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Plant Microbiology
 Plant Microbiology
Michael Gillings and Andrew Holmes
Department of Biological Sciences, Macquarie University,
Sydney,
NSW 2109, Australia

LONDON AND NEW YORK

 © Garland Science/BIOS Scientific Publishers, 2004
First published 2004
This edition published in the Taylor & Francis e-Library, 2005.
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photocopying and recording, or in any information storage or retrieval system, without permission
in writing from the publishers.
A CIP catalogue record for this book is available from the British Library.

ISBN 0-203-50660-X Master e-book ISBN

ISBN 0-203-62500-5 (Adobe e-Reader Format)

ISBN 1 85996 224 6 (Print Edition)
Garland Science/BIOS Scientific Publishers 4 Park Square, Milton Park, Abingdon, Oxon, OX14
4RN, UK and 29 West 35th Street, New York, NY 10001–2299, USA World Wide Web home
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Library of Congress Cataloging-in-Publication Data Plant microbiology/edited by Michael Gillings
and Andrew Holmes. p. cm. ISBN 1-85996-224-6 1. Plants—Microbiology. 2. Plant-microbe
relationsips. 3. Rhizobium. I. Gillings, Michael.II. Holmes, Andrew J.III. Title. QR351.P58325
2004 577.8′52--dc22 2004003943

Production Editor: Catherine Jones

 Contents

Abbreviations vii
Contributors ix
Preface xi

1 The diversity, ecology and molecular detection of arbuscular 1

mycorrhizal fungi
R.Husband
2 Rhizobial signals convert pathogens to symbionts at the legume interface 20
A.Bartsev, H.Kobayashi and W.J.Broughton
3 The root nodule bacteria of legumes in natural systems 35
E.L.J.Walkin
4 Effects of transgenic plants on soil micro-organisms and nutrient 59
dynamics
A.Sessitsch, K.Smalla, E.Kandeler and M.H.Gerzabek
5 Fungal endophytes: hitch-hikers of the green world 81
K.Saikkonen, M.Helander and S.H.Faeth
6 Actinorhizal symbioses: diversity and biogeography 102
D.R.Benson, B.D.Vanden Heuvel and D.Potter
7 Chemical signalling by bacterial plant pathogens 138
C.L.Pemberton, H.Slater and G.P.C.Salmond
8 Quorum quenching—manipulating quorum sensing for disease control 165
L-H.Zhang
9 Plant disease and climate change 175
S.Chakraborty and I.B.Pangga
10 Genetic diversity of bacterial plant pathogens 194
M.Fegan and C.Hayward
11 Genetic diversity and population structure of plant-pathogenic species in 220
the genus Fusarium
B.A.Summerell and J.F.Leslie
12 Genome sequence analysis of prokaryotic plant pathogens 238
D.W.Wood, E.W.Nester and J.C.Setubal
13 Analysis of microbial communities in the plant environment 259
A.J.Holmes
14 The importance of microbial culture collections to plant microbiology 286
E.Cother

Index 302
 Abbreviations
ABC ATP-Binding Cassette
ACL acyl carrier protein
AFLP amplified fragment length polymorphism
AHL acyl homoserine lactone
AM arbuscular mycorrhizal
AOGCM Atmospheric-Ocean General Circulation Model
APG Angiosperm Phylogeny Group
BLAST Basic Local Alignment Search Tool
bp basepair
CGA community genome array
CLPP community level physiological profiling
DF diffusible factor
DGGE denaturing gradient gel electrophoresis
DSF diffusible extracellular factor
EC enzyme commission
ENSO El Nino—Southern Oscillation
EPS exopolysaccharide
FAME fatty acid methyl ester
FGA functional gene array
GMP genetically modified plant
HGT horizontal gene transfer
HR hypersensitive response
INVAM International Culture Collection of Arbuscular and
Vesicular Mycorrhizal Fungi
IS insertion sequence
ITS internal transcribed spacer
LCO lipo-chito-oligosaccharide
LPS lipopolysaccharide
LRR leucine-rich repeat
MGS metabolic group-specific
MYA million years ago
NAO North Atlantic Oscillation
NCBI National Center for Biotechnological Information
NU nodulation unit
ORF open reading frame
OTU operational taxonomic unit
PCR polymerase chain reaction
PGS phylogenetic group-specific
POA phylogenetic oligonucleotide array
R resistance
RFLP restriction fragment length polymorphism
RISA ribosomal intergenic spacer analysis
RNB root nodule bacteria
ROS reactive oxygen species
SAM S-adenosylmethionine
SOI Southern Oscillation Index
SSCP single-strand conformation polymorphism
SSU small subunit
TC transport classification
Ti tumour-inducing
T-RFLP terminal RFLP
TTSS type III secretion systems
VCG vegetative compatibility group
 Contributors

Bartsev, A. LBMPS, l’Université de Genève, 1 ch de l’Impératrice, 1292

Chambésy/Genéve, Switzerland
Benson, D.R. Dept. Molecular and Cell Biology, University of Connecticut, Storrs, CT
06268–3044, USA
Broughton, W.J. LBMPS, l’Université de Genève, 1 ch de l’Impératrice, 1292
Chambésy/Genéve, Switzerland
Chakraborty, S. CSIRO Tropical Agriculture, CRC for Tropical Plant Pathology,
University of Queensland, Qld 4072, Australia
Cother, E. NSW Agriculture, Forest Road, Orange NSW 2800, Australia
Faeth, S.H. Department of Biology, Arizona State University, Tempe, AZ 85287–1501,
USA
Fegan, M. CRC for Tropical Plant Protection, The University of Queensland, St. Lucia,
Queensland, 4072, Australia
Gerzabek, M.H. Institute of Soil Research, University of Agricultural Sciences Vienna,
A-1180 Vienna, Austria
Gillings, M. Molecular Prospecting Group and Key Centre for Biodiversity, Department
of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia
Hayward, C. CRC for Tropical Plant Protection, The University of Queensland, St.
Lucia, Queensland, 4072, Australia
Helander, M. Section of Ecology, Department of Biology, FIN-20014 University of
Turku, Finland
Holmes, A.J. School of Molecular and Microbial Biosciences, The University of Sydney,
Sydney, New South Wales, 2006, Australia
Husband, R. Department of Biology, University of York, PO Box 373, York, YO10
5YW, UK
Kandeler, E. Institute of Soil Science, University of Hohenheim, D-70599 Stuttgart,
Germany
Kobayashi, H. LBMPS, l’Université de Genève, 1 ch de l’Impératrice, 1292
Chambésy/Genéve, Switzerland
Leslie, J.F. Department of Plant Pathology, 4002 Throckmorton Plant Sciences Center,
Kansas State University, Manhattan, Kansas 66506–5502, USA
Nester, E.W. Department of Microbiology, Box 357242, University of Washington,
Seattle, WA 98195–7242, USA
Pangga, I.B. CSIRO Tropical Agriculture, CRC for Tropical Plant Pathology, University
of Queensland, Qld 4072, Australia
Pemberton, C.L. Department of Biochemistry, University of Cambridge, Tennis Court
Road, Building O, Downing Site, Cambridge, CB2 1QW, UK
Potter, D. Dept. Pomology, Univ. of California, Davis, One Shields Avenue, Davis,
California 95616, USA
Saikkonen, K. MTT Agrifood Research Finland, Plant Production Research, Plant
Protection, FIN-31600 Jokioinen, Finland
Salmond, G.P.C. Department of Biochemistry, University of Cambridge, Tennis Court
Road, Building O, Downing Site, Cambridge, CB2 1QW, UK
Sessitsch, A. ARC Seibersdorf research GmbH, Div. of Environmental and Life
Sciences, A-2444 Seibersdorf, Austria
Setubal, J.C. University of Campinas, Bioinformatics Laboratory Institute of
Computing, CP 6176, Campinas, SP 13083–970, Brazil
Slater, H. Department of Biochemistry, University of Cambridge, Tennis Court Road,
Building O, Downing Site, Cambridge, CB2 1QW, UK (now at: New Phytologist
Central Office, Bailrigg House, Lancaster University, Lancaster, LA1 4YE, UK)
Smalla, K. Federal Biological Research Centre for Agriculture and Forestry, Institute for
Plant Virology, Microbiology and Biosafety, D-38104 Braunschweig, Germany
Summerell, B.A. Royal Botanic Gardens and Domain Trust, Mrs. Macquaries Road,
Sydney, New South Wales, 2000, Australia
Vanden Heuvel, B.D. Dept. Pomology, Univ. of California, Davis, One Shields Avenue,
Davis, California 95616, USA
Watkin, E.L.J. School of Biomedical Sciences, Curtin University of Technology, GPO
Box U1987, Perth, WA 6102, Australia
Wood, D.W. University of Washington, Department of Microbiology, 1959 NE Pacific
Street, Box 357242, Seattle, WA 98195, USA
Zhang, L.-H. The Institute for Molecular Agrobiology, 1 Research Link, The National
University of Singapore, 117604, Singapore
 Preface

The last decade has seen major changes in the way that we investigate the varied
interactions between micro-organisms and plants. The widespread adoption of molecular
methods in plant microbiology has given us the opportunity to investigate these
biological systems with unparalleled precision and sensitivity.
We have known for a long time about the many mutually beneficial relationships
between plants and micro-organisms. However, the detailed analysis of these
relationships has often eluded us because of our inability to bring some of the microbial
symbionts into pure culture, and to tease apart the complex biochemical interplay
between mutualists. The first chapters in this book demonstrate how far we have come in
the characterisation of plant mutualisms. We are now in a position to answer questions
about the diversity, ecology and community structure of the most abundant of terrestrial
mutualisms, that of mycorrhizal fungi and their plant hosts. There has been enormous
progress in the characterisation of the molecular signals and other factors controlling host
specificity in rhizobial associations. In a similar vein, the biology and ecology of fungal
endophytes and of Frankia are yielding up their secrets. We now also have the tools to
ask questions about how agricultural practices, and in particular the use of transgenic
organisms, might affect rhizosphere communities.
The identification and characterisation of plant pathogens has been a major focus of
plant microbiology for both economic reasons and international quarantine. A diverse
array of molecular methods is now available for diagnosis and detection of pathogens.
These methods have often revealed unsuspected diversity and led to rearrangements of
taxonomic schemes. The second part of the book deals with some examples of diversity
of pathogens in the fungal and bacterial worlds. It also summarises one of the most
important revolutions in our understanding of bacterial communities, the discovery of
quorum sensing. We must now view bacteria as communities of interacting cells, able to
coordinate their biochemical activities in a manner dependent on the number of cells in
the local environment. This discovery offers deep insights into the mechanisms by which
bacteria cause disease in plants, and also offers opportunities for new methods of disease
control. We must also bear in mind that regardless of our current understanding, global
climate change will have major impacts on the distribution and severity of plant diseases.
The rapid improvements in high-throughput DNA sequencing and analysis are also
poised to rapidly expand our understanding of plant microbiology. Already the entire
genome sequence of several plant pathogens has been obtained, and more are at an
advanced stage. We also now have the ability to investigate the last real biological
frontier, the vast diversity of micro-organisms that have yet to be discovered, let alone
characterised. There are now standard methods to recover microbial genes from
environmental samples, such as soils and sediments, without recourse to standard
laboratory culture. However, characterisation of the biochemistry and physiology of
microbial species may continue to rely on culturable organisms, and for this reason, the
continued existence and support for international microbial culture collections must be a
high priority.
The science of plant microbiology is a diverse and complex one, and we realise that
there are many areas that have not been examined in this book. Nevertheless, we hope
that these contents convey some of the rapid progress and excitement inherent in current
investigations of micro-organisms and their interactions with plants.
Michael Gillings and Andrew Holmes
 1
The diversity, ecology and molecular
detection of arbuscular mycorrhizal fungi
Rebecca Husband

Plant Microbiology, Michael Gillings and Andrew Holmes

© 2004 Garland Science/BIOS Scientific Publishers, Abingdon.

1.1 Introduction

The vast majority of land plants rely on interactions with root symbionts to ensure
adequate nutrient uptake. Of these root symbionts the most widespread and common are
the arbuscular mycorrhizal (AM) fungi, which form associations with ca 60% of plant
species (Smith and Read, 1997). The AM association is also arguably the most successful
root symbiosis in evolutionary terms. Fossil evidence and molecular clock estimates
indicate that the AM symbiosis originated at least 400 MYA (million years ago) and has
not changed appreciably since (Simon et al., 1993a; Taylor et al., 1995). The association
is almost universally distributed in early plant taxa with loss of the symbiosis only
occurring more recently in ca 10% of plant families (Tester et al., 1987; Trappe, 1987). It
is therefore hypothesised that the AM symbiosis was instrumental in ensuring the
successful colonisation of land by plants (Simon et al., 1993a).
The AM symbiosis is widely accepted to be mutualistic. The most obvious benefit to
the fungus is a ready supply of carbon, whilst the plant gains access to nutrients (most
notably phosphorus) that might not be available from root uptake alone (Smith and Read,
1997). Other benefits to the plant include improved water relations and protection against
pathogens (Newsham et al., 1995). Plants associated with AM fungi often exhibit
increased growth and survival (Smith and Read, 1997) although critically the level of
benefit depends on a variety of factors including the particular host-fungal combination
(Helgason et al., 2002; Streitwolf-Engel et al., 1997; van der Heijden et al., 1998a). The
differential effects individual AM fungi have on plant performance are therefore
proposed to have a major influence on ecosystem functioning, from affecting productivity
(Klironomos et al., 2000), to altering competitive interactions (Gange et al., 1993; Grime
et al., 1987; Hartnett and Wilson, 1999; O’Connor et al., 2002), and influencing the
overall diversity of plant communities (van der Heijden et al., 1998b).
Yet despite the important role AM fungi play in ecosystem functioning, little is known
of the community structure and ecology of the fungi themselves. To date, fewer than 200
AM fungal species have been identified (Morton and Benny, 1990). This apparent low
global diversity of AM fungi compared to their associated host plant communities has led
to the widespread belief, only now being challenged, that AM fungi are a functionally
homogeneous group (Smith and Read, 1997). Indeed, with few exceptions, the majority
 Plant microbiology 2

of ecological studies have ignored the functional diversity of individual AM fungi and
grouped all the fungi into a single class. To be fair, this is because AM fungi are
notoriously difficult to study. Firstly, AM fungi are obligate biotrophs and we have yet to
find a way of culturing them independently of their plant hosts. Secondly, AM fungi
exhibit a very low morphological diversity, making the reliable identification of different
species difficult. Fungal structures formed internally within the host root possess few
diagnostic characters; therefore the taxonomy of AM fungi is based on the subcellular
structures of asexual spores. The spores of AM fungi are relatively large, easy to extract
from the soil and have enough characteristics to enable identification to species level by
experienced personnel (see the International Culture Collection of Arbuscular and
Vesicular Mycorrhizal Fungi (INVAM) website http://invam.caf.wvu.edu/ for more
information).
Estimating the diversity of AM fungi in the field has therefore traditionally relied on
detecting the spores present in the soil. Since the spores tend to be ephemeral a
complementary approach involves growing ‘trap’ plants in field soils in the greenhouse
and analysing the spores produced. Unfortunately, these methods are problematic because
fungal sporulation rates are influenced both by the environment and the host plant species
(Bever et al., 1996; Eom et al., 2000; Morton et al., 1995), therefore spore counts are not
a direct measure of diversity. Furthermore, the population of spores in the soil may bear
little relation to the AM fungal populations colonising roots (Clapp et al., 1995). One of
the most important methodological advances in the study of AM communities has been
the application of the polymerase chain reaction (PCR) to directly identify the AM fungi
in planta. Recovering sequence information from the field gives us direct access, without
relying on culturing, to the AM fungi present in roots, the ecologically significant niche.
Sequence information also provides us with the means to consistently distinguish
between morphologically similar taxa. Thus molecular tools now present us with new
opportunities for understanding the role of AM fungi in the environment. In this chapter
some of the methods available to study AM fungal diversity in the field are outlined and
the key findings from such studies are reviewed.

1.1.1 The taxonomy of AM fungi

Based on morphological characters, fewer than 200 AM fungal species have been
described (Morton and Benny, 1990). They can be divided into five families, the
Acaulosporaceae (Acaulospora, Entrophospora), Gigasporaceae (Gigaspora,
Scutellospora) and Glomaceae (Glomus) (Morton and Benny, 1990) plus the newly
described lineages (Archaeosporaceae) and Paraglomaceae (Paraglomus) (Morton and
Redecker, 2001). Traditionally these families have been placed in the order Glomales,
phylum Zygomycota, but in recent years both morphological and molecular evidence
have indicated that the phylum Zygomycota as currently defined cannot be sustained.
Firstly, many of the organisms assigned to it, including AM fungi, are not known to have
a sexual stage, i.e. they do not form zygosporangia (Benny, 1995). Secondly,
phylogenetic analyses based on sequence data have demonstrated that the lineages
ascribed to the Zygomycota do not share a common ancestor, i.e. the phylum
Zygomycota is polyphyletic (O’Donnell et al., 2001; Tehler et al., 2000). Consequently,
based on a small subunit (SSU) ribosomal gene (rDNA) phylogeny, Schüßler et al.
 The diversity, ecology and molecular detection 3

(2001b) have proposed a new classification for the AM fungi, removing them from the
Zygomycota and placing them in a new phylum the Glomeromycota. The analyses of
Schüßler et al. (2001b) indicate that the AM fungi can be separated into a monophyletic
clade that is not closely related to any of the Zygomycota lineages, but is instead
probably diverged from the same common ancestor as the Ascomycota and
Basidiomycota.
At the lower taxonomic level, the SSU rDNA phylogeny indicates a large genetic
diversity within the genus Glomus that is not reflected by any of the morphological
characters available (Schwarzott et al., 2001). Sequences for the AM fungi within this
genus can have genetic distances as large as that between the families Acaulosporaceae
and Gigasporaceae and as a result the classification of Schüßler et al. (2001b) also
proposes a new family and order ranking (see Figure 1.1). The proposed order
Diversisporales contains the two traditional family groupings of the Acaulosporaceae and
Gigasporaceae, plus a new family Diversisporaceae fam. ined. consisting of some of the
AM fungi originally classified within the genus Glomus. The remaining ‘classical’
Glomus spp. have been placed in the order Glomerales, which clearly separates into two
distinct family-ranked clades, Glomus Group A and B. Two further orders are proposed,
the Paraglomerales (single family Paraglomeraceae) and the Archaeosporales (two
families, Archaeosporaceae and Geosiphonaceae). As currently defined, the family
Archaeosporaceae is paraphyletic. The type species for the Geosiphonaceae is a non-
mycorrhizal fungus, Geosiphon pyriforme, which forms an association with
cyanobacteria. Previously it had been proposed that Geosiphon represented the ancestral
precursor to AM fungi (Gehrig et al., 1996), but the recent rDNA data reveal it is in fact
closely related to the Archaeosporaceae and consequently the phylum Glomeromycota, as
defined, includes both mycorrhizal and non-mycorrhizal fungi.
The classification of Schüßler et al. (2001b) will necessarily evolve as more
information becomes available. The lack of convincing morphological characters for
many of the groupings and the presence of multiple sequence variants within single
spores of AM fungi (discussed in Section 1.2), means that more detailed work is needed
before the taxonomy and systematics of AM fungi are truly understood. Nonetheless the
classification of Schüßler et al. (2001b) represents the basis for a new taxonomy for AM
fungi, finally acknowledging what has long been obvious, namely that AM fungi are not
typical Zygomycetes.
 Plant microbiology 4

Figure 1.1. Phylogeny of the

Glomeromycota (neighbour-joining
analysis of SSU rDNA sequences)
indicating the families and orders
proposed by Schüßler et al., (2001b).
Bootstrap values >70% (1000)
replicates are shown. Asterisks (*)
identify the AM fungal taxa previously
classified within the genus Glomus. A
 The diversity, ecology and molecular detection 5

putative choanozoan, C. limacisporum

L42528, (Cavalier-Smith and Allsopp,
1996) is used as an outgroup.
Accession numbers are as follows: Ar.
trappei Y17634, Ge. pyriforme
AJ276074, Ar. leptoticha AJ301861,
P. occultum AJ276082, P. brasilianum
AJ301862, G. υiscosum Y17652, G.
etunicatum Y17639, G. luteum
AJ276089, G. lamellosum AJ276087,
G. mosseae AJ418853, G. sinuosum
AJ133706, G. intraradicesAJ301859
G. clarum AJ276084, E. colambiana
Z14006, A. spinosa Z14004, A.
scrobiculata AJ306442, A. longula
AJ306439, G. etunicatum AJ301860,
G. versiforme X86687, G. spurcum
Y17650, S. dipapillosa Z14013, S.
cerradensis AB041344, S. callospora
AJ306443, S. projecturata AJ242729,
Gi. albida Z14009, Gi. margarita
X58726, Gi. Gigantea Z14010.

1.2 Molecular techniques used to study AM fungi in the field

Molecular techniques have the potential to revolutionise the study of AM fungi in the
environment. Previously we have only had access to those AM fungi amenable to trap
culture or actively sporulating in the field. Now a variety of nucleic acid-based strategies
have been developed that enable the AM fungi to be characterised independently of spore
formation. The majority of sequence information for AM fungi is derived from the
ribosomal RNA genes (rDNA). In most organisms the ribosomal RNA genes are present
in multiple copies arranged in tandem arrays. Each repeat unit consists of genes encoding
a small (SSU or 18S) and a large (LSU or 28S) subunit, separated by an internal
transcribed spacer (ITS), which includes the 5.8S rRNA gene (Figure 1.2). The SSU and
5.8S genes evolve relatively slowly and are useful for studies of distantly related
organisms. The LSU and ITS regions evolve more quickly and are useful for fine-scale
differentiation between species. In most organisms, the process of concerted evolution
ensures that within an individual the multiple copies of rDNA are identical, but early
studies looking at the genetic diversity of AM fungi revealed an unexpectedly high
degree of ITS sequence variation within single spores (Lloyd-MacGilp et al., 1996;
Sanders et al., 1995). Sequence divergence within single spores has since been detected
 Plant microbiology 6

in the ITS of many different species (Antoniolli et al., 2000; Jansa et al., 2002; Lanfranco
et al., 1999; Pringle et al., 2000). The diversity is not restricted to the ITS, but has also
been detected in the SSU (Clapp et al., 1999) and extensive intrasporal diversity appears
to exist in the LSU (Clapp et al., 2001; Rodriguez et al., 2001). Most recently, intrasporal
variation has been detected in a gene encoding a binding protein (BiP) (Kuhn et al.,
2001).
Currently the full implications of the intrasporal variation present within the
Glomeromycota are unclear. One obvious problem is that there is no straight-forward
correlation between sequence identity and species identity, and as a consequence there is
no phylogenetic species concept for AM fungi. Furthermore there is no straightforward
correlation between the number of sequence variants detected within a root and the
number of separate infection events. Thus, ecological studies that use molecular markers
to study AM fungal diversity are limited as to the conclusions they can make. Not only is
it impossible to ascribe a species name to a sequence type, but it is also impossible to
determine how many different AM fungi are colonising each root. As a consequence the
majority of studies reviewed in this chapter have simply placed the AM fungi into groups
based on sequence similarity, making the assumption that the AM fungi within each
grouping share at least some ecological or functional characteristics. Such an assumption
is not necessarily unjustified because in general most of the intrasporal sequence
variation is relatively minor. Usually phylogenetic analyses reveal that the majority of
sequence variants will form a ‘core cluster’ with a minority of the sequence variants
revealing a greater divergence and clustering elsewhere (Clapp et al., 2001, 2002). Even
so, more research is clearly needed in order to understand the genetic organisation of AM
fungi. For a more detailed discussion of the topic see Clapp et al. (2002), and Sanders
(2002).

Figure 1.2. A repeat unit of the fungal

ribosomal RNA genes, showing the
position of primers commonly used for
the study of AM fungi.

1.2.1 Community detection

One of the most common goals in arbuscular mycorrhizal research is to determine the
role AM fungi play in ecosystem functioning. Previously any such research has been
severely limited by the lack of basic information such as the identities and distributions of
 The diversity, ecology and molecular detection 7

the fungi. Whilst molecular techniques can help us gain such information, the methods
themselves are limited by the available molecular markers. As a rule, in the wider field of
molecular ecology, a single set of ribosomal primers is quickly established that is
sufficient for all preliminary investigations of the organism(s) in question (see Avise,
1994; Carvalho, 1998). Not so the arbuscular mycorrhizas; as yet there is no single
method that can reliably measure in planta diversity. Separate from the issue of
intraspecies diversity, we have yet to develop markers that can differentiate all AM
fungal sequences from non-AM fungal and plant sequences. With the discovery of the
highly divergent families of the Archaeosporaceae and Paraglomeraceae the sequence
divergence within the Glomeromycota is considerable, so it may never be possible to rely
on a single molecular marker. Currently, a compromise must be made between the level
of genetic resolution and the number of lineages detected.
The first paper to apply molecular techniques to AM research appeared 10 years ago
(Simon et al., 1992). The authors used general eukaryotic primers to amplify SSU
sequences from spores and, based on this information, designed the primer VANS1
which they hoped to be a general AM fungal primer. They subsequently designed family-
specific primers (VAGIGA, VAGLO, VAACAU) which, when teamed with VANS1,
enabled the direct amplification of AM fungi from within plant roots (Simon et al.,
1993b). Although these primers appeared to work well on roots from microcosm studies,
in the field they were more problematic (Clapp et al., 1995). It was later revealed that the
VANS1 site is not well conserved throughout the Glomeromycota (Clapp et al., 1999;
Schüßler et al., 2001a).
Helgason et al. (1998) also targeted the SSU when they designed the AM1 primer to
exclude plant sequences and preferentially amplify AM fungal sequences. Coupled with a
general eukaryotic primer, it has been successfully utilised in the field to determine the
diversity of AM fungi from many different habitats (Daniell et al., 2001; Helgason et al.,
1998, 1999, 2002; Husband et al., 2002a, b; Kowalchuk et al., 2002; Vandenkoornhuyse
et al., 2002). However, new sequence data have revealed that the AM1 primer is not well
conserved in certain divergent lineages, the Archaeosporaceae and the Paraglomeraceae
(Morton and Redecker, 2001). The AM1 primer also contains two mis-matches for
sequences belonging to the Glomus group B clade defined by Schüßler et al. (2001b). In
addition, in some habitat types, relatively high proportions (up to 30%) of non-AM fungi,
mainly pyrenomycetes, are co-amplified (Daniell et al., 2001). Even so, at this present
time AM1 remains the most broadly applicable single primer suitable for field studies,
reliably detecting the three traditional families, and having numerous studies that provide
useful comparisons.
In contrast, Kjø1ler and Rosendahl, (2001) have designed LSU primers specific to a
subgroup in the Glomeraceae. The primers LSURK4f and LSURK7r are used in a nested
PCR following amplification with general eukaryotic primers (LSU0061 and LSU0599;
van Tuinen et al., 1998) and are designed to amplify a lineage within the Glomus group A
clade, including G.mosseae, G.caledonium and G.geosporum. The authors took this
approach because preliminary characterisation of the spore populations in their field site
determined that the AM fungal community was dominated by many very closely related
Glomus species that would be very difficult to distinguish using the SSU gene. Therefore
they utilised the higher diversity of the LSU to separate the different species, enabling
community comparisons to be made within this subgroup. A series of group-specific
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