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 Pythium
Diagnosis, Diseases and Management

Editors
Mahendra Rai
Department of Biotechnology, SGB Amravati University
Amravati, Maharashtra, India
Kamel A. Abd-Elsalam
Unit of Excellence in Nano-Molecular Plant Pathology
Plant Pathology Research Institute, Giza, Egypt
Avinash P. Ingle
Department of Biotechnology, Engineering School of Lorena,
University of Sao Paulo, Lorena, SP, Brazil

p,
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Cover illustration provided by Prof. Francois Barja. Designing assistance from Bengisu Sengun.
Mostowfizadeh-Ghalamfarsa and Fatemeh Salmaninezhad, Department of Plant Protection, Shiraz University, Shiraz, Iran.

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Preface
The genus Pythium is one of the most important fungi of class Oomycota. It is a well-known soil
borne phytopathogenic fungus causing significant damage in agriculture, forest, nurseries, etc. and
is ubiquitously found across the world. It also affects the seed germination and attacks the seedlings
both at pre- and post-emergence stage. It is an unseen enemy of the root zone of various plants and
is hence considered as “hidden terror” for a number of plants. The accurate diagnosis of Pythium
causing root rot in plants is very important because it is often confused with root rots caused by
various other fungi such as Phytophthora, Rhizoctonia, Chalara, Cylindrocladium, Fusarium and
Aphanomyces. Taxonomic identification of Pythium species is also important as they vary in their
host range and temperature requirements. It is proposed that Pythium root rot is difficult to control
once rot has begun; therefore, its effective and eco-friendly management is a major concern. In
addition, Pythium is responsible for causing infections in different animals including horses, dogs,
human being, etc. and such infections are known as Pythiosis.
Considering these facts, the present book is focused on all the important aspects related to
Pythium biology. Broadly, this book is divided into four parts: Part I deals with an overview, host
range and plant diseases, Part II presents various challenges in the taxonomy and diagnosis, and
also current technological developments in detection and diagnosis of Pythium, Part III is devoted
to the role of Pythium as human pathogen and Part IV is focused on the management of Pythium.
The text in each chapter is supported by numerous clear, informative tables and figures. Each chapter
contains relevant references of published articles, which offers a potentially large amount of primary
information and further links to a nexus of data and ideas.
All the chapters included in the present book have been written by the specialists, experts in
the concerned topic and these chapters are highly informative and detailed. Therefore, we believe
that this book will serve as a rich guide for undergraduate or graduate students of various disciplines
like agriculture, plant pathology, plant physiology, mycology, molecular biology, biotechnology,
and allied subjects. In addition, the book will be useful for researchers in these fields and the people
working in various agro-based industries, regulatory bodies, food and agriculture organizations.
The editors are highly thankful to all the contributors for their outstanding efforts to provide
state-of-the-art information on the subject matter for their respective chapters. The collective efforts
taken by all the authors will help to enhance and update the knowledge of the readers, particularly
about Pythium and its pathogenesis in both plants and animals. We express our sincere thanks to the
publisher and the authors of the chapters, whose research work have been cited in the book. We are
also thankful to the entire team at CRC Press for their generous cooperation and efforts in producing
this book. MR wishes to thank University Grants Commission, New Delhi for the award of BSR
Faculty Fellowship.
We hope that the book will be useful for all the readers to find the required information on the
latest research and advances in the field of Pythium biology.

Mahendra Rai
Kamel A. Abd-Elsalam
Avinash P. Ingle
Contents
Preface iii

Part I: Incidence, Host-range and Diseases

1. The Genus Pythium: An Overview 3
Mahendra Rai, Kamel A. Abd-Elsalam, Avinash P. Ingle, Priti Paralikar
and Pramod Ingle
2. The Genus Pythium in Three Different Continents 15
Hani Mohamed Awad Abdelzaher, Shaima Mohamed Nabil Moustafa and Hashem
Al-Sheikh
3. Pythium: Diseases and Their Management 30
Patrycja Golińska and Magdalena Świecimska
4. The Genus Phytopythium 45
Abdul Mubeen Lodhi, Saleem Shahzad and Rehana Naz Syed
5. Top Three Plant Pathogenic Pythium Species 77
Amal-Asran and Kamel A. Abd-Elsalam
6. Pythium Species Associated With Die-back Apple Trees and Citrus
Gummosis in Tunisia 92
Naima Boughalleb-M’Hamdi, Najwa Benfradj and Souli Mounira
7. Pathogenic and Beneficial Pythium Species in China: An Updated Review 107
Hon H. Ho and Kamel A. Abd-Elsalam
8. The Pythium Complex of the Mid-North Region of Brazil 123
Janete Barros da Silva, Douglas Henrique Trigueiro Silva, Francynara Pontes Rocha,
Givanilso Cândido Leal, Helanny Márcia Ribeiro Trajano, Joseane Lustosa Machado,
Laércio de Sousa Saraiva, Maria do Amparo de Moura Macêdo, Osiel César da
Trindade Júnior, José de Ribamar de Sousa Rocha, Nayara Dannielle Costa de
Sousa and Tamyres Lopes Rios
9. Pythium spp. on Vegetable Crops: Research Progress and Major Challenges 136
Pratibha Sharma, Prashant P. Jambhulkar, Raja, M. and Shaily Javeria
10. Host Plants and Specificity of the Genus Pythium 162
Kamel A. Abd-Elsalam and Amal-Asran

Part II: Identity and Taxonomy

11. Taxonomic Challenges in the Genus Pythium 179
Reza Mostowfizadeh-Ghalamfarsa and Fatemeh Salmaninezhad
vi Pythium: Diagnosis, Diseases and Management

12. Diagnosis of Pythium by Classical and Molecular Approaches 200

Shivannegowda Mahadevakumar and Kandikere Ramaiah Sridhar

Part III: As a Human Pathogen

13. Pythium insidiosum – An Emerging Mammalian Pathogen 227
Erico S. Loreto, Juliana S.M. Tondolo and Janio M. Santurio

Part IV: Management of Diseases Caused by Pythium

14. Damping-off Caused by Pythium Species: Disease Profile and Management 257
Mohammad Imad Khrieba
15. The Genus Pythium: Genomics and Breeding for Resistance 270
Ramadan A. Arafa, Said M. Kamel and Kamel A. Abd-Elsalam
16. Role of Phytochemicals in Plant Diseases Caused by Pythium 287
Rajendra M. Gade, Mahendra Rai, Ranjit S. Lad and Amol V. Shitole
17. Pythium aphanidermatum and Its Control Measures 299
Tahira Parveen, Mukesh Meena, Tripta Jain, Kavita Rathore, Surbhi Mehta and
Kanika Sharma
18. Management of Pythium Diseases 314
Rehana Naz Syed, Abdul Mubeen Lodhi and Saleem Shahzad
19. Management of Pythium spp. by Arbuscular Mycorrhizal Fungi 344
Sarika R. Bhalerao, Pratiksha R. Gund, Sunita D. Bansod and Mahendra Rai
20. Pythium Species as Biocontrol Agents 360
Mousa Alghuthaymi, Khaled Kasem, Omar Atik and Kamel A. Abd-Elsalam

Index 379
About the Editors 381
Color Section 383
 CHAPTER

1
The Genus Pythium: An Overview
Mahendra Rai1*, Kamel A. Abd-Elsalam2, Avinash P. Ingle3, Priti Paralikar1 and Pramod Ingle1
1 Nanobiotechnology Lab, Department of Biotechnology, SGB Amravati University,
Amravati - 444602, Maharashtra, India
2 Unit of Excellence in Nano-Molecular Plant Pathology, Plant Pathology Research Institute,
9 Gamaa St., 12619 Giza, Egypt
3 Department of Biotechnology, Engineering School of Lorena, University of Sao Paulo,
Estrada municipal do Campinho, sn, 12602-810 Lorena, SP, Brazil

Introduction
Pythium is a soil-borne pathogen which contains more than 300 species and the majority of them are
plant pathogenic. Pythium is classified into 10 different clades based on morphological and genetic
characteristics (Rossman et al. 2017). It is commonly found in soil, sand, various water sources and
dead and decaying part of plants. It is ubiquitously distributed across the globe including America,
Asia, Africa and Australia. The old taxonomic criteria proposed was confusing and there were some
difficulties in the validation of various Pythium species. Pringsheim discovered the genus Pythium
for the first time in 1858, and placed it in the family Saprolegniaceae (Pringsheim 1858). Thereafter,
a number of attempts were made to propose different taxonomic systems for the classification
of Pythium but all such systems were rejected time to time by various taxonomists (Ho 2018).
According to current taxonomic system, Pythium is placed in family Pythiaceae, order Pythiales,
class Oomycetes, phylum Oomycota, and kingdom Chromista (Kirk et al. 2008).
As discussed earlier, Pythium spp. are mainly pathogenic to a wide variety of crop plant
families and is a major problem in greenhouses and nurseries. Root rot and damping-off are the most
important diseases caused by Pythium (Rai et al. 2018). It is reported that Pythium can infect some
important stages of plant’s growth which mainly include infection to the seed before germination or
during germination. It may attack the young seedlings before or just after emergence. The causative
agents generally feed on the root system causing damping-off, which ultimately results in poor
germination and spindly plants (McKellar and Nelson 2003). The infection also leads to shortened
or distorted leaves, fewer tillers and smaller heads; collectively, it leads to a great loss in crop yield
and economic loss (http://www.syngenta-us.com/prodrender/imagehandler.ashx?ImID=37907769-
8aca-45f1-9ed2-71ad09069464&fTy=0&et=8).
In addition, it is proposed that, among different species of Pythium, P. insidiosum is the most
prevalent etiologic agent responsible for pythiosis in mammals (Krajaejun et al. 2018). However,
among mammals, pythiosis is commonly reported in dogs, horses and humans. Apart from these,
sometimes it is also observed in other animals, such as calves (Perez et al. 2005), cats (Rakich et al.
2005), sheep (Santurio et al. 2008), a bird (Pesavento et al. 2008), etc. It is also responsible for rare,
non-transmissible disease generally found in tropical, subtropical and temperate regions (Mendoza

*Corresponding author: [email protected]; [email protected]

4 Pythium: Diagnosis, Diseases and Management

2005). However, recently it was reported that the infections are not only restricted to these climatic
conditions, but also observed in regions like California and Arizona, where the climate does not fit
this description. It was clearly indicated that the environmental niche for P. insidiosum is expanding,
possibly due to outcome of environmental changes like unnecessary flooding of rice fields or
irrigated landscape development (Gaastra et al. 2010).
Apart from the pathogenesis, there are two important concerns about Pythium which are
always raised in many available reports, one is about the confirm identifications of Pythium species
and another is about its management (Sutton et al. 2006, Tambong et al. 2006). The overlapping
mycelial and sporangial characters mainly hinder the morphological identification of Pythium spp.
Some conventional identification methods are available which include identification of species on
the basis of morphology of antheridia, oogonia and sporangia, but it greatly varies under different
cultural conditions. In addition, these approaches are time-consuming and require highly expert
hands (Kumar et al. 2008). It has been proved that management of Pythium is very difficult once
the infection is established. There are some traditional approaches involving the use of chemical
fungicides (antifungal agents), phytochemicals and biological agents which are routinely in practice
for the management of Pythium; however, still there is need to develop more sensitive, eco-friendly
and economically viable methods for the efficient management of Pythium (Rai et al. 2018).
Considering all these aspects, in the present chapter we have focused on overview of Pythium,
which mainly includes its recent taxonomic status, worldwide distribution, pathogenesis and
management.

Taxonomic challenges
Traditionally, the identification of Pythium species is based on morphological characteristics (Dick
1990, Van der Plaats-Niterink 1981) such as sporangia, shape and size of oogonia, antheridia
and, sporangia, oospores, and rate of growth on culture medium. However, being biologically
and ecologically diverse species, the morphological characteristics are highly variable and hence
determination of identity is a major challenge (Ghalamfarsa 2015). Considering these difficulties,
morphological characteristics can not be used for identification of Pythium species (Lévesque and
de Cock 2004).
Mycelia of Pythium species branch out apically at right angles; hyphae are hyaline, with
mostly 5-7 µm wide; cross septa are generally present in old culture and not in new ones (Vander
PlaatsNiterink 1981). Streaming of protoplast is often conspicuous in newly formed hyphae. The
hyphal walls of Pythium are mainly composed of polysaccharides (80-90%) like ß1-6 linked glucans
and ß1-3 and ß1-4 (cellulose) (Postma et al. 2009). Interestingly, hyphal wall of Pythium spp. does
not contain chitin or chitosan, whereas it consists of varying concentration of protein (3-8%) and lipid
(1-3%) (Postma et al. 2009). These characteristics are variable under different culture conditions,
and therefore, many species are morphologically similar. Some of these characteristics can also
change or be acquired or lost readily (Lévesque and De Cock 2004). In addition, the morphological
characteristics used for species differentiation has not always correlated with the major clades in
Pythium determined by molecular methods (e.g. Lévesque and De Cock 2004). Therefore, molecular
markers have been found to be essential tools for determining identity of Pythium spp.
Molecular markers are important tools for confirming the identity of the fungi being fast,
authentic, specific and sensitive as compared to the use of morphological markers. The molecular
techniques are promising alternatives to determine the identity of fungi even without the knowledge
of taxonomy. Many researchers used Internal transcribed spacer (ITS) region for determination
of identity of a particular pathogen (Robideau et al. 2011, Rai et al. 2014, Salmaninezhad and
Mostowfizadeh-Ghalamfarsa 2019). In spite of much variation in sequences in ITS region, the
availability of primers that provide sequence data is also essential (Lévesque and de Cock 2004).
The Genus Pythium: An Overview 5

Based on phylogenetic study, polyphyletic nature of Pythium has been suggested because
its species have originated from two or more independent ancestors. Considering these facts,
it is felt that taxonomic revisions of the genus Pythium are necessary, while some authors have
recommended creation of five new genera (Uzuhashi et al. 2010). Lévesque and de Cock (2004)
studied 116 species and varieties of Pythium on the basis of ITS rDNA sequencing and further, based
on phylogeny, classified these species in 11 major clades (i.e. A to K). The authors further confirmed
that the Pythium species in clade K are genetically different from the rest of the genus. Interestingly,
the members of this clade demonstrated morphology intermediary between Phytophthora and
Pythium and therefore, this group has been designated as a new genus termed as Phytopythium. The
morphological and molecular studies of clade K together with improved taxon sampling, led to its
reassignment as genus Phytopythium (Bala et al. 2010, Marano et al. 2014, De Cock et al. 2015,
Jesus et al. 2016). Phytopythium generally showed morphological characteristics similar to both
Pythium and Phytophthora as they proliferate internally similar to some species of Phytophthora;
however, development and release of zoospores is external (De Cock et al. 2015) or partly internal
and partly external to sporangia (Marano et al. 2014, Jesus et al. 2016). The important morphological
difference between these clades is due to the sporangial morphology (ovoid, globose, elongated or
filamentous) (Levesque and de Cock 2004, Uzuhashi et al. 2010)

Pathogenicity of Pythium
Many members of the genus Pythium cause infections and diseases in plants, animals and human
beings. The pathogenicity of Pythium to plants, animals and humans has been briefly discussed
below.

Diseases in plants
As discussed above, the genus Pythium is a readily recognized plant pathogen with a very wide host
range and distribution (Van Buyten and Höfte 2013). Certainly, all the members are not pathogenic
but most of them cause serious loss to crops under favorable conditions like susceptible host,
environment and geographical range. Pythium spp. primarily causes infection to the juvenile or
succulent tissues, limiting their damage to seedlings or feeder roots. In non-seedling plant hosts like
grass, tomato transplants, peanuts, and chrysanthemums, most affected parts are stems and foliage
leaves. A fruit rot was also seen in crops like beans, squash, and watermelon (Hendrix and Campbell
1973). Pythium spp. involve in the destruction of the fine roots and root tips of trees (Lorio 1966),
curtailing the inability of roots to absorb sufficient nitrogen from the soil (Campbell and Otis 1954).
Peach and citrus decline also hampers the production which is associated with Pythium spp. (Sleeth
1953, Hendrix et al. 1966).
Michigan is the third largest producer of floriculture in the USA. The million dollar business
is mainly affected by various diseases caused by Pythium which mainly includes damping-off,
crown and root rot. Del Castilo Munera and Hausbeck (2016) worked on the isolation of pathogenic
fungi from various flowering plants and identified them on the basis of morphology and ITS
sequencing. Among the various isolates, 287 isolates were obtained from poinsettias, 726 from
geranium and other greenhouse floral cultures. P. aphanidermatum, P. ultimum, P. cylindrosporum,
and P. irregulare were the most commonly reported species (Del Castilo Munera and Hausbeck
2016). Seed rot, root rot, seedling damping off, flower rot and black leg in ornamental plants are
profoundly caused by Pythium spp. (Martin and Loper 1999, Moorman et al. 2002). Stephen and
Powell (1982) reported different Pythium spp. such as P. aphanidermatum, P. ultimum, P. spinosum
and P. debaryanum associated with damping-off of impatiens, vinca and celosia. In case of crop
plants, Pythium infects both underground and aerial parts. P. aphanidermatum and P. myriotylum
are the major crop pathogenic Pythium spp. (Agrios 2005). Pythium infections are confined to the
6 Pythium: Diagnosis, Diseases and Management

meristematic tissues of mature plants. They cause necrotic lesions on root tips and less commonly
affect the tap roots. But the deeper invasion may cause infection of vascular parts (Watanabe et al.
2008).
Studies on seedling diseases of corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) in
Ohio showed the predominance of Pythium spp. Broders et al. (2007) reported eleven Pythium
spp.: P. attrantheridium, P. dissotocum, P. echinulatum Matthews, P. graminicola, P. inflatum,
P. irregulare, P. helicoides Drechs., P. sylvaticum, P. torulosum, P. ultimum var. ultimum, and P.
ultimum Trow var. sporangiiferum Drechs. associated with corn and soybean seeds rot. Out of these,
six species were pathogenic to corn and nine species were pathogenic to soybean seeds. According
to van der Plaats-Niterink (1981), Pythium spp. commonly infects seedlings, tap roots, root tips or
feeder roots and also mature plants which leads to death of respective plant. P. aphanidermatum
is reported to cause damping-off of seedlings, root and crown rots of mature cucumber (Cucumis
sativus L.) plants (Zitter et al. 1996, Al-Sa’di et al. 2007). Pegg and Manners (2014) reported the
association of Pythium spp. with the nursery plants like blueberries, causing cutting and stem rot,
and aerial rot. Rai et al. (2018) have summarized the Pythium spp. and Fusarium spp. responsible
for the soft rot of ginger (Zingiber officinale Rosc.). P. aphanidermatum and P. myriotylum are the
majorly reported Pythium spp. responsible for soft rot in ginger rhizome (Dohroo 2005, Le et al.
2014, 2016).

Diseases in animals and humans

P. insidiosum is an oomycete pathogen in mammals in the tropical and subtropical region. The
disease is reported throughout the world. Mostly pythiosis is reported in horses and dogs. Hasika
and colleagues (2019) studied the demographic distribution of Pythium keratitis in South India and
reported the large series of patients with Pythium keratitis. This indicated the devastating need of
diagnosis, treatment and awareness in clinicians (Mendoza and Newton 2005, Wilson 2012, Hasika
et al. 2019) and rarely in rabbits (Gaastra et al. 2010, Botton et al. 2011). The systemic infections are
observed in horses involving ulcerative, proliferative, pyogranulomatous lesion (Reis et al. 2003).
As mentioned earlier, Pythium species are present in soil and water resources, when animals like
horses with open wound come in contact with these Pythium species get infected through such
wounds. Similarly, Pythium causes infections in humans also in the same way through severely
injured tissues. Apart from these, Pythium was also reported to infect the gastrointestinal tract of
cats (Prasertwitayakij et al. 2003, Rakich et al. 2005, Bosco et al. 2005, Badenoch et al. 2009, Fortin
et al. 2017). In case of dogs, cutaneous/subcutaneous and gastrointestinal lesions are seen (Miller
1985, Grooters 2003).
Pythiosis has also been reported in other domestic animals such as cats (nasal cavity, retrobulbar
space, skin and subcutaneous tissues) (Foil et al. 1984, Bissonnette et al. 1991, Thomas and Lewis
1998), a dromedary camel (skin, gastric) (Wellehan et al. 2004), cattle (skin/subcutaneous) (Miller
et al. 1985), and sheep (skin/subcutaneous, disseminated) (Tabosa et al. 2004). The first case of
human pythiosis was reported from patients in Thailand in 1985 (Imwidthaya 1994).
Commonly, three forms of pythiosis have been reported in humans: (i) granulomatous and
ulcerative lesions of skin and subcutaneous tissues of face and limbs (ii) Ophthalmic pythiosis
responsible for keratitis (Pal and Mahendra 2014, Garg 2019), and (iii) systemic pythiosis
responsible for vasculitis, thrombosis and aneurysms (Tanphaichitra 1989, Thianprasit et al. 1996,
Prasertwitayakij et al. 2003). Pupaibool et al. (2006) reported P. insidiosum as a predominant
causative agent in humans, leading to human pythiosis and ultimately death.
The animals from tropical, subtropical and temperate regions are mostly susceptible to Pythium
infections; moreover, these region mainly include countries like Australia, Argentina, Brazil, Costa
Rica, Colombia, Egypt, Greece, Haiti, Indonesia, India, Japan, Thailand, Papua New Guinea and
the USA (Mendoza et al. 1996). First case of Pythium infection in animals (pythiosis) was reported
in late 1800s for the first time. A controversy was reported when the similarity between lesions due
The Genus Pythium: An Overview 7

to pythiosis and equine cutaneous habronemiasis was explained by Miller (1983). The infection
caused by P. insidiosum triggers the T helper 2 [Th2] subset present in the host which further leads
to inflammatory reaction which occurs mainly in eosinophils and mast cells. Later, these cells
degranulate around the hyphal elements of P. insidiosum where a Splendore-Hoeppli-like reaction
occurs (Mendoza and Newton 2005). Periorbital cellulitis caused by P. insidiosum was reported in a
two-year child in the US, which extended to the nasopharynx and compromised airway, leading to
gastrostomy (Shenep et al. 1998).

Different approaches for management of Pythium spp.

Chemical and physical agents in Pythium management
The genus Pythium is one of the most important plant pathogenic genera responsible for causing
infections in broad host range of plants. Due to its ubiquitous nature, it is very difficult to control
using chemical and physical methods. As it becomes resistant to chemical antifungals, researchers
are encouraged to find new alternatives for its management. Pythium spp. are the ubiquitous water
molds with habitats ranging from terrestrial to aquatic. It causes multibillion dollar losses of the crop
yield worldwide every year. P. aphanidermatum is one of the most destructive member among the
various Pythium species which causes many economically important diseases (van West et al. 2003,
Parveen and Sharma 2015). Most widely used control method by farmers is a soil solarization, i.e.
hydrothermal heating of soil during hot months, when soil is exposed to direct sunlight and change
in soil environment can help in controlling Pythium progression (Katan 2000). Chemical methods
usually used for the control of Pythium involves the use of different fungicides like mancozeb,
carbendazim, ridomil, mancozeb, and topsin, etc. (Elliott 2003, Anomynous 2005, ANSAB 2011,
Poduyal 2011). These synthetic fungicides are more effective because they control pathogen by
either destroying cell membrane or increasing its permeability or by inhibiting their metabolic
processes (Osman and Al-Rehiayam 2003).

Phytochemicals in Pythium management

The use of organic materials can also be a good option for control of Pythium infections because
of its unique composition and microbial activity. There are many reports available claiming the
management of P. aphanidermatum and some other Pythium species using certain waste materials
like sugarcane residues, poultry slurry and municipal bio-solids. This supplementary organic matter
contributes higher nutrient content to soil, ultimately increasing the yield (Parveen and Sharma 2015).
Antimicrobial components of plant extracts can be used as biocontrol agents as they bring about the
change in proton flux across the membrane, thus changing the cell environment and ultimately
death of cell (Omidbeygi et al. 2007, Pane et al. 2011). Plant extracts rich in hydrophobic lipids
can be deleterious to fungus as they partition the fungal cell wall and mitochondria, disturbing their
structure, rendering the leakage of cellular contents and finally death of cell (Burt 2004, Gonçalves
et al. 2017). The extracts of Zygophyllum fabago, Azadirachta indica, Allium sativum and Curcuma
longa were reported to have significant inhibition of P. aphanidermatum in in vitro study (Dana et
al. 2010, Singh et al. 2010). However, the leaf extract of Zimmu (Allium cepa × Allium sativum)
showed potential antifungal efficacy against Pythium in vivo (Muthukumar et al. 2010). Nowadays,
it is possible to use extracts of various plants such as: Thymus vulgaris and Zingiber officinale
instead of chemical fungicides for the control of tomato damping-off diseases caused by Pythium
and Fusarium species. Vinayaka et al. (2014) reported antifungal activity of aqueous extract of
Usnea pictoides against P. aphanidermatum isolated from rotten ginger rhizome.
In addition, methanolic extract of Vitex agnus-castus showed potential antifungal activity
against P. ultimum causing infection in tomato under both in vitro and in vivo conditions. The
involvement of pathogenesis-related (PR) genes in delayed infection was demonstrated by treating
8 Pythium: Diagnosis, Diseases and Management

tomato plants with Vitex agnus-castus methanolic extract and/or P. ultimum. The expression of
PR genes, i.e. PR-1, PR-2, PR-5 and PR-6 involved in plant defense mechanism was monitored
(Svecová et al. 2013). More et al. (2017) reported the effect of different medicinal plant waste
extracts in various solvents against the soil borne pathogen P. debaryanum, which was isolated from
infected tomato on Vaartaza’s medium. Various solvent-extract system used were include acetone,
ethanol, methanol and chloroform extracts of A. marmelos, S. cumini and P. pinnata. One of the
study includes evaluation of pathogenicity of P. debaryanum in some varieties of tomato by the
soil inoculation technique. After inoculation, the initial symptoms were observed between 7-21
days. Pre-damping off seed decay and post-damping off stem lesions were observed in addition to
top rot and root rot. Further, authors tested the efficacy of methanolic, ethanolic and chloroform
extracts of leaves and fruits of A. marmelos against this pathogen. Maximum growth inhibition of
pathogen was reported in case of methanolic extract of leaves and fruits of A. marmelos followed by
ethanolic extract; however, minimum antifungal activity was reported in chloroform extract (More
et al. 2017).

Biological methods of Pythium management

Biological control of Pythium involves the accumulation of antagonistic metabolites, spatial
competition and nutrients diversification, hyphal interactions, mycoparasitism, enzymes secretion
and microbes feeding on Pythium propagules. Use of Trichoderma harzianum against Pythium was
reported by Elad (1982) and it was proposed that it can be used in disease reduction and increased
seed germination (Shanmugam et al. 1999). Lumsden and Locke (1989) demonstrated significant
control of damping-off caused by P. ultimum in various crops like cotton, cabbage and zinnia using
Gliocladium virens. In addition, different species of Trichoderma are known for their antagonistic
activity and are hence commonly used as potential biocontrol agents. Manoranjitham and Prakasam
(2000) demonstrated the significant efficacy of T. hamatum,T. harzianum, T. reesai, T. viridae and
Psudomonas fluorescens against P. aphanidermatum. Similarly, in another study, Ram (2000)
evaluated potential of T. harzianum, T. aureoviride and T. virens against Pythium spp. and reported
significant reduction in the disease and increased the yield. P. fluorescens controlled the damping-
off in sugar beet (Bardin et al. 2004). Management of pre- and post-emergence of P.
aphanidermatum mediated damping-off in chilli was reported by Haritha et al. (2010). T. hamatum
and T. harzianum were also reported to be antagonistic against P. aphanidermatum and P. ultimum
in chilli (Kamala and Indira 2011, Sharma et al. 2014). Biological control methods cannot be
considered as curative methods of disease control because they are efficient only when pathogen
load is low to moderate. Biocontrol methods may become ineffective in case the crop is already
infected with the pathogen.

Nanotechnological approaches in Pythium management

In present days, nanotechnology is expected to turn into the innovative frontier in agriculture
practices by contributing novel application. Nanoparticles have benefits depending upon type,
concentration, size and mode of synthesis in agriculture application (Singh et al. 2016, 2017,
Tripathi et al. 2017, Yang et al. 2017, Elmer et al. 2018). Among the different bioactivities possessed
by nanoparticles, antimicrobial activity is important because there is a harmful impact of plant
pathogens in agriculture and to tackle this problem nanotechnology is extensively used. Increased
application of bactericides and fungicides for plant pathogens management caused harmful effect by
contaminating vulnerable ecosystem. Furthermore, the use of nanomaterial offers many additional
properties like improved solubility, long lasting residual activity of agrichemical, and delivery can
be exploited in plant health management (Fig. 1.1). The application of nanoparticles in agriculture
not only suppresses the growth of pathogen but also promotes the growth of plant (Pourkhaloee et
The Genus Pythium: An Overview 9

Figure 1.1. Nanomaterials for delivery of fungicides

Color version at the end of the book

al. 2011, Hu et al. 2014, Shinde et al. 2018, Mohamed et al. 2018). Different classes of nanoparticles
are used in agriculture depending upon their mode of action. For the antimicrobial application in
plant pathogen management, nanoparticles such as silver, copper, sulfur, zinc, carbon nanotubes,
etc. are widely used (Fosso-Kankeu et al. 2016, Athawale et al. 2018, Rai et al. 2018). Nanoparticles
were not only reported to show strong antifungal activity but they also help to maintain soil nutrients
status (Ponmurugan et al. 2016, Prasad et al. 2017).

Conclusion
Pythium is ubiquitous in distribution, basically a soil-borne fungus with a large number of pathogenic
species and polyphyletic. It is responsible for damping-off, soft-rot, and blight; consequently, there is
a huge economic loss of crops in agriculture. Pythium also causes pythiosis in mammals, particularly
horses, dogs and humans. The identification and differentiation of different Pythium spp. is an
arduous task based on morphological characters, such as antheridia, oogonia and sporangia, which
are not stable in different culture media. Therefore, the use of molecular markers for confirmation of
species are essentially required. Moreover, these markers are rapid, accurate, sensitive and specific
as compared to the morphological markers. Another important problem with Pythium spp. is its
management. Although various chemicals/fungicides, phytochemicals are commonly used for the
management of different Pythium spp., none of them is effective for complete management of
the disease. The biological methods or green methods have advantage over the chemical methods
because these are eco-friendly and economically viable. However, these methods are useful when
the pathogen load is low and their efficacy reduces when pathogen is already present in crops
or soils. There is a greater need to apply nanotechnological strategies which are emerging with
remarkable efficacy.
10 Pythium: Diagnosis, Diseases and Management

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 CHAPTER

2
The Genus Pythium in Three Different
Continents
Hani Mohamed Awad Abdelzaher1,2*, Shaima Mohamed Nabil Moustafa1,2 and Hashem Al-Sheikh3
1 Biology Department, College of Science, Jouf University, P.O. Box: 2014, Sakaka, Saudi Arabia
2 Department of Botany & Microbiology, Faculty of Science, Minia University, El-Minia City, Egypt
3 Department of Biology, College of Science, King Faisal University, Al-Hassa, Saudi Arabia

Introduction
Pythium species belong to Oomycetes which are fungal like microorganisms located under the
kingdom Straminipila (Uzuhashi 2015). So far, more than 150 species of Pythium have been
reported (Uzuhashi 2015). Pythium is a unique fungus in many respects. The members of this genus
can be terrestrial and aquatic, parasitic, phytopathogenic and biocontroller, beneficial and harmful,
psychrophilic and thermophiles, and ultimately friend and foe. The study of Pythium began after its
definition by the biologist Pringsheim in 1858. Serial studies and amendments in the classification
and definition have been performed until we reached the current situation by dividing the genus into
four (Ovatisporangium, Elongisporangium, Globisporangium and Pilasporangium) distinct genera
(Abdelzaher et al. 1995, Abdelzaher 1999, Rahman et al. 2015, Uzuhashi et al. 2015).
Hyphae of Pythium spp. are characterized by the absence of cross-sectional walls (Coenocytic)
only at the limits of sexual structure (antheridia and oogonia) and the boundaries of zoosporangia
and hyphal swellings. Incidental cross walls may be formed at the edge of the colony. These
fungi have multiple forms of zoosporangia: filamentous, lobulated, spherical, oval and internal
to external proliferated ones. Some species do not produce zoospores. Swimming biflagellated
zoospores develop in a transparent thin vesicle, from where many zoospores release after maturity
in appropriate water medium. Sexual reproduction takes place in Pythium by means of antheridia
and oogonia. There are many forms of antheridia, including terminal and intercalary, and small and
large. Oogonia are of various shapes, which mainly include spherical and oval, having smooth and
rough surface, some are spiny and some are without spines. The antheridia are intertwined with
oogonia in several ways: front and side, broad fusion and fine fusion, single antheridium with one
oogonium, and several antheridia with one oogonium. As a result of the fusion of the two gametes,
zygote is formed, and subsequently one or more oospores are produced inside each oogonium.
These oospores are either thin or thick walled, and may fill or not fill the vicinity of the oogonia
(Plaats-Niterink 1981).
Pythium is composed of many morphological groups. Modern molecular analyses have
revealed that the genus Pythium exists as a polyphyletic group that contains several monophyletic
groups. Uzuhashi et al. (2010) limited the genus Pythium to those species that possess filamentous
zoosporangia and generated four new genera to represent fungi with non- filamentous zoosporangia.
These genera were (1) Ovatisporangium, (2) Elongisporangium, (3) Globisporangium and (4)

*Corresponding author: [email protected]

16 Pythium: Diagnosis, Diseases and Management

Pilasporangium. Similarly, Bala (2010) reported a new genus Phytopythium, the members of which
are with globose to ovoid, often papillate and internally proliferating zoosporangia. Lately, de Cock
et al. (2015) published molecular-based proof that members of Pythium clade K as described by
Lévesque and de Cock (2004) belong to the genus Phytopythium while spotting the genus status of
remaining species of Pythium.
The purpose of this chapter is to provide historical account of the evolution of the study of the
genus Pythium as a result of research work during nearly three decades in three different continents.

Pythium species in Japan

During the period of the doctoral study in Japan, many species of Pythium were isolated and
identified, including those that were isolated for the first time outside of Ireland, and new recordings
in Japan as well as other fungi registered in the past (Abdelzaher 1994). From 1992 to the end
of 1994, the use of molecular methods to assist in the identification of species of Pythium was
not known. Therefore, identification of Pythium species was only by means of morphological
identification, which was, in my personal opinion, sufficient at that time. What is noteworthy is that
the molecular identification is not sufficient in itself, but it is useful in confirming the morphological
identification and the separation of very similar species (Abdelzaher et al. 1994a, b, c, d, e, f, 1995,
Uzuhashi et al. 2015, Chenari et al. 2015).

Methods for isolation of Pythium species from aquatic habitats

Many methods have been proposed for the isolation of aquatic Pythium spp. However, the best way
is to use baits from various parts of plants. It was found that the best baits for isolation of these fungi
were parts of the leaf blade of the seedlings of maize plant, as well as the internal pectin rind of
orange or mandarin fruits. What is important for the great potential for isolation of aquatic pythia
is the selection of stagnant water such as pond water, lakes, streams and small water pools, and
not fast runoff. However, there are some Pythium spp. which can be found in flowing river water,
most of which are species of filamentous zoosporangia with oospores that are difficult to detect. It
is preferable to study the presence of Pythium spp. in soil adjacent to water basins. For isolation of
pythia, 15 ml pond water or 5 gm soil plus 10 ml pond water is placed in deep Petri dishes together
with 10 sterile baits as mentioned above, and then incubated at temperatures between 20-25ºC, for
nearly 5 days (Abdelzaher et al. 1995). For the use of different baits than that mentioned, filter paper
discs were used to isolate specific types of Pythia such as P. fluminum var. fluminum which can
utilize complex cellulose (Abdelzaher et al. 1994d).

Suitable selective medium for isolation and purification

of Pythium spp.
It is very important to use a selective medium to isolate and cultivate Pythium spp. Several antibiotics
can be used to eradicate and inhibit growth of bacteria and other non-pythiaceous fungi. Over time,
vancomycin, ampicillin, streptomycin, nystatin, pimarcin, rifampicin, pentachloronitrobenzene and
miconazole were used as antibacterial and antifungal (rather than Pythium) agents. Recently, some
of the above-mentioned antibiotics have been banned because they cause serious diseases. The
problem with the commercial type of the fungicide like Pancreatoronetropenazine is to contain
hexachlorobenzene (HCB), which possesses carcinogenic activity. The problem with Pimarcin is
the difficulty of getting it in many countries of the world because of the food sanitation law (Tojo
2017). For this reason, it became necessary to use a selective medium that did not contain the
previously mentioned banned antibiotics. Therefore, the use of selective media of NARF (nysta
tin+ampicillin+rifampicin+fluazinam) or NARM (nystatin+ampicillin+rifampicin+miconazole) is
recommended. NARF selective medium was prepared by mixing antibiotics of 50 mg of nystatin
The Genus Pythium in Three Different Continents 17

dissolved in 1 ml C2H5OH, 250 mg of ampicillin dissolved in 1 ml distilled H2O, 10 mg of rifampicin

dissolved in 1 ml dimethylsulphoxide, 0.5 mg of fluazinam dissolved in 1 ml of autoclaved water
agar (0.1%), all then thoroughly added to 1 liter sterilized corn meal agar medium that is cooled to
50 °C. NARM selective medium was prepared by mixing antibiotics of 10 mg of nystatin dissolved
in 1ml C2H5OH, 250 mg of ampicillin dissolved in 1 ml distilled H2O, 10 mg of rifampicin dissolved
in 1 ml dimethylsulphoxide, 1 mg of miconazole dissolved in 1 ml dimethylsulphoxide, all then
thoroughly added to 1 liter sterilized corn meal agar medium that is cooled to 50 °C. Researchers
who wish to isolate Pythium spp. can choose one of the previous mentioned selective media based
on the availability of these antibiotics.

Pythium spp. from aquatic habitats in Osaka

From 1942 to 1994, 24 Pythium species and groups of fungi were isolated from aquatic environments
in Osaka (Abdelzaher et al. 1995) (Fig. 2.1). All of these fungi were identified on the basis of
morphological criteria, including shape and thickness of mycelia, shape of zoosporangia, male
(antheridia) and female (oogonia) structures, and profile of oospores (Abdelzaher et al. 1994a, b,
f, 1995). During 1992-1994, the identification was only based on morphological characteristics
because groups of Pythium were known for lack of sexual reproduction. Different Pythium spp. like
P. carolinianum Matthews, P. catenulatum Matthews, P. coloratum Vaataja, P. deliense Meurs, P.
diclinum Tokunaga, P. dissotocum Drechsler, P. fluminum Park var. fluminum, P. irregular Buisman,
P. marsipium Drechsler, P. middletonii Sparrow, P. monosspermum Pringsh., P. myriotylum
Drechsler, P. papillatum Matthews, P. pleroticum T. Ito, P. spinosum Sawada, P. sylvaticum
Campbell et Hendrix, P. torulosum Coker et Patterson, P. ultimum Trow var. ultimum, P. undulatum
H. E. Petersen, P. vexans de Bary, Pythium ‘group F’, Pythium ‘group HS’, Pythium ‘group P’ and
Pythium ‘group T’ were recorded and identified from aquatic habitats in Osaka, Japan. It is worth
mentioning that after application of molecular criteria in identification process, the identification

Figure 2.1. One of the ponds used in isolation of Pythium spp. in Osaka, Japan, in 1993,
is a pond used to irrigate crop plants on adjacent farms
18 Pythium: Diagnosis, Diseases and Management

of the majority of these fungi was confirmed, as well as renaming of a few, especially Pythium
groups. Some of these fungi were previously isolated in Japan and others were isolated for the first
time in Japan, one of which was isolated for the first time outside its native habitat and another was
reassigned as a new species of a new genus (Abdelzaher et al. 1994a, b, c, d, e, f, 1995, Uzuhashi
et al. 2019).

P. fluminum var. fluminum from pond water in Osaka

Pythium fluminum var. fluminum was isolated from cellulose in fresh water habitats in Northern
Ireland and registered as a new species in 1977 (Park 1977). This species of Pythium was then
considered as type locality of North Ireland. No one has been able to isolate this fungus since its
first isolation until 1994. It was again isolated from a fresh water lake named “Tatsumi” located in
Sakai district of Osaka city, Japan (Abdelzaher et al. 1994d). However, after 1994 there is no report
regarding the isolation of this fungus till today. This might be due to the following reasons:
• Vast majority of Pythium spp. are not capable of degrading complex cellulose, such as filter
papers and the like, but they can degrade cellulose compounds of relatively small molecular
weight, and soften cellulose and pectin walls to facilitate penetration with their penetration
pegs.
• This species of Pythium can easily degrade cellulose as a result of secretion of C1 and Cx
cellulases and can therefore be isolated using filter paper discs, which most researchers do not
use as a bait.
• This taxon is one of the major cellulose decomposers in the water bodies, so it was found
in a lake next to a field of rice that produced large quantities of rice straw during harvesting
time. Therefore, we advise researchers to try to isolate this fungus from places similar to that
mentioned previously.

P. marsipium from pond water in Osaka

During the study of occurrence of pythiacous fungi in three ponds in Osaka, from 1992 to 1994, P.
Marsipim was isolated from one of the ponds using baits of mandarin internal rind (Abdelzaher et
al. 1994e). At that time, the fungus was considered a rare occurrence in Japan, isolated a few times
since it was first isolated by Ito in 1936 from a pond water in Kyoto, Japan. This fungus showed
unique structures of the genus Pythium as it possesses swimming zoospores differentiating in a
distinct transparent vesicle, characteristic of pythiaceous fungi. This fungus is rarely present or
rather difficult to isolate, since it has been isolated only a few times, and may be due to the use of
certain baits of pectinaceous layer of internal mandarin rind.
Moreover, due to the discovery of the molecular biology methods for identification, this P.
marsipium has been converted into a new species of the genus Globisporangium. At the time of
writing this chapter, the process of publishing a scientific paper on the transformation of P. marsipium
to Globisporangium lacustre sp. nov. is now proceeding. This was done after the use of molecular
phylogenetic analysis based on the internal transcribed spacer regions (ITS) of the nuclear ribosomal
RNA as well as cytochrome c oxidase subunit 1 genes of fungal mitochondria (our new paper).
Since I first isolated this fungus in 1992, I (the first author) found some morphological structures of
P. marsipium that differed, in part, from characters of the genus Pythium, such as the bladder-shaped
zoosporangium, which is similar to that in the genus Pythiogeton (Uzuhashi et al. 2019). However,
a transparent vesicle emerges from the zoosporangia through evacuation tubes and remains for
a period of time in which zoospores are differentiated. The vesicles explode and zoospores are
released. Differentiation of zoospores inside vesicles are an inherent characteristic of this genus. I
recently contributed to a research on converting P. marsipium to Globisporangium lacustre using
the above-mentioned molecular criteria.
The Genus Pythium in Three Different Continents 19

Groups of Pythium
Pythium spp. from water bodies are particularly those fungi which do not produce sexual reproductive
structures (Abdelzaher et al. 1995). Such fungi are therefore defined on the basis of zoosporangial
forms. The group of filamentous zoosporangia was defined as Pythium ‘group of F’, the group of
lobulated zoosporangia was defined as Pythium ‘group of T’, the group of proliferated zoosporangia
was defined as Pythium ‘group of P’, the group of globose zoosporangia was defined as Pythium
‘group of G’, and the group of fungi with no zoosporangia and only hyphal swellings was defined
as Pythium ‘group of HS’.
Differences have been found in the physiology and virulence between isolates of Pythium
‘group F’, which led us to try to distinguish between 10 isolates within this group (Abdelzaher et al.
1994f). Total soluble protein and ioszymes were used as tools available at the time to differentiate
these isolates. Subsequently, it has been proved that there were differences between these isolates
on the basis of total soluble protein and ioszymes. It should be noted that subsequent recent studies
on individuals belonging to those groups proved that these fungi belong to already known and
identified Pythium species. These fungi have lost their ability to reproduce sexually. This may be
partly due to the availability of appropriate conditions for fungi during their presence in aquatic
environments, which does not make it necessary for sexual reproduction. To name a few, one of
the isolates of Pythium ‘group F’ was converted to P. dissotocum after following the methods of
molecular biology known in the genetic identification of pythiaceous fungi.
One of the most important advices for those interested in identification of Pythium species is to
concentrate well on the forms and shapes of zoosporangia. Researchers must make sure that there
are transparent vesicles emerging from zoosporangia, whereas zoospores differentiate inside these
vesicles and then release.

Pathogenicity of Pythium species

It was found that isolated Pythium spp. caused serious diseases to many crop plants. These diseases
ranged from damping-off of seedling to root-rot of adult plants. It was also found that many of
the crop plants that produce a weak production are due to attack on their feeder roots by Pythium
species. Therefore, the ability of these plants to absorb water and salts from the soil is reduced that
leads to weak plant and low yield. Because Pythium spp. are facultative parasites, they are already
saprophytes, but when conditions become suitable for the fungus and unfavorable for the host plant,
they become pathogenic and virulent, causing serious diseases of germinating seeds, seedling and
even roots of adult plants. Of the conditions not suitable for the plant, but suitable for fungus are:
increase in the proportion of water in the soil with bad drainage, inappropriate climatic conditions
for plant growth and abundance of insects and worms in the vicinity of the plant. Any plant in
the stage of germinating seeds and seedling is susceptible to infection by Pythium. It has been
studied that diseases of bush okra, cauliflower, cucumber, maize, melon, soybean, spinach, tomato
and wheat (Ichitani et al. 1993, Abdelzaher et al. 1994c, 1997a, d, Abdelzaher and Elnaghy 1998,
Abdelzaher et al. 2000, Abdelzaher 2003, 2004, 2006, Elnaghy et al. 2003, 2014, Abdelzaher et al.
2004a, Feng et al. 2019).
Many chemicals are used to control the infection of crop plants by Pythium. One of the most
famous of these chemicals is metalaxyl. Studies have found that this fungicide kills fungi that are
harmful and also beneficial, causing an imbalance in the environment (Abdelzaher et al. 2004c).
Therefore, studies have sought to find a safe alternative way of biological control. It has been
found that there are many natural enemies of the pathogenic pythia, including one from the same
genus which is Pythium oligandrum. Several biological control applications using P. oligandrum to
overcome infection by plant pathogens have been successful. However, application of P. oligandrum
to plants must be confirmed prior to infection in the expected infested fields (Elnaghy et al. 2014).
20 Pythium: Diagnosis, Diseases and Management

Pythium spp. from different habitats in Gifu and Rishri, Japan,

during 2008
Now-a-days, there is a trend to use molecular tools for identification as a means of confirming the
morphological identification and separation of closely related Pythium spp. (Kageyama et al. 2007,
Tojo et al. 2012, Chenari et al. 2015, Uzuhashi et al. 2015, Shiba et al. 2018).
Pythium adhaerens, P. aquatile, P. diclinum, P. dissotocum, P. pachycaule and P. torulosum as
well as asexual isolate of P. dissotocum (Pythium “group F” and Pythium “group P” were isolated
from six rivers and a lake in Gifu, Japan during spring and summer of 2007 (Fig. 2.2)). All of
the isolated species have been previously recorded from aquatic habitats except of P. pachycaule.
Sequencing of the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) including
the 5.8SrDNA of these pythia confirmed identification based on morphological characteristics
(Kageyama et al. 2011).
A scientific mission to study biodiversity was conducted on the island of Rishiri in northern part
of Japan in the summer of 2007. The first author collaborated with Professor Koji Kageyama, for
isolation and identification of Pythium spp. in water and soil of this island.

Figure 2.2. Collection of water samples for isolation of aquatic Pythium spp. from rivers and
a lake of the city of Gifu, Japan, during spring and summer of 2007

Aquatic pythia from Rishiri Island

Some Pythium were isolated from aquatic habitats of the island of Rishiri (Rahman et al. 2015) as
in the following table (Table 2.1 and Fig. 2.3).

Table 2.1. Distribution of different Pythium spp. in Hime-numa pond, Otatomari-numa pond, Minamihara-
shitsugen pond, Tanetomi-shitugen marsh, Yamunai-sawa river, Fureai Land Shukei pond and Loge Yukiguni
river, located in Rishiri Island, Japan (24-27 July, 2007)

Sampling site Bait (grass) Bait (filter paper disks)

Hime-numa pond *“Group T” (100%)a) -
Otatomari-numa pond *P. undulatum (75%) -
*Saprolegnia sp. (25%)
Minamihara-shitsugen *New species *P. myriotylum (25%)
(later named: Pythium rishiriense Rahman MZ,
Abdelzaher HMA and Kageyama K. sp. nov.)
(Rahman et al. 2015)
Tanetomi-shitugen marsh *P. dissotocum (50%) -
*P. torulosum (50%)
Yamunai-sawa river *“Group F” (100%) *“Group F” (25%)
Fureai Land Shukei pond *“Group F” (100%) *“Group F” (25%)
Loge Yukiguni river *“Group F” (100%) *“Group F” (25%)
(a) Each calculation entry represents the percentage (%) of colonies obtained on NARM selective medium,
using 4 baits for each sample.
The Genus Pythium in Three Different Continents 21

Figure 2.3. The first author collects water samples for isolation of aquatic Pythium spp. in Hime-numa pond,
Otatomari-numa pond, Minamihara-shitsugen pond, Tanetomi-shitugen marsh, Yamunai-sawa river, Fureai
Land Shukei pond and Loge Yukiguni river, located in Rishiri Island, Japan (24-27 July, 2007)

Terrestrial pythia from Rishiri Island

The following Pythium spp. were isolated from soils of the island of Rishiri (Table 2.2).

Table 2.2. Distribution of Pythium spp. in soils of different locations in Rishiri Island on July (24-27, 2007)

Sampling site Bait (grass) Bait Bait Surface soil dilution

(hemp-seed) (mandarin method
internal rind)
Beside Hime-numa pond - - *“Group HS”
(60%)a)
Beside Otatomari-numa - - -
pond
Minamihara-shitsugen - - -
Mikaeridai Picnic Site Saprolegnia sp. - -
(100%)
Mt. Rishiri Kutsugata - - -
Course, 5-goume
Mt. Rishiri Kutsugata - - -
Course, Goyou-no-saka,
6-goume
Mt. Rishiri Kutsugata - - -
Course, Rebun-iwa,
6-goume
Hokuroku Camping Site *P. ultimum var. *“Group HS” *“Group HS” New species
sporangiiferum (40%) (60%) (100%) (later named: Pythium
*“group HS” (60%) alternatum Rahman
MZ, Abdelzaher HMA
and Kageyama K sp.
nov.) (Rahman et al.
2015)
Pon-yama - *“Group HS” -
(100%)
Tanetomi-shitugen *P. dissotocum
(100%)
(a) Each calculation entry represents the percentage (%) of colonies obtained on NARM selective medium,using
5 baits for each sample.
22 Pythium: Diagnosis, Diseases and Management

Heterothalic species isolated from Rishiri soil

Some heterothalic species of Pythium were isolated. The compatible strains were found alone, and
when they meet, sexual reproduction occurs and the ovarian microbes are formed. These species are
abundant in forests and natural vegetation. Many have been found in the forests and jungles of the
island of Rishiri (Fig. 2.4). P. intermedium and P. sylvaticum were isolated during study in Rishiri
Island.

Figure 2.4. Reaction between compatible stains of heterothallic Pythium spp.

Isolation of two new species of Pythium from water and soil

of Rishiri Island
During the search of Pythium species in the natural environments of Rishiri Island, two new species
of Pythium rishiriense and P. alternatum, were isolated and identified based on morphological and
molecular criteria (Rahman et al. 2015). It should be noted that they were initially recognized as
Pythium but we were unable to identify them morphologically at the species level. For this reason,
we expected them to be new types. The first new species of Pythium rishiriense was completely
different from other similar Pythium species on morphological basis by its shape and structure of
 Another Random Document on
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Qui, dépouillé des traits de Charles Quint joué,
Et du manteau tragique aussitôt secoué,
A repris des Démons les gigantesques formes,
Et leurs mains, et leurs pieds, armés d'ongles énormes.
Ce mime est de l'enfer, où son art s'enflamma,
Le sublime Lekain, le terrible Talma;
Sensible et déchirant, nul ne fut plus habile
A peindre l'ame humaine en sa face mobile;
Son vaste sein, foyer d'un cœur tout véhément,
De pathétique empli, l'épanche largement:
S'il imite l'effroi, le remords sur le trône,
Son front pâle ressemble au front de la Gorgone:
S'il veut des passions exhaler les douleurs,
Brisée en longs sanglots, sa voix se fond en pleurs:
Le parterre, frappé de sa magie extrême,
Pense, au malheur qu'il feint, voir le malheur lui-même.
L'acteur ouvrit la bouche, et crut, par ses accents,
Surmonter la hauteur des bruits retentissants:
Mais ses lèvres formaient des paroles perdues.
Ainsi des noirs hivers quand les neiges fondues
Sur les flancs des rochers tombent avec fracas,
Si, du torrent grossi traversant les éclats,
Les voix de deux pasteurs s'appellent des deux rives,
Son cours emporte et rompt leurs clameurs fugitives.
La fureur des Démons, et huppés, et titrés,
Descend de loge en loge aux plus bas des degrés:
Là, des derniers lutins l'épaisse populace
Autour des cabaleurs, et se rue, et s'entasse.
Ainsi, lorsque les grands accordent aux petits
Ces jeux, payés si cher, qu'on leur donne gratis,
Du plus vil peuple on voit la multitude immense
Couvrir un cirque entier, sali de sa présence:
Ainsi l'amas infect de Diables tout fangeux
Formait le centre obscur du parterre orageux.
Ce sont bandits, experts en tous métiers perfides:
Les uns, noirs recruteurs, sont fumants d'homicides;
D'autres en plein marché vendeurs non scrupuleux
D autres, en plein marché, vendeurs non scrupuleux,
Ont des litres menteurs, et des poids frauduleux:
Ceux-là chez nos Thémis s'inscrivent en faussaires;
Ceux-ci, sur leurs fourneaux, impurs apothicaires,
Dosent leur arsenic en de coupables mains,
Et de l'humeur de vivre ils purgent les humains.
D'autres, fatals Hermès, altèrent la monnaie;
D'autres sont croque-morts, le sépulcre les paie:
Apprentis carabins, ceux-là, d'un coup mortel,
Hâtent l'agonisant, convoité du scalpel.
Huissiers, greffiers, et clercs, engeance de vampires,
Ivrognes, débauchés, filoux, escrocs et sbires,
Sirènes des égoûts, harangères Vénus,
Sous les bourgeons en fleurs vendant leurs charmes nus;
Des enfers, en un mot, la plus vile canaille
Tout-à-coup se déchaîne, et hue, et siffle, et braille.
Elle garda long-temps un silence hébété,
Muette d'ignorance et de stupidité:
Ces ressorts que chez nous le vulgaire idolâtre,
Les éclatants décors, les beaux coups de théâtre,
Et le lustre étoilé des princes histrions,
Avaient conquis, ravi ses admirations:
Mais, répondant aux cris des nobles galeries,
Jusqu'aux voûtes monta le cri de ses furies.
Telle, quand des états les chefs ambitieux
Donnent le premier branle aux partis factieux,
L'écume des ruisseaux, la plèbe enorgueillie
Gronde, fait bouillonner sa plus infâme lie,
S'emporte, se déborde; et, sous le joug des lois,
De la démagogie hurlent toutes les voix:
Telle, de ces damnés la cohue insolente
Au vaste amphithéâtre imprime l'épouvante.
Tout rugit: cependant le Stentor des Démons
Fait sortir ce discours de ses larges poumons;
Perché sur un haut banc, en épervier farouche,
Qu'attache un pied crochu sur une vieille souche:
«Par un juste suffrage accueillons notre acteur »
 «Par un juste suffrage accueillons notre acteur,»
Dit-il, «mais que du drame il nous taise l'auteur.
«Son ouvrage sans goût, sans règle, sans morale,
«N'a qu'une vérité hideuse ou triviale.
«J'ai frémi, mais d'horreur; j'ai ri, mais de pitié.
«Le monstre qui le fit doit être châtié,
«Écorché, scié, cuit ... il faut que sur la claie
«On le traîne, percé d'une éternelle plaie;
«Ou qu'il soit à l'oubli condamné sans retour:
«L'orgueil est d'un auteur le plus cruel vautour.
«Mais non, de notre enfer déchaînons la critique;
«Qu'il se torde à jamais sous sa dent satirique,
«Et que, de tous les sens en lambeaux déchiré,
«Il rende au noir chaos ce qu'il en a tiré.»

Il dit, roulant un œil où pétille sa rage,

Qui des autres lutins recherche le suffrage:
Mais l'un des plus bouillants, qui veut lui répliquer,
Sentant à ses esprits les paroles manquer,
Pour mieux humilier sa critique verbeuse,
Lui tire, en grimaçant, une langue moqueuse.
Celui-ci, pour punir ce dédain trivial,
Se tourne, en lui montrant son anti-facial.
Le bruit s'accroît. Voici qu'un autre Diable grimpe,
Ami du nourrisson de l'infernal Olympe:
Son aigre voix glapit sur le vacarme entier.
Tel entre des tambours perce un fifre guerrier.

«Est-ce en vain qu'en ces vers, peintre de la nature,

«Le poëte, arrachant tout masque à l'imposture,
«Produit, s'écria-t-il, sans peur, sans préjugé,
«Du fécond univers un vivant abrégé?
«L'abandonnera-t-on aux cris de la cabale?
«Comment du goût, des mœurs, est-il donc le scandale?
«Il ne saurait blesser les règles des rhéteurs,
«Étant hors de la loi des classiques auteurs;
«Non moins original que le furent eux-mêmes
«Ces hardis inventeurs de nos doctes systêmes,
«On les siffla jadis; on le hue à son tour:
«De l'avenir peut-être il deviendra l'amour.
«Son style, en descendant du ton noble au vulgaire,
«Évite mieux l'ennui qu'en un mode ordinaire.
«A quoi bon asservir l'esprit, né dans son sein,
«Au modèle idéal de l'antique dessin?
«La nature est diverse, immense, affreuse, et belle:
«Son tableau grand, bizarre, et varié comme elle,
«Alliant tous les tons, rompant chaque unité,
«Échappe à la froideur de l'uniformité.
«Les peuples, qu'instruirait le cours d'un tel ouvrage,
«Voyant périr deux rois, les plus grands de leur âge,
«L'un, en cerveau brûlé, l'autre, d'un mal impur,
«Sentiraient que des lois le seul empire est sûr.
«N'est-ce rien que d'avoir calculé dans sa tête
«Ce vaste plan moral? l'auteur est-il si bête?
«Sa fable, dites-vous, mérite un châtiment:
«Que peint-il? ce qu'au monde on fait impunément.
«Ne frémissons-nous pas, tout damnés que nous sommes,
«Lorsqu'il nous faut, témoins des cruautés des hommes,
«Voir les tigres, les ours, orner leurs écussons,
«Et leur gloire nourrir et corbeaux et poissons?
«Voir les peuples agneaux immolés en hosties;
«Le crime sur l'autel asseoir ses dynasties;
«Haine, avarice, orgueil, sous de saints capuchons,
«Dans nos ardents brasiers attiser les brandons;
«Voir le rire apprêter la corde aux calvinistes,
«Et la pudeur en proie au viol des papistes;
«Voir baptiser de sang d'incrédules beautés,
«Dont la Luxure en froc fouette les nudités:
«Des bibles, des missels, voir les sinets mystiques
«Cousus, d'un doigt railleur, aux fesses hérétiques,
«Par d'enjoués bourreaux, par de gais assassins...
«Ah! nous-mêmes, près d'eux, nous serions de vrais saints!
«Osons dire tout...! Non. Notre pudeur m'arrête;
«Je vous ferais dresser les cornes sur la tête!
«L'antropophage impie, en son acharnement,
«Ne fait pas ce qu'ils font, religieusement.
«Quoi! ces hommes, d'un Dieu se prétendant l'image,
«L'un par l'autre écrasés, n'écoutent que leur rage!
«Quoi! ces monstres pourront, dans leurs hideux transports,
«Percer de traits aigus les ames et les corps,
«Et viendront nous chanter ces mots, Indépendance,
«Charité, Sainteté, Chasteté, Tolérance!
«Oh! préférons l'horreur de nos punitions
«A ce qu'ont inventé leurs noires passions!
«Souffrons donc qu'un spectacle aux enfers nous retrace
«Les vices que sur terre on envisage en face.
«Craignez-vous que, honteux d'être moqué de nous,
«L'homme ne se corrige?... Ah! tranquillisez-vous;
«Ses mœurs seront toujours criminelles, infâmes,
«Dût-on, chez les mortels, jouer même nos drames.
«Là, qui les jugerait? un famélique essaim,
«Vendant le fiel jaloux qui bouillonne en son sein,
«Dont l'immoralité, ne prêchant que morale,
«Noie honneur et bon sens dans son encre vénale.
«Qui les écouterait? des spectateurs légers,
«Faibles cerveaux, émus par des traits passagers,
«Et de qui la mémoire, en sa marche incertaine,
«Oublie où s'attacha le long fil d'une scène;
«Peu faits pour mesurer par quels puissants efforts
«Vers un seul but profond tendent de grands ressorts.
«Honneur à ce travail! il est digne d'un Diable.
«Craignons que la colère injuste, impitoyable,
«Comme chez les humains, ne dicte nos arrêts,
«Dont l'affront éternel nous flétrisse à jamais.
«Un ouvrage a, par-fois, les beautés qu'on lui nie.
«Gare au sot tribunal qui proscrit le génie!»
A ce mot, ô discorde! ô désordre! ô terreur!
Le cirque est une arène où combat la fureur.
 Les princes infernaux lancent dans le parterre
Trente griffons armés, pour terminer la guerre:
La rage s'en accroît; on mugit autour d'eux.
Les Diablesses, fuyant ce spectacle hideux,
Volent, jetant des cris en nocturnes chouettes.
Des loges et du cintre on perce les retraites;
Et se précipitant des plus hauts des balcons
Sur les derniers des bancs roulent mille Démons.
Tous ceux de qui la foudre avait brûlé les ailes,
Titans, demi-roués en leurs chûtes cruelles,
Bondissent en tombant: telle, d'un pesant choc,
Si du sommet d'un mont le temps détache un roc,
Sa masse retentit sur la plaine ébranlée.

Figure, si tu peux, cette horrible mêlée,

O Muse! aide ma vue à mesurer le tour
Du parquet infernal éclairé d'un faux jour,
Plus vaste que ne sont les abymes stériles
Des ardents souterrains, dévorateurs des villes;
Et non moins spacieux que le cercle étoilé
Qu'embrasse un esprit docte, à qui rien n'est voilé;
Hauteur, d'où les humains, bornés dans leurs limites,
Paraissent à son œil des mouches et des mites.

Misérables damnés! votre dernier loisir

S'écoule en ces fureurs promptes à vous saisir:
L'inflexible Destin déja commande aux Heures
De vous rendre aux tourments de vos tristes demeures.

Xiphorane descend, et s'écriant trois fois:

«Anarchie!» Oh! quel monstre apparut à sa voix!
Hydre informe et sans yeux, de ses mains furieuses,
Elle-même abattant ses têtes odieuses,
En nourrit une seule; et d'un bandeau sanglant
Sur ses propres débris la couronne en hurlant:
Cette tête aggrandie, et d'elle encor frappée,
Tombe et l'hydre renaît de sang toujours trempée
Tombe, et l hydre renaît de sang toujours trempée.
Tel est le monstre. «Accours, épouse du Chaos,
«Toi qui souffles la guerre, et qui hais le repos,
«Des équitables lois ennemie éternelle,
«Dans tes cent mains, dit-il, que la flamme étincelle.»
L'hydre aveugle l'entend, plane, et d'un vague essor
S'abat des hauts plafonds sur les balustres d'or:
Des décorations la rougeâtre lumière
Allume tout-à-coup sa torche incendiaire.
Sous vingt trombes de feu, piliers, voûtes, lambris,
Croulent sur les démons embrasés et meurtris;
Et, tel qu'un puits sans fond, le gouffre à ces ruines
Ouvre, en les entraînant, ses routes intestines.
Leur immense théâtre en cendres se réduit,
Et ne laisse après soi que le vide et la nuit.

Sauve-moi de leur gouffre, ô Dieu vengeur du crime!

Dieu, pour qui notre monde est un point dans l'abyme!
Théose! être éternel, présent à l'infini!
A tout ce qui se meut ton mystère est uni.
Être que tout ignore, et que pourtant mon ame
Invoque, et sent par-tout quand s'élève sa flamme!
Dieu, principe sans forme, inaccessible à tous,
Créateur des soleils qui rayonnent sur nous,
Auteur de tant de cieux inconnus de la terre,
Tu formas les tissus de la mouche éphémère;
Tu n'as pas négligé le ressort palpitant
De son corps invisible, atôme d'un instant;
Et la moindre vapeur, globule de rosée,
Suit ta loi souveraine aux sphères imposée.
Tout n'est que profondeur qui cache ton pouvoir.
Toi, que j'ose implorer, te puis-je concevoir?
Sais-je ce que je suis? pourquoi j'entends et pense?
Si ton souffle bientôt retire ma présence
Du théâtre vivant où chacun est acteur,
Ah! que de l'ordre au moins un moment spectateur,
Je voie, avant ma mort, l'homme sincère et libre,
Je o e, a a t a o t, o e s cè e et b e,
Des lois, reines du monde, observer l'équilibre,
Saper du fol orgueil l'édifice abattu,
N'aspirer qu'aux grandeurs de la noble vertu,
Gouverner par Thémis république ou royaume,
Juger d'un œil égal le palais et le chaume,
Ouvrir son toit, son cœur, à l'humble adversité,
Ne plus, d'un joug sanglant, fouler l'humanité,
Enrichir par le fer la seule agriculture,
Paisible conquérant, explorer la Nature,
Et des Arts, du Commerce, étendant le pouvoir,
Envahir hardiment les trésors du savoir!
Dieu! qu'au néant, enfin, rentre l'Hypocrisie,
Qui change en un enfer le trajet de la vie;
Et je rendrai sans peine, au sein de l'univers,
Cette ame qui te cherche, et qui dicta mes vers.

FIN.
 ON TROUVE DU MÊME AUTEUR,
Chez BARBA, libraire, galerie du Palais-Royal, derriere le Théâtre-
Français.
Agamemnon
Ophis
Isule et Orovèse } tragédies en 5 actes.

Charlemagne
Pinto, ou la journée d'une conspiration, comédie historique, en 5
actes et en prose.
Le Frère et la Sœur jumeaux,
Le faux Bon-Homme,
Le Complot domestique,
} comédies en 3 actes.

Les Ages Français, poëme en strophes et en 15 chants.

Chez NEPVEU, libraire, passage des Panoramas, nº 26.

L'Atlantiade, ou la Théogonie Newtonnienne, poëme en 6

chants.
Homère,
}
Alexandre, poëmes en 4 chants.
L'Homme renouvelé, récit moral, en vers.
Agar et Ismaël, scène orientale.
La Méroveïde, poëme héroï-comique, en octaves, et en 14
chants.
La Panhypocrisiade, ou le Spectacle infernal du seizième siècle,
comédie-épique, en 16 chants.
Cours analytique de Littérature générale, prononcé à l'Athénée
de Paris, 4 vol. in-8º.

Chez LALOY, libraire, rue de Richelieu, vis-à-vis la rue Feydeau.

Les quatre Métamorphoses, poëmes.
Chez FIRMIN-DIDOT, imprimeur du Roi, de l'Institut, et de la Marine,
rue Jacob, nº 24.

La Méroveïde.
La Panhypocrisiade, ou le Spectacle infernal du seizième siècle.

Les Éditions de Plaute et de Christophe-Colomb, comédies en 3 actes

et en vers, et de Baudouin, empereur, tragédie en 3 actes, sont à
refaire, ayant été détruites dans un incendie.
 Corrections.
La premiere ligne indique l'original, la seconde la correction:
p. 256:

LA VIELLE.
LA VIEILLE.
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