Blok
mechanistic studies will enable scientists to 2001). For example, root-knot nematodes and
another >4000 plant-parasitic nematodes cost at
284 G. Vidal-Diez de Ulzurrun et al.

least US$80 billion per year in crop losses (Nicol on nematodes using complex strategies. These
et al. 2011). nematophagous fungi share their ecological
Soil nematodes can be found worldwide, with niche with the well-developed model nematode
the highest nematode density being found in the C. elegans, providing an unrivalled opportunity
tundra and the lowest in deserts, ranging from to study predator–prey coevolution in detail using
2000 to 80 worms per 100 g of soil, respectively the power of the comprehensive toolbox
(van den Hoogen et al. 2019). To survive in these associated with this model organism.
environments, they have diversified considerably Some characteristics, alluded to earlier, that
in terms of morphological features such as length make a model organism ideal for laboratory
(from millimeters to several meters), anatomy work are a short lifecycle, ease of cultivation,
(cuticular decorations, vulva, and mouthparts), small size, and large populations. C. elegans
lifecycle (hermaphroditism, larval stages, and possesses all of these characteristics, which is
parasitism), and feeding habits (bacterivory, why it has become such a well-studied model
fungivory, herbivory, omnivory, and predatory) organism. C. elegans can be fed on Escherichia
(Kiontke and Fitch 2013; Ridall and Ingels 2021; coli both in agar plates and liquid cultures,
Yeates et al. 1993). All of these nematodes inter- making it easy to culture. This nematode is pri-
act with their respective neighbors. For example, marily self-fertilized, hermaphroditic, and
the predatory nematode Pristionchus preys on generates brood sizes of ~300 within a 3-day
other nematodes or even on their own species, lifecycle (Corsi et al. 2015), enabling researchers
and the roundworm Ascaris infects 1.2 billion to conduct genomics analyses easily. In addition,
people through a fecal-oral cycle. Oscheius is C. elegans has a fully differentiated body of 1 mm
one of the most common soil nematodes, feeding in length comprising only a thousand or so cells,
on bacteria (Dold and Holland 2011; Felix 2006; of which 302 in adult hermaphrodites are
Sommer 2006) and playing a central role in soil sensory-, inter-, and motor neurons (White et al.
ecosystems, so that it is used as a biological 1986). Furthermore, a unique characteristic that
indicator of soil environments (Ferris 2010; differentiates these nematodes from other such
Neher 2001). organisms is that they have transparent bodies
The interactions between fungi and nematodes throughout their lifecycle, thus permitting
are diverse and cover the full range, spanning scientists to observe their insides through a micro-
predator to prey (Yang et al. 2020; Baynes et al. scope (Corsi et al. 2015) (Fig. 11.6).
2012; Giannakis and Sanders 1990). Fungivorous For all of these reasons, Sydney Brenner and
nematodes play diverse roles in ecosystems his collaborators began developing C. elegans as
(Baynes et al. 2012). The nematodes a novel model organism in the 1960s. They
Aphelenchoides spp. and Aphelenchus avenae conducted the first EMS mutagenesis screening
feed on pathogenic fungi, thus aiding host plants and characterized its genetics in 1973 (Brenner
fight fungal infections, and they have, therefore, 1974). Decades of extensive study since then
proven of interest as biocontrol agents (Lagerlof have led to major scientific findings, including
et al. 2011; Okada 2006). Other nematodes feed the genetic regulation of development and
on plant-beneficial fungi such as of the genera programmed cell death (Sulston and Horvitz
Gliocladium and Trichoderma (Hypocreales), 1977; Ellis and Horvitz 1986; Yuan and Horvitz
thereby limiting the beneficial effects that these 1992), the mechanism of RNA interference (Fire
fungi have on plants (Hasna et al. 2007). On the et al. 1998), and construction of a fluorescent
other hand, nematodes can also be the host or protein as a reporter (Chalfie et al. 1994). More-
prey of fungi. The nematode-pathogenic fungi over, C. elegans became the first multicellular
Aspergillus spp. Fusarium spp., and Penicillium organism to have its whole genome sequenced,
spp. can infect various species of nematodes representing the strongest genetic resource yet
(Zhang et al. 2020b). Additionally, other fungi and opening up a new era in genetic research
(such as NTF) are carnivorous, actively preying (C. elegans Sequencing Consortium 1998).
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 285

Fig. 11.6 Live-imaging of a cytosolic-GFP labeled Arthrobotrys oligospora growing inside Caenorhabditis elegans.
The colonization process was recorded by Andor Revolution WD and was analyzed with MetaMorph. Scale bars, 50μm

Today, WormBase (https://wormbase.org/) 2009). The sexual dimorphic nociceptive behav-

provides an annotated C. elegans genome with ior arose from sex-specific connection in sensory
49,176 genes, including 19,981 protein-coding to interneuron that triggers dimorphic interneuron
genes. This extensive scientific resource collates responses (Pechuk et al. 2022). Recently, whole-
knowledge on C. elegans, including gene brain GCaMP activity recording in vivo was
descriptions, expression data, single-cell neuronal achieved by means of a polychromatic neuronal
expression maps, ontology, protein features, and identification tool (Yemini et al. 2021).
related publications. Other resources include extensive databases,
Its early establishment as a model species such as the Caenorhabditis natural diversity
resulted in several methods and tools being devel- resource (CaeNDR). This database comprises
oped to facilitate the study of C. elegans in >1500 wild-isolate strains collected worldwide
laboratories. For instance, microinjection and their respective genomes, enabling study of
techniques allow researchers to conduct genetic natural variations and the performance of
manipulation on these worms by injecting DNA genome-wide association mapping with
(including PCR products or plasmids) directly web-based tools (https://caendr.org) (Cook et al.
into their gonads, resulting in extrachromosomal 2017). From an evolutionary and ecological per-
arrays that remain heritable (Stinchcomb et al. spective, CaeNDR alone has wild-isolate strains
1985). The diverse applications of CRISPR-Cas covering large genetic and geographic
gene-editing systems are also widely being distributions. Population genetics and genome-
deployed (Dickinson and Goldstein 2016). wide association studies on these wild isolates
C. elegans also provides considerable resources have revealed that natural variations influence
for neurobiology research, with the whole-animal several C. elegans behaviors, such as foraging,
and sex-specific connectomes, as well as a neuro- pheromone responses, starvation resistance, and
transmitter map, quantitatively illustrating infor- metabolism (Cook et al. 2017; Fox et al. 2022;
mation flow (Cook et al. 2019; Gendrel et al. Webster et al. 2022; Greene et al. 2016; Lee et al.
2016; Pereira et al. 2015; Serrano-Saiz et al. 2019). In nature, wild C. elegans are often found
2013). The mating behavior of male C. elegans in the dauer stage that occurs in response to stress,
is mostly encode by male-specific neurons. starvation, and high population density (Barriere
Males have 385 neurons while and Felix 2007), indicating that the growth
hermaphrodites only have 302 neurons and conditions of C. elegans are far from optimal in
81 out of the 91 male specific neurons are their natural habitats. These habitats include both
involved in the mating circuit (Lints and Hall urban and rural areas, and several substrates such
286 G. Vidal-Diez de Ulzurrun et al.

as rotten fruits and flowers, soil, arthropods, and Two major fungal pathogens that infect
mollusks, supporting that C. elegans forms differ- C. elegans, as well as other nematodes, in nature
ent types of biological interactions with various are Drechmeria and Harposporium. The ascomy-
species in nature (Frezal and Felix 2015). Overall cete Drechmeria coniospora
then, the efforts expended in studying C. elegans (Ophiocordycipitaceae) differs from oomycetes
have resulted in a diversity of tools and rich in that its conidia target natural openings in the
resources, rendering it an excellent model for nematode, such as the mouth and vulva rather
studying its interactions with other organisms, than penetrating the cuticle (Felix and Duveau
including fungi. 2012; Schulenburg and Felix 2017). Infection by
Drechmeria kills the nematodes within 4 days
and, once inside its host, the fungus releases
11.5 The Behavior of C. elegans thousands of spores through the cuticle to the
and Its Responses to Biological outside environment (Jansson 1994).
Interactions Transcriptomic characterization of C. elegans
during Drechmeria infection has revealed a fun-
C. elegans is often the target of infection by damental defense response that involves antimi-
pathogenic viruses, bacteria, and fungi, as well crobial peptides, including two major families of
as being the prey of NTF. Together with the neuropeptide-like proteins (NLPs) and
various tools and resources specific to structurally-related caenacins (CNCs) (Pujol
C. elegans, this susceptibility makes it an excel- et al. 2008).
lent model for studying pathogen–host and Pathogen–host interactions have been studied
predator–prey interactions. Some of these extensively using C. elegans and their natural
pathogens are specific to C. elegans, e.g., Orsay pathogens. Comprehensive analyses of
virus only infects this species and no other in the transcriptomic data have suggested that genes
genus Caenorhabditis (Felix et al. 2011). encoding antimicrobial peptides (AMPs) are
Myzocytiopsis humicola, one of the oomycetes upregulated in response to both bacterial and fun-
with chitin contained in cellulin granules, is a gal infections in vitro, demonstrating antimicro-
natural pathogen that has been isolated mainly bial ability at least under those conditions
from wild C. elegans strains. Zoospores of this (Engelmann et al. 2011; Couillault et al. 2004).
oomycete develop adhesive buds that attach to the In addition, several conserved pathways
nematode cuticle, which then penetrate the nema- regulating the innate immune response in
tode body through narrow germ tubes. Each zoo- C. elegans have been identified, including the
spore produces a thallus that becomes p38 and PMK-1 MAPK pathways, as well as the
compartmentalized at maturity to appear as a DAF-2/DAF-16 and TGF-β pathways, which are
string of pearl-like structures to fill the entire activated following both bacterial and fungal
organism (Osman et al. 2018). Infection triggers infections, though some genes only play a role
a pathogen-specific transcriptomic response in in pathogen-specific responses (Pujol et al. 2008;
C. elegans, characterized by upregulation of the Engelmann et al. 2011; Couillault et al. 2004;
chitinase-like (chil) gene family upon exposure to Kawli and Tan 2008; Zugasti and Ewbank
this pathogen (Osman et al. 2018). Further 2009). In order to avoid pathogen infections,
research has revealed that oomycete recognition C. elegans has acquired avoidance strategies
by C. elegans is mediated by chemosensory against bacterial pathogens such as Pseudomonas
neurons that modulate upregulation of chil aeruginosa strain PA14. This avoidance behavior
genes. This allows the nematode to limit zoospore is inherited across up to four generations by
attachment and increase its survival via the establishing transgenerational epigenetic memory
defense mechanism of inducing chil genes in the via a PIWI-interacting RNA (piRNA) mechanism
epidermis (Fasseas et al. 2021). that regulates neuronal gene expression (Moore
et al. 2019). This learned behavior involves
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 287

foreign small RNA from the PA14 strain pairing between predator and prey species has resulted
with the nematode’s maco-1 gene to elicit avoid- in an evolutionary arms race (Burak et al. 2018).
ance behavior (Kaletsky et al. 2020). This and Classical studies of predator–prey systems were
other stress-induced learning behaviors might focused on population dynamics, with various
release C. elegans from the stress of heat-shock, mathematical models showing that population
starvation, and infections to increase survival and size oscillates between species (Wilks and Lotka
reproductive fitness (Moore et al. 2019; Wan et al. 1941; Volterra 1926). For instance, the lynx–hare
2021; Zhang et al. 2021; Rechavi et al. 2014). cycle showed a counterclockwise pattern,
The predator–prey interactions of NTF and whereby the prey population peaks prior to that
nematodes rely on bilateral communication. The of the predator (Elton and Nicholson 1942),
prey C. elegans produces ascarosides rotifer–algal populations exhibit an antiphase pat-
pheromones, small molecules defined as tern (Becks et al. 2010; Yoshida et al. 2003), and
glycosides of the dideoxysugar ascarylose that the phage-bacteria model uncovered cryptic
differ mainly in the length of the ascarylose car- cycles (Bohannan and Lenski 1999). Recent stud-
bon chain (Ludewig and Schroeder 2013). They ies have indicated that evolution of one species,
regulate dauer formation, mating, development, as well as the coevolution of two species, can
and social interactions (Butcher et al. 2007; shape the pattern of predator–prey cycles (Cortez
Srinivasan et al. 2008; Macosko et al. 2009; and Weitz 2014). Over the past few decades,
Kim et al. 2009). Interestingly, NTF such as scientists have studied predator–prey interactions
A. oligospora can sense several types of at the population level, with these interactions
ascarosides such as ascr#1, ascr#3, and ascr#7 leading to an arms race and often prompting
triggering the most significant trap-producing behavioral changes. Nevertheless, the molecular
response (Hsueh et al. 2017). The predator details of such evolutionary races remain elusive.
A. oligospora produces several volatile organic Recently, a study on lion–zebra and cheetah–
compounds that are attractive to worms, including impala interactions demonstrated that the
MMB (Hsueh et al. 2017). This fatal attractant is a predators have greater muscle power, resulting
thousand-fold stronger than the well-studied in better athletic capabilities, whereas the prey
C. elegans attractant isoamyl alcohol, eliciting have better maneuverability, giving them the abil-
both sex- and stage-specific nematode attraction ity to evade their major predator and match their
and disrupting mating behavior. This finding locomotion. That study has provided physiologi-
indicates that MMB might mimic a cal evidence that predator–prey coevolution leads
Caenorhabditis sex hormone to lure the to a sustainable success rate (both in terms of
nematodes (Hsueh et al. 2017). Behavioral capture and evasion), and thus survival of both
responses to MMB are regulated by the species (Wilson et al. 2018).
C. elegans AWC olfactory neuron. Forward The predator–prey system of the NTF
genetic screening has identified two mutants that A. oligospora and the nematode C. elegans
exhibit strongly reduced chemotaxis toward facilitates study of their molecular interactions
MMB, both of which possessed mutations in the from the perspectives of both predator and prey.
same transcription factor, odr-7, implying that New tools developed for A. oligospora—such as
chemotactic MMB attraction might involve mul- genetic knockout, RNA sequencing, and fluores-
tiple redundant receptors (Hsueh et al. 2017). cence imaging—permit several aspects of this
Predator–prey interactions are universal across fungus to be studied in detail not previously
the tree of life, playing a critical role in stabilizing attainable. Together with the discovery of an effi-
ecosystems and maintaining diversity and popu- cient model strain of NTF (Yang et al. 2020) and
lation sizes. To survive, prey species have the long-established resources available for
evolved strategies to lower predation, but preda- C. elegans, these tools will serve as the perfect
tory species have in turn coevolved more efficient basis for studying the enigmatic molecular mech-
predatory behaviors. The resulting feedback anism driving the nematode-trapping process.
288 G. Vidal-Diez de Ulzurrun et al.

Moreover, how NTF sense their prey and respond Burak MK, Monk JD, Schmitz OJ (2018)
to these signals, as well as how the prey responds Eco-evolutionary dynamics: the predator-prey adap-
tive play and the ecological theater. Yale J Biol Med
to predators, including chemotaxis and defense 91:481–489
responses, remain to be explored. We believe Butcher RA, Fujita M, Schroeder FC, Clardy J (2007)
that this unique predator–prey model might shed Small-molecule pheromones that control dauer devel-
light on the molecular details of cross-kingdom opment in Caenorhabditis elegans. Nat Chem Biol 3:
420–422
communication and how predator–prey C. elegans Sequencing Consortium (1998) Genome
interactions have coevolved. sequence of the nematode C. elegans: a platform for
investigating biology. Science 282:2012–2018
Acknowledgments The authors thank Meredith Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC
Blackwell for providing the overview of this chapter. (1994) Green fluorescent protein as a marker for gene-
expression. Science 263:802–805
Chen S-A, Lin H-C, Schroeder FC, Hsueh Y-P (2021)
Prey sensing and response in a nematode-trapping
References fungus is governed by the MAPK pheromone response
pathway. Genetics 217:iyaa008
Åhman J et al (2002) Improving the pathogenicity of a Chen S-A, Lin H-C, Hsueh Y-P (2022) The cAMP-PKA
nematode-trapping fungus by genetic engineering of a pathway regulates prey sensing and trap morphogene-
subtilisin with nematotoxic activity. Appl Environ sis in the nematode-trapping fungus Arthrobotrys
Microbiol 68:3408–3415 oligospora. G3 Genes|Genomes|Genetics. https://doi.
Ahrén D, Tunlid A (2003) Evolution of parasitism in org/10.1093/g3journal/jkac217
nematode-trapping fungi. J Nematol 35:194–197 Cook DE, Zdraljevic S, Roberts JP, Andersen EC (2017)
Alvaro CG, Thorner J (2016) Heterotrimeric G protein- CeNDR, the Caenorhabditis elegans natural diversity
coupled receptor signaling in yeast mating pheromone resource. Nucleic Acids Res 45:D650–D657
response. J Biol Chem 291:7788–7795 Cook SJ et al (2019) Whole-animal connectomes of both
Balan J, Križková L, Nemec P, Vollek V (1974) Produc- Caenorhabditis elegans sexes. Nature 571:63
tion of nematode-attracting and nematicidal substances Corsaro D et al (2018) New insights from molecular
by predacious fungi. Folia Microbiol 19:512–519 phylogenetics of amoebophagous fungi
Balogh J, Tunlid A, Rosén S (2003) Deletion of a lectin (Zoopagomycota, Zoopagales). Parasitol Res 117:
gene does not affect the phenotype of the nematode- 157–167
trapping fungus Arthrobotrys oligospora. Fungal Corsi AK, Wightman B, Chalfie M (2015) A transparent
Genet Biol 39:128–135 window into biology: a primer on Caenorhabditis
Barriere A, Felix MA (2007) Temporal dynamics and elegans. Genetics 200:387–407
linkage disequilibrium in natural Caenorhabditis Cortez MH, Weitz JS (2014) Coevolution can reverse
elegans populations. Genetics 176:999–1011 predator-prey cycles. Proc Natl Acad Sci U S A 111:
Barron GL (2003) Predatory fungi, wood decay, and the 7486–7491
carbon cycle. Biodiversity 4:3–9 Couillault C et al (2004) TLR-independent control of
Bartnicki-Garcia S, Eren J, Pramer D (1964) Carbon innate immunity in Caenorhabditis elegans by the
dioxide-dependent morphogenesis in Arthrobotrys TIR domain adaptor protein TIR-1, an ortholog of
conoides. Nature 204:804–804 human SARM. Nat Immunol 5:488–494
Baynes MA et al (2012) A mutualistic interaction between Cox GN, Kusch M, Edgar RS (1981) Cuticle of
a fungivorous nematode and a fungus within the endo- Caenorhabditis elegans: its isolation and partial char-
phytic community of Bromus tectorum. Fungal Ecol 5: acterization. J Cell Biol 90:7–17
610–623 de Ulzurrun GV-D, Lee Y-Y, Stajich JE, Schwarz EM,
Becks L, Ellner SP, Jones LE, Hairston NG Jr (2010) Hsueh Y-P (2020) Genomic analyses of two Italian
Reduction of adaptive genetic diversity radically alters oyster mushroom Pleurotus pulmonarius strains.
eco-evolutionary community dynamics. Ecol Lett 13: bioRxiv. https://doi.org/10.1101/2020.09.03.281089
989–997 Dickinson DJ, Goldstein B (2016) CRISPR-based
Bethony J et al (2006) Soil-transmitted helminth methods for Caenorhabditis elegans genome engineer-
infections: ascariasis, trichuriasis, and hookworm. Lan- ing. Genetics 202:885–901
cet 367:1521–1532 Dold C, Holland CV (2011) Ascaris and ascariasis.
Bohannan BJM, Lenski RE (1999) Effect of prey hetero- Microbes Infect 13:632–637
geneity on the response of a model food chain to Ellis HM, Horvitz HR (1986) Genetic-control of
resource enrichment. Am Nat 153:73–82 programmed cell-death in the nematode C-elegans.
Brenner S (1974) The genetics of Caenorhabditis elegans. Cell 44:817–829
Genetics 77:71–94
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 289

Elton C, Nicholson M (1942) Fluctuations in numbers of cascade integrate osmolarity and carbon sensing in
the muskrat (Ondatra zibethica) in Canada. J Anim Neurospora crassa. Proc Natl Acad Sci U S A 114:
Ecol 11:96–126 E8665–E8674
Engelmann I et al (2011) A comprehensive analysis of Hwang J, Pallas DC (2014) STRIPAK complexes: struc-
gene expression changes provoked by bacterial and ture, biological function, and involvement in human
fungal infection in C. elegans. PLoS One 6:e19055 diseases. Int J Biochem Cell Biol 47:118–148
Fasseas MK et al (2021) Chemosensory neurons modulate Irniger S (2011) The Ime2 protein kinase family in fungi:
the response to oomycete recognition in more duties than just meiosis. Mol Microbiol 80:1–13
Caenorhabditis elegans. Cell Rep 34:108604 Jaffee BA (2003) Correlations between most probable
Felix MA (2006) Oscheius tipulae. WormBook, pp 1–8. number and activity of nematode-trapping fungi. Phy-
10.1895/wormbook.1.119.1 topathology 93:1599–1605
Felix MA, Duveau F (2012) Population dynamics and Jansson HB (1982) Predacity by nematophagous fungi and
habitat sharing of natural populations of its relation to the attraction of nematodes. Microb Ecol
Caenorhabditis elegans and C. briggsae. BMC Biol 8:233–240
10:59 Jansson HB (1994) Adhesion of conidia of drechmeria-
Felix MA et al (2011) Natural and experimental infection coniospora to Caenorhabditis-elegans wild-type and
of Caenorhabditis nematodes by novel viruses related mutants. J Nematol 26:430–435
to nodaviruses. PLoS Biol 9:e1000586 Jasmer DP, Goverse A, Smant G (2003) Parasitic nema-
Ferris H (2010) Contribution of nematodes to the structure tode interactions with mammals and plants. Annu Rev
and function of the soil food web. J Nematol 42:63–67 Phytopathol 41:245–270
Field JI, Webster J (1977) Traps of predacious fungi attract Jones JT et al (2013) Top 10 plant-parasitic nematodes in
nematodes. Trans Br Mycol Soc 68:467–469 molecular plant pathology. Mol Plant Pathol 14:946–
Fire A et al (1998) Potent and specific genetic interference 961
by double-stranded RNA in Caenorhabditis elegans. Kaletsky R et al (2020) C. elegans interprets bacterial
Nature 391:806–811 non-coding RNAs to learn pathogenic avoidance.
Fleißner A et al (2005) The so locus is required for vege- Nature 586:445–451
tative cell fusion and postfertilization events in Neu- Kawli T, Tan MW (2008) Neuroendocrine signals modu-
rospora crassa. Eukaryot Cell 4:920–930 late the innate immunity of Caenorhabditis elegans
Fox BW et al (2022) C. elegans as a model for inter- through insulin signaling. Nat Immunol 9:1415–1424
individual variation in metabolism. Nature 607:571 Kim K et al (2009) Two chemoreceptors mediate develop-
Frezal L, Felix MA (2015) C. elegans outside the Petri mental effects of dauer pheromone in C-elegans. Sci-
dish. Elife 4 ence 326:994–998
Friman E, Olsson S, Nordbring-Hertz B (1985) Heavy trap Kiontke K, Fitch DH (2013) Nematodes. Curr Biol 23:
formation by Arthrobotrys oligospora in liquid culture. R862–R864
FEMS Microbiol Ecol 31:17–21 Klironomos JN, Hart MM (2001) Food-web dynamics.
Gendrel M, Atlas EG, Hobert O (2016) A cellular and Animal nitrogen swap for plant carbon. Nature 410:
regulatory map of the GABAergic nervous system of 651–652
C. elegans. Elife 5 Kück U, Beier AM, Teichert I (2016) The composition and
Giannakis N, Sanders FE (1990) Interactions between function of the striatin-interacting phosphatases and
mycophagous nematodes, mycorrhizal and other soil kinases (STRIPAK) complex in fungi. Fungal Genet
fungi. Agr Ecosyst Environ 29:163–167 Biol 90:31–38
Greene JS, Dobosiewicz M, Butcher RA, McGrath PT, Kück U, Radchenko D, Teichert I (2019) STRIPAK, a
Bergmann CI (2016) Regulatory changes in two che- highly conserved signaling complex, controls multiple
moreceptor genes contribute to a Caenorhabditis eukaryotic cellular and developmental processes and is
elegans QTL for foraging behavior. eLife 5 linked with human diseases. Biol Chem 400:1005–
Hasna MK, Insunza V, Lagerlöf J, Rämert B (2007) Food 1022
attraction and population growth of fungivorous Kuo C-Y, Chen S-A, Hsueh Y-P (2020) The high osmo-
nematodes with different fungi. Ann Appl Biol 151: larity glycerol (HOG) pathway functions in
175–182 osmosensing, trap morphogenesis and conidiation of
Hedbacker K, Carlson M (2008) SNF1/AMPK pathways the nematode-trapping fungus Arthrobotrys
in yeast. Front Biosci 13:2408 oligospora. J Fungi 6:191
Hsueh YP, Mahanti P, Schroeder FC, Sternberg PW Lagerlof J, Insunza V, Lundegardh B, Ramert B (2011)
(2013) Nematode-trapping fungi eavesdrop on nema- Interaction between a fungal plant disease, fungivorous
tode pheromones. Curr Biol 23:83–86 nematodes and compost suppressiveness. Acta Agric
Hsueh Y-P et al (2017) Nematophagous fungus Scand Sect B Plant Soil Sci 61:372–377
Arthrobotrys oligospora mimics olfactory cues of sex J. R. Lawton (nee Ress) JR (1957) The formation of
and food to lure its nematode prey. Elife 6 constricting rings in nematode-catching hyphomycetes
Huberman LB, Coradetti ST, Glass NL (2017) Network of grown in pure culture. J Exp Bot 8:50–54
nutrient-sensing pathways and a conserved kinase
290 G. Vidal-Diez de Ulzurrun et al.

Lee D et al (2019) Selection and gene flow shape niche- Meerupati T et al (2013) Genomic mechanisms accounting
associated variation in pheromone response. Nat Ecol for the adaptation to parasitism in nematode-trapping
Evol 3:1455–1463 fungi. PLoS Genet 9:e1003909
Lee C-H et al (2020) Sensory cilia as the Achilles heel of Mihaylova MM, Shaw RJ (2011) The AMPK signalling
nematodes when attacked by carnivorous mushrooms. pathway coordinates cell growth, autophagy and
Proc Natl Acad Sci 117:6014–6022 metabolism. Nat Cell Biol 13:1016–1023
Lee YY, Vidal-Diez de Ulzurrun G, Schwarz EM, Stajich Minglian Z, Minghe M, Keqin Z (2004) Characterization
JE, Hsueh YP (2021) Genome sequence of the oyster of a neutral serine protease and its full-length cDNA
mushroom Pleurotus ostreatus strain PC9. G3 from the nematode-trapping fungus Arthrobotrys
(Bethesda) 11 oligospora. Mycologia 96:16–22
Li Y et al (2005) Phylogenetics and evolution of Monod M et al (2002) Secreted proteases from pathogenic
nematode-trapping fungi (Orbiliales) estimated from fungi. Int J Med Microbiol 292:405–419
nuclear and protein coding genes. Mycologia 97: Moore RS, Kaletsky R, Murphy CT (2019) Piwi/PRG-1
1034–1046 argonaute and TGF-beta mediate transgenerational
Li L et al (2011) Induction of trap formation in nematode- learned pathogenic avoidance. Cell 177:1827–1841
trapping fungi by a bacterium. FEMS Microbiol Lett e1812
322:157–165 Neher DA (2001) Role of nematodes in soil health and
Li J et al (2015) Molecular mechanisms of nematode- their use as indicators. J Nematol 33:161–168
nematophagous microbe interactions: basis for Nicol JM et al. (2011) Current nematode threats to world
biological control of plant-parasitic nematodes. Annu agriculture. In: Genomics and molecular genetics of
Rev Phytopathol 53:67–95 plant-nematode interactions, pp 21–43. doi: https://
Li L et al (2016) Nematode-trapping fungi and fungus- doi.org/10.1007/978-94-007-0434-3_2
associated bacteria interactions: the role of bacterial Nordbring-Hertz B (1973) Peptide-induced morphogene-
diketopiperazines and biofilms on Arthrobotrys sis in the nematode-trapping fungus Arthrobotrys
oligospora surface in hyphal morphogenesis. Environ oligospora. Physiol Plant 29:223–233
Microbiol 18:3827–3839 Nordbring-Hertz B (2004) Morphogenesis in the
Liang L et al (2015) A proposed adhesin AoMad1 helps nematode-trapping fungus Arthrobotrys oligospora--
nematode-trapping fungus Arthrobotrys oligospora an extensive plasticity of infection structures. Mycolo-
recognizing host signals for life-style switching. Fun- gist 18:125–133
gal Genet Biol 81:172–181 Nordbring-Hertz B, Mattiasson B (1979) Action of a
Lints R, Hall DH (2009) Male neuronal support cells, nematode-trapping fungus shows lectin-mediated
overview. WormAtlas. doi:10.3908/wormatlas.2.9 host–microorganism interaction. Nature 281:477–479
Liu K, Tian J, Xiang M, Liu X (2012) How carnivorous Nordbring-Hertz B, Friman E, Veenhuis M (1989) Hyphal
fungi use three-celled constricting rings to trap fusion during initial stages of trap formation in
nematodes. Protein Cell 3:325–328 Arthrobotrys oligospora. A Van Leeuw J Microb 55:
Liu K et al (2014) Drechslerella stenobrocha genome 237–244
illustrates the mechanism of constricting rings and the Nordbring-Hertz B, Jansson H-B, Tunlid A (2011)
origin of nematode predation in fungi. BMC Genomics Nematophagous fungi. In: eLS. Wiley, Chichester, pp
15:114 1–13. https://doi.org/10.1002/9780470015902.
Lopez-Llorca LV, Maciá-Vicente JG, Jansson HB (2007) a0000374.pub3
Mode of action and interactions of nematophagous Okada H (2006) Ecology of fungivorous nematodes and
fungi. In: Ciancio A, Mukerji KG (eds) Integrated their use for suppression of plant diseases. Bulletin of
management and biocontrol of vegetable and grain the National Agricultural Research Center for Tohoku
crops nematodes, vol 2. Springer Netherlands, Region (Japan)
Dordrecht, pp 51–76 Osman GA et al (2018) Natural infection of C. elegans by
Lorenzen S (1994) The phylogenetic systematics of an oomycete reveals a new pathogen-specific immune
freeliving nematodes. Ray Society, London response. Curr Biol 28:640–648 e645
Ludewig AH, Schroeder FC (2013) Ascaroside signaling Page AP, Johnstone IL (2007) The cuticle. WormBook, pp
in C. elegans. WormBook, pp 1–22. 10.1895/ 1–15. https://doi.org/10.1895/wormbook.1.138.1
wormbook.1.155.1 Park H-S, Yu J-H (2012) Genetic control of asexual spor-
Ma N et al (2021) Functional analysis of seven regulators ulation in filamentous fungi. Curr Opin Microbiol 15:
of G protein signaling (RGSs) in the nematode- 669–677
trapping fungus Arthrobotrys oligospora. Virulence Pechuk V et al (2022) Reprogramming the topology of the
12:1825–1840 nociceptive circuit in C elegans reshapes sexual behav-
Macosko EZ et al (2009) A hub-and-spoke circuit drives ior. Curr Biol. https://doi.org/10.1016/j.cub.2022.
pheromone attraction and social behaviour in 08.038
C-elegans. Nature 458:1171–U1110 Pereira L et al (2015) A cellular and regulatory map of the
cholinergic nervous system of C. elegans. Elife 4
11 Nematode-Trapping Fungi and Caenorhabditis elegans as a Model. . . 291

Pfister DH (1997) Castor, Pollux and life histories of fungi. Su H et al (2017) Trapping devices of nematode-trapping
Mycologia 89:1–23 fungi: formation, evolution, and genomic perspectives.
Pramer D, Stoll NR (1959) Nemin: a morphogenic sub- Biol Rev Camb Philos Soc 92:357–368
stance causing trap formation by predaceous fungi. Sulston JE, Horvitz HR (1977) Post-embryonic cell
Science 129:966–967 lineages of the nematode, Caenorhabditis elegans.
Pujol N et al (2008) Anti-fungal innate immunity in Dev Biol 56:110–156
C. elegans is enhanced by evolutionary diversification Tatebayashi K et al (2007) Transmembrane mucins Hkr1
of antimicrobial peptides. PLoS Pathog, e1000105 4 and Msb2 are putative osmosensors in the SHO1
Rechavi O et al (2014) Starvation-induced branch of yeast HOG pathway. EMBO J 26:3521–
transgenerational inheritance of small RNAs in 3533
C. elegans. Cell 158:277–287 Tosi S, Annovazzi L, Tosi I, Iadarola P, Caretta G (2002)
Reed RH, Chudek JA, Foster R, Gadd GM (1987) Osmotic Collagenase production in an antarctic strain of
significance of glycerol accumulation in exponentially Arthrobotrys tortor Jarowaja. Mycopathologia 153:
growing yeasts. Appl Environ Microbiol 53:2119– 157–162
2123 Trudgill DL, Blok VC (2001) Apomictic, polyphagous
Ridall A, Ingels J (2021) Suitability of free-living marine root-knot nematodes: exceptionally successful and
nematodes as bioindicators: status and future damaging biotrophic root pathogens. Annu Rev
considerations. Front Mar Sci 8 Phytopathol 39:53–77
Rodríguez-Peña JM, García R, Nombela C, Arroyo J Tunlid A, Jansson S (1991) Proteases and their involve-
(2010) The high-osmolarity glycerol (HOG) and cell ment in the infection and immobilization of nematodes
wall integrity (CWI) signalling pathways interplay: a by the nematophagous fungus Arthrobotrys
yeast dialogue between MAPK routes. Yeast 27:495– oligospora. Appl Environ Microbiol 57:2868–2872
502 Tunlid A, Jansson HB, Nordbring-Hertz B (1992) Fungal
Rosen S, Sjollema K, Veenhuis M, Tunlid A (1997) A attachment to nematodes. Mycol Res 96:401–412
cytoplasmic lectin produced by the fungus Tunlid A, Rosén S, Ek B, Rask L (1994a) Purification and
Arthrobotrys oligospora functions as a storage protein characterization of an extracellular serine protease
during saprophytic and parasitic growth. Microbiol-Uk from the nematode-trapping fungus Arhtrobotrys
143:2593–2604 oligospora. Microbiol-Uk 140:1687–1695
Saxena G, Dayal R, Mukerji KG (1987) Interaction of Tunlid A, Rosén S, Ek B, Rask L (1994b) Purification and
nematodes with nematophagus fungi: induction of characterization of an extracellular serine protease
trap formation, attraction and detection of attractants. from the nematode-trapping fungus Arthrobotrys
FEMS Microbiol Lett 45:319–327 oligospora. Microbiology 140:1687–1695
Schenck NC, Graham SO, Green NE (1975) Temperature van den Hoogen J et al (2019) Soil nematode abundance
and light effect on contamination and spore germina- and functional group composition at a global scale.
tion of vesicular-arbuscular mycorrhizal fungi. Nature 572:194–198
Mycologia 67:1189–1192 Veenhuis M, Nordbring-Hertz B, Harder W (1985a)
Scholler M, Rubner A (1994) Predacious activity of the Development and fate of electron-dense microbodies
nematode-destroying fungus Arthrobotrys oligospora in trap cells of the nematophagous fungus Arthrobotrys
in dependence of the medium composition. Microbiol oligospora. A Van Leeuw J Microb 51:399–407
Res 149:145–149 Veenhuis M, Nordbring-Hertz B, Harder W (1985b) An
Schulenburg H, Felix MA (2017) The natural biotic envi- electron-microscopical analysis of capture and initial
ronment of Caenorhabditis elegans. Genetics 206:55– stages of penetration of nematodes by Arthrobotrys
86 oligospora. A Van Leeuw J Microb 51:385–398
Serrano-Saiz E et al (2013) Modular control of Veenhuis M, Van Wijk C, Wyss U, Nordbring-Hertz B,
glutamatergic neuronal identity in C. elegans by dis- Harder W (1989) Significance of electron dense
tinct homeodomain proteins. Cell 155:659–673 microbodies in trap cells of the nematophagous fungus
Shepherd AM (1955) Formation of the infection bulb in Arthrobotrys oligospora. A Van Leeuw J Microb 56:
Arthrobotrys oligospora Fresenius. Nature 175:475– 251–261
475 Volterra V (1926) Variazioni e fluttuazioni del numero
Sommer RJ (2006) Pristionchus pacificus. WormBook, pp d’individui in specie animali conviventi (Società
1–8. https://doi.org/10.1895/wormbook.1.102.1 anonima tipografica “Leonardo da Vinci”)
Srinivasan J et al (2008) A blend of small molecules Wan QL et al (2021) N-6-methyldeoxyadenine and his-
regulates both mating and development in tone methylation mediate transgenerational survival
Caenorhabditis elegans. Nature 454:1115–U1146 advantages induced by hormetic heat stress. Sci Adv 7
Stinchcomb DT, Shaw JE, Carr SH, Hirsh D (1985) Extra- Wang RB, Yang JK, Lin C, Zhang Y, Zhang KQ (2006)
chromosomal DNA transformation of Caenorhabditis Purification and characterization of an extracellular
elegans. Mol Cell Biol 5:3484–3496 serine protease from the nematode-trapping fungus
Dactylella shizishanna. Lett Appl Microbiol 42:589–
594
292 G. Vidal-Diez de Ulzurrun et al.

Wang X et al (2014) Bacteria can mobilize nematode- strain for nematode-trapping fungi. Proc Natl Acad Sci
trapping fungi to kill nematodes. Nat Commun 5:5776 U S A 117:6762–6770
Wang R, Wang J, Yang X (2015) The extracellular bioac- Yeates GW, Bongers T, De Goede RG, Freckman DW,
tive substances of Arthrobotrys oligospora during the Georgieva SS (1993) Feeding habits in soil nematode
nematode-trapping process. Biol Control 86:60–65 families and genera-an outline for soil ecologists. J
Wang R et al (2018) Comparative genomic analyses reveal Nematol 25:315–331
the features for adaptation to nematodes in fungi. DNA Yemini E et al (2021) NeuroPAL: a multicolor atlas for
Res 25:245–256 whole-brain neuronal identification in C. elegans. Cell
Wang W, Zhao Y, Bai N, Zhang K-Q, Yang J (2022) 184:272
AMPK is involved in regulating the utilization of car- Yoshida T, Jones LE, Ellner SP, Fussmann GF, Hairston
bon sources, conidiation, pathogenicity, and stress NG Jr (2003) Rapid evolution drives ecological
response of the nematode-trapping fungus arthrobotrys dynamics in a predator-prey system. Nature 424:303–
oligospora. Microbiol Spectr. e02225-02222 306
Warcup JH (1957) Studies on the occurrence and activity Youssar L et al (2019) Intercellular communication is
of fungi in a wheat-field soil. Trans Br Mycol Soc 40: required for trap formation in the nematode-trapping
237–259 fungus Duddingtonia flagrans. PLoS Genet 15:
Webster AK et al (2022) Using population selection and e1008029
sequencing to characterize natural variation of starva- Yu X et al (2021) Fatal attraction of Caenorhabditis
tion resistance in Caenorhabditis elegans. Elife 11 elegans to predatory fungi through 6-methyl-salicylic
Wernet V, Wäckerle J, Fischer R (2022) The STRIPAK acid. Nat Commun 12:1–10
component SipC is involved in morphology and cell- Yuan JY, Horvitz HR (1992) The Caenorhabditis-elegans
fate determination in the nematode-trapping fungus cell-death gene ced-4 encodes a novel protein and is
Duddingtonia flagrans. Genetics 220:iyab153 expressed during the period of extensive programmed
White JG, Southgate E, Thomson JN, Brenner S (1986) cell-death. Development 116:309–320
The structure of the nervous-system of the nematode Zhang G et al (2019) The velvet proteins VosA and VelB
caenorhabditis-elegans. Philos T R Soc B 314:1–340 play different roles in conidiation, trap formation, and
Wilks S, Lotka AJ (1941) Théorie analytique des pathogenicity in the nematode-trapping fungus
associations biologiques. Bull Am Math Soc 47:532– Arthrobotrys oligospora. Front Microbiol 10:1917
534 Zhang W et al (2020a) Comparative genome and
Wilson AM et al (2018) Biomechanics of predator-prey transcriptome analysis of the nematode-trapping fun-
arms race in lion, zebra, cheetah and impala. Nature gus Duddingtonia flagrans reveals high pathogenicity
554:183–188 during nematode infection. Biol Control 143:104159
Xie H et al (2010) Trap induction and trapping in eight Zhang Y et al (2020b) Fungi-nematode interactions: diver-
nematode-trapping fungi (Orbiliaceae) as affected by sity, ecology, and biocontrol prospects in agriculture. J
juvenile stage of Caenorhabditis elegans. Fungi 6
Mycopathologia 169:467–473 Zhang Q et al (2021) The memory of neuronal mitochon-
Xie M et al (2020) Protein kinase Ime2 is required for drial stress is inherited transgenerationally via elevated
mycelial growth, conidiation, osmoregulation, and mitochondrial DNA levels. Nat Cell Biol 23:870
pathogenicity in nematode-trapping fungus Zhen Z et al (2018) MAP kinase Slt2 orthologs play
Arthrobotrys oligospora. Front Microbiol 10:3065 similar roles in conidiation, trap formation, and patho-
Yang J, Tian B, Liang L, Zhang KQ (2007) Extracellular genicity in two nematode-trapping fungi. Fungal Genet
enzymes and the pathogenesis of nematophagous Biol 116:42–50
fungi. Appl Microbiol Biotechnol 75:21–31 Zopf WF (1888) Zur Kenntniss der Infections-Krankheiten
Yang J et al (2011) Genomic and proteomic analyses of the niederer Thiere und Pflanzen, vol 52. E. Blochmann
fungus Arthrobotrys oligospora provide insights into Zugasti O, Ewbank JJ (2009) Neuroimmune regulation of
nematode-trap formation. PLoS Pathog 7:e1002179 antimicrobial peptide expression by a noncanonical
Yang C-T et al (2020) Natural diversity in the predatory TGF-beta signaling pathway in Caenorhabditis elegans
behavior facilitates the establishment of a robust model epidermis. Nat Immunol 10:249–256
 When a Mind Is Not Its Own: Mechanisms
of Host Behavior Control by Parasitic 12
Fungi

Carolyn Elya

Abstract 12.1 Introduction

Many animal parasites can alter the behavior
of animals in ways that benefit their own fit- Insects are recognized as the largest and most
ness. Insects are a frequent target for behavior varied group of eukaryotic organisms on the
manipulation by various parasites, especially planet, with 900,000 recognized species
fungi. Despite many decades of observing (Grimaldi et al. 2005) and an expected 5.5 million
altered behavior in insects parasitized by total species (Stork 2018). The fungal kingdom is
fungi, our understanding of the mechanisms also incredibly diverse and species-rich. The
underpinning this behavior manipulation number of fungal species has been proposed to
remains largely incomplete. This chapter be anywhere between 2 and 12 million
explores our current understanding of the (Hawksworth and Lücking 2017; Wu et al.
mechanistic basis of behavior manipulations 2019) (though this remains a controversial topic
by these so-called zombie fungi, key of active discussion, see Bhunjun et al. (2022)). It
considerations in continuing to make progress is therefore, perhaps, unsurprising that the major-
in our understanding, and where I think the ity of fungal phyla contain entomopathogens,
next advances will come. species that rely on insects for nutrition (Araújo
and Hughes 2016), and that the ability to use
Keywords insects as food has arisen multiple times over
the earth’s evolutionary past (Hughes 2011).
Behavior manipulation · Entomopathogens · Indeed, approximately 65% of all insect orders
Zombie fungi · Cadaver transmission · Active have been observed to be infected by fungi
host transmission · Summit disease · (Araújo and Hughes 2016). Entomopathogenic
Entomophthora · Ophiocordyceps · fungi use a variety of strategies in utilizing insects
Massospora for nutrition. Some entomopathogenic fungi are
generalists: they are able to infect and draw
resources from a variety of host species [e.g.,
Metarhizium robertsii, Beauveria bassiana
(St Leger and Wang 2020; Ortiz-Urquiza
2021)], leveraging the large available insect bio-
mass in their environment. Others are specialists,
C. Elya (✉)
targeting a narrow range of host species. Many
Department of Organismic and Evolutionary Biology,
Harvard University, Cambridge, MA, USA specialist entomopathogenic fungi have evolved
e-mail: [email protected] to manipulate host behavior. By doing so, the
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 293
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_12
294 C. Elya

Fig. 12.1 Four examples of zombie fungi, their mode of Spatafora et al. (2016). Underlined clades (Phylum
dispersal and their relative phylogenetic placement within Ascomycota and Subphylum Entomophthoromycotina)
the fungal kingdom. Clockwise from upper right: are home to most zombie fungi. Funnels adjacent to
Ophiocordyceps spp., aka zombie ant fungus, infect car- these clades indicate taxonomic placement for the
penter ants and spread via cadaver transmission (CT); examples shown in this figure. Ophiocordyceps is an asco-
Strongwellsea castrans infects Delia platura and spreads mycete fungus (Order Hypocreales); all other examples
via active host transmission (AHT); Massospora cicadina are in phylum Zoopagomycota (Order Entomophthorales).
infects 13- and 17-year periodical cicadas and spreads via Tree branch lengths are not proportional to phylogenetic
AHT; Entomophthora muscae infects various dipterans distance
(true flies) and spreads via CT; fungal phylogeny after

fungus increases the likelihood that it will transmission (CT) or active host transmission
encounter a suitable new host that lies within its (AHT). In cadaver-transmitting systems, the fun-
target host range. These behavior-manipulating gus coerces the host to die in an elevated location
fungi, also referred to as “zombie fungi,” have and then sporulates from the dead host from this
been observed predominantly in two fungal high vantage point. In active host transmitting
phyla, Ascomycota and Zoopagomycota (Hughes systems, the fungus alters the abdominal structure
et al. 2016). Like entomopathogenicity itself, the of a living host (either completely replacing it
ability to hijack insect behavior has evolved more with fungal tissue or creating an opening through
than once, at least twice within the subphylum which fungal tissue can emerge) and uses the
Entomophthoromycotina and several times movement of the host to its advantage for dispers-
among Ascomycota (Hughes et al. 2016) ing spores.
(Fig. 12.1). Perhaps the most widely known example of a
Broadly speaking, zombie fungi utilize one of cadaver-transmitting fungus is the
the two strategies to access new hosts: cadaver Ophiocordyceps-infected zombie ant (Fig. 12.1).
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 295

Though often referred to as a single phenomenon, since the mid-1800s (Cohn 1855; Fawcett 1886;
there are actually many different species of Thaxter 1888). Ever since this time they have
Ophiocordyceps that each target a different host fascinated scholars and non-scholars alike,
ant species and elicit their own variation on a recently making several appearances in the popu-
behavioral theme (Evans et al. 2011; de Bekker lar media (Nuwer By Rachel Nuwer on May
et al. 2014; Loreto et al. 2018; Sakolrak et al. 27 2021; Yong 2017; Langley 2018; Lu 2019;
2018). In these systems, Ophiocordyceps- Zimmer 2019; Iati 2021). Yet despite
infected ant hosts show abnormal behaviors late longstanding fascination in zombie fungi, we are
in infection. Whereas healthy ants are usually only just now beginning to uncover the
found in the nest or on foraging trails, zombie mechanisms by which they elicit behaviors in
ants aberrantly leave their nests and wander off their unwitting hosts.
the foraging trail to climb vegetation. After their
climb, zombie ants affix themselves to the plant
Call It Ophiocordyceps
substrate, typically by biting down and
Once there was a genus of 400 species of
subsequent “lock-jaw,” then die (Hughes et al.
insect pathogens, Cordyceps, classified in
2011). Curiously, death in this manner occurs
the order Clavicipitales. Use of DNA
with specific circadian timing, with zombie ants
sequencing and phylogenetic analysis to
exhibiting the final climbing and biting behavior
study the insect associated species
specifically around solar noon (Hughes et al.
of Cordyceps revealed that the ecology
2011). Over several days, Ophiocordyceps erupts
was more ancient than previously realized
from the dead ant’s head, producing a fruiting
and that these insect pathogenic species did
body that will eject infectious fungal spores into
not comprise a monophyletic genus. The
the environment (Hughes et al. 2011). Since ants
species previously placed in a single genus
tend to die elevated above foraging trails, this
now are classified in three families
ideally positions Ophiocordyceps to infect addi-
(Clavicipitaceae, Cordycipitaceae,
tional ants. The majority of described behavior-
Ophiocordycipitaceae) of Hypocreales
manipulating entomopathogens rely on similar
reflecting their independent lineages. The
behavior manipulations and cadaver transmission
phylogenetic classification of these species
to complete their life cycle.
now includes more than 20 genera and
There are far fewer examples of active host
combines ascospore- and conidium-
transmitting zombie fungi. Among these is
producing species within common genera
Massospora cicadina which infects periodical
based on morphological and DNA criteria.
cicadas, insects that only emerge from dormancy
Some species now placed in the
in the soil every 13 or 17 years (Fig. 12.1). Cicada
genus Ophiocordyceps are insect pathogens
nymphs acquire Massospora infection via resting
highlighted in this chapter and in current
spores deposited in the soil (Lovett et al. 2020).
dystopian fiction. Others are known
Once the infected adult emerges, Massospora
throughout the world in traditional medi-
grows in its posterior end, ultimately sloughing
cine. Less well known are yeast-like
off the posterior segments of the abdomen and
members of the Ophiocordyceps lineage,
replacing these tissues with a fungal plug (Lovett
including yeast-like symbionts in mutualis-
et al. 2020). Massospora then disseminates spores
tic associations with bacteria and insects of
to other cicadas during flight and copulation
the order Hemiptera. The evolutionary
attempts by the infected individuals, effectively
pathway from virulent pathogen to obligate
using the host as a mobile transmission vector
symbiont remains to be explained.
(Lovett et al. 2020).
Zombie fungi and their pitied insect victims
have been documented in the scientific literature
296 C. Elya

12.2 Common Themes in Behavioral (Table 12.1). Here, I will summarize these
Manipulation by Zombie Fungi behaviors and discuss the mechanisms by which
they are thought to occur. Where possible, I will
There are five major recurring themes in the discuss occurrences of similar behaviors that are
behaviors elicited by zombie fungi: hyperactivity, elicited by non-fungal parasites and available
summit disease, perimortem postural changes, mechanistic evidence for these systems.
circadian timing, and sexual attraction

Table 12.1 Recurring themes in behaviors elicited by zombie fungi

Possible host Hypothesized
Behavior Description targets fungal action Examples References
Hyperactivity Increase in Nutrient Secretion of Ophiocordyceps Will et al. (2020),
locomotion depletion, fungal spp. Trinh et al. (2021),
locomotion effectors Massospora Cooley et al. (2018),
circuits (metabolites, cicadina Boyce et al. (2019)
SSPs)
Summit Climbing to an Phototactic Secretion of Ophiocordyceps Hughes et al. (2011),
disease elevated location and/or fungal spp. Krasnoff et al.
right before death gravitactic effectors Entomophthora (1995), Pickford and
pathways (metabolites, muscae Riegert (1964),
SSPs) Entomophaga Csata et al. (2021),
grylli Steinkraus et al.
Pandora (2017)
formicae
Eryniopsis
lampyridarum
Postural Specific positioning Invasion and/or Mechanical Ophiocordyceps Hughes et al. (2011),
changes of body parts to restriction of force; spp. Will et al. (2020),
maintain cadaver’s musculature modulation of Entomophthora Trinh et al. (2021),
elevation and/or motor neuron muscae Krasnoff et al.
accommodate activity Entomophaga (1995), Pickford and
fungal dispersal grylli Riegert (1964),
Pandora Csata et al. (2021),
formicae Steinkraus et al.
Eryniopsis (2017)
lampyridarum
Circadian Behaviors only Host circadian Secretion of Ophiocordyceps Hughes et al. (2011),
timing occur within network, fungal spp. Milner et al. (1984),
specific time pathways effectors, Entomophthora Krasnoff et al.
window relative to underlying perhaps spp. (1995), Elya et al.
environmental cues behaviors that according to Entomophaga (2018), Pickford and
occur with fungal grylli Riegert (1964),
circadian timing molecular Entomophaga Nielsen and Hajek
clock maimaiga (2006), Dustan
Erynia (1924)
neoaphidis
Sexual Increased Olfactory and Production of Massospora Boyce et al. (2019),
attraction frequency of gustatory volatile cicadina Naundrup et al.
attempted mating systems, chemical Entomophthora (2021)
with infected hormonal axes attractants, muscae
individuals stimulant
metabolites,
hormonal shifts
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 297

12.2.1 Hyperactivity Will et al. 2020), the latter of which have been
repeatedly implicated in mediating interactions
Hyperactivity, alternatively referred to as between fungi and their host plants and animals
enhanced locomotor activity or ELA, refers to a (Kim et al. 2016). Genomic analysis has found
sustained increase in locomotion and is a behav- that orthologues of the enterotoxin are conserved
ior observed within hosts infected by both CT and only among ant-manipulating members of genus
AHT fungi. Carpenter ants infected with Ophiocordyceps (de Bekker et al. 2017a). Addi-
Ophiocordyceps exhibit hyperactivity prior to tionally, many of the SSPs appear to be species-
their final climb (Trinh et al. 2021). specific, suggesting that they contribute to the
Massospora-infected cicadas exhibit hyperactiv- unique pathology of these fungi (de Bekker
ity after their posterior abdomen has been et al. 2017a). These studies also observed a dra-
replaced with a spore-releasing fungal plug matic upregulation of genes in a cluster predicted
(Boyce et al. 2019). In both instances, hyperac- to synthesize an aflatrem-like alkaloid at the point
tivity likely plays a role in positioning the host for of manipulation (de Bekker et al. 2015; Will et al.
spore dispersal. For the ant, hyperactivity may 2020). Aflatrem has known neurobiological activ-
play a role in driving the ant away from its nest ity, often observed to cause tremors and seizures
and to an elevated location from which it is ideal in livestock (Valdes et al. 1985). In cicadas, a
to eject spores. For the cicada, hyperactivity is recent study investigating the metabolome of
hypothesized to increase the number of Massospora-infected individuals found the
conspecifics encountered, and thus increase the amphetamine relative cathinone (Boyce et al.
number of newly infected hosts (Cooley et al. 2019). Amphetamines have similar structures to
2018; Lovett et al. 2020). the hormones adrenaline and epinephrine and
The molecular drivers of zombie fungus- have been well established to exhibit stimulatory
induced hyperactivity are unclear, but a variety effects on a variety of animals (Randrup and
of mechanisms have been proposed. First, hyper- Munkvad 1967). Overall, many exciting
activity could result from more general changes in compounds have been identified as correlates of
host physiology. Consistent with their mechanism hyperactivity. It will be up to future experiments
of action (i.e., consuming host resources for fuel), to demonstrate their causality.
zombie fungi have been observed to elicit Hyperactivity is also elicited in insect hosts by
starvation-like states in their host (Elya et al. several non-fungal parasites. While the specificity
2018; Will et al. 2020). As many insects have of our mechanistic insight varies among these
been observed to increase locomotion during systems, what we have learned so far suggests
times of starvation (Browne and Evans 1960; that several diverse mechanisms, rather than a
Leonard 1970; C. P. Wheater 1991; Yang et al. common pathway, underlie this behavior alter-
2015), hyperactivity could potentially arise from ation. In crickets infected with hairworms (Phy-
general nutrient depletion. lum Nematomorpha) and sandhoppers infected
Alternatively, hyperactivity could be elicited with mermithids (Phylum Nematoda), hyperac-
via compounds produced and secreted by the tivity occurs prior to a suicidal plunge into water
fungus. Genomic, transcriptomic, and (Herbison et al. 2019). Proteomics studies in
metabolomic studies in zombie ants have these systems have led to the hypothesis that
identified several candidates. In Ophiocordyceps hyperactivity occurs as a result of dysregulation
spp.-infected ants, two independent studies in of energy utilization and/or neurophysiology
distinct experimental systems examined gene (e.g., synaptic vesicle packaging and release,
expression at the point of manipulation (i.e., bit- maintenance of neuronal compartments)
ing). The authors observed upregulation of a puta- (Herbison et al. 2019). In several species of lepi-
tive enterotoxin as well as several putative small dopteran larvae infected with
secreted proteins (SSPs) (de Bekker et al. 2015; nucleopolyhedroviruses (NPVs), pre-death
hyperactivity has been elegantly demonstrated to
298 C. Elya

depend on a virally-encoded protein tyrosine consistent behavioral motifs observed in

phosphatase (ptp) (Kamita et al. 2005; van cadaver-transmitting fungal parasites
Houte et al. 2012). However, a recent study (Table 12.1). While the mechanistic basis of this
demonstrated that ptp is dispensable for hyperac- behavior remains to be determined, there is a
tivity in at least one case (Kokusho and Katsuma short list of host pathways hypothesized to serve
2021). Thus, even among NPVs it appears that as fungal targets to elicit this behavior. The first of
there may be diverse mechanisms underlying these is phototaxis, orientation, and movement
hyperactivity. with respect to light. In field studies,
In contrast to the increased activity seen in Ophiocordyceps-infected ants have been
zombie crickets, sandhoppers, and caterpillars, observed to die more frequently in sunny versus
jewel wasps (Ampulex compressa) evoke hypo- shaded regions of observation plots (Andriolli
activity (reduced locomotion) in their cockroach et al. 2019) and to perish with their heads oriented
hosts. After an initial non-specific sting that toward opening in the forest canopy (Chung et al.
serves to temporarily paralyze its cockroach 2017). These observations suggest that light plays
host, the wasp targets a second injection into the an important role in the end-of-life behaviors
cockroach brain (Haspel et al. 2003). Following exhibited by zombie ants. As light appears to
this second injection the cockroach engages in influence where ants summit and die, the host
approximately 25 min of grooming behaviors phototaxis pathway has been hypothesized to be
and fails to initiate walking behavior (Haspel involved in summiting behavior. Gravitaxis, the
et al. 2003). Work in this system has found that orientation and movement in response to earth’s
the venom cocktail for the second injection is a gravitational pull, is another pathway proposed to
complex mixture of small molecules, peptides play a role in summit behavior. The hallmark of
and proteins (Arvidson et al. 2019), that specifi- summit behavior is death following elevation,
cally targets the insect central complex and achieving elevation requires navigating
(a conserved brain region important for against the earth’s gravitational pull. Thus, it
coordinating and planning motor output) (Haspel seems reasonable that fungal parasites might
et al. 2003). These behavioral effects are drive summiting behavior via altering their
mediated by D1-like dopamine receptors in the host’s gravitactic preferences or circuitry.
cockroach (Nordio et al. 2022), inducing host How fungal parasites might alter phototaxis
hyperactivity, and, more broadly, alter host activ- and gravity sensing in summiting or other host
ity levels, apparently, a frequent strategy pathways is still unclear, although, again, several
employed by behavior-manipulating parasites. hypotheses have arisen from multi-omic
approaches in Ophiocordyceps-ant systems. As
previously discussed, a putative fungal entero-
12.2.2 Summit Disease toxin, several SSPs, and a gene cluster responsi-
ble for synthesizing a related compound to the
Summit disease, also referred to as tree-top dis- tremorgenic aflatrem have been found to be
ease or Wipfelkrankheit (Hofmann 1891), refers highly expressed during Ophiocordyceps-elicited
to the behavior in which an insect climbs to an biting behavior in ants (de Bekker et al. 2015;
elevated location immediately prior to its death. Will et al. 2020). Immediately preceding biting,
The insect then perishes at this high perch, typi- ants had exhibited both hyperactivity and
cally held in place by its mouthparts (Hughes summiting behaviors. It seems likely that these
et al. 2011; Csata et al. 2021), limbs (Carruthers behaviors likely overlap temporally, thus the
et al. 1997), and/or fungal holdfasts (Chung et al. compounds implicated in manipulation could be
2017). Summit disease is elicited by several fun- important for driving one or both of hyperactivity
gal parasites including Ophiocordyceps (Hughes and summiting.
et al. 2011) and Entomophthora muscae There are also several non-fungal parasites that
(Krasnoff et al. 1995) and is one of the most elicit summit disease which can provide insight
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 299

into the mechanistic underpinnings of this behav- a result (O’Reilly and Miller 1989). Thus, egt is
ior. The trematode Dicrocoelium dendriticum hypothesized to drive summiting behavior in
infects ants and coerces them to climb to the top spongy moths partly by blocking the normal
of surrounding flora that, like Ophiocordyceps- occurrence of pupation: infected caterpillars con-
infected ants, they attach via biting (van Paridon tinue feeding on plants rather than returning to the
et al. 2017). Recent structural analysis of ground to undergo metamorphosis, leading them
D. dendriticum-infected ants revealed that at to be elevated at death. However, egt does not
least one of the infected host’s resident worms appear to be required to drive summiting behavior
consistently invades the host’s subesophageal in other NPV-larval systems (Ros et al. 2015). In
ganglion (SOG) (Martín-Vega et al. 2018). addition, various studies have shown that light
Among other functions, the SOG contains cues and perception thereof are required for
descending neurons, neurons that send informa- summiting, and that summiting hosts have aber-
tion from the brain to the ventral nerve cord (the rant expression of circadian genes (Han et al.
insect analog of the vertebrate spinal cord), that 2017; Bhattarai et al. 2018). All told, it seems
are important for coordinating motor output, such likely that there are several overlapping pathways
as walking (Hsu and Bhandawat 2016). The loca- involved in summit disease that vary even among
tion of the worm by itself is unlikely to drive a related viruses.
behavior as complex as summiting but does place
the worm in an ideal position for altering the
activity of host neurons either through chemical 12.2.3 Perimortem Postural Changes
or mechanical action. Notably, Ophiocordyceps
does not invade ant brain tissue (Fredericksen Many zombie fungus-infected insects exhibit
et al. 2017); however, like D. dendriticum, some perimortem postural changes ranging from the
species of zombie fungi (e.g., Entomophthora positioning of mouth parts to the splaying of
muscae, Entomophaga grylli, Pandora formicae, legs and wings. In cadaver-transmitting systems,
Strongwellsea castrans) have been observed to these postural changes typically occur after
occupy the host brain (Humber 1976; Brobyn summiting but before death. In Ophiocordyceps-
and Wilding 1977, 1983; Funk et al. 1993; Elya infected ants, for example, manipulated
et al. 2018; Csata et al. 2021). That brain occu- individuals maintain elevation by biting onto an
pancy is not a consistent feature of behavior- available substrate immediately prior to death
manipulating parasites likely reflects that there (Hughes et al. 2016). These animals exhibit lock-
are multiple routes (e.g., direct and indirect) to jaw, which recent work suggests may be achieved
modifying host behavior. by penetration of the muscles by fungal tissue as
As with parasite-induced hyperactivity, our well as the formation of a fungal cage-like struc-
most thorough understanding of a mechanism ture surrounding mandibular muscle
underlying parasite-elicited summit disease (Fredericksen et al. 2017; Mangold et al. 2019).
comes from baculoviruses. Several baculoviruses In Entomophthora-infected flies, a manipulated
drive summiting in their larval hosts immediately individual extends its proboscis and is then
before death, which is thought to facilitate the “glued” where standing via secretions that are
enhanced dispersal of viral particles (Vasconcelos thought to be produced by the fungi (Brobyn
et al. 1996). Elegant work in Lymantria dispar and Wilding 1983). Proboscis extension has
NPV determined that the gene ecdysteroid uridine been proposed to arise from mechanical force
4′-diphosphate glucosyltransferase (egt) was nec- (i.e., the body cavity has become so full that the
essary and sufficient to elicit summit disease in proboscis cannot remain contracted due to steric
spongy moths (Hoover et al. 2011). The enzyme hindrance) (Brobyn and Wilding 1983). More
encoded by egt catalyzes a reaction that leads to recent observations have reported that the probos-
the inactivation of the larval hormone cis both extends and retracts in the dying fly,
20-hydroxyecdysone (20E) blocking pupation as suggesting that proboscis movement could be
300 C. Elya

driven either by impingement upon underlying timing. For example, flies infected with
muscles or by directly or indirectly perturbing E. muscae only exhibit summit disease and
the action of motor neurons that directly innervate subsequent postural changes at sunset (Krasnoff
musculature and drive changes in contractility et al. 1995; Elya et al. 2018) and, in the field, as
(Elya et al. 2018). mentioned earlier, ants infected with
After summiting and adherence in place, zom- Ophiocordyceps summit and die around solar
bie flies and beetles exhibit a further postural noon (Hughes et al. 2011). How this timing is
change, positioning their wings up and away achieved is particularly curious as there are likely
from the dorsal abdomen (Krasnoff et al. 1995; two molecular clocks at play in each case—one
Steinkraus et al. 2017; Elya et al. 2018). The belonging to each the host and the fungus. Molec-
repositioning of the wings serves to create a direct ular clocks are cellular mechanisms that can
“line of fire” for the fungal spores that will be maintain circadian rhythms in the absence of
ejected from the dorsal abdomen. In the case of environmental cues, or Zeitgebers, such as light
flies, wing-raising is rapid, typically fully or temperature. Clocks synchronize, or entrain, to
executed in 15 min (Krasnoff et al. 1995). environmental signals to align with the earth’s
Wing-raising in zombie flies seems that it could daily cycles and are usually driven by core tran-
possibly arise by several different mechanisms. scription/translation feedback loops. Clocks have
The fungus could impinge on musculature been demonstrated in organisms ranging from
directly as it grows in the body cavity or could bacteria to animals, though their core molecular
indirectly alter posture by rapid uptake of components vary along mostly phylogenetic lines
remaining liquid (dehydration). Alternatively, (Dunlap and Loros 2017). Details of how timing
the fungus could alter the activity of motor from the clock confers various behaviors and
neurons, either directly or indirectly. In the case phenotypes are continuously emerging.
of Eryniopsis lampyridarum-infected beetles with In the case of E. muscae-infected flies, the
wings that take many hours to raise, a mechanical favored explanation of how this timing is
explanation is the more likely. achieved is that it is not dictated by the host
Similar postural changes have been observed clock. This is derived from the two observations.
in insects parasitized by non-fungal organisms. First, flies infected with E. muscae in constant
Ants infected by D. dendriticum also bite down darkness (referred to as “free-running
to maintain an elevated position. These ants do conditions”) do not die with rhythmic timing,
not die in place, but instead remain motionless in even though their fly hosts are known to maintain
situ for several days (Martín-Vega et al. 2018). circadian periodicity in the absence of environ-
This behavior is thought to benefit the trematode mental cues (Krasnoff et al. 1995). This suggests
by making infected ants more likely to be con- that the host clock alone is insufficient to drive the
sumed by their grazing herbivores, which can timing of death. Second, flies housed in darkness
then spread the parasite via droppings back to 72 h after fungal exposure do show rhythmic
the primary snail host (Goater and Colwell timing of death, which suggests that there is
2007). A mechanistic understanding of biting some machinery present capable of driving
behavior in D. dendriticum-infected ants is timed death in the absence of immediate cues,
lacking, although the presence of flukes in the but that this machinery can only entrain after the
SOG may play a role (Martín-Vega et al. 2018). onset of infection (Krasnoff et al. 1995). The most
likely identity of this mystery machinery is a
fungal clock. Though it is very likely that
12.2.4 Circadian Timing E. muscae has a clock of its own, this has not
yet been experimentally demonstrated.
An extremely common theme among zombie On the other hand, the prevailing hypothesis
fungi is that they drive changes in host behavior for timing in Ophiocordyceps-infected ants is that
(and ultimately cause death) with stereotyped this is driven by the host clock, which in turn is
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 301

manipulated by the fungus (de Bekker and Das leading to alterations in sexual behavior. At pres-
2022). Transcriptomic studies have shown that ent, the mechanistic basis of this manipulation is
the expression of various clock genes are unknown.
perturbed at the moment of manipulation Entomophthora muscae, on the other hand,
(de Bekker et al. 2015; Will et al. 2020) and that enhances transmission by appealing to healthy
foraging rhythms observed in healthy ants are hosts (Moller 1993; Watson and Petersen 1993;
essentially absent in infected individuals (Trinh Zurek et al. 2002; Naundrup et al. 2021). Male
et al. 2021). The observation that Ophiocordyceps house flies show a remarkable attraction to late-
continues to produce transcripts in a circadian stage female cadavers, an effect that appears to be
manner under free-running conditions strongly mediated by the combined effects of fungal-
supports the notion that this fungus has its own produced compounds (notably sesquiterpenes),
molecular timekeeping mechanism (de Bekker visual cues related to the characteristic death
et al. 2017b). Details as to how fungal clock pose as well as endogenous female pheromones
activity is linked to host behavior are lacking, (Naundrup et al. 2022). Here, there is no host
but it seems reasonable to hypothesize that host fitness benefit from attracting mates, as the host
behavior might be altered by the secretion of is dead, strongly arguing that, again, this is a
compounds that alter host physiology, ranging fungus-driven manipulation rather than a host
from specifically in neuronal populations to response to infection. Overall, this work is con-
more generally to trigger internal state changes, sistent with many previous observations of fungi
leading to behavioral outputs. employing chemical mimicry to attract hosts, to
serve as either transmission vectors or prey
(Ngugi and Scherm 2006; Matsuura et al. 2009;
12.2.5 Sexual Attraction Hsueh et al. 2017).
Insect sexual behaviors have also been
Some zombie fungi have been observed to alter observed to change in the presence of
host sexual behavior, a change that is non-fungal parasites, though we have little insight
hypothesized to increase the likelihood of trans- into their mechanistic underpinnings. One likely
mission. In the case of Massospora, the fungus manipulated behavior occurs in corn earworm
appears to alter sexual behaviors in infected female moths infected with the Hz-2V virus
animals: infected male cicadas produce female- with a primary site of replication in reproductive
like wing flicks (Cooley et al. 2018) or tolerate tissues (Burand et al. 2005). These females pro-
physical contact by male conspecifics (Cooley duce up to seven times the amount of pheromones
1999), behaviors that do not occur in healthy made by uninfected females and exhibit hyper-
males. An increase in sexual behaviors could be sexual behaviors, continuing to initiate contact
an attempt by the host to increase reproductive with males and resume calling to attract male
output before death, but, curiously, these conspecifics after mating, behaviors which are
behaviors are only observed in Massospora- typically not observed in healthy individuals
infected males that actively transmit to another (Burand et al. 2005). In addition, healthy females
round of hosts and not in infected males that that mated with infected males were more likely
produce resting spores (Cooley et al. 2018). This to resume calling after copulation than if they
observation suggests that hypersexuality is driven mated with uninfected males (Burand and Tan
by the fungus rather than the host. Increased 2006), suggesting that there is some alteration to
sexual behavior could be related to fungal pro- male reproductive chemistry to dampen normal
duction of cathinone (Boyce et al. 2019), as female refractory period post-mating. As copula-
amphetamine intake has been associated with tion requires close physical contact with a partner,
increased sexual behaviors in other animals these changes in female pheromone production
(Frohmader et al. 2010). Alternatively, the fungus and sexual behavior are likely to drive increased
could modulate hormonal levels in the host transmission of the virus to male partners and
302 C. Elya

their subsequent consorts. However, it is possible adaptive advantage to the parasite and is specifi-
that these effects could be driven by the host in cally elicited as a result of infection by that para-
response to infection as an attempt to increase site, not just a general sickness response of the
reproductive output before sterilization takes host. In general, it is wise to remain skeptical that
hold. That the host may be driving these changes a behavior reflects manipulation unless there is
might be supported by the observation that infec- overwhelming evidence to suggest this is the
tion by another virus, Gonad-specific virus case. As there are already great pieces on this
(GSV), also leads to increased pheromonal pro- topic (Poulin 1995; Bhattarai et al. 2021), it will
duction (Raina et al. 2000), although, unlike not be fully discussed here.
Hz-2 V females, GSV-infected females refuse to If all signs point to a behavior being truly the
mate (Raina et al. 2000). There are other instances result of parasitic manipulation, it is important to
of infection by parasites being correlated with check our innate tendencies to ascribe agency or
increased sexual behavior (milkweed beetle intent of the hijacking organism, and to keep in
Labidomera clivicollis and mite Chrysomelboai mind that all these organisms developed their
labidomerae, mite Unionicola ypsilophora and present methods of host behavior manipulation
midge Paratrichocladius rufiventris, cricket following broad evolutionary principles. That is,
Gryllus texensis and iridovirus IIV-6/CrIV while it may be our impulse to look at a behavior
(McLACHLAN 1999; Abbot and Dill 2001; hijacking system and be initially struck by its
Adamo et al. 2014)), but, again, it is not yet seeming cleverness or insidiousness, we should
clear which organisms are driving these changes. resist the urge to embrace complex hypotheses
The argument has been made that any increase in explaining manipulated behaviors in favor of con-
sexual behavior only benefits the parasite, since sidering simpler ones first. While chemical sig-
these parasites induce sterility in their hosts. naling may underlie a behavior in some cases,
However, I do not think we can formally exclude mechanical forces and physical limitations of the
the possibility that these sterile hosts fail to “rec- fungus and animal can be just as crucial in driving
ognize” their impotency and are driving their own behavior. Along these lines, a priori reasoning
futile attempts to reproduce. predicts that manipulated behaviors are most
likely to occur by ectopic activation of existing
host circuitry, not by cobbling together novel
12.3 General Considerations circuits. Importantly, all the behaviors that a
for Understanding manipulated host exhibits are things the host can
Manipulated Behaviors already do—for example, a fly can climb, an ant
can bite, a cicada can mate. The fungus is not
The five themes I introduced early in this chapter causing new behaviors to occur but altering the
(see Common themes in behavioral manipula- timing of these behaviors to align with promoting
tion by zombie fungi) encapsulate many of the the fitness of the fungus rather than the host.
behaviors elicited by zombie fungi, but they are In addition to allowing ourselves to be guided
by no means an exhaustive list. In addition, there by parsimony, it is also important to avoid assum-
are likely many more parasitic puppeteers to be ing that just because two behaviors look alike that
described in our world, and more manipulated they are driven by the same or similar underlying
behaviors out there to be discovered. However, mechanisms (de Bekker et al. 2021). That is,
as we continue to broaden our understanding in parallel behaviors do not necessarily reflect paral-
this realm, it is critical to be guided by parsimony. lel pathways. This does not mean to suggest that
Just because a behavior occurs in an infected each parasite uses a completely different mecha-
animal and has some apparent level of sophistica- nism to drive similar behavior, but it means that
tion, we should not assume that this behavior is we cannot take for granted that just because we
manipulated by the pathogen. At a minimum, a have figured out a mechanism in one system that
truly manipulated behavior is one that confers an it will be generally applicable to others. Behavior
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 303

manipulations that look the same but are driven challenging for many reasons, the future of the
by different parasites probably reflect one of two field is brighter than ever before.
possibilities: either these behaviors are so advan- Historically, one of the main impediments to
tageous to the pathogen that they are heavily our mechanistic understanding of the behaviors
selected for, or these behaviors are relatively elicited by zombie fungi has been the lack of
easy to elicit (either there are many ways to experimentally tractable systems. However, nota-
drive the same behavior, so there are many local ble progress has been made on at least two fronts:
minima that a parasite might stumble across both Ophiocordyceps and Entomophthora
and/or the behavior is governed by readily manip- muscae infections can now take place entirely in
ulable circuit elements). Understanding the mech- a laboratory setting (Elya et al. 2018; Will et al.
anistic underpinnings of similar behaviors in 2020). A recently isolated strain of E. muscae
different systems can help us differentiate infects the fruit fly Drosophila melanogaster
between these possibilities. As much as possible, (Elya et al. 2018), an insect that has been exten-
research efforts should be geared toward parallel sively studied and developed as a model organism
progress in multiple systems to allow for this to study neurobiology and behavior. In the
comparative analysis. E. muscae-fruit fly system, researchers can lever-
Finally, as we pursue a deeper understanding age the D. melanogaster genetic toolkit address a
of the mechanistic basis of parasite manipulated virtually endless number of hypotheses, including
behaviors, it’s important to keep in mind that whether gravitactic or phototactic pathways are
these behaviors are likely to be the product of involved in summiting behavior and whether the
alterations in many parallel pathways or many fly’s clock is involved in determining the time of
systems targeted simultaneously (Adamo 2013). its death. In addition, the universal genomic
That is, while it is tempting to hypothesize simple editing tool CRISPR-Cas9 holds great promise
feed-forward mechanisms, evolution is not a lin- to be applied to lab-grown organisms, such as
ear process, and the robustness of zombified E. muscae and Ophiocordyceps, to test the causal
behaviors is very likely a reflection of redundancy role of fungal genes in myriad host–pathogen
in their mechanisms of action. We may find interactions, including manipulated behaviors.
examples where a single gene appears to have a Taking advantage of these laboratory systems
large effect on behavior [e.g., egt in baculovirus will be key in demonstrating causal relationships
(Hoover et al. 2011)], but it is very likely that between host and fungal genotypes and behav-
what remains to be discovered are smaller pieces ioral phenotype and allow us to improve our
of the whole manipulation story. Ultimately, understanding in ways that simply were not pos-
though this may be a more challenging course to sible before.
understanding these compelling phenomena, per- Even for systems that are not yet laboratory
haps it will be more satisfying when we finally ready, the ongoing improvement of existing and
crack the case. development of new technologies has opened
research avenues previously inaccessible. For
example, long read and chromatin conformation
12.4 Onward and Upward: capture sequencing is enabling the assembly of
Prospects in Understanding bloated, repeat-rich entomophthoralean genomes
Behaviors Driven by (Hajek et al. 2022), and improved metabolomics
Zombie Fungi detection technology recently allowed the discov-
ery of mind-altering compounds in Massospora-
Despite our keen interest and immense efforts, we infected cicadas (Boyce et al. 2019). In addition,
still have a long way to go toward fully under- improved imaging methods have offered insight
standing how zombie fungi alter the behavior of into the structure of fungal and host tissues
their hosts. Although demonstrating the mecha- (Fredericksen et al. 2017) and offer the future
nistic basis of manipulated behavior has proved discovery of morphological correlates of
304 C. Elya

parasitized insects through additional structural how behavior is generated and executed in
analyses. The recent discoveries in the field have animals.
in turn generated a level of excitement that seems
likely to translate to expansion of the zombie
fungus field. With additional brains and hands References
working together on these systems, it seems inev-
itable that we will encounter additional culturing Abbot P, Dill LM (2001) Sexually transmitted parasites
and sexual selection in the milkweed leaf beetle,
breakthroughs to bring new systems into the lab-
Labidomera clivicollis. Oikos 92:91–100. https://doi.
oratory. And so, our efforts will continue to feed org/10.1034/j.1600-0706.2001.920111.x
forward. Adamo SA (2013) Parasites: evolution’s neurobiologists. J
Another challenge within the realm of behav- Exp Biol 216:3–10. https://doi.org/10.1242/jeb.
073601
ior manipulation has been the difficulty of observ-
Adamo SA, Kovalko I, Easy RH, Stoltz D (2014) A viral
ing and analyzing zombified behaviors aphrodisiac in the cricket Gryllus texensis. J Exp Biol
efficiently. Many of these behaviors are not only 217:1970–1976. https://doi.org/10.1242/jeb.103408
subtle but also occur clustered in daily bursts, Andriolli FS, Ishikawa NK, Vargas-Isla R et al (2019) Do
zombie ant fungi turn their hosts into light seekers?
making them challenging to study by traditional
Behav Ecol 30:609–616. https://doi.org/10.1093/
analog ethological methods. The widespread use beheco/ary198
of computational methods to track and analyze Araújo JPM, Hughes DP (2016) Diversity of
animal behavior offers a huge opportunity to help entomopathogenic fungi: which groups conquered the
insect body? Adv Genet 94:1–39. https://doi.org/10.
move our understanding forward. For
1016/bs.adgen.2016.01.001
lab-tractable zombie systems, automated ethol- Arvidson R, Kaiser M, Lee SS et al (2019) Parasitoid jewel
ogy can help us with establishing objective wasp mounts multipronged neurochemical attack to
criteria for staging organisms, which can allow hijack a host brain. Mol Cell Proteomics 18:99–114.
https://doi.org/10.1074/mcp.RA118.000908
us to perform comparative analyses of Bhattarai UR, Li F, Katuwal Bhattarai M et al (2018)
metabolites and gene expression and allow us to Phototransduction and circadian entrainment are the
run behavioral screens under different abiotic key pathways in the signaling mechanism for the
conditions (in non-model hosts) or in various baculovirus induced tree-top disease in the lepidop-
teran larvae. Sci Rep 8:17528. https://doi.org/10.
host genotypes (in model hosts). Even for
1038/s41598-018-35885-4
systems that are not yet lab-tractable, we can Bhattarai UR, Doherty J-F, Dowle E, Gemmell NJ (2021)
marry ethological platforms with field work to The adaptiveness of host behavioural manipulation
expand our understanding of typical host behav- assessed using Tinbergen’s four questions. Trends
Parasitol. https://doi.org/10.1016/j.pt.2021.01.006
ior (e.g., better understand circadian patterns) and Bhunjun C, Senanayake I, Hyde KD, et al (eds) (2022)
test specific mechanistic hypotheses (e.g., does Numbers of fungal species. Fungal Divers 114
the application of cathinone or psilocybin to Boyce GR, Gluck-Thaler E, Slot JC et al (2019) Psycho-
healthy cicadas recapitulate either of the locomo- active plant- and mushroom-associated alkaloids from
two behavior modifying cicada pathogens. Fungal Ecol
tion or mating phenotypes observed in 41:147–164. https://doi.org/10.1016/j.funeco.2019.
Massospora-infected individuals?) We have 06.002
already begun to see the promise of these Brobyn PJ, Wilding N (1977) Invasive and developmental
approaches in the Ophiocordyceps-ant system processes of Entomophthora species infecting aphids.
Trans Br Mycol Soc 69:349–366. https://doi.org/10.
(Trinh et al. 2021). 1016/S0007-1536(77)80072-7
Ultimately, the prospect of understanding how Brobyn PJ, Wilding N (1983) Invasive and developmental
zombie fungi elicit behavioral changes offers processes of Entomophthora muscae infecting
much more than just satiating scientific curiosity. houseflies (Musca domestica). Trans Br Mycol Soc
80:1–8. https://doi.org/10.1016/S0007-1536(83)
Taking advantage of “zombie” insects as model 80157-0
systems (i.e., studying hijacked behaviors) will Browne LB, Evans DR (1960) Locomotor activity of the
serve to complement traditional neuroscience blowfly as a function of feeding and starvation. J Insect
approaches (i.e., studying intrinsic behaviors) Physiol 4:27–37. https://doi.org/10.1016/0022-1910
(60)90065-2
and will serve to enrich our understanding of
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 305

Burand JP, Tan W (2006) Mate preference and mating kimflemingiae. PLoS One 12:e0187170. https://doi.
behavior of male Helicoverpa zea (Lepidoptera: org/10.1371/journal.pone.0187170
Noctuidae) infected with the sexually transmitted de Bekker C, Beckerson WC, Elya C (2021) Mechanisms
insect virus Hz-2V. Ann Entomol Soc Am 99:969– behind the madness: how do zombie-making fungal
973. https://doi.org/10.1603/0013-8746(2006)99[969: entomopathogens affect host behavior to increase
MPAMBO]2.0.CO;2 transmission? MBio 12:e01872–e01821. https://doi.
Burand JP, Tan W, Kim W et al (2005) Infection with the org/10.1128/mBio.01872-21
insect virus Hz-2v alters mating behavior and phero- Dunlap JC, Loros JJ (2017) Making time: conservation of
mone production in female Helicoverpa zea moths. J biological clocks from fungi to animals. Microbiol
Insect Sci 5:6. https://doi.org/10.1093/jis/5.1.6 Spectr 5. https://doi.org/10.1128/microbiolspec.
Carruthers RI, Ramos ME, Larkin TS et al (1997) The FUNK-0039-2016
Entomophaga Grylli (Fresenius) Batko species com- Dustan AG (1924) Studies on a new species of empusa
plex: its biology, ecology, and use for biological con- parasitic on the green apple bug (Lygus communis var.
trol of pest grasshoppers1. Mem Entomol Soc Can novascotiensis Knight) in the Annapolis Valley. Acad
129:329–353. https://doi.org/10.4039/ Entomol Soc, pp 14–36
entm129171329-1 Elya C, Lok TC, Spencer QE et al (2018) Robust manipu-
Chung T-Y, Sun P-F, Kuo J-I et al (2017) Zombie ant lation of the behavior of Drosophila melanogaster by a
heads are oriented relative to solar cues. Fungal Ecol fungal pathogen in the laboratory. elife 7. https://doi.
25:22–28. https://doi.org/10.1016/j.funeco.2016. org/10.7554/eLife.34414
10.003 Evans HC, Elliot SL, Hughes DP (2011) Hidden diversity
Cohn F (1855) Empusa muscae und die Krankheit der behind the zombie-ant fungus Ophiocordyceps
Stubenfliegen. Ein Beitrag zur Lehre von den durch unilateralis: four new species described from carpenter
parasitische Pilze charakterisierten Epidemien. Nova ants in Minas Gerais, Brazil. PLoS One 6:e17024.
acta Academiae Caesareae Leopoldino-Carolinae https://doi.org/10.1371/journal.pone.0017024
Germanicae Naturae Curiosorum 25:299–360 Fawcett W (1886) XXXII.—an entomogenous fungus.
Cooley JR (1999) Sexual behavior in North American Ann Mag Nat Hist 18:316–318. https://doi.org/10.
cicadas of the genera Magicicada and Okanagana. 1080/00222938609459974
PhD, University of Michigan Fredericksen MA, Zhang Y, Hazen ML et al (2017) Three-
Cooley JR, Marshall DC, Hill KBR (2018) A specialized dimensional visualization and a deep-learning model
fungal parasite (Massospora cicadina) hijacks the sex- reveal complex fungal parasite networks in behavior-
ual signals of periodical cicadas (Hemiptera: ally manipulated ants. Proc Natl Acad Sci U S
Cicadidae: Magicicada). Sci Rep 8:1432. https://doi. A. https://doi.org/10.1073/pnas.1711673114
org/10.1038/s41598-018-19813-0 Frohmader KS, Wiskerke J, Wise RA et al (2010) Meth-
Csata E, Billen J, Barbu-Tudoran L, Markó B (2021) amphetamine acts on subpopulations of neurons
Inside Pandora’s box: development of the lethal regulating sexual behavior in male rats. Neuroscience
myrmecopathogenic fungus Pandora formicae within 166:771–784. https://doi.org/10.1016/j.neuroscience.
its ant host. Fungal Ecol 50:101022. https://doi.org/10. 2009.12.070
1016/j.funeco.2020.101022 Funk CJ, Ramoska WA, Bechtel DB (1993) Histopathol-
de Bekker C, Das B (2022) Hijacking time: How ogy of Entomophaga grylli Pathotype 2 Infections in
Ophiocordyceps fungi could be using ant host clocks Melanoplus differentialis. J Invertebr Pathol 61:196–
to manipulate behavior. Parasite Immunol e12909. 202. https://doi.org/10.1006/jipa.1993.1035
https://doi.org/10.1111/pim.12909 Goater CP, Colwell DD (2007) Epidemiological
de Bekker C, Quevillon LE, Smith PB et al (2014) characteristics of an invading parasite: Dicrocoelium
Species-specific ant brain manipulation by a dendriticum in sympatric wapiti and beef cattle in
specialized fungal parasite. BMC Evol Biol 14:166. southern Alberta, Canada. J Parasitol 93:491–494.
https://doi.org/10.1186/s12862-014-0166-3 https://doi.org/10.1645/GE-1060R.1
de Bekker C, Ohm RA, Loreto RG et al (2015) Gene Grimaldi D, Engel MS, Engel MS, Senior Curator and
expression during zombie ant biting behavior reflects Professor Michael S Engel (2005) Evolution of the
the complexity underlying fungal parasitic behavioral Insects. Cambridge University Press
manipulation. BMC Genomics 16:620. https://doi.org/ Hajek A, Gryganskyi A, Stajich J (2022) Entomophaga
10.1186/s12864-015-1812-x maimaiga ARSEF 7190 v1.0. https://mycocosm.jgi.
de Bekker C, Ohm RA, Evans HC et al (2017a) doe.gov/Entmai1/Entmai1.info.html. Accessed
Ant-infecting Ophiocordyceps genomes reveal a high 12 Aug 2022
diversity of potential behavioral manipulation genes Han Y, van Houte S, van Oers MM, Ros VID (2017)
and a possible major role for enterotoxins. Sci Rep 7: Timely trigger of caterpillar zombie behaviour: tempo-
12508. https://doi.org/10.1038/s41598-017-12863-w ral requirements for light in baculovirus-induced tree-
de Bekker C, Will I, Hughes DP et al (2017b) Daily top disease. Parasitology 1–6. https://doi.org/10.1017/
rhythms and enrichment patterns in the transcriptome S0031182017001822
of the behavior-manipulating parasite Ophiocordyceps
306 C. Elya

Haspel G, Rosenberg LA, Libersat F (2003) Direct injec- Entomophthora muscae on its host Musca domestica:
tion of venom by a predatory wasp into cockroach Postural changes in dying hosts and gated pattern of
brain. J Neurobiol 56:287–292. https://doi.org/10. mortality. J Insect Physiol 41:895–903. https://doi.org/
1002/neu.10238 10.1016/0022-1910(95)00026-Q
Hawksworth DL, Lücking R (2017) Fungal diversity Langley L (2018) These zombie flies are being mind-
revisited: 2.2 to 3.8 million species. Microbiol Spectr controlled by a fungus. Natl Geogr
5. https://doi.org/10.1128/microbiolspec.FUNK- Leonard DE (1970) Effects of starvation on behaviour,
0052-2016 number of larval instars, and developmental rate of
Herbison R, Evans S, Doherty J-F et al (2019) A Porthetria dispar. J Insect Physiol 16:25–31. https://
molecular war: convergent and ontogenetic evidence doi.org/10.1016/0022-1910(70)90109-5
for adaptive host manipulation in related parasites Loreto RG, Araújo JPM, Kepler RM et al (2018) Evidence
infecting divergent hosts. Proc Biol Sci 286: for convergent evolution of host parasitic manipulation
20191827. https://doi.org/10.1098/rspb.2019.1827 in response to environmental conditions: convergent
Hofmann O (1891) Insektentotende Pilze mit besonderer evolution of host manipulation. Evolution 174:424.
Berucksichtigung der Nonne. Zeitschrift fur https://doi.org/10.1111/evo.13489
Naturwissenschaften 64:384–385 Lovett B, Macias A, Stajich JE et al (2020) Behavioral
Hoover K, Grove M, Gardner M et al (2011) A gene for an betrayal: how select fungal parasites enlist living
extended phenotype. Science 333:1401. https://doi. insects to do their bidding. PLoS Pathog 16:
org/10.1126/science.1209199 e1008598. https://doi.org/10.1371/journal.ppat.
Hsu CT, Bhandawat V (2016) Organization of descending 1008598
neurons in Drosophila melanogaster. Sci Rep 6:20259. Lu J (2019) How a parasitic fungus turns ants into
https://doi.org/10.1038/srep20259 “zombies.” National Geographic
Hsueh Y-P, Gronquist MR, Schwarz EM et al (2017) Mangold CA, Ishler MJ, Loreto RG et al (2019) Zombie
Nematophagous fungus Arthrobotrys oligospora ant death grip due to hypercontracted mandibular
mimics olfactory cues of sex and food to lure its muscles. J Exp Biol 222. https://doi.org/10.1242/jeb.
nematode prey. elife 6. https://doi.org/10.7554/eLife. 200683
20023 Martín-Vega D, Garbout A, Ahmed F et al (2018) 3D
Hughes B (2011) Conquer or die!: Wellington’s Veterans virtual histology at the host/parasite interface:
and the liberation of the new world. Bloomsbury visualisation of the master manipulator, Dicrocoelium
Publishing dendriticum, in the brain of its ant host. Sci Rep 8:
Hughes DP, Andersen SB, Hywel-Jones NL et al (2011) 8587. https://doi.org/10.1038/s41598-018-26977-2
Behavioral mechanisms and morphological symptoms Matsuura K, Yashiro T, Shimizu K et al (2009) Cuckoo
of zombie ants dying from fungal infection. BMC Ecol fungus mimics termite eggs by producing the
11:13. https://doi.org/10.1186/1472-6785-11-13 cellulose-digesting enzyme beta-glucosidase. Curr
Hughes DP, Araújo J, Loreto R et al (2016) From so Biol 19:30–36. https://doi.org/10.1016/j.cub.2008.
simple a beginning: the evolution of behavioral manip- 11.030
ulation by fungi. In: Advances in genetics. Academic McLACHLAN A (1999) Parasites promote mating suc-
Humber RA (1976) The systematics of the genus cess: the case of a midge and a mite. Anim Behav 57:
Strongwellsea (Zygomycetes: Entomophthorales). 1199–1205. https://doi.org/10.1006/anbe.1999.1087
Mycologia 68:1042–1060 Milner RJ, Holdom DG, Glare TR (1984) Diurnal patterns
Iati M (2021) A fungus could turn some cicadas into of mortality in aphids infected by entomophthoran
sex-crazed “salt shakers of death.” The Washington fungi. Entomol Exp Appl 36:37–42. https://doi.org/
Post 10.1111/j.1570-7458.1984.tb03404.x
Kamita SG, Nagasaka K, Chua JW et al (2005) A Moller AP (1993) A fungus infecting domestic flies
baculovirus-encoded protein tyrosine phosphatase manipulates sexual behaviour of its host. Behav Ecol
gene induces enhanced locomotory activity in a lepi- Sociobiol 33. https://doi.org/10.1007/bf00170255
dopteran host. Proc Natl Acad Sci U S A 102:2584– Naundrup A, Bohman B, Kwadha C et al (2021) A patho-
2589. https://doi.org/10.1073/pnas.0409457102 genic fungus uses volatiles to entice male flies into fatal
Kim K-T, Jeon J, Choi J et al (2016) Kingdom-wide matings with infected female cadavers.
analysis of fungal small secreted proteins (SSPs) bioRxiv:2021.10.21.465334
reveals their potential role in host association. Front Naundrup A, Bohman B, Kwadha CA et al (2022) Patho-
Plant Sci 7:186. https://doi.org/10.3389/fpls.2016. genic fungus uses volatiles to entice male flies into fatal
00186 matings with infected female cadavers. ISME J 16:
Kokusho R, Katsuma S (2021) Bombyx mori 2388–2397. https://doi.org/10.1038/s41396-022-
nucleopolyhedrovirus ptp and egt genes are dispens- 01284-x
able for triggering enhanced locomotory activity and Ngugi HK, Scherm H (2006) Mimicry in plant-parasitic
climbing behavior in Bombyx mandarina larvae. J fungi. FEMS Microbiol Lett 257:171–176. https://doi.
Invertebr Pathol 107604. https://doi.org/10.1016/j.jip. org/10.1111/j.1574-6968.2006.00168.x
2021.107604 Nielsen C, Hajek AE (2006) Diurnal pattern of death and
Krasnoff SB, Watson DW, Gibson DM, Kwan EC (1995) sporulation in Entomophaga maimaiga-infected
Behavioral effects of the entomopathogenic fungus,
12 When a Mind Is Not Its Own: Mechanisms of Host Behavior Control by Parasitic Fungi 307

Lymantria dispar. Entomol Exp Appl 118:237–243. Entomol 63:31–45. https://doi.org/10.1146/annurev-

https://doi.org/10.1111/j.1570-7458.2006.00382.x ento-020117-043348
Nordio S, Kaiser M, Adams ME, Libersat F (2022) Para- Thaxter R (1888) The entomophthoraeae of the United
sitoid wasp venom manipulates host innate behavior States
via subtype-specific dopamine receptor activation. J Trinh T, Ouellete R, de Bekker C (2021) Getting lost: the
Exp Biol 225. https://doi.org/10.1242/jeb.243674 fungal hijacking of ant foraging behaviour in space and
Nuwer R (By Rachel Nuwer on May 27 2021) How this time. Anim Behav 181:165–184
zombie fungus turns cicadas into horror-movie sex Valdes JJ, Cameron JE, Cole RJ (1985) Aflatrem: a
bots. Scientific American tremorgenic mycotoxin with acute neurotoxic effects.
O’Reilly DR, Miller LK (1989) A baculovirus blocks Environ Health Perspect 62:459–463. https://doi.org/
insect molting by producing ecdysteroid 10.1289/ehp.8562459
UDP-glucosyl transferase. Science 245:1110–1112. van Houte S, Ros VID, Mastenbroek TG et al (2012)
https://doi.org/10.1126/science.2505387 Protein tyrosine phosphatase-induced hyperactivity is
Ortiz-Urquiza A (2021) The split personality of Beauveria a conserved strategy of a subset of baculoviruses to
bassiana: understanding the molecular basis of fungal manipulate lepidopteran host behavior. PLoS One 7:
parasitism and mutualism. mSystems 6:e0076621. e46933. https://doi.org/10.1371/journal.pone.0046933
https://doi.org/10.1128/mSystems.00766-21 van Paridon BJ, Gilleard JS, Colwell DD, Goater CP
Pickford R, Riegert PW (1964) The fungous disease (2017) Life cycle, host utilization, and ecological fit-
caused by Entomophthora grylli Fres., and its effects ting for invasive lancet liver fluke, Dicrocoelium
on grasshopper populations in Saskatchewan in 1963. dendriticum, emerging in Southern Alberta, Canada. J
Can Entomol 96:1158–1166 Parasitol 103:207–212. https://doi.org/10.1645/16-140
Poulin R (1995) “Adaptive” changes in the behaviour of Vasconcelos SD, Cory JS, Wilson KR et al (1996)
parasitized animals: a critical review. Int J Parasitol 25: Modified behavior in Baculovirus-infected Lepidop-
1371–1383. https://doi.org/10.1016/0020-7519(95) teran larvae and its impact on the spatial distribution
00100-x of inoculum. Biol Control 7:299–306. https://doi.org/
Raina AK, Adams JR, Lupiani B et al (2000) Further 10.1006/bcon.1996.0098
characterization of the gonad-specific virus of corn Watson DW, Petersen JJ (1993) Sexual activity of male
earworm, Helicoverpa zea. J Invertebr Pathol 76:6– musca domestica (Diptera: Muscidae) infected with
12. https://doi.org/10.1006/jipa.2000.4942 Entomophthora muscae (Entomophthoraceae:
Randrup A, Munkvad I (1967) Stereotyped activities pro- Entomophthorales). Biol Control 3:22–26. https://doi.
duced by amphetamine in several animal species and org/10.1006/bcon.1993.1004
man. Psychopharmacologia 11:300–310. https://doi. Wheater CP (1991) Effect of starvation on locomotor
org/10.1007/BF00404607 activity in some predacious Coleoptera (Carabidae,
Ros VID, van Houte S, Hemerik L, van Oers MM (2015) Staphylinidae). Coleopt Bull 45:371–378
Baculovirus-induced tree-top disease: how extended is Will I, Das B, Trinh T, et al (2020) Genetic underpinnings
the role of egt as a gene for the extended phenotype? of host manipulation by Ophiocordyceps as revealed
Mol Ecol 24:249–258. https://doi.org/10.1111/mec. by comparative transcriptomics. G3: Genes, Genomes,
13019 Genetics 2020.01.03.893917
Sakolrak B, Blatrix R, Sangwanit U, Kobmoo N (2018) Wu B, Hussain M, Zhang W et al (2019) Current insights
Experimental infection of the ant Polyrhachis furcata into fungal species diversity and perspective on nam-
with Ophiocordyceps reveals specificity of behavioural ing the environmental DNA sequences of fungi.
manipulation. Fungal Ecol 33:122–124. https://doi. Mycology 10:127–140. https://doi.org/10.1080/
org/10.1016/j.funeco.2018.03.001 21501203.2019.1614106
Spatafora JW, Chang Y, Benny GL et al (2016) A phylum- Yang Z, Yu Y, Zhang V et al (2015) Octopamine mediates
level phylogenetic classification of zygomycete fungi starvation-induced hyperactivity in adult Drosophila.
based on genome-scale data. Mycologia 108:1028– Proc Natl Acad Sci U S A. https://doi.org/10.1073/
1046. https://doi.org/10.3852/16-042 pnas.1417838112
St Leger RJ, Wang JB (2020) Metarhizium: jack of all Yong E (2017) How the zombie fungus takes over ants’
trades, master of many. Open Biol 10:200307. https:// bodies to control their minds. The Atlantic
doi.org/10.1098/rsob.200307 Zimmer C (2019) After this fungus turns ants into
Steinkraus DC, Hajek AE, Liebherr JK (2017) Zombie zombies, their bodies explode. The New York Times
soldier beetles: epizootics in the goldenrod soldier Zurek L, Wes Watson D, Krasnoff SB, Schal C (2002)
beetle, Chauliognathus pensylvanicus (Coleoptera: Effect of the entomopathogenic fungus,
Cantharidae) caused by Eryniopsis lampyridarum Entomophthora muscae (Zygomycetes:
(Entomophthoromycotina: Entomophthoraceae). J Entomophthoraceae), on sex pheromone and other
Invertebr Pathol 148:51–59. https://doi.org/10.1016/j. cuticular hydrocarbons of the house fly, Musca
jip.2017.05.002 domestica. J Invertebr Pathol 80:171–176
Stork NE (2018) How many species of insects and other
terrestrial arthropods are there on earth? Annu Rev
 Genetics and Infection Biology
of the Entomopathogenic Fungi 13
Song Hong, Junmei Shang, Yanlei Sun, and Chengshu Wang

Abstract especially in terms of the similar expansion

Insects and fungi, both megadiverse groups of protease and chitinase gene families in
of organisms, have a long history of EPF for targeting the protein- and chitin-rich
associations with each other. Among the insect cuticles. Functional genetic studies have
fungi, Hypocreales (Ascomycota) and unveiled an array of EPF genes involved in
Entomophthoromycotina (Zoopagomycota), mediating host recognition and interactions
contain the most common pathogens of with insect hosts, especially mechanisms of
insects. Ascomycete entomopathogenic fungi invading or evading host immunity to facilitate
(EPF) are mostly well-studied and have been fungal colonization of insect body cavities.
found to be either heterothallically or The discovery of small molecules and their
homothallically sexual with a divergent num- essential roles in mediating EPF associations
ber of genes at the mating-type loci of different with insect hosts also have been advanced. In
species. So far more than 100 strains of >60 addition to advancing the mechanisms of
EPF species have been genome sequenced fungus–insect interactions, these works highly
which demonstrated substantial variations in benefit the development of cost-effective
genome size and gene content among each mycoinsecticides and the protection of
other. However, convergent evolution of fun- ecologically and economically important ben-
gal entomopathogenicity has been coined, eficial insects.

Keywords
S. Hong · J. Shang · Y. Sun
Key Laboratory of Insect Developmental and Entomopathogenic fungi · Genome variation ·
Evolutionary Biology, CAS Center for Excellence in Divergent sexuality · Convergent evolution ·
Molecular Plant Sciences, Shanghai Institute of Plant Insect-host specificity · Cuticle penetration ·
Physiology and Ecology, Chinese Academy of Sciences, Hemocoel colonization · Chemical biology
Shanghai, China
C. Wang (✉)
Key Laboratory of Insect Developmental and
Evolutionary Biology, CAS Center for Excellence in 13.1 Introduction
Molecular Plant Sciences, Shanghai Institute of Plant
Physiology and Ecology, Chinese Academy of Sciences, The largest number of eukaryotes on earth are
Shanghai, China insects, while fungi are second in estimated spe-
School of Life Science and Technology, ShanghaiTech cies; exact numbers, however, remain a matter of
University, Shanghai, China debate (Blackwell 2011; Mora et al. 2011;
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 309
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_13
310 S. Hong et al.

Hawksworth and Lücking 2017; Hyde 2022). numbers of EPF of Microsporidia

Fungi and insects have close associations with (Cryptomycota), Blastocladiomycota,
each other including fungal infection of insects, Basidiomycota, and Mucoromycota as well as
and numerous fungus–insect interactions the fungus-like oomycete insect pathogens
including insects using fungi as food and vec- (Humber 2008). Except for the per os infection
toring fungal propagules for dispersal or trans- of microsporidian parasites, other EPF infect
mission. Of these, the fungal species that infect insects largely through cuticle penetration
and kill insects play a pivotal role in (Wang and Wang 2017; Sheng et al. 2022).
manipulating insect population density in differ- Species of ascomycetes, especially the
ent environments (Savory et al. 2015; Lovett cordycipitoid fungi in Hypocreales (Sung et al.
and St Leger 2017; St. Leger 2021). Species of 2007) such as Beauveria (Cordycipitaceae),
entomopathogenic fungi (EPF) such as the Metarhizium (Clavicipitaceae), and
ascomycetes Beauveria bassiana, Metarhizium Ophiocordyceps (Ophiocordycipitaceae) are
anisopliae, and M. acridum have long been common EPF, and new species continue to be
under development for use as biocontrol agents reported frequently (Humber 2017; Imoulan
for eco-friendly management of insect pests et al. 2017; Mongkolsamrit et al. 2020; Wang
(de Faria and Wraight 2007; Wang and Wang et al. 2020a). Phylogenetic analysis of these
2017). Species of the fungi in Entomophthorales hypocrealean pathogens suggests that some EPF
(Entomophthoromycotina) like Entomophaga generally have closer relationships to the plant
maimaiga have also been successfully used in pathogenic fungi than to mammalian pathogens,
biological control of the gypsy moth (Lymantria implying that some EPF diverged from plant
dispar) in North America after introduction of pathogens (Spatafora et al. 2007; Shang et al.
the pathogen from Europe (Andreadis and 2016; Wang and Wang 2017). In particular, the
Weseloh 1990). hypocrealean EPF such as Metarhizium and
By using the model organism Drosophila Beauveria species have recently been explored
melanogaster as a fungal infection model, the as plant endophytes or rhizosphere fungi for
basic knowledge of host antifungal immune mediating nutrient exchanges with plants to pro-
response has been advanced, especially for the mote plant growth and defenses against pests
discovery and elucidation of the innate antifungal (Behie et al. 2012; Moonjely et al. 2016; Sheng
Toll immune pathway (Ferrandon et al. 2007; et al. 2022; Luo et al. 2023) and as biofertilizers
Buchon et al. 2014). The investigation of fungal (Litwin et al. 2020).
associations with insect hosts thus has both In contrast, there are relatively fewer species
applied and basic research importance. This chap- of fungi other than Hypocreales and
ter will summarize the advances of EPF genetics Entomophthoromycotina known to kill insects.
and infection biology largely over the last decade Exceptions include certain microsporidian spe-
or so. cies that infect silkworms (Nosema bombycis),
honeybees (Nocardia cereanae and N. apis),
and shrimp (e.g., Nos. granulosis) that have
13.2 Fungal Species that Infect caused huge economic and ecological losses.
Insects Another microsporidian species Antonospora
locustae (previously called Nos. locustae) has
There are more than 1500 species of fungi been used successfully in the biological control
reported to infect insects (Vega et al. 2009; of locust pests (Pan et al. 2018). Although many
Vega and Kaya 2012; Shang et al. 2015; Araújo species of insects are hosts for microsporidia, few
and Hughes 2016). These EPF are largely are killed. Except for a few Septobasidium and
distributed in phylum Ascomycota and subphy- Uredinella species, it is rare to find basidiomycete
lum Entomophthoromycotina (Zoopagomycota), fungi that can parasitize insects, although seldom
followed by species in order of descending kill them. The species Sporodiniella umbellata
13 Genetics and Infection Biology of the Entomopathogenic Fungi 311

(Mucorales) is one of the few known species of cellulose (1,3) and (1,6)-β-glucans, and hydroxy-
Mucoromycota known to be a specialized patho- proline. The cell walls of fungi are quite differ-
gen of insects (Humber 2008). ent from those of oomycetes, composed of β
Obligate zoosporic parasites in Coelomomyces (1,3)-D-glucan and chitin. The cell wall
and Catenaria (Blastocladiomycota) largely differences also provide markers to selectively
infect mosquito larvae or Daphnia hosts in fresh- target each group when developing pesticides and
water (Gleason et al. 2010). It has been interest- therapeutics (Cortés et al. 2019; Usman et al.
ingly found that some Coelomomyces species 2023).
such as C. psorophorae require two The differences between oomycetes and fungi
phylogenetically distinct hosts to complete their reflect the different evolutionary histories of the
full life cycle: the diploid (or sporophyte) phase is two groups. Although the heterotrophic
in mosquitos, while the haploid (or gametophyte) oomycetes lack plastids, they evolved within a
phase is in a copepod or ostracod host (obligate lineage that includes photosynthetic brown and
alternate host) (Whisler et al. 1975; Gleason et al. golden-brown algae and diatoms (Thakur et al.
2010). Reported oomycete insect pathogens, such 2019). Fungi and animals are opisthokonts, more
as the species Leptolegnia chapmanii, closely related to each other than to other
Lagenidium giganteum, and Pythium organisms such as oomycetes and green plants.
guiyangense, also infect mosquito larvae in Increasing evidence from genomics strongly
water (Shen et al. 2020). Of these, supports that plant pathogenicity of oomycetes
P. guiyangense has been tried for mass produc- such as Phytophthora spp. is the result of hori-
tion and application for mosquito control (Shen zontal gene transfer from plant pathogenic fungi
et al. 2019). Pythium guiyangense kills mosquitos (Richards et al. 2006; Thakur et al. 2019;
whereas many Pythium species are devastating RoyChowdhury et al. 2022).
plant pathogens (Shen et al. 2019), another exam- As in the case of certain plant pathogens, one
ple of a close association between EPF and plant of the typical features of EPF is their host speci-
pathogens. ficity. In addition to the intracellular and highly
insect-host-specific microsporidian parasites, the
zoosporic, and entomophthoralean EPF and
13.3 Fungi Versus Oomycetes oomycetes are also highly host-specific, targeting
certain species or a narrow range of insect hosts
Oomycetes and fungi inhabit similar aquatic and (Roy et al. 2006; Arnesen et al. 2018). The
terrestrial habitats. Both groups have members diverse ascomycete EPF include specialists and
with filamentous growth and sexual and asexual generalists that kill a wide range of different
reproduction by spores. Both groups are hetero- orders of insect hosts. In particular, the zombie-
trophic with lifestyles that include saprobes, ant fungi, Ophiocordyceps species, are highly
parasites, and pathogens of plants and animals. host-specific even to the level of one fungus for
They have osmotrophic nutrition with excreted one ant species (Kobmoo et al. 2012; Luangsa-
extracellular enzymes to break down complex Ard et al. 2018). The divergent Metarhizium spe-
molecules in the surrounding environment from cies, however, evolved as either specialists such
which they absorb smaller molecules. as M. acridum and M. album or generalists such
Oomycetes were long regarded as “lower as M. robertsii, M. anisopliae, and M. brunneum
fungi,” but with technological advances (Hu et al. 2014; Mongkolsamrit et al. 2020).
differences between the two groups became Metarhizium host recognition is typically
clear. Primary differences include diploid nuclei detected by observing the formation of
in the oomycete somatic body and often haploid appressoria by the spores germinated on host
or alternating haploid and diploid nuclei in the cuticles (Shang et al. 2021). For example,
body of the fungi described in this chapter. M. acridum conidia can only form appressoria
Oomycete cell walls are largely composed of on locust hosts but not on cockroaches. In
312 S. Hong et al.

Fig. 13.1 Microscopic imaging of Metarhizium spore could form the infection structure appressoria (AP) on
differentiation on different insects. The conidia (CO) of both insects while the specialist could only form
the generalist M. robertsii and locust-specific species appressoria on the locust host, not the non-host cockroach.
M. acridum were inoculated on the hind wings of cock- Bar, 5 μm
roach and locust for 18 h. It was evident that M. robertsii

contrast, the generalist species M. robertsii can in controlling host muscles and evident invasion
produce appressoria on both insects (Fig. 13.1). of the host nervous system by Entomophthora
Variation in host preference and virulence has muscae (Fredericksen et al. 2017; Elya et al.
been used in evaluating biocontrol agents of dif- 2018), mechanisms in controlling the behaviors
ferent strains of the same EPF species, e.g., the of dying insects remain objects of research.
same strain of B. bassiana may have varied Entomophthoralean Massospora species infect
abilities to kill different species of insects insects (largely cicadas) that remain active while
(Wraight et al. 2010; Mei et al. 2020). sporulating and spread their infective spores in
A typical feature of some EPF species that has flight (Boyce et al. 2019). Continuing research on
brought widespread interest to these fungi is their infections by the diverse EPF should answer
ability to manipulate host behavior. Some questions about the similar behaviors of the dis-
entomophthoralean EPF cause “submit disease” tinct hosts.
to drive the dying insects to climb upward to the
top of plants or alter host copulation behaviors to
benefit the transmission of fungal spores (Roy 13.4 Divergent Reproduction
et al. 2006; Hansen and De Fine Licht 2019; Controls
Lovett et al. 2020). The infection of some
Ophiocordyceps species also causes climbing Similar to other fungi, many EPF species undergo
behaviors of infected ants and insects before the asexual, sexual, and/or parasexual reproduction at
formation of incapacitated zombies (de Bekker different developmental stages. Except for
2019). Phylogenetic analysis of zombie-ant C. militaris and Ophiocordyceps fungi, the sexual
fungi suggests that these fungi might have state of other cordycipitoid fungi is largely cryptic
diverged from non-manipulating and beetle- and rare in nature (Hu et al. 2013; Zheng et al.
infecting ancestors (Araujo and Hughes 2019). 2013; Mei et al. 2020). However, genomic analy-
Except for the implications of zombie-ant fungi sis revealed that the haploids of Cordyceps sensu
13 Genetics and Infection Biology of the Entomopathogenic Fungi 313

lato largely contain the idiomorphic mating type 1 loci of Metarhizium species contain three genes,
(MAT) MAT1-1 or MAT1-2 locus, indicating that i.e., MAT1-1-1 (encoding an α-domain protein),
these EPF species may undergo heterothallic sex- MAT1-1-2 (encoding a protein that contains the
ual cycles (Fig. 13.2a). Intriguingly, the highly conserved proline, proline, and phenylalanine
duplicated genome of O. sinensis contains both residues) and MAT1-1-3 [(encoding a high mobil-
the MAT1-1 and MAT1-2 loci (Fig. 13.2b). This ity group (HMG) domain protein], while the
fungus is homothallic (Hu et al. 2013; Zheng et al. MAT1-1 loci of Cordyceps and Beauveria species
2013; Xia et al. 2017a). It is noteworthy that both only have MAT1-1-1 and MAT1-1-2. The MAT1-
the MAT1-1 and MAT1-2 idiomorphs have also 2 loci of the haploid EPF strains also differ from
been detected in the single spore isolate of each other. The MAT1-2 idiomorph of C. militaris
M. majus (Hu et al. 2014). Since the sexual state contains only MAT1-2-1 (encoding an HMG
of this fungus remains unclear, it is still hard to domain protein) whereas an additional MAT1-2-
conclude that M. major is a homothallic fungus. 3 (encoding a function unknown protein) gene is
For the two strains of M. acridum with full present at the MAT1-2 locus of M. acridum while
genomes sequenced, CQMa102 is a clear an extra of MAT1-2-8 (functionally unknown)
MAT1-2-type haploid (Gao et al. 2011); however, occurs in B. bassiana (Valero-Jimenez et al.
the repetitive strain ARSEF 324 was suggested to 2016). Intriguingly, the sequenced MAT1-2 hap-
be diploid/heterozygous with the alternative loid of C. cicadae contains a pseudo MAT1-1-1
insertion of transposons at the MAT1-2 loci gene that encodes a protein short of the critical
(Nielsen et al. 2021). α-domain (Lu et al. 2017). Gene transcription
In addition to the reproductive style difference analysis revealed that this pseudogene is silent
between the closely-related EPF, the structures of during fungal development, and the deletion of
their MAT loci are also highly divergent from both this gene and MAT1-2-1 did not prevent the
each other (Fig. 13.2a). For example, the MAT1- formation of asexual-like fruiting bodies.

Fig. 13.2 Schematic

structuring of the MAT loci
encoded in different species
of hypocrealean
ascomycete
entomopathogenic fungi.
(a) Divergent MAT
structures of the selected
heterothallic species. (b)
Syntenic relationship of the
MAT loci between the
homothallic O. sinensis and
heterothallic C. militaris.
The MAT1-1 idiomorph
comprises the α-domain
gene (MAT1-1-1), the
proline-proline-
phenylalanine-rich domain
gene (MAT1-1-2), and with
or without an HMG-box
domain gene (MAT1-1-3).
The MAT1-2 idiomorph
includes an HMG domain
gene (MAT1-2-1) with or
without a small open
reading frame (e.g., MAT1-
2-8 in B. bassiana) of
unclear function
314 S. Hong et al.

Despite the essential role of MAT loci in haplogroup 1 of M. robertsii isolates (n = 216) all
controlling fungal sexuality, the induction of belong to the MAT1-1 genotype while the ratios
fruiting body formation revealed that both the of the haplogroup 2 (MAT1-1/MAT1-2 = 70/16)
MAT1-1 and MAT1-2 isolates of C. militaris and 3 (MAT1-1/MAT1-2 = 6/58) isolates are
could fruit without the production of sexual biased to be either MAT1-1 or MAT1-2 idiomorph.
perithecia and ascospores, which suggests that The isolates of M. brunneum collected in the USA
fungal fruiting is independent of mating (Zheng are skewed to be MAT1-2 (30/37) genotype
et al. 2011). Similar asexual fruiting has been (Rehner 2020). The cause and effect of disparate
observed in ascomycetes such as Morchella mating-type distributions in fungal populations
sextelata (Liu et al. 2022). However, the pairing are still puzzling. As for entomophthoralean
test of the MAT1-1 and MAT1-2 deletion mutants EPF, except for the known sexual form of
failed to obtain fruiting bodies of C. militaris zygospores, the MAT loci and reproductive con-
(Lu et al. 2016). Otherwise, the cross of the trol of these fungi are still unclear due to the
ΔMAT1-1-1 with MAT1-2 isolates abolished the complexity of genome architecture in these fungi.
production of sexual perithecia and ascospores
while the mating of ΔMAT1-1-2 and MAT1-
2 led to fruiting body formation with barren 13.5 Genomic Advances
perithecia (Lu et al. 2016). It is still enigmatic of the Entomopathogenic
that the MAT1-1/MAT1-2 ratio of the C. militaris Fungi
conidia formed by newly collected wild and sex-
ual samples failed to reach a 50:50 rate but is By taking advantage of the technical advances in
highly biased toward the MAT1-1 type (Zheng genome sequencing, genomic studies of EPF spe-
et al. 2011). Unequal prevalence of mating types cies have thrived for more than 10 years (Wang
also occurs after the sub-culturing of C. militaris et al. 2016; Wang and Wang 2017). To date, there
strains, which was found in association with fun- are more than 110 strains belonging to 65 species
gal culture degeneration (Chen et al. 2017). of EPF with genome sequences reported
Even infrequently occurring, the sexual state (Table 13.1). Of these most belong to the
of B. bassiana (Cordyceps bassiana) has been hypocrealean ascomycete pathogens followed by
observed and was inducible under laboratory the entomophthoralean and microsporidian spe-
conditions (Li et al. 2001). Population genomic cies. A few species of oomycete insect pathogens
studies revealed that B. bassiana largely have also been sequenced with different genome
maintains clonal reproduction in nature, and the sizes, while none of the blastocladian insect
MAT1-1/MAT1-2 ratio of the isolates collected parasites has been sequenced. Even though fungal
from forest environments of southern China was entomopathogenicity has diverged multiple times
close to 50% (Mei et al. 2020). In contrast, the (Wang and Wang 2017), it is still surprising to see
mating types were highly biased toward the the considerable differences in genome sizes and
MAT1-1 type (65%) for the B. bassiana popula- contents among the inter- and intra-phylum of
tion sampled in Denmark (Meyling et al. 2009), these fungi (Table 13.1). In particular, the
and extremely skewed (MAT1-1/MAT1-2 = 146/ genome sizes of the entomophthoralean species
4) for the populations sampled in Cuba (Ramos such as Massospora platypediae (>2 gigabytes,
et al. 2017). Variation of MAT ratios was also Gb), Massospora cicadina (ca. 1.5 Gb), and
found among sub-populations of B. bassiana Entomophthora maimaiga (ca. 1.2 Gb) reach the
(Cai et al. 2022). Likewise, the population survey gigabyte levels whereas those of the strict intra-
of Metarhizium species also suggests a domi- cellular microsporidia EPE are usually less than
nance of clonal reproduction of these fungi. Inter- 10 megabytes (Mb). Huge genome size variations
estingly, however, the distribution pattern of are also present among the intra-phylum species.
MAT1-1/MAT1-2 ratios is associated with the iso- For example, small genomes are also present for
late haplogroups (Rehner 2020). For example, the entomophthoralean fungi such as
13 Genetics and Infection Biology of the Entomopathogenic Fungi 315

Table 13.1 Information of the entomopathogenic fungal or oomycete species and strains with sequenced genomes
Genome
size Gene
Species Strain (Mb) number NCBI Accessions References
Ascomycetes
Akanthomyces lecanii RCEF 1005 35.6 11,030 AZHF00000000 Shang et al. (2016)
(Lecanicillium lecanii)
Akanthomyces lecanii UM487 32.6 8126 LUKN00000000 NP
(Lecanicillium lecanii)
Aschersonia aleyrodi RCEF 2490 30.9 8461 AZGY00000000 Shang et al. (2016)
(Moelleriella libera)
Aschersonia badia MTCC 10142 28.8 9292 JMQE00000000 Agrawal et al. (2016)
(Hypocrella siamensis)
Ascosphaera apis ARSEF 7405 20.4 6442 AZGZ00000000 Shang et al. (2016)
Beauveria bassiana ARSEF 2860 33.7 10,366 ADAH00000000 Xiao et al. (2012)
Beauveria bassiana D1-5 34.7 11,861 ANFO00000000 NP
Beauveria bassiana ARSEF 5078 34.6 10,831 JTCZ00000000 Valero-Jimenez et al.
Beauveria bassiana ARSEF 1520 35.1 10,599 JTCW00000000 (2016)
Beauveria bassiana ARSEF 4305 34.7 10,513 JTCY00000000
Beauveria bassiana ARSEF 8028 34.7 10,210 JRHA00000000
Beauveria bassiana ARSEF 2579 36.7 10,753 JTCX00000000
Beauveria bassiana ARSEF 3097 32.2 NA JABAOI000000000 NP
Beauveria bassiana JEF-007 36.4 10,695 MRVG00000000 Lee et al. (2018)
Beauveria bassiana ERL836 35.45 10,399 PPTI00000000 NP
Beauveria bassiana JEF-350 35.66 11,225 JAAZTN000000000 Kim et al. (2022)
Beauveria bassiana BV-ECA0 32.8 9593 JACVNG000000000 Castro-Vásquez
Beauveria bassiana BV-ECA1 33 9636 JACVNF000000000 et al. (2022)
Beauveria bassiana BV-ECA13 32.8 9566 JACVNE000000000
Beauveria bassiana BV-ECA26 32.16 9465 JACVND000000000
Beauveria bassiana BV-ECA27 32.97 9648 JACVNC000000000
Beauveria bassiana BV-ECA31 33 9546 JACVNB000000000
Beauveria bassiana BV-ECA43 34.5 10,210 JACVNA000000000
Beauveria bassiana BV-ECA44 34.8 10,453 JACVMZ000000000
Beauveria brongniartii RCEF 3172 32.4 9595 AZHA00000000 Shang et al. (2016)
Beauveria felina SYSU-MS7908 29.4 NA JAEMHR000000000 NP
Beauveria rudraprayagi MTCC 8017 36.6 NA JMNB00000000 NP
Beauveria pseudobassiana KACC 47484 34.51 NA LUDX00000000 NP
Beauveria pseudobassiana RGM 2184 34.5 8469 JAKJXD000000000 Altimira et al. (2022)
Cordyceps cicadae CC02 33.3 9701 MWMN00000000 Lu et al. (2017)
Cordyceps cicadae BA-001 33.3 9627 AEIW00000000 NP
Cordyceps militaris Cm01 32.2 9684 AEVU00000000 Zheng et al. (2011)
Cordyceps militaris ATCC 34164 33.6 9371 PRJNA323705 Kramer and Nodwell
(2017)
Cordyceps militaris HN01 32.3 10,095 MQTM00000000 Chen et al. (2019)
Cordyceps GD15 29.05 9150 NRQP00000000 Zhang et al. (2018)
guangdongensis
Keithomyces neogunnii Cg7.2a 32.6 8641 JAIOWQ000000000 Long et al. (2022)
(Metacordyceps
neogunnii)
Hirsutella thompsonii MTCC 3556 34.6 9756 APKB00000000 Agrawal et al. (2015)
Hirsutella thompsonii MTCC 6686 34.7 10,259 APKU00000000
(continued)
316 S. Hong et al.

Table 13.1 (continued)

Genome
size Gene
Species Strain (Mb) number NCBI Accessions References
Isaria ARSEF 2679 33.5 10,060 AZHB00000000 Shang et al. (2016)
fumosorosea (Cordyceps
fumosorosea)
Isaria IJ1G 34.93 11,441 SPUK00000000 Lin et al. (2019)
javanica (Cordyceps
javanica)
Isaria javanica IJ2G 34.97 11,143 SPUL00000000
(Cordyceps javanica)
Metarhizium acridum CQMa 102 39.4 9849 ADNI00000000 Gao et al. (2011)
Metarhizium acridum ARSEF 324H1 44.7 12,536 JAGRQK000000000 Nielsen et al. (2021)
Metarhizium acridum ARSEF 324H2 39.2 10,197 JAGRQL000000000
Metarhizium album ARSEF 1941 30.5 8472 AZHE00000000 Hu et al. (2014)
Metarhizium anisopliae BRIP 53293 38.6 11,415 APNB00000000 Pattemore et al.
(2014)
Metarhizium anisopliae BRIP 53284 37.9 11,456 APNC00000000 NP
Metarhizium anisopliae ARSEF 549 38.5 10,891 AZNF00000000 Hu et al. (2014)
Metarhizium anisopliae CQMa421 45.3 NA PRJNA644049 NP
Metarhizium anisopliae JEF-290 42.9 13,646 JABUBV000000000 Lee et al. (2019)
Metarhizium anisopliae E6 38.4 10,817 JNNZ00000000 Staats et al. (2014)
Metarhizium brunneum ARSEF 3297 37.1 10,689 AZNG00000000 Hu et al. (2014)
Metarhizium brunneum ARSEF 4556 37.4 11,420 PRJNA608152 Saud et al. (2021)
Metarhizium RCEF 6700 32.1 8596 PRJNA817331 NP
cylindrosporum
Metarhizium RCEF 0880 30.1 8652 PRJNA817331 NP
guniujiangensis
Metarhizium frigidum ARSEF 4124 50.03 13,547 PRJNA272623 NP
Metarhizium guizhouense ARSEF 977 43.2 11,787 AZNH00000000 Hu et al. (2014)
Metarhizium humberi ESALQ1638 38.3 10,633 JACEFI000000000 Iwanicki et al. (2022)
Metarhizium majus ARSEF 297 42.1 11,535 AZNE00000000 Hu et al. (2014)
Metarhizium rileyi RCEF 4871 32 8764 AZHC00000000 Shang et al. (2016)
Metarhizium robertsii ARSEF 23 41.7 11,689 ADNJ02000000 Gao et al. (2011)
Metarhizium robertsii ARSEF 2575 39.7 12,384 JELW00000000 NP
Ophiocordyceps sinensis CO 18 120 6972 ANOV00000000 Hu et al. (2013)
Ophiocordyceps sinensis 1229 114 6972 LKHE00000000 Li et al. (2016)
Ophiocordyceps sinensis ZJB12195 101.1 9961 LWBQ00000000 NP
Ophiocordyceps sinensis IOZ07 110.8 8916 JAAVMX000000000 Shu et al. (2020)
Ophiocordyceps sinensis CC1406–203 119.2 8621 NGJJ00000000 Liu et al. (2020b)
Ophiocordyceps sinensis Nyingchi 124.8 7939 PRJNA382001 Xia et al. (2017a)
Ophiocordyceps EC05 30.5 7455 JAACLJ000000000 Will et al. (2020)
camponoti-floridani
Ophiocordyceps BCC 80369 37.91 7046 PDHP00000000 Kobmoo et al.
camponoti-leonardi (2018)
Ophiocordyceps BCC 79314 49.27 6946 PDHQ00000000
camponoti-saundersi
Ophiocordyceps BCC 54312 43 6799 LKCN00000000
polyrhachis-furcata
Ophiocordyceps Map-16 21.9 7621 NJES00000000 de Bekker et al.
camponoti-rufipedis (2015), de Bekker
Ophiocordyceps 1348a 22.2 7995 NJEU00000000 et al. (2017)
unilateralis
(continued)
13 Genetics and Infection Biology of the Entomopathogenic Fungi 317

Table 13.1 (continued)

Genome
size Gene
Species Strain (Mb) number NCBI Accessions References
Ophiocordyceps Map-64 23.3 8174 NJET00000000
unilateralis
Ophiocordyceps SC16a 23.9 8629 LAZP00000000
unilateralis
Ophiocordyceps 1346 32.3 11,275 NA
subramanianii
Ophiocordyceps CCMB 661 30.4 NA JACJUF000000000 NP
unilateralis
Ophiocordyceps BCC 54312 43.3 10,146 LKCN00000000 Wichadakul et al.
polyrhachis-furcat (2015)
Ophiocordyceps HCMA 001 23.97 NA JACYFI000000000 NP
xuefengensis
Sporothrix insectorum RCEF 264 34.7 9496 AZHD00000000 Shang et al. (2016)
Tolypocladium inflatum NRRL 8044 30.4 9998 AOHE00000000 Bushley et al. (2013)
Torrubiella hemipterigena BCC 1449 28.5 11,471 CDHN00000000 Horn et al. (2015)
Microsporidia
Nosema ceranae BRL01 7.86 2614 ACOL00000000 Cornman et al.
(2009)
Nosema ceranae PA08 1199 5.69 3209 JPQZ00000000 Pelin et al. (2015)
Nosema ceranae BRL 8.82 2288 MUP00000000 Huang et al. (2021)
Nosema bombycis CQ1 15.69 4458 ACJZ00000000 Pan et al. (2013)
Nosema antheraeae YY 6.6 3413 NA
Nosema apis BRL 01 8.57 2771 ANPH00000000 Chen et al. (2013)
Antonospora locustae CLX 3.17 1857 MPSM00000000 Chen et al. (2020b)
(Nosema locustae)
Nosema granulosis Ou3-Ou53 8.86 3639 SBJO00000000 Cormier et al. (2021)
Edhazardia aedis USNM 41457 51.35 4190 AFBI00000000 Desjardins et al.
Vavraia culicis Floridensis 6.12 2773 AEUG00000000 (2015)
Tubulinosema Franzen 7.56 3016 RCSS00000000 NP
ratisbonensis
Entomophthorales
Basidiobolus CBS 931.73 88.8 16,111 MCFE00000000 Mondo et al. (2017),
meristosporus Arnesen et al. (2018)
Basidiobolus heterosporus B8920 44.05 NA JNET00000000 NP
Conidiobolus coronatus NRRL 28638 39.9 10,635 JXYT00000000 Chang et al. (2015)
Conidiobolus incongruus B7586 93.9 NA JNEM00000000 NP
Neoconidiobolus FSU 785 24.4 8867 PRJNA196084 NP
thromboides
(Conidiobolus
thromboides)
Capillidium heterosporum CGMCC3.15857 33.4 10,857 WBMQ00000000 Wang et al. (2020b)
(Conidiobolus
heterosporus)
Entomophthora muscae Berkeley 1228 21,712 PRJNA479887 Wang et al. (2018)
Entomophthora muscae Air source 24.29 NA FCOS00000000 NP
Entomophthora maimaiga ARSEF 7190 1177.4 14,701 MycoCosm, JGI NP
Massospora cicadina MCPNR19 1488.88 5453 JAKSZP000000000 Boyce et al. (2019)
Massospora platypediae NA 2054.3 NA QKRY000000000
Massospora cicadina MICH 231384 766.6 NA QMCF00000000 NP
Zoophthora radicans ATCC 208865 655.2 14,479 MycoCosm, JGI NP
(continued)
318 S. Hong et al.

Table 13.1 (continued)

Genome
size Gene
Species Strain (Mb) number NCBI Accessions References
Mucoromycetes
Sporodiniella umbellata MES 1446 26.24 10,781 MycoCosm, JGI NP
Oomycetes
Pythium guiyangense Su 110 30,943 QXDM00000000 Shen et al. (2019)
Lagenidium giganteum ARSEF 373 126.3 NA NSDO00000000 NP
Lagenidium giganteum MTLA-03 70.79 NA PSQM00000000 NP
NA not available, NP not published

Neoconidiobolus thromboides (previously (CD) chromosomes among the strains of Fusar-

Conidiobolus thromboides, 24.4 Mb) and ium oxysporum (Ma et al. 2010), CD-like chro-
Capillidium heterosporum (previously mosome loss was also observed in M. anisopliae
Conidiobolus heterosporus, 33.4 Mb). Except (Wang et al. 2003). It is a bit surprising that the
for the highly repetitive (>40%) and expanded small-sized microsporidian genomes have higher
genomes of Ophiocordyceps sinensis (>100 Mb) numbers of chromosomes than these two ascomy-
(Hu et al. 2013; Xia et al. 2017a; Shu et al. 2020), cete EPF. For example, the locust parasite
the ascomycete EPF have typical genome sizes of Antonospora locustae has 17 chromosomes
30–50 Mb similar to most filamentous plant and (Chen et al. 2020b), and the silkworm
saprobic fungi. Due to the losses of essential microsporidia Nosema bombycis has
metabolic pathways for the synthesis of amino 18 chromosomes (Pan et al. 2013). It is also
acids, nucleotides, and even ATP (Nakjang et al. noteworthy that, in contrast to the approximately
2013), the compact genomes of obligate 50% GC content of filamentous fungal genomes,
entomopathogenic microsporidia are largely the genomes of some microsporidian parasites are
smaller than those of other EPF. However, size highly AT rich, e.g., a ratio of 74% AT for the
variations also exist between the microsporidian genome of the honeybee parasite N. ceranae
parasites of mosquitos from a few megabytes (Cornman et al. 2009), 78% AT for the mosquito
(e.g., Vavraia culicis, 6.12 Mb) to >50 Mb parasite E. aedis (Desjardins et al. 2015), and
(e.g., Edhazardia aedis, 51.3 Mb). 69% AT for the genome of N. bombycis (Pan
Until the employment of the third generation et al. 2013). Even the genome of A. locustae
of sequencing techniques, it was technically diffi- having an overall ratio of 42% GC content, the
cult and costly to obtain the full genome ultra-low GC region (ca. 20% of 25 kb) is present
sequences of EPF, as with other eukaryotes. in most chromosomes (Chen et al. 2020b). The
Among these genome-sequenced EPF, only two selection and function of this kind of evolutionary
ascomycete strains, i.e., Metarhizium brunneum output remain elusive. Similar to the other early
ARSEF 4556 (Saud et al. 2021) and Cordyceps diverged fungi, the genome of the
militaris ATCC 34164 (Kramer and Nodwell entomophthoralean fungus Basidiobolus
2017) have been sequenced and assembled with meristosporus is rich with methylated adenines
seven chromosomes each from telomere to telo- (i.e., N6-methyldeoxyadenine, 6 mA), a modifi-
mere. Surely, it does not mean that the closely- cation associated with the magnitude of gene
related cordycipitoid EPF would have the same expressions (Mondo et al. 2017).
number of chromosomes. At least, similar to the Comparative genomic analysis of ascomycete
presence or absence of conditionally dispensable EPF revealed that fungal entomopathogenicity is
13 Genetics and Infection Biology of the Entomopathogenic Fungi 319

characteristically associated with their genomic C. militaris that commonly perform sexual repro-
expansion of protease and chitinase protein duction is short of highly similar paralogous
families to degrade the protein- and chitin-rich genes and maintains a high-value RIP index
insect cuticles (Shang et al. 2016). Serine (Zheng et al. 2011). In contrast, the largely asex-
proteases are also rich in the entomophthoralean ual relative B. bassiana lacks the RIP mechanism
pathogens (Arnesen et al. 2018), and in the of genome defense and encodes many duplicated
oomycete pathogen Pythium guiyangense (Shen paralogues (Xiao et al. 2012; Valero-Jimenez
et al. 2019). In contrast, plant pathogens typically et al. 2016). It is interesting to note that, unex-
evolved the expanded families of carbohydrate- pectedly, the Aedes aegypti-specific
active enzyme genes for targeting plant cell walls. microsporidian Edhazardia aedis has a much
However, an array of putative effector genes is larger genome and many more genes (51.3 Mb;
similarly present in the genomes of EPF and plant 4190 genes) than those of Vavraia culicis
pathogens (Shang et al. 2016; Wang 2020). (6.1 Mb; 2773 genes), a microsporidian generalist
Regarding the evolution of EPF host specific- of mosquitos. Nevertheless, E. aedis apparently
ity, comparative and phylogenomic analysis of diverged earlier than did the generalist V. culicis
Metarhizium species with different host ranges (Desjardins et al. 2015).
revealed a speciation trajectory of Metarhizium As shown in Table 13.1, genomes of multiple
with species diverging from specialists to strains have been sequenced for quite a few EPF
generalists via intermediate transitional species species. Besides the alternative MAT idiomorph
with narrow host ranges (Hu et al. 2014). During of sequenced strains, the data reveal the varied
this process, extensive protein family expansions degree of genome size and gene content
occurred largely through gene duplications, differences among strains. In particular, the
which includes obtaining secondary metabolic genome size of Massospora cicadina strain
gene clusters in the generalist Metarhizium spe- MCPNR19 (ca. 1.5 Gb) almost doubles that of
cies such as the biosynthetic gene cluster (BGC) the strain MICH 231384 of M. cicadina
of non-selective insecticidal destruxins (Wang (ca. 766.6 Mb). A similar pattern is observed
et al. 2012; Hu et al. 2014). Duplication of poly- between the two sequenced strains of the
ketide synthase (PKS) BGC in Metarhizium spe- oomycete Lagenidium giganteum (Table 13.1).
cies can benefit the fungi to adapt to diverse For the two sequenced strains of M. acridum,
environments for stress resistance (Chen et al. strain CQMa102 has a clear haploid genomic
2015; Zeng et al. 2018). In particular, the architecture (Gao et al. 2011). In contrast, the
generalists evolved higher numbers of G-protein sequence data of M. acridum ARSEF 324 strain
coupled receptors (GPCRs) for the recognition of were assembled into two hybrid genomes H1 and
diverse hosts than the specialist species did H2, which have different genome sizes and gene
(Wang et al. 2016; Shang et al. 2021; Shang contents from each other (Nielsen et al. 2021).
et al. 2022). Overall, comparative genomic studies of inter-
The genome-wide analysis of the index of and intra-species of EPF are still required. Taken
repeat-induced point (RIP) mutations for each together with functional genetic investigations,
Metarhizium species suggested that the RIP the diverse biology of these fungi can then be
genome-defense mechanism against gene dupli- mapped to their genomic blueprints.
cation would occur in specialist but not generalist
Metarhizium species (Hu et al. 2014; Nielsen
et al. 2021). Since the RIP process occurs only 13.6 Molecular Interactions Among
after fertilization and immediately prior to meio- Fungi and Insects
sis (van Wyk et al. 2021), the findings suggest
that the specialist Metarhizium would maintain By benefiting from the advent of EPF genomics
the ability to reproduce sexually (St Leger and as well as tractable transformation techniques,
Wang 2020). In support to some extent, functional genetic studies have been greatly
320 S. Hong et al.

increased in recent years to unveil the molecular and the formation of biofilms on hydrophobic
associations between fungi and insects (Wang surfaces (Mei et al. 2021).
and Wang 2017; Li and Xia 2022; Tang et al. Aside from the differentiation of infection
2022). By using the species of B. bassiana, structures and secretion of cuticle-degradation
M. robertsii, M. anisopliae, and M. acridum as enzymes (Gao et al. 2020), the generation of
models, main efforts have focused on the proper turgor pressure within infection cells is
mechanisms in invading or evading host immu- also a critical prerequisite for the EPF penetration
nity involved in fungal penetration of insect of insect cuticles, which is similar to plant
cuticles and colonization of insect body cavities. pathogens such as Magnaporthe oryzae (Wilson
Shortly after spores land on and adhere to and Talbot 2009). However, in contrast to the
insect cuticles, the spores of EPF germinate to requirement of glycogen in M. oryzae
initiate host recognition, followed by penetration (Badaruddin et al. 2013), the degradation of
of cuticles (Shang et al. 2023). A finely-tuned triacylglycerol (TAG) instead of glycogen is the
cascade has been unveiled in M. robertsii to dis- main source for the solute buildup in M. robertsii
tinguish cuticle and insect-hemocoel micro- (Huang et al. 2019; Li et al. 2022). Thus, the
niches during fungal infection of insects (Zhang genes involved in TAG biogenesis and degrada-
et al. 2021). In observations of this process, tion (into glycerol) are critically needed in
GPCRs have been demonstrated to have differen- M. robertsii for cuticle penetration and therefore
tial expression in different Metarhizium species pathogenicity (Gao et al. 2013, 2016; Chen et al.
when induced on different insects (Gao et al. 2018a).
2011). Loss-of-function investigation of different Similar to animal cells, TAG is stored in liquid
GPCRs in M. robertsii revealed that alternative droplets (LDs) in fungi. The key LD-surface pro-
members contribute at varied levels to the fungal tein, perilipin-like protein Mpl1, was found in
recognition of different insect species, which was Metarhizium to contribute to maintaining the
reflected by the ratio of appressorium formation. homeostasis of LDs (Wang and St Leger
Interestingly, one of the ten-GPCR gene deletion 2007b). Insect cuticle surfaces are nutrient poor,
mutants lost its ability to form appressoria on all which will induce autophagic responses at the
hosts examined due to the impairment of the early stage of EPF infection (Duan et al. 2013;
nucleus translocation of the mitogen-activated Ying and Feng 2019). LD isolations followed by
protein kinase (MAPK) Mero-Fus3 (Shang et al. lipidomics and proteomics analysis of
2021). This behavior is consistent with the M. robertsii uncovered the potential mechanisms
finding that Mero-Fus3 has been verified to be of a short period of TAG increase in fungal cells
only one of four MAPKs required for mediating (Chen et al. 2018a, b). In particular, upon nitro-
appressorium formation in M. robertsii (Chen gen starvation, the core enzyme MrATG8 and the
et al. 2016). The histone lysine methyltransferase stress-related heat shock proteins (HSPs) were
of M. robertsii is also involved in the epigenetic activated, whereas the turnover of lipases and
regulation of appressorium formation (Lai et al. the ATG proteins such as ATG1, ATG7,
2020). Coupled with host recognition, EPF will ATG11, and ATG20 involved in the internaliza-
secrete different proteases and chitinases into the tion of LDs into vacuoles were downregulated
micro-niche of mucilage for host cuticle degrada- when referred to fungal growth in a nutrient-rich
tion (Ortiz-Urquiza and Keyhani 2013; Zhang medium (Fig. 13.3). In addition, the species of
et al. 2021). The formation of mucilage was phospholipids were differentially regulated by
unveiled in Metarhizium species through the pro- different proteins (Chen et al. 2018a). Consistent
duction of the exopolysaccharide with this notion, the machinery of
galactosaminogalactan (GAG) by a five-gene microlipophagy (i.e., LD entry into vacuoles
cluster. On top of the function of adhesin Mad1 without the function of autophagosomes) instead
(Wang and St Leger 2007a), GAG production can of macroautophagy, will be followed after TAG
additively facilitate spore adherence to cuticles accumulation to degrade LDs for building up the
appressorial turgor pressure (Li et al. 2022).
13 Genetics and Infection Biology of the Entomopathogenic Fungi 321

immunity once the cells of M. anisopliae enter

the caterpillar body cavity (Wang and St Leger
2006). Otherwise, similar to the function of the
LysM effectors of plant pathogens (Kombrink
and Thomma 2013), the LysM-like proteins
encoded by B. bassiana can bind the cell wall
chitin or chitin oligomers to evade insect immu-
nity (Cen et al. 2017). The study has also shown
that B. bassiana can employ the microRNA-like
RNA to target and suppress the Toll receptor
ligand Spätzle 4 of mosquitos to invade host
immunity (Cui et al. 2019). The M35 family of
metalloprotease of M. robertsii requires
degrading host prophenoloxidases to suppress
insect cuticular and hemocyte melanization
activities (Huang et al. 2020). Consistent with
the finding of the requirement of Niemann-Pick
disease type C2 (Npc2) proteins for antibacterial
response in Drosophila melanogaster (Shi et al.
2012), an insect-like Npc2 horizontally obtained
Fig. 13.3 Lipid metabolism implicated at the early stage in M. robertsii was required for fungal virulence
of M. robertsii infections. TAG triacylglycerol, SE steryl
to sequester host cholesterol for fungal sterol
esters, AB autophagic body, LC lysophosphatidylcholine,
PC phosphatidylcholine, PE phosphatidylethanolamine, biosynthesis during its quick replication in insect
PS phosphatidylserine, PA phosphatidic acid, PI hemocoels (Zhao et al. 2014).
phosphatidylinositol, PG phosphatidylglycerol, HSPs As indicated above, similar to plant pathogens,
heat shock proteins, LIPs lipases, LD lipid droplets. Up
EPF genomes also encode an array of effector-
arrows, increase; down arrows, decrease
like proteins (Shang et al. 2016). However, due to
It is noteworthy that the microbiotas assem- the lack of plant apoplast structures and the short/
bled on insect cuticles have great potential in lack of the intracellular nucleotide-binding and
mediating colonization resistance against fungal oligomerization domain NOD-like receptors
parasite infections (Bruner-Montero et al. 2021; (NLRs) in animal cells (Jones et al. 2016; Wang
Pessotti et al. 2021; Hong et al. 2022; Janke et al. 2020), the effector-like interactive mechanisms
2022). Fungal pathogens have evolved additional have been questioned in fungus–animal
abilities or strategies by producing antimicrobial interactions (Lowe and Howlett 2012). Increasing
peptides and/or antibiotic compounds to outcom- evidence, however, has shown that the patho-
pete insect surface microorganisms to facilitate genic fungi of insects, nematodes, and mammals
fungal penetration of insect cuticles (Sun et al. may similarly employ the secreted effectors as
2022a, b; Hong et al. 2023). virulence factors to suppress host immunity
Once reaching insect hemocoels, EPF cells (Stuart et al. 2013; Lopes Fischer et al. 2020;
encounter the extensive host cellular and humoral Wang 2020; Fischer and Requena 2022). For
immune defenses (Wang and Wang 2017; Qu and example, the secreted effector-like proteins of
Wang 2018). Different studies have revealed the the human pathogens Candida albicans and
diverse strategies employed by EPF to counteract Cryptococcus neoformans can manipulate host
insect antifungal immune responses. For exam- immune responses via toll-like receptors (Wang
ple, cell wall remodeling to reduce glucan content et al. 2019b; Dang et al. 2022). A cytolytic pep-
and mask cell wall components with a collagen- tide candidalysin secreted by C. albicans can
like protein Mcl1, occur to evade cellular damage epithelial cell membranes by pore forma-
tion (Moyes et al. 2016). It is anticipated that the
322 S. Hong et al.

EPF effectors may similarly effect in invading other fungi (Keller et al. 2005; Palmer and Keller
and/or evading insect-host immunity. 2010). In particular, the homologous
PKS-terpene Sub cluster for the biosynthesis of
the meroterpene subglutinols (Kato et al. 2016),
13.7 Chemical Genetics and Biology and the DtxS BGC for the production of the
of the Entomopathogenic insecticidal cyclopeptide destruxins (Wang et al.
Fungi 2012) are located on either end of chromosome
7 of M. brunneum. In contrast, the telomere/sub-
Apart from the functional insights of EPF genes/ telomere location of BGCs is not apparent in
enzymes involved in mediating the interactions C. militaris (Fig. 13.4b).
with insect hosts summarized above, different Chemical genetic studies revealed that the Swn
studies have shown that the small molecules pro- BGC contains a core NRPS-PKS (non-ribosomal
duced by EPF secondary metabolic BGCs also peptide synthetase-polyketide synthase) hybrid
contribute to mediating fungal infection of insect enzyme gene SwnK for the biosynthesis of the
hosts. Prior to chemical genetic studies, a plethora alkaloid mycotoxin swainsonine. The precursor
of bioactive metabolites have been identified pipecolic acid can be produced through multiple
from different EPF species, including pathways, including the lysine aminotransferase
polyketides, non-ribosomal peptides, alkaloids, (SwnA) and/or lysine cyclodeaminase activities
pyridones, and terpenes (Zhang et al. 2020a). (Luo et al. 2020). It has been recently found that
The early purposes of compound identifications an Upm supercluster of Metarhizium species
were largely the searches for novel chemicals and contains three BGCs including the intertwined
drug screenings (Newman and Cragg 2020). pseurotin/ovalicin BGCs that are
Except for antibiotic activity assays, the mesosyntencially conserved to the pseurotin/
biological functions of fungal metabolites were fumagillin BGCs of Aspergillus fumigatus. The
considered dispensable and largely ignored. The former, however, has the additional ustilaginoidin
lack of gene/genomic sequence information and BGC, and the supercluster is jointly controlled by
tractable genetic transformation systems also lim- a master regulator to mediate the biosynthesis of
ited the chemical, genetic, and biological function four classes of compounds, i.e., ustilaginoidin D,
studies of EPF in the early days. indigotides, pseurotin A, and hydroxyl-ovalicin
Different numbers of BGCs have been (Sun et al. 2022b). The SerS (also called
predicted from each new EPF genome acquired, MaNPS1) BGC was evident for the biosynthesis
which vary from ca. 30 to >60 BGCs in of the cyclic heptapeptide serinocyclins (Krasnoff
Cordyceps and Metarhizium species (Shang et al. 2007; Moon et al. 2008). However, the
et al. 2016; Chen et al. 2020a; Zhang et al. detailed biosynthetic mechanism of serinocyclins
2020b). Big gaps still exist in terms of the remains unclear to date. In particular, the
connections between the identified BGCs and enzymes involved in production of four
compounds that have been structurally identified non-proteinogenic amino acids:
and between the predicted BGCs and unknown 1-aminocyclopropane-1-carboxylic acid,
compounds that can be biosynthesized. For exam- hydroxyproline, γ-hydroxylysine, and β-alanine,
ple, the seven chromosomes of M. brunneum are still unclear. In contrast, the DtxS BGC has
encode 66 BGCs, while only seven conserved been well characterized to produce the
BGCs have been genetically elucidated, hexapeptide destruxins with the functions of
completely or in part in different Metarhizium four genes, and the core NRPS DtxS1 contains
species (Fig. 13.4a). Likewise, only three out of six modules for the linear assembly of individual
35 BGCs have been characterized in C. militaris substrate into cyclopeptides (Wang et al. 2012).
(Fig. 13.4b). The distribution patterns follow to Differently, the hexapeptide beauvericin is
some extent the telomere/sub-telomere biosynthesized by an NRPS BbBEAS that
localizations of BGCs that have been reported in contains only two modules to mediate the
13 Genetics and Infection Biology of the Entomopathogenic Fungi 323

Fig. 13.4 Mapping of the secondary metabolic each species. The clusters highlighted in bold with
(SM) gene clusters on the chromosomes of M. brunneum named genes have been characterized for biosynthetic
(a) and C. militaris (b) strains with their genomes finished. mechanism and/or chemical biology, which have been
The sequential digits show the number of biosynthetic indicated in this context
gene clusters encoded on individual chromosomes of

interactive biosynthesis of beauvericin from a characterized in C. militaris, interestingly, which

putative N-methyl-dipeptidol intermediate follows a protector-protégé strategy that the pro-
(Xu et al. 2008). duction of the deaminase inhibitor pentostatin can
Although it cannot be predicted by different protect the stability of cordycepin from deamina-
programs such as antiSMASH (Blin et al. 2021), tion into 3-deoxyinosine (Xia et al. 2017b). The
the Cns cluster containing four genes for the cyclodepsipeptide beauveriolides can by pro-
coupled biosynthesis of the adenosine anticancer duced by the related species B. bassiana,
compounds cordycepin and pentostatin has been B. brongniartii, and C. militaris that all contain
324 S. Hong et al.

the conserved BesA-BesB NRPS-PKS BGC. On top of the evident insecticidal activities, sur-
However, uncommon chemical diversity has vival assays using the WT and mutants of differ-
been found among these species; the rather diver- ent fungal species against alternative insect
gent B. brongniartii and C. militaris species pro- species also found the non-obvious virulence
duce the same analogs such as beauveriolides I, contributions of destruxins (Donzelli et al. 2012)
II, and III, whereas B. bassiana produces the main and tenellin (Eley et al. 2007) for M. robertsii and
derivatives of beauveriolides Ba, Ka, and Ca (Yin B. bassiana, respectively. Dtx A was implicated
et al. 2020). The conserved Dcs PKS cluster is in targeting insect immunophilins (Wang et al.
also present in B. bassiana, C. militaris, and 2019a), and Dtxs could suppress both cellular
C. fumosorosea, which is responsible for the pro- and humoral immune responses to assist the prop-
duction of nonanolides such as decarestrictine agation of fungal cells in insects (Wang et al.
C1. It was interesting to find a thioesterase func- 2012; Han et al. 2022). It is also interesting to
tioning as a polyketide cyclase to catalyze the find that the DtxS cluster is only present in the
formation of medium-ring lactones (Gao et al. generalist species of Metarhizium that can infect a
2021). Biosynthetic elucidation of the wide range of insect hosts (Wang et al. 2012; Hu
bibenzoquinone oosporein revealed the function et al. 2014; Xu et al. 2015).
of the OpS gene cluster for the sequential biosyn- Oosporein can inhibit the activation of
thesis of the intermediates orsellinic acid, prophenoloxidases and transcription of antimi-
benzenetriol, benzenetetrol, and finally the crobial peptide genes in insects (Feng et al.
dimerized pigment oosporein. The pathway is 2015). Consistent with its potent antibiotic
tightly controlled by a pathway-specific transcrip- activities, oosporein production in B. bassiana
tion factor OpS3 (Feng et al. 2015). It is notewor- has the ability to inhibit bacterial proliferation
thy that the genes located outside some BGCs of within insects before and after fungal killing of
entomopathogens can also function as tailoring the insect hosts (Fan et al. 2017; Wei et al. 2017).
enzymes in catalyzing the biosynthesis of Interestingly, it has recently been found that
derivatives. For example, the tandemly-linked M. robertsii contains the HelA BGC that is
glycosyltransferase-methyltransferase gene pair conserved to that of Aspergillus fumigatus for
BbGT1/MT1 outside the TenS BGC catalyze the the production of the triterpene helvolic acid
methylglucosylation of 15-hydroxytenellin, the (HA) (Lv et al. 2017). HA is a potent antibiotic
product of the TenS cluster in B. bassiana, into and abundantly accumulated in conidial spores to
glycosides (Chen et al. 2021). The BbGT1/MT1 facilitate fungal infection of insects by
orthologues MrGT1/MT1 function outside the “outwitting” the host cuticular microbiotas (Sun
Upm supercluster in M. robertsii for the produc- et al. 2022a). Likewise, the production of multiple
tion of indigotides, i.e., the methylglucosylated classes of compounds with antibiotic activities by
monomeric naphtho-γ-pyrones (Sun et al. 2022b). the Upm BGC can assist M. robertsii to counter-
By using the null mutants obtained in chemical act different antagonistic bacteria to facilitate fun-
genetic studies, insect bioassays with the wild gal adaptations to diverse niches (Sun et al.
type (WT) and mutant strains of B. bassiana or 2022b). In short, it is not surprising that fungal
Metarhizium species facilitated the identification small molecules play essential roles in mediating
of which metabolites are required for EPF infec- EPF associations with insect hosts, an area that
tion of insect hosts. For example, virulence deserves further investigation.
contributions have been found for the
cyclopeptides beauvericin (Xu et al. 2008),
destruxins (Wang et al. 2012), cyclosporine 13.8 Summary and Perspectives
(Yang et al. 2018), and beauveriolides (Yin
et al. 2020); the bibenzoquinone oosporein In summary, the genetics and molecular biology
(Feng et al. 2015; Mc Namara et al. 2019); and of fungal associations with insects have been well
2-pyridones tenellin analogs (Chen et al. 2021). explored and advanced in the past decade or
13 Genetics and Infection Biology of the Entomopathogenic Fungi 325

so. The obtaining of the high-quality genomes of Agrawal Y, Narwani T, Subramanian S (2016) Genome
representative EPF species has facilitated the sequence and comparative analysis of clavicipitaceous
insect-pathogenic fungus Aschersonia badia with
understanding of the evolution of fungal Metarhizium spp. BMC Genomics 17:367
entomopathogenicity and the functional genetic Altimira F, Arias-Aravena M, Jian L et al (2022) Genomic
investigations of fungus–insect interactions. and experimental analysis of the insecticidal factors
However, compared to the well-advanced effector secreted by the entomopathogenic fungus Beauveria
pseudobassiana RGM 2184. J Fungi (Basel) 8:253
mechanisms of fungus–plant interactions (Jones Andreadis TG, Weseloh RM (1990) Discovery of
et al. 2016), the virulence requirement of effectors Entomophaga maimaiga in North American gypsy
in fungal suppression of insect immunity is still moth, Lymantria dispar. Proc Natl Acad Sci U S A
poorly understood (Wang 2020). Future efforts 87:2461–2465
Araújo JP, Hughes DP (2016) Diversity of
should be directed toward this area. Following entomopathogenic fungi: which groups conquered the
the de novo genomic studies of different EPF insect body? Adv Genet 94:1–39
species, population genomic, and pan-genomic Araujo JPM, Hughes DP (2019) Zombie-ant fungi
investigations by re-sequencing of diverse intra- emerged from non-manipulating, beetle-infecting
ancestors. Curr Biol 29:3735–3738
and interspecific genomes are still needed to Arnesen JA, Malagocka J, Gryganskyi A, Grigoriev IV,
unveil the genetic diversity as well as the hidden Voigt K, Stajich JE, De Fine Licht HH (2018) Early
drivers of selection leading to the varied degree of diverging insect-pathogenic fungi of the order
host specificity or host preference among differ- Entomophthorales possess diverse and unique
subtilisin-like serine proteases. G3 (Bethesda) 8:
ent strains or species of entomopathogens. Con- 3311–3319
sistent with the protective functions of human Badaruddin M, Holcombe LJ, Wilson RA, Wang ZY,
skin and plant phyllosphere microbiotas (Liu Kershaw MJ, Talbot NJ (2013) Glycogen metabolic
et al. 2020a; Harris-Tryon and Grice 2022), recent genes are involved in trehalose-6-phosphate synthase-
mediated regulation of pathogenicity by the rice blast
studies have shown that insect ectomicrobiomes fungus Magnaporthe oryzae. PLoS Pathog 9:
can similarly mediate the colonization resistance e1003604
against fungal parasites (Hong et al. 2022; Janke Behie SW, Zelisko PM, Bidochka MJ (2012) Endophytic
et al. 2022). Future investigation of fungus–insect insect-parasitic fungi translocate nitrogen directly from
insects to plants. Science 336:1576–1577
interactions will have to be expanded beyond Blackwell M (2011) The Fungi: 1, 2, 3 . . . 5.1 million
bilateral regimes to better understand the species? Am J Bot 98:426–438
mechanisms of multilateral interactions among Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van
fungi, bacteria, and insects. The knowledge Wezel GP, Medema MH, Weber T (2021) antiSMASH
6.0: improving cluster detection and comparison
obtained may benefit the development of cost- capabilities. Nucleic Acids Res 49:W29–w35
effective mycoinsecticides as well as the protec- Boyce GR, Gluck-Thaler E, Slot JC et al (2019) Psycho-
tion of ecologically and economically important active plant- and mushroom-associated alkaloids from
beneficial insects like bees and silkworms. two behavior modifying cicada pathogens. Fungal Ecol
41:147–164
Bruner-Montero G, Wood M, Horn HA, Gemperline E,
Acknowledgment The authors are highly appreciative of Li L, Currie CR (2021) Symbiont-mediated protection
Drs. Meredith Blackwell and Yen-Peng Hsueh for their of Acromyrmex leaf-cutter ants from the
help in preparing this chapter. This work was supported by entomopathogenic fungus Metarhizium anisopliae.
the National Key R&D Program of China mBio 12:e0188521
(2022YFD1400700 and 2022YFD1400500). Buchon N, Silverman N, Cherry S (2014) Immunity in
Drosophila melanogaster--from microbial recognition
to whole-organism physiology. Nat Rev Immunol 14:
796–810
References Bushley KE, Raja R, Jaiswal P et al (2013) The genome of
Tolypocladium inflatum: evolution, organization, and
Agrawal Y, Khatri I, Subramanian S, Shenoy BD (2015) expression of the cyclosporin biosynthetic gene clus-
Genome sequence, comparative analysis, and evolu- ter. PLoS Genet 9:e1003496
tionary insights into chitinases of entomopathogenic Cai Y, Nie Y, Gao Y, Huang B (2022) Natural populations
fungus Hirsutella thompsonii. Genome Biol Evol 7: of the entomopathogenic fungus Beauveria bassiana in
916–930 Chinese forest ecosystems are diverse and reveal equal
326 S. Hong et al.

frequencies of mating types within phylogenetic spe- Cornman RS, Chen YP, Schatz MC et al (2009) Genomic
cies. Fungal Ecol 56:101139 analyses of the microsporidian Nosema ceranae, an
Castro-Vásquez R, Solano-González S, Molina-Bravo R, emergent pathogen of honey bees. PLoS Pathog 5:
Montero-Astúa M (2022) Draft genome sequences of e1000466
eight isolates of Beauveria bassiana of neotropical Cortés JCG, Curto M, Carvalho VSD, Pérez P, Ribas JC
origin. Microbiol Resour Announc 11:e00267–e00222 (2019) The fungal cell wall as a target for the develop-
Cen K, Li B, Lu YZ, Zhang SW, Wang CS (2017) Diver- ment of new antifungal therapies. Biotechnol Adv 37:
gent LysM effectors contribute to the virulence of 107352
Beauveria bassiana by evasion of insect immune Cui C, Wang Y, Liu J, Zhao J, Sun P, Wang S (2019) A
defenses. PLoS Pathog 13:e1006604 fungal pathogen deploys a small silencing RNA that
Chang Y, Wang SS, Sekimoto S et al (2015) attenuates mosquito immunity and facilitates infection.
Phylogenomic analyses indicate that early fungi Nat Commun 10:4298
evolved digesting cell walls of algal ancestors of land Dang EV, Lei S, Radkov A, Volk RF, Zaro BW, Madhani
plants. Genome Biol Evol 7:1590–1601 HD (2022) Secreted fungal virulence effector triggers
Chen Y, Pettis JS, Zhao Y et al (2013) Genome sequenc- allergic inflammation via TLR4. Nature 608:161–167
ing and comparative genomics of honey bee de Bekker C (2019) Ophiocordyceps-ant interactions as an
microsporidia, Nosema apis reveal novel insights into integrative model to understand the molecular basis of
host-parasite interactions. BMC Genomics 14:451 parasitic behavioral manipulation. Curr Opin Insect Sci
Chen YX, Feng P, Shang YF, Xu YJ, Wang CS (2015) 33:19–24
Biosynthesis of non-melanin pigment by a divergent de Bekker C, Ohm RA, Loreto RG et al (2015) Gene
polyketide synthase in Metarhizium robertsii. Fungal expression during zombie ant biting behavior reflects
Genet Biol 81:142–149 the complexity underlying fungal parasitic behavioral
Chen X, Xu C, Qian Y et al (2016) MAPK cascade- manipulation. BMC Genomics 16:620
mediated regulation of pathogenicity, conidiation and de Bekker C, Ohm RA, Evans HC, Brachmann A, Hughes
tolerance to abiotic stresses in the entomopathogenic DP (2017) Ant-infecting Ophiocordyceps genomes
fungus Metarhizium robertsii. Environ Microbiol 18: reveal a high diversity of potential behavioral manipu-
1048–1062 lation genes and a possible major role for enterotoxins.
Chen A, Wang Y, Shao Y, Huang B (2017) A novel Sci Rep 7:12508
technique for rejuvenation of degenerated caterpillar de Faria MR, Wraight SP (2007) Mycoinsecticides and
medicinal mushroom, Cordyceps militaris mycoacaricides: a comprehensive list with worldwide
(Ascomycetes), a valued traditional Chinese medicine. coverage and international classification of formulation
Int J Med Mushrooms 19:87–91 types. Biol Control 43:237–256
Chen YX, Li B, Cen K, Lu YZ, Zhang SW, Wang CS Desjardins CA, Sanscrainte ND, Goldberg JM et al (2015)
(2018a) Diverse effect of phosphatidylcholine biosyn- Contrasting host-pathogen interactions and genome
thetic genes on phospholipid homeostasis, cell evolution in two generalist and specialist
autophagy and fungal developments in Metarhizium microsporidian pathogens of mosquitoes. Nat
robertsii. Environ Microbiol 20:293–304 Commun 6:7121
Chen YX, Cen K, Lu Y, Zhang S, Shang YF, Wang CS Donzelli BGG, Krasnoff SB, Moon YS, Churchill AC,
(2018b) Nitrogen-starvation triggers cellular accumu- Gibson DM (2012) Genetic basis of destruxin produc-
lation of triacylglycerol in Metarhizium robertsii. Fun- tion in the entomopathogen Metarhizium robertsii.
gal Biol 122:410–419 Curr Genet 58:105–116
Chen Y, Wu Y, Liu L et al (2019) Study of the whole Duan ZB, Chen YX, Huang W, Shang YF, Chen PL,
genome, methylome and transcriptome of Cordyceps Wang CS (2013) Linkage of autophagy to fungal
militaris. Sci Rep 9:898 development, lipid storage and virulence in
Chen B, Sun YL, Luo FF, Wang CS (2020a) Bioactive Metarhizium robertsii. Autophagy 9:538–549
metabolites and potential mycotoxins produced by Eley KL, Halo LM, Song Z et al (2007) Biosynthesis of the
Cordyceps fungi: a review of safety. Toxins 12:410 2-pyridone tenellin in the insect pathogenic fungus
Chen L, Gao X, Li R et al (2020b) Complete genome of a Beauveria bassiana. Chembiochem 8:289–297
unicellular parasite (Antonospora locustae) and tran- Elya C, Lok TC, Spencer QE, McCausland H, Martinez
scriptional interactions with its host locust. Microb CC, Eisen M (2018) Robust manipulation of the
Genom 6:mgen000421 behavior of Drosophila melanogaster by a fungal path-
Chen B, Sun YL, Li SQ, Yin Y, Wang CS (2021) Induc- ogen in the laboratory. elife 7:e34414
tive production of the iron-chelating 2-pyridones Fan Y, Liu X, Keyhani NO, Tang G, Pei Y, Zhang W,
benefits the producing fungus to compete for diverse Tong S (2017) Regulatory cascade and biological
niches. mBio 12:e0327921 activity of Beauveria bassiana oosporein that limits
Cormier A, Chebbi MA, Giraud I et al (2021) Comparative bacterial growth after host death. Proc Natl Acad Sci
genomics of strictly vertically transmitted, feminizing U S A 114:E1578–E1586
microsporidia endosymbionts of amphipod Feng P, Shang Y, Cen K, Wang C (2015) Fungal biosyn-
crustaceans. Genome Biol Evol 13:evaa245 thesis of the bibenzoquinone oosporein to evade insect
13 Genetics and Infection Biology of the Entomopathogenic Fungi 327

immunity. Proc Natl Acad Sci U S A 112:11365– entomopathogenic fungus Verticillium

11370 hemipterigenum. Genome Announc 3:e0143914
Ferrandon D, Imler JL, Hetru C, Hoffmann JA (2007) The Hu X, Zhang YJ, Xiao GH et al (2013) Genome survey
Drosophila systemic immune response: sensing and uncovers the secrets of sex and lifestyle in caterpillar
signalling during bacterial and fungal infections. Nat fungus. Chin Sci Bull 58:2846–2854
Rev Immunol 7:862–874 Hu X, Xiao G, Zheng P et al (2014) Trajectory and
Fischer R, Requena N (2022) Small-secreted proteins as genomic determinants of fungal-pathogen speciation
virulence factors in nematode-trapping fungi. Trends and host adaptation. Proc Natl Acad Sci U S A 111:
Microbiol 30:615–617 16796–16801
Fredericksen MA, Zhang Y, Hazen ML, Loreto RG, Huang W, Hong S, Tang G, Lu Y, Wang C (2019)
Mangold CA, Chen DZ, Hughes DP (2017) Three- Unveiling the function and regulation control of the
dimensional visualization and a deep-learning model DUF3129 family proteins in fungal infection of hosts.
reveal complex fungal parasite networks in behavior- Philos Trans R Soc Lond Ser B Biol Sci 374:20180321
ally manipulated ants. Proc Natl Acad Sci U S A Huang A, Lu M, Ling E, Li P, Wang CS (2020) A M35
114:12590–12595 family metalloprotease is required for fungal virulence
Gao Q, Jin K, Ying SH et al (2011) Genome sequencing against insects by inactivating host prophenoloxidases
and comparative transcriptomics of the model and beyond. Virulence 11:222–237
entomopathogenic fungi Metarhizium anisopliae and Huang Q, Wu ZH, Li WF et al (2021) Genome and
M. acridum. PLoS Genet 7:e1001264 evolutionary analysis of Nosema ceranae: A
Gao Q, Shang YF, Huang W, Wang CS (2013) Glycerol- microsporidian parasite of honey bees. Front Microbiol
3-phosphate acyltransferase contributes to 12:645353
triacylglycerol biosynthesis, lipid droplet formation, Humber RA (2008) Evolution of entomopathogenicity in
and host invasion in Metarhizium robertsii. Appl Envi- fungi. J Invertebr Pathol 98:262–266
ron Microbiol 79:7646–7653 Humber RA (2017) Back to the future: cordycipitoid fungi
Gao Q, Lu Y, Yao H, Xu YJ, Huang W, Wang C (2016) in a postgenomic world. Mycology 8:267–275
Phospholipid homeostasis maintains cell polarity, Hyde KD (2022) The numbers of fungi. Fungal Divers
development and virulence in Metarhizium robertsii. 114:1
Environ Microbiol 18:3976–3990 Imoulan A, Hussain M, Kirk PM, El Meziane A, Yao Y-J
Gao BJ, Mou YN, Tong SM, Ying SH, Feng MG (2020) (2017) Entomopathogenic fungus Beauveria: Host
Subtilisin-like Pr1 proteases marking evolution of specificity, ecology and significance of morpho-
pathogenicity in a wide-spectrum insect-pathogenic molecular characterization in accurate taxonomic clas-
fungus. Virulence 11:365–380 sification. J Asia-Pacif Entomol 20:1204–1212
Gao DW, Jamieson CS, Wang G, Yan Y, Zhou J, Houk Iwanicki NSA, Botelho A, Klingen I, Junior ID,
KN, Tang Y (2021) A polyketide cyclase that forms Rossmann S, Lysoe E (2022) Genomic signatures and
medium-ring lactones. J Am Chem Soc 143:80–84 insights into host niche adaptation of the
Gleason FH, Marano AV, Johnson P, Martin WW (2010) entomopathogenic fungus Metarhizium humberi. G3
Blastocladian parasites of invertebrates. Fungal Biol 12:jkab416
Rev 24:56–67 Janke RS, Kaftan F, Niehs SP et al (2022) Bacterial
Han P, Gong Q, Fan J, Abbas M, Chen D, Zhang J (2022) ectosymbionts in cuticular organs chemically protect
Destruxin A inhibits scavenger receptor B mediated a beetle during molting stages. ISME J 16:2691–2701
melanization in Aphis citricola. Pest Manag Sci 78: Jones JD, Vance RE, Dangl JL (2016) Intracellular innate
1915–1924 immune surveillance devices in plants and animals.
Hansen AN, De Fine Licht HH (2019) Why are there so Science 354:aaf6395
few examples of entomopathogenic fungi that manipu- Kato H, Tsunematsu Y, Yamamoto T, Namiki T,
late host sexual behaviors? Fungal Ecol 38:21–27 Kishimoto S, Noguchi H, Watanabe K (2016) New
Harris-Tryon TA, Grice EA (2022) Microbiota and main- natural products isolated from Metarhizium robertsii
tenance of skin barrier function. Science 376:940–945 ARSEF 23 by chemical screening and identification of
Hawksworth DL, Lücking R (2017) Fungal diversity the gene cluster through engineered biosynthesis in
revisited: 2.2 to 3.8 million species. Microbiol Spectr Aspergillus nidulans A1145. J Antibiot 69:561–566
5:FUNK-0052-2016 Keller NP, Turner G, Bennett JW (2005) Fungal secondary
Hong S, Sun Y, Sun D, Wang CS (2022) Microbiome metabolism - from biochemistry to genomics. Nat Rev
assembly on Drosophila body surfaces benefits the Microbiol 3:937–947
flies to combat fungal infections. iScience 25:104408 Kim JC, Park SE, Lee SJ, Kim JS (2022) Whole-genome
Hong S, Sun YL, Chen HM, Wang CS (2023) Suppression sequence of Beauveria bassiana JEF-350, a strain with
of the insect cuticular microbiomes by a fungal high insecticidal activity against melon thrips (Thrips
defensin to facilitate parasite infection. ISME J 17:1– palmi). Microbiol Resour Announc 11:e0047022
11 Kobmoo N, Mongkolsamrit S, Tasanathai K,
Horn F, Habel A, Scharf DH et al (2015) Draft genome Thanakitpipattana D, Luangsa-Ard JJ (2012) Molecu-
sequence and gene annotation of the lar phylogenies reveal host-specific divergence of
328 S. Hong et al.

Ophiocordyceps unilateralis sensu lato following its Liu Q, Qu S, He G, Wei J, Dong C (2022) Mating-type
host ants. Mol Ecol 21:3022–3031 genes play an important role in fruiting body develop-
Kobmoo N, Wichadakul D, Arnamnart N, Rodriguez De ment in Morchella sextelata. J Fungi (Basel) 8:564
La Vega RC, Luangsa-Ard JJ, Giraud T (2018) A Long L, Liu Z, Deng C, Li C, Wu L, Hou B, Lin Q (2022)
genome scan of diversifying selection in Genomic Sequence and Transcriptome Analysis of the
Ophiocordyceps zombie-ant fungi suggests a role for Medicinal Fungus Keithomyces neogunnii. Genome
enterotoxins in co-evolution and host specificity. Mol Biol Evol 14:evac033
Ecol 27:3582–3598 Lopes Fischer N, Naseer N, Shin S, Brodsky IE (2020)
Kombrink A, Thomma BP (2013) LysM effectors: Effector-triggered immunity and pathogen sensing in
secreted proteins supporting fungal life. PLoS Pathog metazoans. Nat Microbiol 5:14–26
9:e1003769 Lovett B, St Leger RJ (2017) The insect pathogens.
Kramer GJ, Nodwell JR (2017) Chromosome level assem- Microbiol Spectr 5:FUNK-0001-2016
bly and secondary metabolite potential of the parasitic Lovett B, Macias A, Stajich JE, Cooley J, Eilenberg J, de
fungus Cordyceps militaris. BMC Genomics 18:912 Fine Licht HH, Kasson MT (2020) Behavioral
Krasnoff SB, Keresztes I, Gillilan RE, Szebenyi DM, betrayal: How select fungal parasites enlist living
Donzelli BG, Churchill AC, Gibson DM (2007) insects to do their bidding. PLoS Pathog 16:e1008598
Serinocyclins A and B, cyclic heptapeptides from Lowe RG, Howlett BJ (2012) Indifferent, affectionate, or
Metarhizium anisopliae. J Nat Prod 70:1919–1924 deceitful: lifestyles and secretomes of fungi. PLoS
Lai Y, Cao X, Chen J, Wang L, Wei G, Wang S (2020) Pathog 8:e1002515
Coordinated regulation of infection-related morpho- Lu Y, Xia Y, Luo F, Dong C, Wang C (2016) Functional
genesis by the KMT2-Cre1-Hyd4 regulatory pathway convergence and divergence of mating-type genes ful-
to facilitate fungal infection. Sci Adv 6:eaaz1659 filling in Cordyceps militaris. Fungal Genet Biol 88:
Lee SJ, Lee MR, Kim S et al (2018) Genomic analysis of 35–43
the insect-killing fungus Beauveria bassiana JEF-007 Lu Y, Luo F, Cen K et al (2017) Omics data reveal the
as a biopesticide. Sci Rep 8:12388 unusual asexual-fruiting nature and secondary meta-
Lee MR, Li D, Lee SJ et al (2019) Use of Metarhizum bolic potentials of the medicinal fungus Cordyceps
aniopliae s.l. to control soil-dwelling longhorned tick, cicadae. BMC Genomics 18:668
Haemaphysalis longicornis. J Invertebr Pathol 166: Luangsa-Ard J, Tasanathai K, Thanakitpipattana D,
107230 Khonsanit A, Stadler M (2018) Novel and interesting
Li J, Xia Y (2022) Host-pathogen interactions between Ophiocordyceps spp. (Ophiocordycipitaceae,
Metarhizium spp. and locusts. J Fungi 8:602 Hypocreales) with superficial perithecia from
Li ZZ, Li CR, Huang B, Fan MZ (2001) Discovery and Thailand. Stud Mycol 89:125–142
demonstration of the teleomorph of Beauveria Luo FF, Hong S, Chen B et al (2020) Unveiling of
bassiana (Bals.) Vuill., an important entomogenous swainsonine biosynthesis via a multi-branched path-
fungus. Chin Sci Bull 46:751–753 way in fungi. ACS Chem Biol 15:2476–2484
Li Y, Hsiang T, Yang RH et al (2016) Comparison of Luo F, Tang G, Hong S, Gong T, Xin X-F, Wang CS
different sequencing and assembly strategies for a (2023) Promotion of Arabidopsis immune responses
repeat-rich fungal genome, Ophiocordyceps sinensis. by a rhizosphere fungus via supply of pipecolic acid to
J Microbiol Methods 128:1–6 plants and selective augment of phytoalexins. Sci
Li B, Song S, Wei X, Tang G, Wang CS (2022) Activation China Life Sci 66:1119–1133
of microlipophagy during early infection of insect Lv JM, Hu D, Gao H et al (2017) Biosynthesis of helvolic
hosts by Metarhizium robertsii. Autophagy 18:608– acid and identification of an unusual C-4-demethyla-
623 tion process distinct from sterol biosynthesis. Nat
Lin R, Zhang X, Xin B et al (2019) Genome sequence of Commun 8:1644
Isaria javanica and comparative genome analysis Ma LJ, van der Does HC, Borkovich KA et al (2010)
insights into family S53 peptidase evolution in fungal Comparative genomics reveals mobile pathogenicity
entomopathogens. Appl Microbiol Biotechnol 103: chromosomes in Fusarium. Nature 464:367–373
7111–7128 Mc Namara L, Dolan SK, Walsh JMD, Stephens JC, Glare
Litwin A, Nowak M, Rozalska S (2020) TR, Kavanagh K, Griffin CT (2019) Oosporein, an
Entomopathogenic fungi: unconventional applications. abundant metabolite in Beauveria caledonica, with a
Rev Environ Sci Biotechnol 19:23–42 feedback induction mechanism and a role in insect
Liu H, Brettell LE, Singh B (2020a) Linking the virulence. Fungal Biol 123:601–610
phyllosphere microbiome to plant health. Trends Mei LJ, Chen M, Shang Y et al (2020) Population geno-
Plant Sci 25:841–844 mics and evolution of a fungal pathogen after releasing
Liu J, Guo L, Li Z et al (2020b) Genomic analyses reveal exotic strains to control insect pests for 20 years. ISME
evolutionary and geologic context for the plateau fun- J 14:1422–1434
gus Ophiocordyceps sinensis. Chin Med 15:107 Mei L, Wang X, Yin Y, Tang G, Wang CS (2021) Con-
servative production of galactosaminogalactan in
Metarhizium is responsible for appressorium mucilage
13 Genetics and Infection Biology of the Entomopathogenic Fungi 329

production and topical infection of insect hosts. PLoS global populations of the honeybee pathogen Nosema
Pathog 17:e1009656 ceranae. Environ Microbiol 17:4443–4458
Meyling NV, Lubeck M, Buckley EP, Eilenberg J, Rehner Pessotti RC, Hansen BL, Reaso JN, Ceja-Navarro JA, El-
SA (2009) Community composition, host range and Hifnawi L, Brodie EL, Traxler MF (2021) Multiple
genetic structure of the fungal entomopathogen lineages of Streptomyces produce antimicrobials
Beauveria in adjoining agricultural and seminatural within passalid beetle galleries across eastern North
habitats. Mol Ecol 18:1282–1293 America. elife 10:e65091
Mondo SJ, Dannebaum RO, Kuo RC et al (2017) Wide- Qu S, Wang S (2018) Interaction of entomopathogenic
spread adenine N6-methylation of active genes in fungi with the host immune system. Dev Comp
fungi. Nat Genet 49:964–968 Immunol 83:96–103
Mongkolsamrit S, Khonsanit A, Thanakitpipattana D et al Ramos Y, Portal O, Lysoe E, Meyling NV, Klingen I
(2020) Revisiting Metarhizium and the description of (2017) Diversity and abundance of Beauveria bassiana
new species from Thailand. Stud Mycol 95:171–251 in soils, stink bugs and plant tissues of common bean
Moon YS, Donzelli BG, Krasnoff SB et al (2008) from organic and conventional fields. J Invertebr
Agrobacterium-mediated disruption of a nonribosomal Pathol 150:114–120
peptide synthetase gene in the invertebrate pathogen Rehner SA (2020) Genetic structure of Metarhizium spe-
Metarhizium anisopliae reveals a peptide spore factor. cies in western USA: finite populations composed of
Appl Environ Microbiol 74:4366–4380 divergent clonal lineages with limited evidence for
Moonjely S, Barelli L, Bidochka MJ (2016) Insect patho- recent recombination. J Invertebr Pathol 177:107491
genic fungi as endophytes. Adv Genet 94:107–135 Richards TA, Dacks JB, Jenkinson JM, Thornton CR,
Mora C, Tittensor DP, Adl S, Simpson AG, Worm B Talbot NJ (2006) Evolution of filamentous plant
(2011) How many species are there on Earth and in pathogens: gene exchange across eukaryotic
the ocean? PLoS Biol 9:e1001127 kingdoms. Curr Biol 16:1857–1864
Moyes DL, Wilson D, Richardson JP et al (2016) Roy HE, Steinkraus DC, Eilenberg J, Hajek AE, Pell JK
Candidalysin is a fungal peptide toxin critical for (2006) Bizarre interactions and endgames:
mucosal infection. Nature 532:64–68 entomopathogenic fungi and their arthropod hosts.
Nakjang S, Williams TA, Heinz E et al (2013) Reduction Annu Rev Entomol 51:331–357
and expansion in microsporidian genome evolution: RoyChowdhury M, Sternhagen J, Xin Y, Lou B, Li X, Li
new insights from comparative genomics. Genome C (2022) Evolution of pathogenicity in obligate fungal
Biol Evol 5:2285–2303 pathogens and allied genera. PeerJ 10:e13794
Newman DJ, Cragg GM (2020) Natural products as Saud Z, Kortsinoglou AM, Kouvelis VN, Butt TM (2021)
sources of new drugs over the nearly four decades Telomere length de novo assembly of all
from 01/1981 to 09/2019. J Nat Prod 83:770–803 7 chromosomes and mitogenome sequencing of the
Nielsen KN, Salgado JFM, Natsopoulou ME, model entomopathogenic fungus, Metarhizium
Kristensen T, Stajich JE, De Fine Licht HH (2021) brunneum, by means of a novel assembly pipeline.
Diploidy within a haploid genus of entomopathogenic BMC Genomics 22:87
fungi. Genome Biol Evol 13:evab158 Savory F, Leonard G, Richards TA (2015) The role of
Ortiz-Urquiza A, Keyhani NO (2013) Action on the sur- horizontal gene transfer in the evolution of the
face: entomopathogenic fungi versus the insect cuticle. oomycetes. PLoS Pathog 11:e1004805
Insects 4:357–374 Shang Y, Feng P, Wang C (2015) Fungi that infect insects:
Palmer JM, Keller NP (2010) Secondary metabolism in altering host behavior and beyond. PLoS Pathog 11:
fungi: does chromosomal location matter? Curr Opin e1005037
Microbiol 13:431–436 Shang YF, Xiao GH, Zheng P, Cen K, Zhan S, Wang CS
Pan G, Xu J, Li T et al (2013) Comparative genomics of (2016) Divergent and convergent evolution of fungal
parasitic silkworm microsporidia reveal an association pathogenicity. Genome Biol Evol 8:1374–1387
between genome expansion and host adaptation. BMC Shang JM, Shang YF, Tang GR, Wang CS (2021) Identi-
Genomics 14:186 fication of a key G-protein coupled receptor in
Pan G, Bao J, Ma Z et al (2018) Invertebrate host mediating appressorium formation and fungal viru-
responses to microsporidia infections. Dev Comp lence against insects. Sci China Life Sci 64:466–477
Immunol 83:104–113 Shang J, Tang G, Lu M, Wang C (2022) Host and envi-
Pattemore JA, Hane JK, Williams AH, Wilson BA, Stodart ronmental sensing by entomopathogenic fungi to infect
BJ, Ash GJ (2014) The genome sequence of the bio- hosts. Curr Clin Microbiol Rep 9:69–74
control fungus Metarhizium anisopliae and compara- Shang JM, Tang GR, Yang J, Lu M, Wang CZ, Wang CS
tive genomics of Metarhizium species. BMC (2023) Sensing of a spore surface protein by a Dro-
Genomics 15:660 sophila chemosensory protein induces behavioral
Pelin A, Selman M, Aris-Brosou S, Farinelli L, Corradi N defense against fungal parasitic infections. Curr Biol
(2015) Genome analyses suggest the presence of poly- 33:276–286
ploidy and recent human-driven expansions in eight Shen D, Tang Z, Wang C et al (2019) Infection
mechanisms and putative effector repertoire of the
330 S. Hong et al.

mosquito pathogenic oomycete Pythium guiyangense Valero-Jimenez CA, Faino L, Spring In’t Veld D, Smit S,
uncovered by genomic analysis. PLoS Genet 15: Zwaan BJ, van Kan JA (2016) Comparative genomics
e1008116 of Beauveria bassiana: uncovering signatures of viru-
Shen D, Nyawira KT, Xia A (2020) New discoveries and lence against mosquitoes. BMC Genomics 17:986
applications of mosquito fungal pathogens. Curr Opin van Wyk S, Wingfield BD, De Vos L, van der Merwe NA,
Insect Sci 40:111–116 Steenkamp ET (2021) Genome-wide analyses of
Sheng H, McNamara PJ, St Leger RJ (2022) Metarhizium: repeat-induced point mutations in the Ascomycota.
an opportunistic middleman for multitrophic lifestyles. Front Microbiol 11:622368
Curr Opin Microbiol 69:102176 Vega FE, Kaya HK (2012) Insect pathology, 2nd edn.
Shi XZ, Zhong X, Yu XQ (2012) Drosophila Academic, San Diego, CA
melanogaster NPC2 proteins bind bacterial cell wall Vega FE, Goettel MS, Blackwell M et al (2009) Fungal
components and may function in immune signal entomopathogens: new insights on their ecology. Fun-
pathways. Insect Biochem Mol Biol 42:545–556 gal Ecol 2:149–159
Shu R, Zhang J, Meng Q et al (2020) A new high-quality Wang CS (2020) Grand challenges in the research of
draft genome assembly of the Chinese Cordyceps fungal interactions with animals. Front Fungal Biol 1:
Ophiocordyceps sinensis. Genome Biol Evol 12: 602032
1074–1079 Wang C, St Leger RJ (2006) A collagenous protective coat
Spatafora JW, Sung GH, Sung JM, Hywel-Jones NL, enables Metarhizium anisopliae to evade insect
White JF Jr (2007) Phylogenetic evidence for an ani- immune responses. Proc Natl Acad Sci U S A 103:
mal pathogen origin of ergot and the grass endophytes. 6647–6652
Mol Ecol 16:1701–1711 Wang C, St Leger RJ (2007a) The MAD1 adhesin of
St Leger RJ, Wang JB (2020) Metarhizium: jack of all Metarhizium anisopliae links adhesion with blasto-
trades, master of many. Open Biol 10:200307 spore production and virulence to insects, and the
St. Leger RJ (2021) Insects and their pathogens in a MAD2 adhesin enables attachment to plants. Eukaryot
changing climate. J Invertebr Pathol 184:107644 Cell 6:808–816
Staats CC, Junges A, Guedes RL et al (2014) Comparative Wang CS, St Leger RJ (2007b) The Metarhizium
genome analysis of entomopathogenic fungi reveals a anisopliae perilipin homolog MPL1 regulates lipid
complex set of secreted proteins. BMC Genomics 15: metabolism, appressorial turgor pressure, and viru-
822 lence. J Biol Chem 282:21110–21115
Stuart LM, Paquette N, Boyer L (2013) Effector-triggered Wang CS, Wang SB (2017) Insect pathogenic fungi: geno-
versus pattern-triggered immunity: how animals sense mics, molecular interactions, and genetic
pathogens. Nat Rev Immunol 13:199–206 improvements. Annu Rev Entomol 62:73–90
Sun YL, Hong S, Chen HM, Yin Y, Wang CS (2022a) Wang C, Skrobek A, Butt TM (2003) Concurrence of
Production of helvolic acid in Metarhizium contributes losing a chromosome and the ability to produce
to fungal infection of insects by bacteriostatic inhibi- destruxins in a mutant of Metarhizium anisopliae.
tion of the host cuticular microbiomes. Microbiol FEMS Microbiol Lett 226:373–378
Spectr 10:e0262022 Wang B, Kang Q, Lu Y, Bai LQ, Wang CS (2012)
Sun YL, Chen B, Li XL, Yin Y, Wang CS (2022b) Unveiling the biosynthetic puzzle of destruxins in
Orchestrated biosynthesis of the secondary metabolite Metarhizium species. Proc Natl Acad Sci U S A 109:
cocktails enables the producing fungus to combat 1287–1292
diverse bacteria. mBio 13:e0180022 Wang JB, St Leger RJ, Wang C (2016) Advances in
Sung GH, Hywel-Jones NL, Sung JM, Luangsa-Ard JJ, genomics of entomopathogenic fungi. Adv Genet 94:
Shrestha B, Spatafora JW (2007) Phylogenetic classi- 67–105
fication of Cordyceps and the clavicipitaceous fungi. Wang Y, Stata M, Wang W, Stajich JE, White MM,
Stud Mycol 57:5–59 Moncalvo JM (2018) Comparative genomics reveals
Tang D, Tang X, Fang W (2022) New downstream signal- the core gene toolbox for the fungus-insect symbiosis.
ing branches of the mitogen-activated protein kinase mBio 9:e0063618
cascades identified in the insect pathogenic and plant Wang J, Weng Q, Hu Q (2019) Effects of Destruxin A on
symbiotic fungus Metarhizium robertsii. Front Fungal silkworm's immunophilins. Toxins (Basel) 11:349
Biol 3:911366 Wang W, Deng Z, Wu H et al (2019b) A small secreted
Thakur R, Shiratori T, Ishida KI (2019) Taxon-rich protein triggers a TLR2/4-dependent inflammatory
multigene phylogenetic analyses resolve the phyloge- response during invasive Candida albicans infection.
netic relationship among deep-branching Nat Commun 10:1015
Stramenopiles. Protist 170:125682 Wang Y-B, Wang Y, Fan Q et al (2020a) Multigene
Usman S, Ge X, Xu Y et al (2023) Loss of phylogeny of the family Cordycipitaceae
phosphomannose isomerase impairs growth, perturbs (Hypocreales): new taxa and the new systematic posi-
cell wall integrity, and reduces virulence of Fusarium tion of the Chinese cordycipitoid fungus Paecilomyces
oxysporum f. sp. cubense on banana plants. J Fungi hepiali. Fungal Divers 103:1–46
(Basel) 9:478
13 Genetics and Infection Biology of the Entomopathogenic Fungi 331

Wang Y, Nie Y, Yu D, Xie X, Qin L, Yang Y, Huang B associated with fungal infection. Anal Bioanal Chem
(2020b) Genome-wide study of saprotrophy-related 407:4815–4821
genes in the basal fungus Conidiobolus heterosporus. Yang X, Feng P, Yin Y, Bushley K, Spatafora JW, Wang
Appl Microbiol Biotechnol 104:6261–6272 C (2018) Cyclosporine biosynthesis in Tolypocladium
Wei G, Lai Y, Wang G, Chen H, Li F, Wang S (2017) inflatum benefits fungal adaptation to the environment.
Insect pathogenic fungus interacts with the gut MBio 9:e01211–e01218
microbiota to accelerate mosquito mortality. Proc Yin Y, Chen B, Song SX, Li B, Yang XQ, Wang CS
Natl Acad Sci U S A 114:5994–5999 (2020) Production of diverse beauveriolide analogs in
Whisler HC, Zebold SL, Shemanchuk JA (1975) Life closely related fungi: a rare case of fungal
history of Coelomomyces psorophorae. Proc Natl chemodiversity. mSphere 5:e0066720
Acad Sci U S A 72:693–696 Ying SH, Feng MG (2019) Insight into vital role of
Wichadakul D, Kobmoo N, Ingsriswang S, autophagy in sustaining biological control potential of
Tangphatsornruang S, Chantasingh D, Luangsa-Ard fungal pathogens against pest insects and nematodes.
JJ, Eurwilaichitr L (2015) Insights from the genome Virulence 10:429–437
of Ophiocordyceps polyrhachis-furcata to pathogenic- Zeng G, Zhang P, Zhang Q et al (2018) Duplication of a
ity and host specificity in insect fungi. BMC Genomics Pks gene cluster and subsequent functional diversifica-
16:881 tion facilitate environmental adaptation in Metarhizium
Will I, Das B, Trinh T, Brachmann A, Ohm RA, de Bekker species. PLoS Genet 14:e1007472
C (2020) Genetic Underpinnings of Host Manipulation Zhang C, Deng W, Yan W, Li T (2018) Whole genome
by Ophiocordyceps as Revealed by Comparative sequence of an edible and potential medicinal fungus,
Transcriptomics. G3 (Bethesda) 10:2275–2296 Cordyceps guangdongensis. G3 (Bethesda) 8:1863–
Wilson RA, Talbot NJ (2009) Under pressure: 1870
investigating the biology of plant infection by Zhang L, Fasoyin OE, Molnar I, Xu Y (2020a) Secondary
Magnaporthe oryzae. Nat Rev Microbiol 7:185–195 metabolites from hypocrealean entomopathogenic
Wraight SP, Ramos ME, Avery PB, Jaronski ST, fungi: novel bioactive compounds. Nat Prod Rep 37:
Vandenberg JD (2010) Comparative virulence of 1181–1206
Beauveria bassiana isolates against lepidopteran Zhang L, Yue Q, Wang C, Xu Y, Molnar I (2020b)
pests of vegetable crops. J Invertebr Pathol 103:186– Secondary metabolites from hypocrealean
199 entomopathogenic fungi: genomics as a tool to eluci-
Xia EH, Yang DR, Jiang JJ et al (2017a) The caterpillar date the encoded parvome. Nat Prod Rep 37:1164–
fungus, Ophiocordyceps sinensis, genome provides 1180
insights into highland adaptation of fungal pathogenic- Zhang X, Meng Y, Huang Y, Zhang D, Fang W (2021) A
ity. Sci Rep 7:1806 novel cascade allows Metarhizium robertsii to distin-
Xia YL, Luo FF, Shang YF, Chen PL, Lu YZ, Wang CS guish cuticle and hemocoel microenvironments during
(2017b) Fungal cordycepin biosynthesis is coupled infection of insects. PLoS Biol 19:e3001360
with the production of the safeguard molecule Zhao H, Xu C, Lu HL, Chen X, St Leger RJ, Fang W
pentostatin. Cell Chem Biol 24:1479–1489 (2014) Host-to-pathogen gene transfer facilitated infec-
Xiao G, Ying SH, Zheng P et al (2012) Genomic tion of insects by a pathogenic fungus. PLoS Pathog
perspectives on the evolution of fungal 10:e1004009
entomopathogenicity in Beauveria bassiana. Sci Rep Zheng P, Xia Y, Xiao G et al (2011) Genome sequence of
2:483 the insect pathogenic fungus Cordyceps militaris, a
Xu Y, Orozco R, Wijeratne EM, Gunatilaka AA, Stock SP, valued traditional Chinese medicine. Genome Biol
Molnar I (2008) Biosynthesis of the cyclooligomer 12:R116
depsipeptide beauvericin, a virulence factor of the Zheng P, Xia Y, Zhang S, Wang C (2013) Genetics of
entomopathogenic fungus Beauveria bassiana. Chem Cordyceps and related fungi. Appl Microbiol
Biol 15:898–907 Biotechnol 97:2797–2804
Xu Y-J, Luo F, Gao Q, Shang Y, Wang C (2015)
Metabolomics reveals insect metabolic responses
 Xylaria Sclerotia Formed Within Termite
Nests: A Review of Their Biology 14
and Human Uses

Huei-Mei Hsieh and Yu-Ming Ju

Abstract 14.1 Introduction

Certain Xylaria species form massive sclerotia
of several centimeters in diameter within the Massive sclerotia of several centimeters in diam-
underground nests of macrotermitine termites eter formed within abandoned subterranean ter-
after the termites abandon the nest. Vernacu- mite nests are known in traditional medicine in
larly the sclerotia are called Wulingshen in China as Wulingshen and Puttumanga in India.
China or Nilamanga and Puttumanga in south- The sclerotia are produced by fungi of the genus
ern India. Xylaria nigripes once was com- Xylaria Hill ex Schrank (Xylariaceae,
monly regarded as the sole producer of the Sordariomycetes, Ascomycota). The nests are
massive sclerotia, which are in fact produced built by macrotermitine termites (subfamily
by at least six Xylaria species, including X. Macrotermitinae), the fungus-growing termites,
nigripes and close relatives. Xylaria sclerotia which have a broad geographical distribution in
have been used in folk medicine for treating a tropical and subtropical regions of Asia and
wide range of ailments, whereas dried mycelia Africa (Aanen et al. 2009; Mueller et al. 2005).
from cultures are used mainly for soothing The fungi cultivated by macrotermitine termites
anxiety, depression, insomnia, and some neu- are species of the genus Termitomyces Heim
rological disorders. Here, we review the (Lyophyllaceae, Agaricomycetes, Basidiomycota),
biological functions, pharmacological which form a mutually symbiotic relationship with
properties, and beneficial implications for the termites (Batra and Batra 1979; Sangvichien
human health from cultured materials prepared and Taylor-Hawksworth 2001). Xylaria species
from mycelia or stromata produced in cultures are not cultivated by termites but enter termite
of sclerotium-forming Xylaria species. nests as stowaways where they are competitors of
Termitomyces (Visser et al. 2009). Nevertheless,
Keywords the exact interactions among Xylaria species,
termites, and Termitomyces mushrooms remain to
Ascomycota · Macrotermitinae · Wuling be clarified (Rogers et al. 2005).
capsule · Wulingshen · Xylariaceae · Xylaria sclerotia (Fig. 14.1a, b) formed within
Medicinal properties subterranean termite nests are massive compared
to those produced by other fungal species.
Sclerotia are resistant structures composed of
H.-M. Hsieh · Y.-M. Ju (✉)
tightly compacted mycelium. Sclerotia of the
Institute of Plant and Microbial Biology, Academia Sinica,
Taipei, Taiwan Xylaria species discussed here vary in shape
e-mail: [email protected] from spherical to ovoid or even irregular and are
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 333
Y.-P. Hsueh, M. Blackwell (eds.), Fungal Associations, The Mycota 9,
https://doi.org/10.1007/978-3-031-41648-4_14
334 H.-M. Hsieh and Y.-M. Ju

Fig. 14.1 Xylaria nigripes, X. neonigripes, and X. grayish powdery conidial mass on the surface. E. Stromata
furcata. A–E. X. nigripes. A. Sclerotia. B. Vertical section produced in nature. F. X. neonigripes, which can be
of sclerotium showing a white interior. C. Colony produc- separated from X. nigripes by the narrowed sterile
ing stromata on a 9-cm Petri dish containing oatmeal agar; stromatal apex. Stromata are produced in nature. G. X.
the culture was obtained from a sclerotium shown in A. D. furcata. Stromata are produced in nature. Bars: A, B, E–
Part of a stroma produced from the colony in C showing a G = 1 cm; D = 2 mm
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 335

several centimeters in diameter. A compacted mentioned that Wulingshen is also called

mass of tasteless white hyphae comprises a core Lei-zhen-zi, which literally means fruits of thun-
covered by a peridium that is black or dark der, apparently because sclerotia hanging from
brown, and smooth on the surface while fresh the roof of nesting chambers twisted as thunder
but wrinkled after drying (Ju et al. 2022). The struck, an extremely difficult if not impossible
top of the sclerotium has a narrow-broken point, observation. The name Lei-zhen-zi seemingly
indicating its former connection point with the implies that the formation of Wulingshen
attached base of the parent stroma (Ju et al. coincides with the thunderstorm season in
2022). Fungal stromata are similar to sclerotia China; 84% of the thunderstorms occur during
because they are composed of tightly organized May and September (Zhang et al. 2017). Field
mycelium, but they differ in function because studies are needed to confirm whether there is a
they are the site of ascospore and conidium pro- connection between the formation of Wulingshen
duction in Xylaria. and thunderstorms. Interestingly, a connection
The British mycologist Miles J. Berkeley was between thunderbolts and fungi was also noted
probably the first to describe massive sclerotia by Lowy (1974) who recorded that Amanita
from termite nests as Sclerotium stipitatum muscaria (L.) Lam. was believed to fruit on the
Berk. & Currey, based on a specimen collected ground in places where thunderbolts struck in the
from southern Lara State, India, formerly known Roman, Hindu, and Mayan cultures. Chinese lit-
as Travancore. It is called Puttumanga in Tamil erature was rather vague as to which species
language, which means termite-hill mango produced Wulingshen until Wu (1984) pointed
(Berkeley 1862). However, the producer of the out that Wulingshen was produced by
sclerotia was unclear for the next 44 years until X. nigripes and an unnamed Xylaria species.
Petch (1906) studied massive sclerotia from ter- Wulingshen is said in folklore to be effective
mite nests in Sri Lanka. He placed them under a in eliminating dampness in body condition, eas-
bell jar on a damp filter paper, where ascospore- ing infantile convulsions, stopping palpitations,
producing stromata of a Xylaria species were promoting lactation, nourishing hearts and
produced. This was the first time massive kidneys, and curing insomnia, nose bleeds, and
sclerotia from termite nests were connected to a hemorrhaging after childbirth (Ying et al. 1987).
Xylaria species. It should be noted that Petch More recently, traditional uses of wild-collected
(1913) referred to the Xylaria species as X. sclerotia in China have progressed to the produc-
nigripes (Klotzsch) M. C. Cooke but his concept tion of commercial products. Today, dried myce-
of X. nigripes was broad, with X. escharoidea lial powder, Wuling powder, prepared from
(Berk.) Sacc. and six other names listed as Xylaria cultures, is encapsulated and referred to
synonyms. Xylaria escharoidea is a distinct spe- as Wuling capsule by Pharmacopoeia Commis-
cies and is more commonly encountered than X. sion of China (2020). Wuling capsule was
nigripes (Rogers et al. 2005). Many specimens approved by the China Food and Drug Adminis-
labeled as X. nigripes should be recognized as X. tration (CFDA) with the authorized document
escharoidea because their ascospores possess a number Z19990048 and has become a leading
distinctive pore-like germination site. Chinese patent medicine for dealing with insom-
Massive Xylaria sclerotia have been consumed nia. Production of Wuling powder is subjected to
as herbal medicine in China. Medicinal uses of standardized cultivation and preparation, and its
these sclerotia from termite nests were first containing mannitol and adenosine have been
reported in “Revised local gazetteer of the district proposed for assaying the product. The
of Guan-xian” from Sichuan, China (Zhuang and recommended dosage of Wuling capsule for
Zheng 1886), where the name Wulingshen was insomnia is three capsules (0.33 g each), followed
introduced for the first time. The medicinal effect by three doses per day taken orally for 20 contin-
of Wulingshen in the gazetteer is brief, only men- uous days (Feng et al. 2016).
tioning it for nourishing kidneys. The gazetteer
336 H.-M. Hsieh and Y.-M. Ju

Information on Xylaria sclerotia and in dry situations or when the nests were aban-
Wulingshen is easily available in internet doned in the dry season. To our knowledge, mas-
searches, but most of the information concerns sive sclerotia are only collected from nature, not
the efficacy of Wulingshen for treating illnesses formed in artificial conditions.
or the high price of Wulingshen (currently several
hundred US dollars per Kg in dry weight). Here,
we will discuss compounds or functional extracts 14.3 Xylaria Species Producing
produced from cultivated mycelium (used inter- Massive Sclerotia in Termite
changeably with Wuling capsule) and stromata, Nests: Systematics
and their potential applications to human health. and Relationship with Termites

Xylaria nigripes was once considered to be the

14.2 Known Habitat and Localities sole producer of massive sclerotia found in ter-
of Massive Xylaria Sclerotia mite nests. However, several studies indicate that
this may not be the case. The studies by Wu
Petch (1913) mentioned that massive Xylaria (1984) and Chen et al. (1988) revealed more
sclerotia had been collected from India, Sri than one Xylaria species capable of producing
Lanka, Java in Indonesia, and Africa. The close massive sclerotia, and Deepna Latha et al.
association between nests of macrotermitine (2015) linked an Indian Xylaria sclerotium to X.
termites and massive Xylaria sclerotia strongly acuminatilongissima Y.-M. Ju & H.-M. Hsieh by
suggests the geographic distribution of the using ITS (Nuc rDNA internal transcribed
sclerotium-producing Xylaria species coincides spacers = ITS1-5.8S-ITS2) sequence.
with that of macrotermitine termites in tropical Wulingshen, a valuable herbal medicine in
and subtropical regions of Asia and Africa. Sichuan Province, China, can be procured from
When the macrotermitine termites properly traditional Chinese medicine markets there. Ju
manage the fungus gardens within termite nests, et al. (2022) obtained ITS sequences from
the dominant fungi are the Termitomyces 54 Wulingshen samples and concluded that they
mushrooms, the cultivated crop of termites. belonged to six different Xylaria species, two of
Once the termite nests are abandoned by the which, X. nigripes and X. neonigripes Y.-M. Ju,
termites, Xylaria species soon gain the chance to H.-M. Hsieh & X.-S. He are shown in Fig. 14.1e,
start exploiting the nutrients of the fungus f. The result was further supported by sequences
gardens by producing hyphae profusely. Massive of three protein-coding loci: β-tubulin (β-TUB)
sclerotia are produced by sclerotium-forming and α-actin (α-ACT), and RNA polymerase II
Xylaria species under proper conditions in the second largest subunit (RPB2). The six Xylaria
fungus gardens, which are gradually shrunk to species connected with Wulingshen from
disappearance after the nutrient is completely Sichuan, China, are X. nigripes, X.
depleted. At this stage, massive sclerotia are rogersionigripes Y.-M. Ju, H.-M. Hsieh & X.-S.
often found hanging from the roof of the under- He, X. neonigripes, X. subescharoidea, and two
ground chambers as if they were formed in the air, unnamed species (X. sp. A, X. sp. B) that are
a phenomenon that Zhuang and Zheng (1886) had known only in their sclerotial forms. About
noted in the original description of Wulingshen. one-half (51.9%) of the massive sclerotia were
Although massive Xylaria sclerotia are produced from X. rogersionigripes, followed by 20.4%
in abandoned termite nests, they are not always from X. nigripes, 13.0% from X. neonigripes,
found when the abandoned nests are excavated; 11.1% from X. sp. A, 1.9% from X.
the proper conditions under which massive subescharoidea, and 1.9% from X. sp. B.
sclerotia are formed remain to be discovered. There are 43 known Xylaria species associated
Petch (1913), based on his field experiences, with termite nests in the world (Hsieh et al. 2020;
believed that massive sclerotia were only formed Ju and Hsieh 2007; Ju et al. 2022; Rogers et al.
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 337

2005; Wangsawat et al. 2021a), which can be and RPB2 in the context of more than a hundred
divided into two general groups by their stromatal Xylaria species from all known substrate types,
morphology: the X. nigripes group (Fig. 14.1e, f) with which Xylaria species are associated. The
and the X. furcata Fr. group (Fig. 14.1g). Species phylogenetic trees had topologies that were simi-
in the X. nigripes group have cylindrical, lar to those shown in Hsieh et al. (2010) and
unbranched stromata that are more robust, U’Ren et al. (2016), with the species associated
ca. 4–8 mm diameter at the perithecium-bearing with termite nests grouped into a single clade.
part. Species in the X. furcata group have antler- Interestingly, within this clade, the six
like, dichotomously branched stromata that are Wulingshen-producing species mentioned
delicate, ca. 1–2 mm diameter at the above, together with X. acuminatilongissima,
perithecium-bearing part. It should be X. escharoidea, and X. cirrata Pat., formed a
emphasized that not every Xylaria species subclade, suggesting that the sclerotium-forming
associated with termite nests can produce massive habit arose only once within Xylaria (Fig. 14.2).
sclerotia, but all known sclerotium-producing Xylaria acuminatilongissima was linked to a mas-
species belong to the X. nigripes group. sive sclerotium from Kerala, India (Deepna Latha
The production of massive sclerotia is unique et al. 2015), while X. escharoidea and X. cirrata
among Xylaria species. Are the sclerotium- have not been linked to massive sclerotia. It is
producing Xylaria species phylogenetically possible that the latter two species may have lost
related? Ju et al. (2022) included the sclerotium- the ability to form massive sclerotia.
producing Xylaria species in phylogenetic In the studies reviewed below, the Xylarias
analyses based on sequences of β-TUB, α-ACT, studied were often assumed to be X. nigripes.

Fig. 14.2 Phylogenetic tree, generated by Maximum- subescharoidea, X. sp. A, and X. sp. B. Wulingshen
Likelihood analysis based on sequences of β-TUB, samples prefixed with “WLS” are framed. While
α-ACT, and RPB2, is modified from Fig. 2 in Ju et al. X. acuminatilongissima was linked to a Xylaria sclerotium
(2022), where the complete tree was fully presented in in India (Deepna Latha et al. 2015), X. escharoidea and X.
Supplementary Fig. 1 of Ju et al. (2022) but is shown cirrata are the only species that have not been linked to
only schematically on the left, with the area within the sclerotia. Numbers at internodes represent bootstrap
rectangle enlarged to show the detail of the clade denoted values and are immediately followed by the posterior
by TE. Within the TE clade, the subclade in green shade probability values greater than 50% with Bayesian infer-
encompassed the Wulingshen-producing species, i.e., X. ence analysis
neonigripes, X. nigripes, X. rogersionigripes, X.
338 H.-M. Hsieh and Y.-M. Ju

ITS sequences from some of them have been system (CNS), are broadly distributed throughout
deposited in GenBank, and the identities of the brain and the spinal cord and are crucial for a
these species were reassessed by comparing healthy CNS (Ginhoux et al. 2013). Microglial
their sequences with those of the sclerotium- response is pathology dependent, affects immu-
forming Xylaria species studied by Ju et al. nity and metabolism, and plays an important role
(2022). In these cases, the results from the in the progression of neurodegenerative diseases,
comparisons are presented here; otherwise, origi- such as Parkinson’s disease, Alzheimer’s disease,
nal identifications are retained. and frontotemporal dementia (Bachiller et al.
2018). Main pathways including JAK/STATs
and NFkB govern microglial pro-inflammatory
14.4 Possible Functions response, and an uncontrolled inflammatory
of Compounds Isolated from response by microglial cells can lead to a detri-
Cultured Sclerotium-Forming mental outcome (Bachiller et al. 2018). An impor-
Xylaria Species tant concern for selecting bioactive compounds
for anti-inflammatory effects concerns their cyto-
Information about compound-producing Xylaria toxic effects on cells treated with these
species, including their morphological state for compounds. Only the bioactive compounds not
compound production, associated GenBank num- causing significant toxicity on cells are reviewed
ber, culture or specimen deposition, and applied herein.
experimental cell lines, and functions of various Eleven compounds were isolated from ethyl
compounds are listed in Table 14.1. A brief intro- acetate extracts of the liquid fermentation of the
duction to applied cell lines is also denoted in X. nigripes culture YMJ 653 (GenBank accession
Table 14.1. number EU179868) (Chang et al. 2017). Of the
compounds tested, nigriterpene C, a sesquiter-
pene, and formannoxin alcohol significantly
14.4.1 Anti-inflammation exhibited inhibitory effects on the expression of
and Neuroprotection iNOS, COX2, and the production of NO by BV-2
Associated microglial cells of murine brain when treated with
with Lipopolysaccharide a lipopolysaccharide (LPS) stimulant and hence
Toxicity and Oxidative Stress were considered as promising candidates for anti-
neuroinflammatory drugs. Nigriterpene C also
Mycelia of most Wulingshen cultures synthesize exhibited concentration-dependent inhibition of
compounds known to have anti-inflammatory iNOS expression, COX2 expression, and NO pro-
effects. Here we briefly describe several signaling duction with the IC50 (the half maximal inhibitory
components of inflammation and the microglial concentration applied for a given biological pro-
responses towards these signaling components. cess) being 8.1 ± 2.3, 16.6 ± 5.5, and
These include inducible nitric oxide synthase 21.7 ± 4.9 μM, respectively, and therefore could
(iNOS), cyclooxygenase (COX2), tumor necrosis be more potent than formannoxin alcohol.
factor TNF-α, interleukin IL-1β, nitric oxide Ergosterol peroxide and ergostarien-3β-ol
(NO), and prostaglandin E2 (PGE2), which are isolated from a hexane fraction of dried stromata
widely known for signaling in inflammation of cultivated X. nigripes exhibited anti-
(Bachiller et al. 2018; Kawahara et al. 2015; inflammatory activities with ergosterol peroxide
Rojo et al. 2008). The activation of inflammatory being stronger in the inhibition of several
molecules is known to have a deleterious role in pro-inflammatory mediators in RAW 264.7
neurodegenerative diseases by expanding the macrophages treated with LPS (Liaw et al.
inflammatory response and increasing neuronal 2017). The pro-inflammatory mediators include
damage (Bachiller et al. 2018). Microglia, a pop- NO, IL-1β, TNF-α, IL-6, and PGE2. Ergosterol
ulation of macrophage cells in the central nervous peroxide (25 μmol/L) also greatly suppressed the
 14
Table 14.1 Information of compound-producing Xylaria species
Compound names Species Suggested or Morphological Strain Deposition GenBank Cell line/
or extracts name confirmed name state number Location no. Function microorganism References
(3β,5α,6β)- X. nigripes N/A Mycelia N/A ZJPCa N/A Cytotoxicity against U2OSb Xiong
Stigmastane-3,5,6- osteosarcoma cells et al.
triol (2016)
(3β,5α,6β, 22E)-6- X. nigripes N/A Mycelia N/A ZJPCa N/A Neuroprotection by SH-SY5Yc Xiong
Methoxyergosta- an increase in cell et al.
7,22-diene-3,5-diol viability (2016)
(3β,5α,6β, 22E)-6- X. nigripes N/A Mycelia N/A ZJPCa N/A Cytotoxicity against A549d Xiong
Methoxyergosta- lung cancer cells et al.
7,22-diene-3,5-diol (2016)
(3β,5α,6β, 22E)-6- X. nigripes N/A Mycelia N/A ZJPCa N/A Cytotoxicity against U2OSb Xiong
Methoxyergosta- osteosarcoma cells et al.
7,22-diene-3,5-diol (2016)
(3β,5α,8α,22E)- X. nigripes N/A Mycelia N/A ZJPCa N/A Anti- BV-2e Xiong
Ergosta-6,22- neuroinflammation et al.
diene-3,5,8-triol (2016)
4,8-Dihydroxy- X. X. escharoidea Mycelia TF18 N/A MN509048 Antimicrobial S. aureus, Nagam
3,4- escharoidea activity B. subtilis, et al.
dihydronaphthalen- P. aeruginosa, (2021)
1(2H)-one and C. albicans
(6S)- Agroclavine X. nigripes N/A Dried stromata CUI KIBf N/A Cholesterol ester N/A Hu and Li
N-oxide 20090818 transfer protein (2017)
inhibitor
8,9-Didehydro-10- X. nigripes N/A Dried stromata CUI KIBf N/A Cholesterol ester N/A Hu and Li
hydroxy-6,8- 20090818 transfer protein (2017)
dimethylergolin inhibitor
Agroclavine X. nigripes N/A Dried stromata CUI KIBf N/A Inhibitory activity on N/A Hu and Li
20090818 acetylcholinesterase (2017)
Agroclavine X. nigripes N/A Dried stromata CUI KIBf N/A Cholesterol ester N/A Hu and Li
Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses

20090818 transfer protein (2017)

inhibitor
Ergostarien-3β-ol X. nigripes X. Cultivated KJ-XN-07-1 KJBCg JQ927570 Anti-inflammation RAW 264.7h Liaw et al.
rogersionigripes stromata (2017)
Ergosterol D X. nigripes N/A Mycelia N/A ZJPCa N/A Cytotoxicity against U2OSb Xiong
osteosarcoma cells et al.
(2016)
339

(continued)
Table 14.1 (continued)
340

Compound names Species Suggested or Morphological Strain Deposition GenBank Cell line/
or extracts name confirmed name state number Location no. Function microorganism References
Ergosterol X. nigripes X. Cultivated KJ-XN-07-1 KJBCg JQ927570 Anti-inflammation RAW 264.7h Liaw et al.
peroxide rogersionigripes stromata (2017)
Fimbriether B X. fimbriata X. fimbriata Mycelia YMJ491 IPMBi GU324753 Anti-inflammation RAW 264.7h Chen et al.
(2019)
Fimbriether E X. fimbriata X. fimbriata Mycelia YMJ491 IPMBi GU324753 Anti-inflammation RAW 264.7h Chen et al.
(2019)
Fimbriether G X. fimbriata X. fimbriata Mycelia YMJ491 IPMBi GU324753 Anti-inflammation RAW 264.7h Chen et al.
(2019)
Formannoxin X. nigripes X. nigripes Mycelia YMJ653 IPMBi EU179868 Anti- BV-2e Chang
alcohol neuroinflammation et al.
(2017)
Nigriterpene C X. nigripes X. nigripes Mycelia YMJ653 IPMBi EU179868 Anti- BV-2e Chang
neuroinflammation et al.
(2017)
Pollenopyrrosides X. nigripes N/A Mycelia N/A ZJPCa N/A Inhibition of A7r5j Li et al.
A oxidative stress (2015)
Pollenopyrrosides X. nigripes N/A Mycelia N/A ZJPCa N/A Inhibition of A7r5j Li et al.
B oxidative stress (2015)
(R)-5- X. nigripes N/A Mycelia N/A ZJPCa N/A Inhibitor against N/A Huang
Methylmellein monoamine oxidase et al.
A (2019)
Water soluble, X. nigripes X. Cultivated N/A KJBCg KJ627787 Anti-inflammation RAW 264.7h Chen et al.
nondigestible rogersionigripes stromata (2018)
polysaccharides
CXNP
Water soluble, X. nigripes X. Cultivated N/A KJBCg KJ627786 Anti-inflammation RAW 264.7h Chen et al.
nondigestible rogersionigripes stromata (2018)
polysaccharides
TXNP
Water soluble, X. nigripes X. Cultivated N/A KJBCg KJ627786 Anti-inflammation RAW 264.7h Jen et al.
nondigestible rogersionigripes stromata (2021)
polysaccharides F1
of XN
H.-M. Hsieh and Y.-M. Ju
 14

Water soluble, X. nigripes X. Cultivated N/A KJBCg KJ627786 Anti-inflammation RAW 264.7h Jen et al.
nondigestible rogersionigripes stromata (2021)
polysaccharides F2
of XN
Xylanigripone A X. nigripes N/A Dried stromata CUI KIBf N/A Cholesterol ester N/A Hu and Li
20090818 transfer protein (2017)
inhibitor
Xylanigripone C X. nigripes N/A Dried stromata CUI KIBf N/A Inhibitory activity on N/A Hu and Li
20090818 acetylcholinesterase (2017)
Xylapyrrosides A X. nigripes N/A Mycelia N/A ZJPCa N/A Inhibition of A7r5j Li et al.
oxidative stress (2015)
Xylapyrrosides B X. nigripes N/A Mycelia N/A ZJPCa N/A Inhibition of A7r5j Li et al.
oxidative stress (2015)
Xylarinaps A X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Antioxidation and PC12l Li et al.
00611 neuroprotection by (2021)
an increase in cell
viability
Xylarinaps B X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Antioxidation and PC12l Li et al.
00611 neuroprotection by (2021)
an increase in cell
viability
Xylarinaps D X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Antioxidation and PC12l Li et al.
00611 neuroprotection by (2021)
an increase in cell
viability
Xylarinaps E X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Antioxidation and PC12l Li et al.
00611 neuroprotection by (2021)
an increase in cell
viability
Xylarinig A X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Neuroprotection by PC12l Li et al.
00611 an increase in cell (2022)
Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses

viability
Xylarinig B X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Neuroprotection by PC12l Li et al.
00611 an increase in cell (2022)
viability
(continued)
341
Table 14.1 (continued)
342

Compound names Species Suggested or Morphological Strain Deposition GenBank Cell line/
or extracts name confirmed name state number Location no. Function microorganism References
Xylarinig C X. nigripes N/A Mycelia CGBWSHF SCUNk N/A Neuroprotection by PC12l Li et al.
00611 an increase in cell (2022)
viability
a
ZJPC: Zhejiang Jolly Pharmaceutical Co., China
b
U2OS: Derived from sarcoma of human tibia and one of its applications is in the study of bone tumorigenicity (Lauvrak et al. 2013)
c
SH-SY5Y: A subline of the SK-N-SH cell line which was from neuroblastoma of bone marrow and widely used in the research of Parkinson’s disease (Xicoy et al. 2017)
d
A549: Isolated from human lung adenocarcinoma and its applications include drug metabolism and tumorigenicity (Croce et al. 1999; Foster et al. 1998)
e
BV-2: Derived from murine microglia and immortalized by transduction of neonatal primary microglia with v-raf/v-myc carrying J2 retrovirus and used for the study of
neuroinflammation and neurodegenerative disorders (Timmerman et al. 2018)
f
KIB: Kunming Institute of Botany, CAS, China
g
KJBC: Kang Jian Biotech Corp., Ltd., Taiwan
h
RAW 264.7: Monocyte/macrophage-like, established from a tumor of BALB/c mice induced with Abelson murine leukemia virus, and the cells increase nitric oxide production
and phagocytosis upon lipopolysaccharide stimulation (Taciak et al. 2018)
i
IPMB: Institute of Plant and Microbial Botany, AS, Taiwan
j
A7r5: Derived from aortic smooth muscle cells of rat and can be used for the study of vascular oxidative stress (Liao et al. 2018; Zhou et al. 2020)
k
SCUN: South-Central University for Nationalities, China
l
PC12: Cells originated from a pheochromocytoma of rat adrenal medulla and are used for studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and
synaptogenesis (Wiatrak et al. 2020)
H.-M. Hsieh and Y.-M. Ju
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 343

expressions of iNOS, COX2, and NF-κB, and mycelia of X. nigripes (Xiong et al. 2016). One of
hence the mechanism of the inhibition was the six steroids, i.e., (3β,5α,8α,22E)-ergosta-6,22-
involved in the regulation of the NF-κB pathway. diene-3,5,8-triol exhibited an inhibitory effect on
BLAST searches using ITS indicated that the NO production in the LPS-induced BV-2
so-called X. nigripes culture (GenBank microglial cells, and its value of IC50 was
JQ927570) used in this study actually showed a 27.6 μM, comparable to NGmonomethyl-L-argi-
99.62% similarity to X. rogersionigripes nine (IC50 = 14.4 μm), an inhibitor of NO
(YMJ1148). synthase. Another steroid, (3β,5α,6β, 22E)-6-
Two water soluble, nondigestible methoxyergosta-7,22-diene-3,5-diol, at the con-
polysaccharides, TXNP and CXNP, extracted centration of 10 μM, exhibited a significant
from stromata formed in two X. nigripes cultures neuroprotective effect on human SH-SY5Y neu-
with GenBank accession numbers KJ627786 and roblastoma cells, showing a 21% increase in cell
KJ627787, were obtained by following steps viability by attenuating β-amyloid25–35-induced
including water extraction, ethanol precipitation, cell damage.
and treatments of α-amylase and α-glucosidase; Four spirocyclic pyrrole alkaloids, including
these two samples were then compared for anti- pollenopyrrosides A and B and xylapyrrosides A
inflammatory activities (Chen et al. 2018). TXNP and B, were isolated from the ethanol extracts of
and CXNP were dose-dependent in suppressing cultured mycelia of X. nigripes (Li et al. 2015). In
NO, IL-1β, TNF-α, and PGE2 productions as well addition, total syntheses for three of the isolated
as iNOS, COX-2, and NF-κB expressions in compounds, i.e., pollenopyrroside B,
LPS-induced RAW264.7 macrophages. TXNP xylapyrrosides A and B, were achieved, and
predominantly composed of glucose (97.8%) three new analogs, xylapyrrosides A1, A2, and
had better suppression of these inflammatory B1 were also produced during the syntheses. The
mediators than CXNP composed of mannose isolated and synthesized pyrrole alkaloids men-
(41.4%), glucose (17.1%), and xylose (41.5%). tioned above as well as an antioxidant flavonoid
Both of the ITS sequences of the two cultures (t)-catechin hydrate inhibited cytotoxicity on
KJ627786 and KJ627787 matched that of X. A7r5 rat vascular smooth muscle cells, treated
rogersionigripes (YMJ1148) with which they with tert-Butyl hydroperoxide (tBHP) for induc-
shared 100% and 99.81% similarities, respec- tion of oxidative stress. Cell viability dropped to
tively. Water soluble, nondigestible 59.6% after tBHP was employed but increased to
polysaccharides from the strain with the GenBank a range of 85.3%–103.8% after the addition of
accession no. KJ627786 were separated and pyrrole alkaloids tested at 100 μM. The antioxi-
purified into two bioactive polysaccharide dant effects of the pyrrole alkaloids are
components F1 and F2, with the former being concentration-dependent with pollenopyrroside
885.2 kDa and composed of predominantly glu- B and xylapyrroside A2 being stronger in the
can (95.5%) and the latter being 24.5 kDa and a suppression of the tBHP-induced apoptosis. In
composition of mannose (13.8%), glucose another study by Verano and Tan (2017), both
(71.9%), and galactose (14.3%) (Jen et al. synthesized pollenopyrroside A and
2021). F1 exhibited better anti-inflammatory xylapyrroside A exhibited an inhibition effect
activities, suppressing the production of NO, with IC50 values being 17 μM and 11 μM, respec-
TNF-α, and IL-6 more strongly, than F2. The tively, on the production of reactive oxygen spe-
combination of F1 and F2 demonstrated synergis- cies (ROS) induced by the treatment of high
tic effects in the expression of anti-inflammatory glucose on rat mesangial cells. Their analogs,
activities that were comparable to that of the the newly synthesized 2-OH pollenopyrroside A
polysaccharide before being fractioned. and 2-OH xylapyrroside, are highly valuable as
Nineteen compounds, including nine pyrrole- antioxidants with IC50 values of 0.52 μM and
containing alkaloids and six steroids, were 0.27 μM, respectively. Both studies bring new
isolated from an ethanol extract of the fermented
344 H.-M. Hsieh and Y.-M. Ju

insights for profound exploration of the unique 14.4.2 Cytotoxic Effects Against
spiro-alkaloids in drug discovery. Cancer Cell Lines
Four out of the five pairs of naphthalenone
derivative enantiomers, i.e., xylarinaps A, B, D, Three of the six steroids from Xiong et al. (2016),
and E, reported in Li et al. (2021) and four new i.e., (3β, 22E)-ergosta-7,9(11),22-trien-3-ol
resorcinol derivatives, i.e., (-)-xylarinig A, (+)- (ergosterol D), (3β,5α,6β, 22E)-6-
xylarinig A, xylarinigs B and C, reported in Li methoxyergosta-7,22-diene-3,5-diol, and
et al. (2022) were isolated from ethyl acetate (3β,5α,6β)-stigmastane-3,5,6-triol, exhibited
extracts of the solid fermentation of X. nigripes, remarkable cytotoxic effects against human
exhibiting neuroprotective effects from the dam- U2OS osteosarcoma cells, with IC50 values
age of PC12 cells after induction with oxygen and being 0.93 μM, 6.0 μM, and 13.2 μM, respec-
glucose deprivation (OGD). The OGD-induced tively. (3β,5α,6β, 22E)-6-methoxyergosta-7,22-
apoptosis of PC12 cells was significantly diene-3,5-diol also exhibited cytotoxicity against
decreased after the treatment of the A549 human lung carcinoma cells
xylarinaps A, B, D, and E, and (-)-xylarinig A, (IC50 = 11.9 μm). Staurosporine was the positive
(+)-xylarinig A, xylarinigs B and C. Superoxide control with IC50 values being 0.01 μM and
dismutase (SOD) and glutathione peroxidase 0.003 μM in the cell line test of U2OS and
(GSH-Px) alleviate oxidative stress through A549, respectively.
detoxification of free radicals and hence are
important components of the major antioxidant
systems (Battin and Brumaghim 2009; Fukai 14.4.3 Antimicrobial Properties
and Ushio-Fukai 2011). The application of
xylarinaps A, B, D, and E also resulted in a 4,8-Dihydroxy-3,4-dihydronaphthalen-1(2H)-
decrease of the malondialdehyde (MDA) level one isolated from methanol extract from X.
and increases of the SOD and GSH-Px levels, escharoidea exhibited antimicrobial activity
which indicates their potential usage in reducing against pathogenic organisms including Staphy-
oxidative stress (Li et al. 2021). The lococcus aureus, Bacillus subtilis, Pseudomonas
concentrations of enhanced cell viability and aeruginosa, and Candida albicans on par with
inhibited apoptosis were effective at 0.1 and standards of ampicillin and amphotericin B
1 μmol/L in studies of Li et al. (2021, 2022). (Nagam et al. 2021). Moreover, molecular
Although sclerotium-forming ability has not docking studies revealed that 4,8-dihydroxy-3,4-
been reported in X. fimbriata, a species associated dihydronaphthalen-1(2H)-one was a prominent
with termite nests, anti-inflammatory function antibacterial agent. The activity was
was also detected from its fermented broth, demonstrated from a marked interaction with
where three isoprenyl phenolic ethers, key residues of binding pockets on DNA binding
fimbriethers B, E, and G, were isolated from its domain (AgrAC) of accessory gene regulator pro-
ethyl acetate extracts of the isolate YMJ491 tein A (AgrA) of S. aureus. The compound
(GenBank GU324753) (Chen et al. 2019). displayed three binding interactions, namely the
Fimbriethers B, E, and G at a concentration of 8-OH bond with Glu188, the 4-OH bond with
100 μM inhibited NO production in cells of Glu144, and the >C = O bond with Lys146.
LPS-induced murine macrophage RAW 264.7 Xylaria escharoidea is not a known sclerotium-
with the Emax values of inhibitory activity being forming species but is mentioned here because it
31.3 ± 1.3%, 38.9 ± 0.1%, and 49.7 ± 0.5%, is in the same species group as other sclerotium-
respectively. Also, in the study, the methyl ben- forming Xylaria species and occurs in termite
zoate moiety and stereochemistry of C-3′ are mounds (Ju et al. 2022).
considered as the possible sites for significant
biological activity.
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 345

14.4.4 Enzyme Inhibitors neurotransmitters; patients with neurological

disorders, such as Parkinson’s disease and multi-
Ergot alkaloids are produced by various fungi in ple system atrophy, can benefit from treatment of
the phylum Ascomycota, but they have been MAOIs (Sub Laban and Saadabadi 2022). Fur-
reported only in Dicyma Boulanger species thermore, MAOIs form a separate class from
(Florea et al. 2017) and X. nigripes (Hu and Li other antidepressants and usually are not the first
2017) within Xylariales. Among six ergot choice in treating mental health disorders as a
alkaloids isolated from dried stromata of result of several dietary restrictions, side effects,
X. nigripes by chloroform/methanol extraction, and safety concerns. Two types of monoamine
both xylanigripone C and agroclavine exhibited oxidase, MAO-A and MAO-B are present, with
inhibition of acetylcholinesterase (AChE) activity the former mostly distributed in the placenta, gut,
with the former being stronger at a concentration and liver, while the latter in the brain, liver, and
of 50 μM and an 84% inhibition rate of the platelets. A phenolic compound, (R)-5-
positive control tacrine (Hu and Li 2017). The methylmellein, extracted from mycelia of
function of AChE involves the termination of cultured X. nigripes, exhibited activity as a selec-
impulse transmission by rapid hydrolysis of the tive inhibitor against MAOI-A with IC50 of
neurotransmitter acetylcholine in numerous cho- 4.6 μM, which, however, was less potent than
linergic pathways in the central and peripheral the known MAO-A inhibiter clorgyline with
nervous systems; a few reversible inhibitors of IC50 of 0.025 μM (Huang et al. 2019). Also, a
AChE are approved drugs in pharmacotherapy variety of (R)-5-methylmellein analogs were
of Alzheimer’s disease (Čolović et al. 2013). synthesized, and an (R)-5-methylmellein analog
The understanding of the reversibility of (13aR) was the most potent and selective MAO-A
xylanigripone C inhibition on AChE will be help- inhibitor with IC50 of 0.06 μm among the
ful for understanding the medicinal utilization of 36 analogs tested.
the compound. Hu and Li (2017) also showed that
four ergot alkaloids, including xylanigripone A,
agroclavine, 8,9-didehydro-10-hydroxy-6,8- 14.5 Protection Effects of Wuling
dimethylergolin, and (6S)-agroclavine N-oxide, Capsule or Fermented Mycelia
at the concentration of 160 μM compared to a of Sclerotium-Producing
water control, resulted in a decrease in the Xylaria Species
activities of cholesterol ester transfer protein
(CETP) at rates of 49%, 73%, 36.5%, and 14.5.1 Regulation of Anxiety
58.5%, respectively. CETP is a plasma protein and Depression
for facilitating the transport of cholesteryl esters
and triglycerides between lipoproteins. The The prevalence of depression in patients with
development of CETP inhibitors aimed at reduc- epilepsy varied in several studies, but significant
ing the risk of coronary heart disease has had a numbers of depressed patients were revealed
long and difficult course, and only Anacetrapib (Fiest et al. 2013; Jackson and Turkington
has gone into the planned completion in clinical 2005). Furthermore, a small subset of epileptic
trials among the four considered inhibitors (Tall patients suffered from lifetime depression (Fiest
and Rader 2018). et al. 2013). In a study of 104 patients with
Monoamine oxidase inhibitors (MAOIs) are comorbidities of epilepsy and depression, Wuling
capable of blocking the monoamine oxidase capsule reduced symptoms of depression (Peng
enzymes, which break down different types of et al. 2015). Compared to the placebo group, the
neurotransmitters including norepinephrine, sero- group taking Wuling capsule had improvements
tonin, dopamine, and tyramine from the brain, in comorbid depression, including a total effec-
thus retaining the availability of these tive rate for depression, scales designed to assess
quality of life, and concerns about future seizures.
346 H.-M. Hsieh and Y.-M. Ju

For both placebo and test groups, differences in randomized controlled trials revealed that treat-
seizure frequency and severity were not observed, ment of Wuling capsule singly when compared to
and most adverse effects were mild and compara- no treatment, or a cotreatment of Wuling capsule
ble. In a study of 64 cases of Type 2 diabetes and an antidepressant when compared to several
mellitus, Wuling capsule ameliorated depression antidepressant therapies alone, were more effec-
and improved the state of inflammation by tive in reducing PSD estimated from diagnostic
decreasing concentrations of cytokines (IL-6 and scores and response rates (Peng et al. 2014).
TNF-α) that contribute to inflammation and The small-scale clinical studies of the Wuling
reduced oxidative stress by increasing the con- capsule reviewed above could help evaluate the
centration of SOD and decreasing the concentra- feasibility, time required, possible adverse events,
tion of MDA (Wang et al. 2020). and effective size of the subject pool for a full-
Major depressive disorder (MDD), is a mental scale project.
disorder that causes a persistent sadness or
depressed mood, anhedonia or decreased interest
in engaging activities, feeling of emptiness or 14.5.2 Possible Mechanism
hopelessness, low motivation, a difficulty in con- for the Alleviation
centration, appetite changes, psychomotor retar- of Depression
dation or irritability, disturbed sleep, apathetic,
negative, or even suicidal thoughts; MDD was Brain-derived neurotrophic factor (BDNF) plays
ranked as the third cause of the burden of disease a critical role in cognition, and its relation to PSD
worldwide in 2008 by WHO, which projected is easily recognized because BNDF serum levels
that MDD would rank first by 2030 (Bains and are lower in PSD patients than in post-stroke
Abdijadid 2022). A systematic review of clinical patients without PSD; certain antidepressants
data of MDD with a total of 880 cases from year could enhance BDNF expression in the brain,
2006 to 2015 and a subsequent meta-analysis of resulting in a relaxation of depression symptoms
the data based on randomized controlled trials (Zhang and Liao 2020). The therapeutic effect
revealed that the cotreatment of Wuling capsule can be eliminated in animals with the BDNF
and an antidepressant exhibited better efficacy gene deletion (Zhang and Liao 2020). Wuling
than antidepressant monotherapy in symptomatic capsule at a dose of 100 mg kg-1b.wt. exhibited
improvement at last-observation endpoint as well an alleviation effect on the level of depression as
as study-defined response and remission, while well as the impairment of hippocampal-
there was no significant difference between dependent learning and memory in PSD model
Wuling capsule and some antidepressants tested rats, the same as the treatment of the antidepres-
as monotherapies (Zheng et al. 2016). Further- sant escitalopram at a dose of 0.2 mg kg-1b.wt.
more, the numbers of adverse drug reactions, for 21 days (Li et al. 2011). Interestingly, the
including nausea, constipation, tachycardia, and recovery function of Wuling capsule on depres-
dry mouth were less reported in patients with sion behaviors and cognitive deficits was not
Wuling capsule treatment when compared to related to the expression of BDNF, as determined
those with antidepressant monotherapy. via immunohistochemical staining and mRNA
Post-stroke depression (PSD), an important amplification, which remained low after Wuling
complication of stroke leading to increased dis- capsule treatment. Similarly, in a study of hippo-
ability and mortality, occurs in a significant num- campal neurogenesis of rats under chronic unpre-
ber of patients and is associated with the severity dictable mild stress, Wuling capsule exhibited a
of impairment in activities of daily living, social mitigation effect on depressed behaviors, and the
functioning, and cognitive function (Robinson antidepressant effect was related to enhancing the
and Jorge 2016). A systematic review and meta- expression of the protein connexin-43 rather than
analysis of clinical data of PSD with a total of through BDNF mediation (Li et al. 2010).
1378 cases from 2006 to 2013 based on
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 347

Translocator protein (TSPO), a five- modulation of synaptic plasticity, perception of

transmembrane structure located at outer and pain, learning, aggression, and depression; con-
inner mitochondrial membrane contact sites, is trol of the synthesis of NO is the key to regulating
critical for brain steroidogenesis and modulates its activity (Esplugues 2002). Evidence revealed
behaviors related to anxiety and depression that the inhibition of neuronal nitric oxide
(Barron et al. 2021). In a study of learned help- synthase in rat hippocampus induced
lessness (LH) model of mice with depression, antidepressant-like effects, an implication of neu-
TSPO and certain mitophagy-related proteins, robiological function of endogenous hippocampal
including voltage-dependent anion channel NO in times of stress and depression (Joca and
1 (VDAC1), PTEN-induced kinase PINK1 and Guimarães 2006). NO activates guanylate
Beclin 1, significantly decreased in LH model cyclase, and the activation enhances the forma-
mice (Li et al. 2016b). Also, kinesin KIFC2, tion of intracellular messenger cyclic guanosine
relevant to mitochondrial transport was down- monophosphate (cGMP) and the effects of cGMP
regulated in the LH mice. Mechanistic steps of a were regulated via mediators, including cGMP-
PINK1/Parkin-directed pathway linking mito- degrading phosphodiesterases which play a role
chondrial damage, ubiquitylation, and autophagy in degradation of cGMP as well as determination
have demonstrated the PINK1/Parkin-mediated of the shape of cGMP signal (Russwurm and
mitophagy is dependent on VDAC1 (Geisler Koesling 2004). L-arginine-nitric oxide-cGMP
et al. 2010). In a study of LH model mice with signaling pathway has been reported in the
depression induced by inescapable e-shock, involvement of the pathogenesis of depression
Wuling powder treatment with a concentration (Calabresi et al. 1999; Dhir and Kulkarni 2007).
ranging from 0.5 to 2.0 g/kg significantly In a study of chronic unpredictable mild stress in
ameliorated the helpless behavior, and shortened rats, a powder prepared from Wuling mycelia
the time of latency to eat and the immobility time exhibited antidepressant-like activity via a sup-
during the forced swimming test (Li et al. 2016a). pression of behavioral changes, including sucrose
This study also demonstrated that Wuling powder preference, food consumption, and crossing num-
exerted neuroprotective effects by altering the ber (locomotor activity and willingness during an
expression level of the proteins related to the open-field test); the activity was possibly
TPSO mediated-mitophagy signaling pathway in involved in the L-arginine-nitric oxide-cGMP sig-
mesencephalon of mice. Wuling powder signifi- naling pathway (Tan et al. 2016). The
cantly upregulated the expression level of TPSO, antidepressant-like effect of Wuling powder was
VDAC1, PINK1, Beclin 1, and KIFC2, which significantly reversed with the pretreatment of L-
were significantly decreased in LH mice when arginine and sildenafil (phosphodiesterase 5 inhib-
compared to the controls. On the other hand, in itor) but was significantly enhanced with
LH mice the increased expression level of Parkin, pretreatment of 7-nitroindazole (a specific neuro-
possibly due to the recruiting effect of Parkin to nal nitric oxide synthase inhibitor) and methylene
the damaged mitochondria, was downregulated blue (direct inhibitor of both nitric oxide synthase
via Wuling powder treatment. Wuling powder and soluble guanylate cyclase). Hence, the
also significantly elevated the integrated optical antidepressant-like effect may be dependent on
density of Synaptogyrin-3-postitive nerve fibers the inhibition of NO synthesis and mediated
in ventral striatum of LH mice exhibiting a through the reduction in cGMP.
decreased optical density and hence expressing
anhedonia to initiate feeding in novelty-
suppressed feeding test and depression. 14.5.3 Regulation of Inflammatory
NO is synthesized from L-arginine by nitric Mediators, Reduced Apoptosis,
oxide synthase throughout the CNS including and Neuroprotection
spinal cord, retina, and brain (Wiesinger 2001).
The role of NO as a neurotransmitter and Both the water extract and the ethanol extract
neuromodulator in the nervous system includes prepared from a culture obtained from the
348 H.-M. Hsieh and Y.-M. Ju

specimen KJ-XN-07-01 of X. nigripes exhibited downregulation of Bax and caspase 3, the

immunomodulatory properties, including a dose- pro-apoptotic members. Compared to the hot
dependent inhibitory effect on NO, PGE2, and water extract, the ethanol extract exhibited better
cytokine (IL-1β, IL-6, TNF-α, and IFN-γ) pro- protective effects on mitochondrial dysfunctions
duction in LPS-stimulated macrophages; water in cells with oxidative damage, and higher anti-
extract was more potent than ethanol extract oxidant activity by scavenging DPPH
(Ko et al. 2011). Compared with the ethanol (2,2-diphenyl-1-picrylhydrazyl) radicals,
extract, the water extract exhibited a greater inhi- inhibiting lipid oxidation, and reducing power.
bition on iNOS and COX2 expressions and a Both extracts contain substantial amounts of
stronger suppression on the LPS-induced IκB-α flavonoids, phenols, and polysaccharides with
phosphorylation and NF-κB activation. Further- phenolic contents being significantly higher in
more, the ethanol extract was higher in total fla- the ethanol extract. In a study of rats with ische-
vonoid and phenol content but lower in β-glucan mic stroke induced by middle cerebral artery
content than the water extract. These results occlusion (MCAO), neuroprotection effects of
revealed that the mechanism of anti-inflammatory the water extract prepared from X. nigripes were
action could be mediated by inhibiting iNOS and reported (Sun et al. 2017). The neuroprotective
COX2 expression via NF-κB signaling pathway, effects included significantly reduced neuronal
and the potent anti-inflammatory activities of injury, relieved neurobehavioral deficits, and
water extract could be associated with the alleviated MCAO-induced reduction of the
β-glucan content. Divate and Chung (2017) expression levels of (i) heme oxygenase-1
reported that both hot water and 70% ethanol (HO-1), (ii) nuclear factor-erythroid 2-related fac-
extracts of X. nigripes mycelia exerted protection tor 2 (Nrf2), and (iii) neurons and astrocyte
against inflammatory stimuli from LPS in marker genes in the ischemic brain. HO-1 and
RAW264.7 cells by reducing the concentrations Nrf2 are two major regulators during the pro-
of NO, TNF-α, IL-6, and COX2. Both extracts cesses of anti-oxidation, anti-inflammation, and
exhibited the anti-inflammation potential to anti-apoptosis. The markers are glial fibrillary
increase phagocytic activity and concentration of acidic protein, nerve growth factor receptor,
anti-inflammatory cytokine IL-10 of RAW 264.7 BDNF, neurotrophic tyrosine receptor kinase
cells against diseased-related stimuli from E. coli. type 1 and type 2. Moreover, the increased levels
The study also revealed that X. nigripes mycelia of ROS, MDA, and cleaved caspase 3 via MCAO
possessed immunomodulatory activities by induction were reduced by the administration of
enhancing splenocyte proliferation against stimu- the water extract. As the finding revealed, the
lation of mitogens, concanavalin A or LPS in neuroprotective effect of X. nigripes following
mice spleens as well as increasing cytotoxicity ischemic insult in the brain is mediated by the
of splenic natural killer cells against YAC-1 lym- anti-oxidative pathway Nrf2/HO-1. The neuro-
phoma cells. pathological features of Alzheimer’s disease
Both the ethanol extract and the hot water (AD) include the accumulation of misfolded
extract from cultured mycelia of X. nigripes β-amyloid (Aβ) peptides in extracellular plaques
exerted neuroprotection against H2O2-induced and phosphorylated tau (P-tau) protein as paired
cell damage of PC12 cells by inhibiting the filaments (PHFs) in intraneuronal neurofibrillary
release of lactate dehydrogenase to reduce cell tangles; Aβ42, P-tau, and total tubulin associated
injury, decreasing DNA damage, and rebuilding unit (T-tau) are core cerebrospinal fluid
mitochondrial membrane potential (Divate et al. biomarkers in AD pathology (Janelidze et al.
2017c). Also, abnormal apoptosis resulting from 2020). In clinical treatment of AD, Wuling cap-
the H2O2-induction was arrested by both the sule combined with Oxiracetam significantly
applied extracts, which regulated Bcl-2 family, improved cognitive function, activities of daily
the regulators of apoptosis by upregulation of living and quality of life, and reduced inflamma-
Bcl-2, the pro-survival member and tory reaction as measured by high sensitivity
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 349

C-reactive protein, IL-1β, and TNF-α when com- extract, which was substantially high in the
pared to Oxiracetam monotherapy (Fu et al. concentrations of epicatechin, catechin, and
2021). Moreover, the reduced Aβ1-42, and coumaric acid among analyzed polyphenolic
increased T-tau and P-tau181 levels in AD compounds. The effective protection of the
patients, when compared to healthy subjects, extract could be through the effects of
were ameliorated via the treatment of Wuling antioxidants on reducing oxidative stress.
capsule combined with Oxiracetam and the X. nigripes purchased from a culture center
ameliorated effects are significantly better than (BCRC 34219) was grown on various solid
Oxiracetam monotherapy. substrates, including wheat bran, soy meal, and
In a study of rats with seizure development and a mixture with ratios of wheat bran to soy meal of
impairment in learning ability induced by 4:1, 1:1, and 1:4; the fermented materials were
pentylenetetrazole injection 12 times within then extracted with hot water or 70% ethanol for
48 d, treatment with Wuling mycelia exhibited the comparison of bioactivities (Divate et al.
neuroprotection effects, including (i) significant 2017b). In general, ethanol extraction was more
shortening of the duration and alleviation of effective than hot water extraction for the acqui-
behavioral changes upon stimulated seizure, and sition of active compounds. The ethanol extract
(ii) the postponement of latency to the onset of from the wheat bran fermentation was the most
myoclonic seizures and generalized seizure after potent in exhibiting antioxidant activity, espe-
specific times of treatment (Chen et al. 2012a). cially DPPH radical scavenging and reducing
Wuling treatment was also beneficial via the power as well as anti-inflammation activities,
effects on reduction of frequency of working measured by the inhibition of COX2 activity
error and reference memory error and partial when compared to the hot water extract and the
reversal of impairment of learning ability. The extracts from other growth substrates. The etha-
pentylenetetrazole induction in rats also resulted nol extract from the substrate containing equal
in a decrease in histamine contents in brain amounts of wheat bran and soy meal was signifi-
including the regions of hippocampus, thalamus, cantly more effective in the prevention of the
and hypothalamus, which were elevated with the H2O2-induced damage in neuronal cells of PC12
administration of Wuling mycelia. Histamine has when compared to the water extract and the
been shown to modulate inflammatory processes extracts from other growth substrates.
and regulate microglia and astrocytes in the brain, Solid-state fermentation was applied to
and a reduction of its synthesis has been achieve synergistic effects of the bioactivities of
associated with the disorders of Tourette’s syn- two components, i.e., X. nigripes mycelium
drome and obsessive-compulsive disorder (YMJ653, EU179868) and growth substrates
(Carthy and Ellender 2021). prepared from whole grains, including brown
Aqueous extract prepared from X. nigripes rice, red beans, mung beans, or soybeans (Divate
exhibited protective effects against hepatotoxicity et al. 2017a). Compared to unfermented grains,
induced via carbon tetrachloride (CCl4) in mice the grains with the mycelial fermentation
(Song et al. 2011). Intragastrication of the aque- exhibited greater antioxidative, anti-
ous extract significantly inhibited the elevation of inflammatory, and neuroprotective potential.
serum glutamate oxalate transaminase, serum glu- The red bean fermentation was more potent than
tamate pyruvate transaminase, and the levels of the other fermentation substrates. The IC50 for the
liver thiobarbituric acid reactive substances. It ethanol extract of the fermented and unfermented
also resulted in an increase in the liver SOD, red beans on DPPH scavenging activity was
catalase, and GSH-Px concentrations as well as 2.0 ± 0.1 and 2.7 ± 0.1 mg/mL, respectively,
serum total antioxidant activity in the CCl4- and on lipid peroxidation inhibition was
induced hepatotoxicity mice. Furthermore, liver 0.97 ± 0.1 and 3.44 ± 0.1 mg/mL, respectively.
conditions, as revealed histologically, were obvi- The ethanol extract of the fermented and unfer-
ously ameliorated with the treatment of the mented red beans exhibited 36.5 ± 0.5% and
350 H.-M. Hsieh and Y.-M. Ju

24.9 ± 1.5% of inhibition on COX2, respectively; and has been marketed for supporting sleep qual-
and 27.2 ± 1.1% and 25.4 ± 2.2% of neuronal ity and relaxation (Deshmukh et al. 2021).
protection in PC12 cells, respectively. Further- Wuling powder in combination with nembutal
more, lipid peroxidation inhibition and reducing sodium, typically used as a sedative, results in
power both exhibited a significant correlation shortening sleep onset and prolonging sleep dura-
with the total phenolic contents of the tested tion when compared to a single treatment of nem-
extracts. butal sodium in mice (Ma et al. 1999). The
The ethyl acetate extracts prepared from X. administration of Wuling powder adjunctive
chaiyaphumensis Wangsawat et al., X. siamemsis resulted in increased concentrations of glutamate
Wangsawat et al., X. subintraflava Wangsawat and GABA in the brain and elevations in the
et al., X. thienhirunae Wangsawat et al., and X. activity of glutamate decarboxylase, responsible
vinacea Wangsawat et al. exhibited antioxidant for GABA synthesis and the binding affinity of
properties, and X. vinacea was the most potent GABA receptors in the cerebral cortex (Ma et al.
with IC50 of 0.194 mg/mL and 0.020 mg/mL, as 1999). Hence, it is suggestive that Wuling powder
measured via DPPH and ABTS [2,2′-azino-bis facilitated the entry of glutamate and GABA into
(3-ethylbenzothiazoline-6-sulfonic acid)] assays, brain, to activate GABA receptors, and thus exert
respectively (Wangsawat et al. 2021b). Although its sedative and sleep-promoting properties.
these five Xylaria species are associated with A systematic review of clinical data for 1850
termite nests, their ability to form sclerotia has individuals with insomnia disorder from 2008 to
not been reported. 2021 and subsequent meta-analysis of the data
based on randomized controlled trials revealed
that sleep quality, assessed as Pittsburgh Sleep
14.5.4 Sedative Effects and Regulation Quality (PSQI), can be safely and effectively
of Insomnia Disorder ameliorated by the treatment of Wuling capsule
monotherapy (Zhou et al. 2022). Additionally, the
The amino acid, γ-aminobutyric acid (GABA), cotreatment of Wuling capsule with a conven-
synthesized in the cytoplasm of the presynaptic tional medication from benzodiazepine drugs,
neuron from glutamate by the enzyme glutamate sedating antidepressants, or cognitive behavior
decarboxylase, is the primary inhibitory neuro- therapy was more effective in PSQI than the
transmitter within the adult CNS, and primarily conventional treatment alone. In the study, the
functions to reduce neuronal excitability by severity of insomnia disorder was measured via
inhibiting nerve transmission (Allen et al. 2022; Sleep Dysfunction Rating Scale and the scale was
Jewett and Sharma 2022). Many illnesses, such as significantly reduced by Wuling capsule when
generalized anxiety, schizophrenia, autism spec- compared to the control, benzodiazepines.
trum disorder, seizures, and epilepsy have been Adverse events also were significantly reduced
associated with low concentrations of GABA and in the trial of Wuling capsule as monotherapy
numerous drugs that modulate GABA signaling when compared to benzodiazepines and in the
have been applied to clinical situations (Allen trial of Wuling capsule as adjuvant therapy with
et al. 2022). The balance between inhibitory neu- sedating antidepressant when compared to sedat-
ronal transmission via GABA and excitatory neu- ing antidepressant monotherapy. The treatment
ronal transmission via glutamate is of great duration, 8 weeks was better than 4 weeks for
importance for proper cell membrane stability the ameliorating effects of Wuling capsule on
and neurologic function (Allen et al. 2022). insomnia disorder. The use of Wuling capsule as
GABA was detected in Wuling capsule (He and a treatment of insomnia disorder required careful
Liu 2010). The product GABA-rich Zylaria™ recommendation, due to the high heterogeneity,
with the inclusion of mycelial extracts of possibly from differences in age and course of
X. nigripes benefits health through its natural disease among 18 studies, as well as the high risk
and gentle tranquillizing effect on human bodies of bias in the included trials with some being
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 351

insufficient in the information of generating ran- as Type 2 diabetes mellitus (T2DM) and obesity
dom number table and stratified blocked random- (Sesti et al. 2001). Glucose transporter type
ization as described in the study. 4 (GLUT4) is responsible for the transportation
of glucose across the plasma membrane, and one
of the potential causes for resistance to insulin-
14.5.5 Regulation of Memory stimulated glucose transport may be related to
Impairment defective intracellular signaling of GLUT4 trans-
location in skeletal muscle from stored intracellu-
Rapid eye movement (REM) sleep which occurs lar vesicles to active components of the plasma
in mammals and birds, is a unique phase of sleep. membrane, a possible inherent impairment in the
REM sleep behavior disorder (RBD) is muscle cells as T2DM is a heritable disease;
characterized by dream enactment and the loss research shows that T2DM conditions can be
of muscle atonia during REM sleep with dreams improved or reversed by the increase of GLUT4
often involving violent and aggressive action. levels (Vargas et al. 2022).
Several synucleinopathies, including Parkinson’s The aqueous extract prepared from stromatal
disease, Lewy Body Dementia, and multiple sys- powder of X. nigripes significantly exhibited a
tem atrophy, are frequently associated with the hypoglycemic effect on normal rats and rats
RBD type classified as “secondary/symptomatic” with insulin resistance (SIIR) resulting from an
(Matar et al. 2021). CREB (cAMP-response ele- administration of steroid (Chen et al. 2015). The
ment binding protein), a transcription factor, decreases in plasma glucose concentration were
promotes the recruitment of the co-activator pro- 26% (from 177.6 ± 12.5 to 133.3 ± 29.7 mg/dL)
tein CBP, a CREB-binding protein required for and 21% (from 128.6 ± 12.5 to 100.9 ± 10.7 mg/
both short- and long-term memory formation dL), respectively, in SIIR and normal rats. The
(Chen et al. 2010). The function of CREB is hypoglycemic effect on both groups of rats was
largely regulated by the phosphorylation at also accompanied by increases in concentrations
Ser133 by protein kinase A (PKA), and the acti- of IRS-1 and GLUT4. Furthermore, the hypogly-
vation of PKA/CREB signaling pathway in the cemic effect of the extract on SIIR rats was
hippocampus is critically important in spatial abolished when a three-day’s injection of seroto-
memory formation (Mizuno et al. 2002). nin antagonist α-p-chlorophenylalanine was
The water extract prepared from X. nigripes applied and followed by the administration of
mitigated an impairment of spatial learning and the extract. Increased insulin sensitivity and the
memory which was induced in Sprague Dawley consequence of a serotonin-related hypoglycemic
rats deprived of REM sleep (Zhao et al. 2014). effect may be related to changes in serotonin
Also, a reduced phosphorylation of CREB was regulation after administration of the extract
detected in the hippocampi of rats deprived of (Chen et al. 2015).
REM sleep, but the intragastric administration of
the water extract reversed the reduced phosphor-
ylation. Phosphorylation of CREB was suggested 14.6 Amino Acids, Minerals,
as one of the mechanisms for mitigation of the Nucleosides, and Others from
impairment. Sclerotia, Mycelia From
Cultures, and Wuling Capsule

14.5.6 Regulation of Insulin Sensitivity Amino acids, including aspartic acid, glutamic
acid, serine, histidine, glycine, arginine, proline,
Insulin receptor substrate (IRS) molecules are alanine, threonine, methionine, valine, phenylala-
significant mediators in insulin signaling, and nine, isoleucine, and leucine, had been identified
defects in muscle IRS-1 expression and function from sclerotia and mycelia of a sclerotium-
have been reported in insulin-resistant states such producing Xylaria species as well as in Wuling
352 H.-M. Hsieh and Y.-M. Ju

capsule (Chen et al. 1990; He and Liu 2010). components of different sclerotium-producing
Additionally, cystine and lysine were detected in Xylaria species should be assessed for the appro-
sclerotia and mycelia (Chen et al. 1990). The priateness of consuming Wulingshen in mixtures.
detection of tyrosine was reported from mycelia Wuling capsule, which has been sold with popu-
(Chen et al. 1990). Mannitol and glucose were larity in Chinese drugstores, is based on the dried
reported from sclerotia and mycelia; additionally, mycelia obtained from a Xylaria species. In some
fructose, lactose, sucrose, and raffinose were published articles related to Wuling capsule, X.
identified from mycelia (Chen et al. 1990). The nigripes was assumed to be the research species,
minerals, Fe, Mn, Sn, Cr, Ni, Mo, V, Cu, Zn, Se, but this requires reconfirmation (Ju et al. 2022).
Mg, B, Ca, K, Na, and P, were detected both in
sclerotia and in mycelia (Chen et al. 1990).
Nucleosides of Wuling capsule include adeno- References
sine, uridine, and guanosine (Lu et al. 2011).
Other identified components of Wuling capsule Aanen DK, de Fine Licht HH, Debets AJ, Kerstes NA,
Hoekstra RF, Boomsma JJ (2009) High symbiont relat-
are 5-methylmellein, 5-hydroxymellein,
edness stabilizes mutualistic cooperation in fungus-
5-carboxylmellein, and genistein (Chen et al. growing termites. Science 326:1103–1106
2012b; Lu et al. 2011). Allen MJ, Sabir S, Sharma S. (2022) GABA receptor. In:
StatPearls [Internet]. StatPearls Publishing, Treasure
Island, FL. Available via https://www.ncbi.nlm.nih.
gov/books/NBK526124/. Accessed 15 Aug 2022
14.7 Epilogue Bachiller S, Jiménez-Ferrer I, Paulus A, Yang YY,
Swanberg M, Deierborg T, Boza-Serrano A (2018)
With more massive Xylaria sclerotia being Microglia in neurological diseases: a road map to
brain-disease dependent-inflammatory response. Front
analyzed, it is likely that there will be more Cell Neurosci 12
sclerotium-producing Xylaria species discovered. Bains N, Abdijadid S. (2022) Major depressive disorder
This work needs to be coupled with the activities [Internet]. Treasure Island, FL: StatPearls Publishing.
of collecting fresh stromata from the wild, from Accessed 15 Aug 2022
Barron AM, Higuchi M, Hattori S, Kito S, Suhara T, Ji B
which morphological characteristics can be (2021) Regulation of anxiety and depression by mito-
described and cultures can be obtained for further chondrial translocator protein-mediated steroidogene-
study. There have been many research reports on sis: the role of neurons. Mol Neurobiol 58:550–563
Wulingshen or Wulingshen-derived materials, Batra LR, Batra SWT (1979) Termite-fungus
mutualism. In: Batra LR (ed) Insect-fungus symbiosis:
mainly on medicinal ingredients, pharmacology, nutrition, mutualism and commensalism. Allanheld,
and clinical applications. In these research Osmun & Co., Montclair, NJ, pp 117–163
reports, most of the research materials used were Battin EE, Brumaghim JL (2009) Antioxidant activity of
identified as X. nigripes. Some of the DNA sulfur and selenium: a review of reactive oxygen spe-
cies scavenging, glutathione peroxidase, and metal-
sequences deposited in GenBank of the National binding antioxidant mechanisms. Cell Biochem
Center for Biotechnology Information (NCBI) Biophys 55:1–23
were labeled as derived from X. nigripes. We Berkeley MJ (1862) On two tuberiform vegetable
noticed that most of these sequences were in fact productions from Travancore. Trans Linn Soc Lond
23:91–92
from X. rogersionigripes rather than X. nigripes Calabresi P, Gubellini P, Centonze D, Sancesario G,
(Ju et al. 2022). Therefore, attention should be Morello M, Giorgi M, Pisani A, Bernardi G (1999) A
paid to which species of sclerotium-producing critical role of the nitric oxide/cGMP pathway in
Xylaria species are used in research in the corticostriatal long-term depression. J Neurosci 19:
2489–2499
published studies on Wulingshen. Wulingshen Carthy E, Ellender T (2021) Histamine,
sold at traditional Chinese medicine markets is neuroinflammation and neurodevelopment: a review.
frequently mixed with massive sclerotia produced Front Neurosci-Switz 15(680214):1–26
by more than one Xylaria species. Are the medic- Chang JC, Hsiao G, Lin RK, Kuo YH, Ju YM, Lee TH
(2017) Bioactive constituents from the termite nest-
inal ingredients of Wulingshen produced by dif-
ferent Xylaria species similar? Chemical
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 353

derived medicinal fungus Xylaria nigripes. J Nat Prod Divate RD, Chung YC (2017) In vitro and in vivo assess-
80:38–44 ment of anti-inflammatory and immunomodulatory
Chen W-R, Li Z-T, Shi M-Z, Ni J-Y (1988) Study on activities of Xylaria nigripes mycelium. J Funct
biological characteristics of fungal Chinese medicine Foods 35:81–89
Wulingshen (in Chinese). Chin J Mod App Pharm 5: Divate RD, Wang CC, Chou ST, Chang CT, Wang PM,
10–13 Chung YC (2017a) Production of Xylaria nigripes-
Chen W-R, Fang H-F, Ma Z-Z, Ding R-R (1990) Chemi- fermented grains by solid-state fermentation and an
cal constituents of Xylaria nigripes (in Chinese). Chin assessment of their resulting bioactivity. Lwt-Food
Pharm J 25:647–649 Sci Technol 81:18–25
Chen GQ, Zou XY, Watanabe H, van Deursen JM, Shen J Divate RD, Wang CC, Chou ST, Chang CT, Wang PM,
(2010) CREB binding protein is required for both Chung YC (2017b) Using wheat bran and soybean
short-term and long-term memory formation. J meal as solid state fermentation substances for the
Neurosci 30:13066–13077 production of Xylaria nigripes with bioactivities. J
Chen GF, Ren GL, Zhang LS, Hu XY (2012a) Effects of Taiwan Inst Chem E 70:127–133
Wuling mycelia on pentylenetetrazole-induced epi- Divate RD, Wang PM, Wang CC, Chou ST, Chang CT,
lepsy in rats (in Chinese). Zhejiang Da Xue Xue Bao Chung YC (2017c) Protective effect of medicinal fun-
Yi Xue Ban 41:647–652 gus Xylaria nigripes mycelia extracts against hydrogen
Chen Y, Lu J, Zhu M, Luo L (2012b) Determination of peroxide-induced apoptosis in PC12 cells. Int J
multiple constituents in Wuling capsules by HPLC Immunopath Ph 30:105–112
simultaneously (in Chinese). China J Chin Mat Med Esplugues JV (2002) NO as a signalling molecule in the
37:218–221 nervous system. Br J Pharmacol 135:1079–1095
Chen YI, Tzeng CY, Cheng YW, Hsu TH, Ho WJ, Liang Feng DD, Tang T, Lin XP, Yang ZY, Yang S, Xia ZA,
ZC, Hsieh CW, Tzen JTC, Chang SL (2015) The Wang Y, Zheng P, Wang Y, Zhang CH (2016) Nine
involvement of serotonin in the hypoglycemic effects traditional Chinese herbal formulas for the treatment of
produced by administration of the aqueous extract of depression: an ethnopharmacology, phytochemistry,
Xylaria nigripes with steroid-induced insulin-resistant and pharmacology review. Neuropsychiatr Dis Treat
rats. Phytother Res 29:770–776 12:2387–2402
Chen CF, Su CH, Lai MN, Ng LT (2018) Differences in Fiest KM, Dykeman J, Patten SB, Wiebe S, Kaplan GG,
water soluble non-digestible polysaccharides and anti- Maxwell CJ, Bulloch AGM, Jette N (2013) Depression
inflammatory activities of fruiting bodies from two in epilepsy: a systematic review and meta-analysis.
cultivated Xylaria nigripes strains. Int J Biol Macromol Neurology 80:590–599
116:728–734 Florea S, Panaccione DG, Schardl CL (2017) Ergot
Chen MC, Wang GJ, Kuo YH, Chiang YR, Cho TY, Ju alkaloids of the family Clavicipitaceae. Phytopathol-
YM, Lee TH (2019) Isoprenyl phenolic ethers from the ogy 107:504–518
termite nest-derived medicinal fungus Xylaria Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL
fimbriata. J Food Drug Anal 27:111–117 (1998) Characterization of the A549 cell line as a type
Čolović MB, Krstić DZ, Lazarević-Pašti TD, Bondžić II pulmonary epithelial cell model for drug metabo-
AM, Vasić VM (2013) Acetylcholinesterase inhibitors: lism. Exp Cell Res 243:359–366
pharmacology and toxicology. Curr Neuropharmacol Fu Y, Tong J, Di L, Zhang J, Liu X (2021) Clinical
11:315–335 observation of Wuling capsules combined with
Croce MV, Colussi AG, Price MR, Segal-Eiras A (1999) Oxiracetam in the treatment of Alzheimer’s disease.
Identification and characterization of different China Pharm 30:69–71
subpopulations in a human lung adenocarcinoma cell Fukai T, Ushio-Fukai M (2011) Superoxide dismutases:
line (A549). Pathol Oncol Res 5:197–204 role in redox signaling, vascular function, and diseases.
Deepna Latha KP, Veluthoor S, Manimohan P (2015) On Antioxid Redox Sign 15:1583–1606
the taxonomic identity of a fungal morph used in Geisler S, Holmström KM, Skujat D, Fiesel FC, Rothfuss
traditional medicine in Kerala State, India. Phytotaxa OC, Kahle PJ, Springer W (2010) PINK1/Parkin-
201:287–295 mediated mitophagy is dependent on VDAC1 and
Deshmukh SK, Sridhar KR, Gupta MK (2021) Applica- p62/SQSTM1. Nat Cell Biol 12:119–131
tion of selected species of the genus Xylaria in tradi- Ginhoux F, Lim S, Hoeffel G, Low D, Huber T (2013)
tional medicine. In: Deshmukh SK, Sridhar KR (eds) Origin and differentiation of microglia. Front Cell
Advances in macrofungi: pharmaceuticals and Neurosci 7(45):1–14
cosmeceuticals. CRC Press, Boca Raton (FL), pp He X, Liu P (2010) Determination of 14 kinds of amino
122–136 acids in Wuling capsule by HPLC-FLD (in Chinese).
Dhir A, Kulkarni SK (2007) Involvement of L-arginine- Chin Tradit Pat Med 32:1358–1361
nitric oxide-cyclic guanosine monophosphate pathway Hsieh H-M, Lin C-R, Fang M-J, Rogers JD, Fournier J,
in the antidepressant-like effect of venlafaxine in mice. Lechat C, Ju Y-M (2010) Phylogenetic status of
Prog Neuro-Psychoph 31:921–925 Xylaria subgen. Pseudoxylaria among taxa of the sub-
family Xylarioideae (Xylariaceae) and phylogeny of
354 H.-M. Hsieh and Y.-M. Ju

the taxa involved in the subfamily. Mol Phylogenet unpredictable mild stress (in Chinese). Zhong Xi Yi Jie
Evol 54:957–969 He Xue Bao 8:662–669
Hsieh H-M, Chou J-C, Ju Y-M (2020) Xylaria insolita and Li DQ, Li XJ, Duan JF, Cai W (2011) Effects of Wuling
X. subescharoidea: two newly described species col- capsule on hippocampal dependent cognitive changes
lected from a termite nesting site in Hua-lien, Taiwan. in post-stroke depression rats. Int J Pharmacol 7:50–57
Bot Stud 61(11):1–9 Li M, Xiong J, Huang Y, Wang LJ, Tang Y, Yang GX, Liu
Hu DB, Li M (2017) Three new ergot alkaloids from the XH, Wei BG, Fan H, Zhao Y et al (2015)
fruiting bodies of Xylaria nigripes (Kl.) Sacc. Chem Xylapyrrosides A and B, two rare sugar-morpholine
Biodivers 14:e1600173:1–7 spiroketal pyrrole-derived alkaloids from Xylaria
Huang C, Xiong J, Guan HD, Wang CH, Lei XS, Hu JF nigripes: isolation, complete structure elucidation,
(2019) Discovery, synthesis, biological evaluation and and total syntheses. Tetrahedron 71:5285–5295
molecular docking study of (R)-5-methylmellein and Li DM, Zheng J, Wang MY, Feng L, Liu YY, Yang N,
its analogs as selective monoamine oxidase A Zuo PP (2016a) Wuling powder prevents the
inhibitors. Bioorgan Med Chem 27:2027–2040 depression-like behavior in learned helplessness mice
Jackson MJ, Turkington D (2005) Depression and anxiety model through improving the TSPO mediated
in epilepsy. J Neurol Neurosur Ps 76:i45–i47 mitophagy. J Ethnopharmacol 186:181–188
Janelidze S, Mattsson N, Palmqvist S, Smith R, Beach TG, Li DM, Zheng J, Wang MY, Feng L, Ren ZL, Liu YY,
Serrano GE, Chai XY, Proctor NK, Eichenlaub U, Yang N, Zuo PP (2016b) Changes of TSPO-mediated
Zetterberg H et al (2020) Plasma P-tau181 in mitophagy signaling pathway in learned helplessness
Alzheimer’s disease: relationship to other biomarkers, mice. Psychiatry Res 245:141–147
differential diagnosis, neuropathology and longitudinal Li J, Li LQ, Long HP, Liu J, Jiang YP, Xue Y, Wang WX,
progression to Alzheimer’s dementia. Nat Med 26: Tan GS, Gong ZC, Liu JK (2021) Xylarinaps A-E, five
379–386 pairs of naphthalenone derivatives with
Jen CI, Su CH, Lu MK, Lai MN, Ng LT (2021) Synergis- neuroprotective activities from Xylaria nigripes. Phy-
tic anti-inflammatory effects of different polysaccha- tochemistry 186(112729):1–9
ride components from Xylaria nigripes. J Food Li LQ, Li J, Long HP, Liu JK, Wang X (2022) Four new
Biochem 45(e13694):1–11 resorcinol derivatives with neuroprotective activities
Jewett BE, Sharma S. (2022) Physiology, GABA. In: from Xylaria nigripes. Nat Prod Res 36:1522–1528
StatPearls [Internet]. StatPearls Publishing, Treasure Liao W, Fan HB, Wu JP (2018) Egg white-derived antihy-
Island, FL. Available via https://www.ncbi.nlm.nih. pertensive peptide IRW (Ile-Arg-Trp) inhibits angio-
gov/pubmed/30020683. Accessed 15 Aug 2022 tensin II-stimulated migration of vascular smooth
Joca SRL, Guimarães FS (2006) Inhibition of neuronal muscle cell via angiotensin type I receptor. J Agric
nitric oxide synthase in the rat hippocampus induces Food Chem 66:5133–5138
antidepressant-like effects. Psychopharmacology 185: Liaw CC, Wu SJ, Chen CF, Lai MN, Ng LT (2017) Anti-
298–305 inflammatory activity and bioactive constituents of
Ju Y-M, Hsieh H-M (2007) Xylaria species associated cultivated fruiting bodies of Xylaria nigripes
with nests of Odontotermes formosanus in Taiwan. (ascomycetes), a Chinese medicinal fungus. Int J Med
Mycologia 99:936–957 Mushrooms 19:915–924
Ju Y-M, Hsieh H-M, He X-S (2022) Wulingshen, the Lowy B (1974) Amanita muscaria and the thunderbolt
massive Xylaria sclerotia used as traditional Chinese legend in Guatemala and Mexico. Mycologia 66:188–
medicine, is produced by multiple species. Mycologia 191
114:175–189 Lu J-X, Zhu M, Chen Y, Zhang P, Fang L (2011) HPLC
Kawahara K, Hohjoh H, Inazumi T, Tsuchiya S, Sugimoto specific chromatogram of chemical constituents of
Y (2015) Prostaglandin E2-induced inflammation: Rel- Wuling capsules (in Chinese). Yao Wu Fen Xi Za
evance of prostaglandin E receptors. Biochim Biophys Zhi 4:764–767
Acta 1851:414–421 Ma Z-Z, Zhang P-P, Chen W-R (1999) Studies on the
Ko HJ, Song AR, Lai MN, Ng LT (2011) Immunomodu- sedative and sleeping effects of Wuling mycelia and
latory properties of Xylaria nigripes in peritoneal mac- its pharmacological mechanism. Chinese Pharm J 34:
rophage cells of Balb/c mice. J Ethnopharmacol 138: 374–347
762–768 Matar E, McCarter SJ, St Louis EK, Lewis SJG (2021)
Lauvrak SU, Munthe E, Kresse SH, Stratford EW, Namlos Current concepts and controversies in the management
HM, Meza-Zepeda LA, Myklebost O (2013) Func- of REM sleep behavior disorder. Neurotherapeutics
tional characterisation of osteosarcoma cell lines and 18:107–123
identification of mRNAs and miRNAs associated with Mizuno M, Yamada K, Maekawa N, Saito K, Seishima M,
aggressive cancer phenotypes. Br J Cancer 109:2228– Nabeshima T (2002) CREB phosphorylation as a
2236 molecular marker of memory processing in the hippo-
Li D-Q, Li X-J, Duan J-F, Cai W (2010) Wuling capsule campus for spatial learning. Behav Brain Res 133:135–
promotes hippocampal neurogenesis by improving 141
expression of connexin 43 in rats exposed to chronic
14 Xylaria Sclerotia Formed Within Termite Nests: A Review of Their Biology and Human Uses 355

Mueller UG, Gerardo NM, Aanen DK, Six DL, Schultz Tall AR, Rader DJ (2018) The trials and tribulations of
TR (2005) The evolution of agriculture in insects. CETP Inhibitors. Circ Res 122:106–112
Annu Rev Ecol Evol S 36:563–595 Tan YF, Liao ZL, Qiu YJ, Zhu JP, Yu EY (2016) Possible
Nagam V, Aluru R, Shoaib M, Dong GR, Li Z, Pallaval involvement of L-arginine-nitric oxide (NO)-cyclic
VB, Ni JF (2021) Diversity of fungal isolates from guanosine monophosphate (cGMP) signaling pathway
fungus-growing termite Macrotermes barneyi and in the antidepressant-like effect of Wuling mycelia
characterization of bioactive compound from Xylaria powder in rat. Biomed Pharmacother 78:60–65
escharoidea. Insect Sci 28:392–402 Timmerman R, Burm SM, Bajramovic JJ (2018) An over-
Peng L, Zhang X, Kang DY, Liu XT, Hong Q (2014) view of in vitro methods to study microglia. Front Cell
Effectiveness and safety of Wuling capsule for post Neurosci 12(242):1–12
stroke depression: a systematic review. Complement U'Ren JM, Miadlikowska J, Zimmerman NB, Lutzoni F,
Ther Med 22:549–566 Stajich JE, Arnold AE (2016) Contributions of North
Peng WF, Wang X, Hong Z, Zhu GX, Li BM, Li Z, Ding American endophytes to the phylogeny, ecology, and
MP, Geng Z, Jin Z, Miao L et al (2015) The anti- taxonomy of Xylariaceae (Sordariomycetes,
depression effect of Xylaria nigripes in patients with Ascomycota). Mol Phylogenet Evol 98:210–232
epilepsy: a multicenter randomized double-blind study. Vargas E, Podder V, Carrillo Sepulveda MA (2022) Phys-
Seizure-Eur J Epilep 29:26–33 iology, glucose transporter type 4 [Internet]. StatPearls
Petch T (1906) The fungi of certain termite nests. Ann Roy Publishing, Treasure Island, FL. Accessed
Bot Gard (Peradeniya) 3:185–270 15 Aug 2022
Petch T (1913) Termite fungi: a résumé. Ann Roy Bot Verano AL, Tan DS (2017) Family-level stereoselective
Gard (Peradeniya) 5:303–341 synthesis and biological evaluation of
Pharmacopoeia Commission of China (2020) Pharmaco- pyrrolomorpholine spiroketal natural product
poeia of the People’s Republic of China, the 2020 antioxidants. Chem Sci 8:3687–3693
edition, vol. 1 (in Chinese). China Medical Science Visser AA, Ros VI, De Beer ZW, Debets AJ, Hartog E,
Press, Beijing, 1902 p + index Kuyper TW, Laessoe T, Slippers B, Aanen DK (2009)
Robinson RG, Jorge RE (2016) Post-stroke depression: a Levels of specificity of Xylaria species associated with
review. Am J Psychiat 173:221–231 fungus-growing termites: a phylogenetic approach.
Rogers JD, Ju Y-M, Lehmann J (2005) Some Xylaria Mol Ecol 18:553–567
species on termite nests. Mycologia 97:914–923 Wang HP, Chen HZ, Gao Y, Wang SJ, Wang X, Tang
Rojo LE, Fernandez JA, Maccioni AA, Jimenez JM, XM, Fang W, Shi XY, Yao J, Chen Q (2020) The
Maccioni RB (2008) Neuroinflammation: implications effect of wuling capsule on depression in Type 2 dia-
for the pathogenesis and molecular diagnosis of betic patients. Biosci Rep 40:BSR20191260:1–9
Alzheimer’s disease. Arch Med Res 39:1–16 Wangsawat N, Ju Y-M, Phosri C, Whalley AJS,
Russwurm M, Koesling D (2004) NO activation of Suwannasai N (2021a) Twelve new taxa of Xylaria
guanylyl cyclase. EMBO J 23:4443–4450 associated with termite nests and soil from northeast
Sangvichien E, Taylor-Hawksworth PA (2001) Thailand. Biology-Basel 10(575):1–33
Termitomyces mushrooms: a tropical delicacy. Mycol- Wangsawat N, Nahar L, Sarker SD, Phosri C, Evans AR,
ogist 15:31–33 Whalley AJS, Choowongkomon K, Suwannasai N
Sesti G, Federici M, Hribal ML, Lauro D, Sbraccia P, (2021b) Antioxidant activity and cytotoxicity against
Lauro R (2001) Defects of the insulin receptor sub- cancer cell lines of the extracts from novel Xylaria
strate (IRS) system in human metabolic disorders. species associated with termite nests and LC-MS anal-
FASEB J 15:2099–2111 ysis. Antioxidants-Basel 10(1557):1–16
Song AR, Ko HJ, Lai MN, Ng LT (2011) Protective Wiatrak B, Kubis-Kubiak A, Piwowar A, Barg E (2020)
effects of Wu-Ling-Shen (Xylaria nigripes) on carbon PC12 cell line: cell types, coating of culture vessels,
tetrachloride-induced hepatotoxicity in mice. differentiation and other culture conditions. Cells-
Immunopharm Immunot 33:454–460 Basel 9(958):1–15
Sub Laban T, Saadabadi A. (2022) Monoamine oxidase Wiesinger H (2001) Arginine metabolism and the synthe-
inhibitors (MAOI) [Internet]. Treasure Island, FL: sis of nitric oxide in the nervous system. Prog
StatPearls Publishing. Accessed 15 Aug 2022 Neurobiol 64:365–391
Sun RZ, Zhang YH, He SS, Li R, Xue F, Chen YC, Yan Wu J-L (1984) Fungal Chinese medicine—Wulingshen
WJ, Wang HN, Peng ZW (2017) Xylaria nigripes (in Chinese). J Chengdu Univ Tradit Chin Med 1984:
protects mice against cerebral ischemic injury by 33–35
activating Nrf2/HO-1 pathway. Int J Clin Exp Med Xicoy H, Wieringa B, Martens GJM (2017) The SH-SY5Y
10:11636–11645 cell line in Parkinson’s disease research: a systematic
Taciak B, Bialasek M, Braniewska A, Sas Z, Sawicka P, review. Mol Neurodegener 12(10):1–11
Kiraga L, Rygiel T, Krol M (2018) Evaluation of Xiong J, Huang Y, Wu XY, Liu XH, Fan H, Wang W,
phenotypic and functional stability of RAW 264.7 Zhao Y, Yang GX, Zhang HY, Hu JF (2016) Chemical
cell line through serial passages. PLoS One 13: constituents from the fermented mycelia of the medici-
e0198943:1–13 nal fungus Xylaria nigripes. Helv Chim Acta 99:83–89
356 H.-M. Hsieh and Y.-M. Ju

Ying J-Z, Mao X-L, Ma Q-M, Zong Y-C, Wen H-A depression disorder: a meta-analysis of randomized
(1987) Icons of medicinal fungi from China. Science controlled trials. E Asian Arch Psychiat 26:87–97
Press, Beijing, p 575 Zhou B, Qiu Y, Wu N, Chen AD, Zhou H, Chen Q, Kang
Zhang E, Liao P (2020) Brain-derived neurotrophic factor YM, Li YH, Zhu GQ (2020) FNDC5 attenuates oxida-
and post-stroke depression. J Neurosci Res 98:537– tive stress and NLRP3 inflammasome activation in
548 vascular smooth muscle cells via activating the
Zhang QH, Ni X, Zhang FQ (2017) Decreasing trend in AMPK-SIRT1 signal pathway. Oxidative Med Cell
severe weather occurrence over China during the past Longev 2020:6384803:1–15
50 years. Sci Rep-Uk 7:42310:1–8 Zhou HF, Zhao Y, Peng WH, Han WB, Wang DY, Wang
Zhao ZQ, Li YP, Chen HY, Huang LQ, Zhao F, Yu Q, ZC, Ren XX, Pan GZ, Lin Q, Wang X (2022) Efficacy
Xiang ZH, Zhao ZX (2014) Xylaria nigripes mitigates and safety of Wuling capsule for insomnia disorder: a
spatial memory impairment induced by rapid eye systematic review and meta-analysis of randomized
movement sleep deprivation. Int J Clin Exp Med 7: controlled trials. Sleep Med 93:1–14
356–362 Zhuang S-H, Zheng T-S (1886) Revised local gazetteer of
Zheng W, Zhang Y-F, Zhong H-Q, Mai S-M, Yang X-H, the district of Guan-xian (in Chinese), vol 12. Guan
Xiang Y-T (2016) Wuling capsule for major County Magistrate, Guanxian, p 162