Biotechnology and Biology of Trichoderma

527 Pages·2014·26.494 MB·English

by  Vijai G. Gupta, Monika Schmoll, Alfredo Herrera-Estrella, R.S. Upadhyay, Irina Druzhinina and Maria Tuohy (Eds.)

BIOTECHNOLOGY
AND BIOLOGY OF
TRICHODERMA
V K. G , M S , a h -e ,
ijai upta oniKa chMoll lfredo errera Strella
r. S. u , i d , M G. t
padhyay rina ruzhinina aria uohy
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Preface
A growing world population and the increased overview of the use of this fungus as a cell factory in
energy consumption caused by a higher standard of biotechnology. The enzyme systems of Trichoderma have
living pose a challenge on current efforts to sustain a even been used for bioremediation, which is a further
healthy environment and counteract climate change in important contribution to environmental sustainability.
the future. Replacing the limited resource of fossil oil Other products of potential relevance for industry are
and related products with renewable, carbon dioxide- the secondary metabolites produced by Trichoderma spp.
neutral resources requires a considerate strategy, as also as well as metabolic byproducts with interesting physi-
renewable biomass is not an unlimited resource. In order ological or chemical functions.
to achieve a sustainable economy, the delicate balance While T. reesei serves as a workhorse for industrial
between use of biomass/land for food production and enzyme production, other species of the genus are used
for use in industry and as an energy resource has to be for plant protection in agriculture. Thereby, these fungi
kept. Species of the genus Trichoderma can play a signifi- play an important role in establishing this important and
cant role in the strategy for a sustainable future and this delicate balance between food production and the use
book summarizes the capabilities these fungi offer. of biomass for energy production and chemical industry.
On the one hand, the metabolic capacities of Tricho- Efficient and sustainable use of biomass requires protec-
derma are of central importance for breakdown of plant tion of energy plants and food crops from pathogens in
cell walls into small compounds that can be utilized by order to guarantee that biomass as a limited resource can
yeast not only for bioethanol production, but also as fulfill the need of both society and industry. Different
building blocks for chemical synthesis. With its potent species of Trichoderma act positively on plant growth and
cellulase system and its versatility for heterologous pro- resistance of plants against disease. The chapters of this
teins, which facilitates complementation of this system book include a thorough summary on mechanisms and
with efficient enzymes from other organisms, Tricho- application of biocontrol, the enhancing effect on plant
derma reesei has become one of the cornerstones for sec- immunity and mycoparasitism.
ond-generation biofuel production. Several chapters of Considering the huge potential of Trichoderma for
this book provide an overview of the enzyme system of use in agriculture and industry, exploration of natural
Trichoderma and its optimization for efficient utilization isolates of the genus is warranted to further increase
and conversion of lignocellulosic material. Additionally, the genomic resources to be exploited. Screening the
novel and established tools for enhancing cellulase pro- biodiversity of different habitats and the ecophysiol-
duction are discussed. However, besides production of ogy of Trichoderma in a genomic perspective as well as
second-generation biofuels from plant material, indus- analysis of this diversity delivers important insights
trial use of Trichoderma also extends to production of into the promises the genus Trichoderma holds for the
silver nanoparticles and applications in beer and wine future.
industry as well as in textile industry. In summary, this book gives a detailed overview of
Trichoderma also serves as a versatile host for expres- the field of industrial and agricultural use as well as
sion of heterologous proteins and a broad array of tools the research with Trichoderma from industrial enzyme
are available for modification of the genome of this fun- production to strain improvement to biocontrol and
gus for improvement of its production capacity. Chapters diversity.
on heterologous protein production with Trichoderma,
secretion and industrial strain improvement provide an Editors
xi
Foreword
Trichoderma exists probably since at least 100 millions need for exchange of molecular genetics research tech-
of years, but it entered the scientific spotlight only in the niques became apparent. Most recently the genomes
late seventies of the last century, when the first oil shock of several Trichoderma biocontrol species have been
prompted governments to look for alternatives for fossil sequenced, and two of them (T. atroviride, T. virens) have
fuel. Researchers at the US military laboratories at Natick, been published.
Massachusetts, then remembered to possess a culture of Yet Trichoderma offers much more to science: its spe-
a green fungus that had been destroying all the cotton cies are among the most frequent mitosporic fungi com-
material (tents, belts, clothes) of the US soldiers during monly detected in cultivation-based surveys. They can
the Second World War in the South Pacific at Guadal- be isolated from an innumerable diversity of natural
canal (Solomon Islands), and who was subsequently and artificial substrata, particularly also from materi-
demonstrated to have exceptional cellulolytic abilities. als infested with xenobiotics, demonstrating their high
This fungus, like any other Trichoderma isolate at that opportunistic potential and adaptability to various eco-
time, was then named “T. viride” because the genus was logical conditions. Consequently, it has broad impacts
believed to consist only of a single species. It was later on mankind: one of the most stimulating recent find-
re-identified as “T. reesei” (in honor of one of the research- ings is that some Trichoderma spp. occur or can occur as
ers exploring its cellulolytic properties, Elwyn T. Reese), symptomless associates of plant-endophytes, thereby
for a few years misnamed as T. longibrachiatum, and stimulating plant growth, delaying the onset of drought
finally found out to be the asexual form of a very com- stress and preventing attacks of pathogens. Yet, there
mon tropical ascomycete, Hypocrea jecorina. The inter- are also negative impacts of Trichoderma on mankind:
est in this organism was of outmost importance to the in a clinical context, a pair of genetically related species
Trichoderma community in general, because it challenged (T. longibrachiatum and T. orientale) have been shown to
researchers to develop a whole toolbox of molecular occur as opportunistic pathogens of immunocompro-
genetics techniques for its manipulation, finally culmi- mised humans, and several Trichoderma spp. can occur as
nating in the sequencing of the genome of the original indoor molds, although their effect on human health is
isolate QM6a and several of its cellulase-producing less severe than that of other fungal species. Finally, some
mutants, which comprise an invaluable aid to study this species like T. aggressivum, T. pleuroticola, T. pleurotum,
organism. and T. mienum have turned their mycoparasitic abilities
While Trichoderma is consequently known to many against commercial mushrooms like Agaricus and Pleuro-
people only as the organism that makes cellulases, a tus, thereby causing large economic losses.
parallel world of Trichoderma started to develop in 1932 All these properties make Trichoderma one of the most
when R. Weindling published the mycoparasitic abilities versatile and intriguing fungal genus, which still offers
of Trichoderma “lignorum” (an illegitimate name) on plant numerous aspects to be dealt with in more detail. This
pathogenic fungi. This biocontrol ability is due to the book has been initiated to describe the current stage of
profound ability of Trichoderma to parasitize or even prey knowledge on Trichoderma from various perspectives,
on other fungi, which today is known to be the innate thereby outlining also those areas where further prog-
nature of the whole genus. Weindling’s findings formed ress is needed.
the basis for a multitude of studies on the potential use
of various Trichoderma spp. for the biological control of Christian P. Kubicek
plant pathogenic fungi, resulting in the commercializa- Professor for Biotechnology and Microbiology
tion of some of them. The cellulase and the biocontrol Department of Chemical Engineering
researchers long formed two isolated communities with Vienna University of Technology
little information exchange, but this improved once the Vienna, Austria
xiii
List of Contributors
Sunil S. Adav School of Biological Sciences, Nanyang Tech­ Luis H.F. Do Vale Department of Cell Biology, University of
nological University, Singapore Brasilia, Brasilia, Federal District, Brazil
Marika Alapuranen Roal Oy, Rajamäki, Finland Zhiyang Dong Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China
Miguel Alcalde Departamento de Biocatálisis, Instituto de
Catálisis y Petroleoquímica, CSIC, Madrid, Spain Sedigheh Karimi Dorcheh Institute for Genetic Microbiol­
ogy, Friedrich­Schiller University Jena, Jena, Germany
N. Aro VTT Technical Research Centre of Finland, Espoo,
Finland Irina Druzhinina Institute of Chemical Engineering, Vienna
University of Technology, Research Area Biotechnology and
Lea Atanasova Research Area Biotechnology and Microbio­
Microbiology, Vienna, Austria
logy, Institute of Chemical Engineering, Vienna University
of Technology, Vienna, Austria Ahmed M.A. El-Bondkly National Research Centre, Dokki,
Giza, Egypt
Antonio Ballesteros Departamento de Biocatálisis, Instituto
de Catálisis y Petroleoquímica, CSIC, Madrid, Spain M.M. Elsharkawy Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Hoda Bazafkan Health and Environment Department,
Japan
Austrian Institute of Technology GmbH (AIT), Tulln, Austria
Carlos Roberto Felix Departamento de Biologia Celular,
Gabriele Berg Graz University of Technology, Environmen­
Universidade de Brasilia, Brasilia, Federal District, Brasil
tal Biotechnology, Graz, Austria
Nicolas Lopes Ferreira IFP Energies nouvelles, Biotechnol­
Jean-Guy Berrin Laboratoire de Biologie des Champignons
ogy Department, Rueil­Malmaison, France
Filamenteux, INRA, Polytech Marseille, Aix Marseille Uni­
versité, Marseille, France Edivaldo X.F. Filho Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Robert Bischof Institute of Chemical Engineering, Vienna
University of Technology and Austrian Centre of Industrial Anli Geng School of Life Sciences and Chemical Technology,
Biotechnology (ACIB), Vienna, Austria Ngee Ann Polytechnic, Clementi, Singapore
Senta Blanquet IFP Energies nouvelles, Biotechnology Roberto J. González-Hernández Departamento de Biología,
Department, Rueil­Malmaison, France Universidad de Guanajuato, Guanajuato, México
Rosa Elena Cardoza Area of Microbiology, University School Sabine Gruber Research Area Biotechnology and Microbiol­
of Agricultural Engineers, University of León, Ponferrada, ogy, Institute of Chemical Engineering, Vienna University of
Spain Technology, Vienna, Austria
Sergio Casas-Flores División de Biología Molecular, IPICyT, Vijai K. Gupta Molecular Glycobiotechnology Group,
San Luis Potosí, México Department of Biochemistry, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland
Warawut Chulalaksananukul Biofuels by Biocatalysts
Research Unit, Chulalongkorn University, Bangkok, Santiago Gutiérrez Area of Microbiology, University School
Thailand; Department of Botany, Chulalongkorn University, of Agricultural Engineers, University of León, Ponferrada,
Bangkok, Thailand Spain
Hexon Angel Contreras-Cornejo Instituto de Investigacio­ Lóránt Hatvani Department of Microbiology, University of
nes Químico­Biológicas, Universidad Michoacana de San Szeged, Szeged, Hungary
Nicolás de Hidalgo, Morelia, Michoacán, México Senta Heiss-Blanquet IFP Energies nouvelles, Biotechnology
Christian Joseph R. Cumagun College of Agriculture, Uni­ Department, Rueil­Malmaison, France
versity of the Philippines Los Baños, Los Baños, Laguna, Rosa Hermosa Centro Hispano­Luso de Investigaciones
Philippines Agrarias (CIALE), University of Salamanca, Salamanca, Spain
Manzoor H. Dar International Rice Research Institute, IRRI, Arturo Hernández-Cervantes Departamento de Biología,
New Delhi, India Universidad de Guanajuato, Guanajuato, México
Marcelo V. de Sousa Department of Cell Biology, University Marco J. Hernández-Chávez Departamento de Biología,
of Brasilia, Brasilia, Federal District, Brazil Universidad de Guanajuato, Guanajuato, México
Christian Derntl Research Area Gene Technology, Institute Isabelle Herpoel-Gimbert Laboratoire de Biologie des
of Chemical Engineering, Vienna University of Technology, Champignons Filamenteux, INRA, Polytech Marseille, Aix
Vienna, Austria Marseille Université, Marseille, France
xv
xvi LIST OF CONTRIBUTORS
Alfredo Herrera-Estrella Laboratorio Nacional de Genómica Valdirene Neves Monteiro Universidade Estadual de Goiás,
para la Biodiversidad, Cinvestav Sede Irapuato, Irapuato, Unidade Universitária de Ciências Exatas e Tecnológicas da
Guanajuato, Mexico Universidade Estadual de Goiás­UnUCET, Anápolis, Goiás,
Robert Hill Bio­Protection Research Centre, Lincoln Univer­ Brazil
sity, Canterbury, New Zealand Héctor M. Mora-Montes Departamento de Biología, Univer­
M. Hyakumachi Laboratory of Plant Pathology, Faculty of sidad de Guanajuato, Guanajuato, México
Applied Biological Sciences, Gifu University, Gifu City, Shahram Naeimi Department of Biological Control Research,
Japan Iranian Research Institute of Plant Protection, Amol,
Katarina Ihrmark Uppsala BioCenter, Department of Forest Mazandaran, Iran
Mycology and Plant Pathology, Swedish University of Agri­ H.A. Naznin Laboratory of Plant Pathology, Faculty of
cultural Sciences, Uppsala, Sweden Applied Biological Sciences, Gifu University, Gifu City,
J.J. Joensuu VTT Technical Research Centre of Finland, Japan
Espoo, Finland Helena Nevalainen Department of Chemistry and Biomo­
Magnus Karlsson Uppsala BioCenter, Department of Forest lecular Sciences, Macquarie University, NSW, Australia;
Mycology and Plant Pathology, Swedish University of Agri­ Biomolecular Frontiers Research Centre, Macquarie Univer­
cultural Sciences, Uppsala, Sweden sity, NSW, Australia
Péter Körmöczi Department of Microbiology, University of Eliane Ferreira Noronha Departamento de Biologia Celular,
Szeged, Szeged, Hungary Universidade de Brasilia, Brasilia, Federal District, Brasil
László Kredics Department of Microbiology, University of Anthonia O’Donovan Molecular Glycobiotechnology
Szeged, Szeged, Hungary Group, Department of Biochemistry, School of Natural Sci­
ences, National University of Ireland Galway, Galway,
Adinarayana Kunamneni Departamento de Biocatálisis,
Ireland
Instituto de Catálisis y Petroleoquímica, CSIC, Madrid,
Spain T. Pakula VTT Technical Research Centre of Finland, Espoo,
Finland
Gang Liu College of Life Science, Shenzhen University,
Shenzhen, China Robyn Peterson Department of Chemistry and Biomolecular
Sciences, Macquarie University, NSW, Australia; Biomolecu­
Jesús Salvador López-Bucio Instituto de Biotecnología,
lar Frontiers Research Centre, Macquarie University, NSW,
Universidad Nacional Autónoma de México, Cuernavaca,
Australia
Morelos, México
Francisco J. Plou Departamento de Biocatálisis, Instituto de
José López-Bucio Instituto de Investigaciones Químico­
Catálisis y Petroleoquímica, CSIC, Madrid, Spain
Biológicas, Universidad Michoacana de San Nicolás de
Hidalgo, Morelia, Michoacán, México Terhi Puranen Roal Oy, Rajamäki, Finland
Robert L. Mach Research Area Gene Technology, Institute of Lina Qin Institute of Microbiology, Chinese Academy of Sci­
Chemical Engineering, Vienna University of Technology, ences, Beijing, China
Vienna, Austria Barbara Reithner Research Area Gene Technology, Institute
Astrid R. Mach-Aigner Research Area Gene Technology, of Chemical Engineering, Vienna University of Technology,
Institute of Chemical Engineering, Vienna University of Vienna, Austria
Technology, Vienna, Austria Carlos A.O. Ricart Department of Cell Biology, University of
Lourdes Macías-Rodríguez Instituto de Investigaciones Brasilia, Brasilia, Federal District, Brazil
Químico­Biológicas, Universidad Michoacana de San María Belén Rubio Centro Hispano­Luso de Investigaciones
Nicolás de Hidalgo, Morelia, Michoacán, México Agrarias (CIALE), University of Salamanca, Salamanca,
László Manczinger Department of Microbiology, University Spain
of Szeged, Szeged, Hungary M.G.B. Saldajeno Laboratory of Plant Pathology, Faculty of
Antoine Margeot IFP Energies nouvelles, Biotechnology Applied Biological Sciences, Gifu University, Gifu City,
Department, Rueil­Malmaison, France Japan
Katoch Meenu Microbial Biotechnology Division, Indian M. Saloheimo VTT Technical Research Centre of Finland,
Institute of Integrative Medicine (CSIR), Jammu, Jammu and Espoo, Finland
Kashmir, India Birinchi K. Sarma Department of Mycology and Plant
Robert N.G. Miller Department of Cell Biology, University Pathology, Institute of Agricultural Sciences, Banaras Hindu
of Brasilia, Brasilia, Federal District, Brazil University, Varanasi, Uttar Pradesh, India
Vianey Olmedo Monfil Departamento de Biología, Univer­ Monika Schmoll Health and Environment Department,
sidad de Guanajuato, Guanajuato, México Austrian Institute of Technology GmbH (AIT), Tulln, Austria
Enrique Monte Centro Hispano­Luso de Investigaciones Bernhard Seiboth Institute of Chemical Engineering, Vienna
Agrarias (CIALE), University of Salamanca, Salamanca, University of Technology and Austrian Centre of Industrial
Spain Biotechnology (ACIB), Vienna, Austria
LIST OF CONTRIBUTORS xvii
Verena Seidl-Seiboth Institute of Chemical Engineering, Siu Kwan Sze School of Biological Sciences, Nanyang Tech­
Vienna University of Technology, Research Area Biotechnol­ nological University, Singapore
ogy and Microbiology, Vienna, Austria Doris Tisch Health and Environment Department, Austrian
Gauri Dutt Sharma Bilaspur University, Bilaspur, Chattis­ Institute of Technology GmbH (AIT), Tulln, Austria
garh, India José E. Trujillo-Esquivel Departamento de Biología, Univer­
M. Shimizu Laboratory of Plant Pathology, Faculty of Applied sidad de Guanajuato, Guanajuato, México
Biological Sciences, Gifu University, Gifu City, Japan Maria G. Tuohy Molecular Glycobiotechnology Group,
Dhara Shukla Facility for Ecological and Analytical Testing, Department of Biochemistry, School of Natural Sciences,
Indian Institute of Technology, Kanpur, Uttar Pradesh, India National University of Ireland Galway, Galway, Ireland
Shafiquzzaman Siddiquee Biotechnology Research Institute, R.S. Upadhyay Department of Botany, Banaras Hindu
University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia University, Varanasi, Uttar Pradesh, India
Roberto Nascimento Silva Department of Biochemistry and Csaba Vágvölgyi Department of Microbiology, University of
Immunology, School of Medicine, University of São Paulo, Szeged, Szeged, Hungary
Ribeirão Preto, São Paulo, Brazil Khabat Vahabi Institute of General Botany and Plant Physi­
Akanksha Singh Department of Botany, Banaras Hindu ology, Friedrich­Schiller University Jena, Jena, Germany
University, Varanasi, Uttar Pradesh, India Padma S. Vankar Facility for Ecological and Analytical
Harikesh B. Singh Department of Mycology and Plant Testing, Indian Institute of Technology, Kanpur, Uttar
Pathology, Institute of Agricultural Sciences, Banaras Hindu Pradesh, India
University, Varanasi, Uttar Pradesh, India Jari Vehmaanperä Roal Oy, Rajamäki, Finland
Gurpreet Singh Microbial Biotechnology Division, Indian R.A. Vishwakarma Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India Kashmir, India
Sudhanshu Singh International Rice Research Institute, Shaowen Wang College of Life Science, Shenzhen Univer­
IRRI, New Delhi, India sity, Shenzhen, China
U.S. Singh International Rice Research Institute, IRRI, New Christin Zachow Austrian Centre of Industrial Biotechnol­
Delhi, India ogy (ACIB GmbH), Graz, Austria; Graz University of Tech­
Andrei Stecca Steindorff Departamento de Biologia Celular, nology, Environmental Biotechnology, Graz, Austria
Universidade de Brasília, Brasília, Distrito Federal, Brazil Najam W. Zaidi International Rice Research Institute, IRRI,
Alison Stewart Bio­Protection Research Centre, Lincoln New Delhi, India
University, Canterbury, New Zealand Susanne Zeilinger Research Area Biotechnology and Micro­
Xiaoyun Su Institute of Microbiology, Chinese Academy of biology, Institute of Chemical Engineering, Vienna Univer­
Sciences, Beijing, China sity of Technology, Vienna, Austria
C H A P T E R
1
Biodiversity of the Genus Hypocrea/Trichoderma
in Different Habitats
László Kredics1, *, Lóránt Hatvani1, Shahram Naeimi2, Péter Körmöczi1,
László Manczinger1, Csaba Vágvölgyi1, Irina Druzhinina3
1Department of Microbiology, University of Szeged, Szeged, Hungary,
2Department of Biological Control Research, Iranian Research Institute of Plant Protection, Amol, Mazandaran, Iran,
3Institute of Chemical Engineering, Vienna University of Technology, Research Area Biotechnology and Microbiology,
Vienna, Austria
*Corresponding author email: [email protected]
O U T L I N E
Introduction 3 Living Plants (Endophytes) 11
Mushroom-Related Substrata 13
Methodology of Studying Trichoderma Biodiversity 3
Human Body 14
Methods for the Identification of Trichoderma Strains 3
Water-Related Environments 14
Evolution of the Approach: From the Culture-Based
Air and Settled Dust 17
Method to Metagenomics 4
Conclusions 18
Trichoderma Diversity in Different Habitats 5
Natural Soils, Decaying Wood and Plant Material 5
Agricultural Habitats 9
INTRODUCTION
METHODOLOGY OF STUDYING
TRICHODERMA BIODIVERSITY
Members of the genus Trichoderma are cosmopolitan
and prevalent components of different ecosystems in a
Methods for the Identification of Trichoderma
wide range of climatic zones (Kubicek et al. 2008). The
Strains
occurrence of Trichoderma species is modulated by sev-
eral factors including microclimate, the availability of Formerly the species-level identification of Tricho-
substrates as well as complex ecological interactions derma/Hypocrea isolates was performed based on exclu-
(Hoyos-Carvajal and Bissett, 2011). Survival in different sively their morphological characteristics (Danielson
geographical habitats can be related to metabolic diver- and Davey, 1973; Summerbell, 2003; Gams and Bissett,
sity, high reproductive capacity and competitive capa- 1998). Different media were used for culturing Tricho-
bilities of Trichoderma strains in nature (Cardoso Lopes derma isolates for the analysis of their morphology and
et al. 2012). The aim of this chapter is to give an overview culture characteristics, e.g. malt extract agar, which is
about the studies aimed at the investigation of Tricho- appropriate for conidium production and the observa-
derma biodiversity in a wide variety of different ecologi- tion of conidiophore branching, or potato dextrose agar,
cal habitats. which proved useful for observing pigment production
3
Biotechnology and Biology of Trichoderma
http://dx.doi.org/10.1016/B978-0-444-59576-8.00001-1 Copyright © 2014 Elsevier B.V. All rights reserved.
4 1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
(Hoyos-Carvayal and Bissett, 2011). The preliminary development and practical applications of ITS barcodes
identification of species based on conidiophore struc- are presented and discussed in chapter 3: DNA barcode
ture, morphology as well as the size and morphology of for species identification in Trichoderma. For the analy-
conidia can be performed with the aid of taxonomic keys sis of tef1, ITS and rpb2 sequences the online programme
and descriptions available in the literature (Bissett, 1984, TrichoBLAST and its updated version, TrichoMARK are
1991a, b, c, 1992; Gams and Bissett, 1998; Chaverri and recommended (www.isth.info; Kopchinskiy et al., 2005).
Samuels, 2003; Jaklitsch, 2009, 2011; Samuels et al., 2006b, TrichoCHIT (www.isth.info), an online barcoding pro-
2012a,b). However, without professional expertise this gramme for the screening and identification of excellent
may often lead to incorrect diagnoses due to the difficul- chitinase producer strains of Hypocrea lixii/Trichoderma
ties of morphology-based species identification, therefore harzianum was developed by Nagy et al. (2007).
the results of early studies must be handled with special The use of species-specific primers in polymerase
care (Kubicek et al., 2008). In order to get around such chain reaction can also lead to quick and precise diagno-
problems and give precise species-level diagnoses, the use sis. For example, Chen et al. (1999a, b) developed a PCR-
of biochemical and molecular methods is recommended. based assay for the fast and specific detection of Th2 and
Among the biochemical methods, the metabolic Th4, the aggressive Trichoderma biotypes causing green
profiling technique of Biolog Incorporated (Hayward, mould disease of Agaricus bisporus, while the method
California)—which provides the possibility of quanti- developed by Kredics et al. (2009) allows the rapid and
tative measurements of growth and the assimilation of specific identification of Trichoderma pleurotum and Trich-
different carbon and nitrogen sources—proved to be a oderma pleuroticola, the causal agents of green mould in
useful tool to aid species identification and provide data the world-wide production of oyster mushroom (Pleu-
on the ecological roles of species (Kubicek et al., 2003; rotus osteatus) even directly from the growing substrate
Hoyos-Carvajal et al., 2009; Atanasova and Druzhinina, without the need of cultivation.
2010).
A cellulose-acetate electrophoresis-based isoenzyme
Evolution of the Approach: From the Culture-
analysis—with the involvement of glucose-6-phosphate
Based Method to Metagenomics
dehydrogenase, glucose-6-phosphate isomerase, 6-phos-
phogluconate dehydrogenase, peptidases A, B and D, Most of the studies about Trichoderma biodiversity
and phosphoglucomutase enzymes (Hebert and Beaton, applied the standard culture-based approach compris-
1993)—was applied by Szekeres et al. (2006) and Kredics ing the collection of samples, isolation of Trichoderma
et al. (2011a, 2012) for the identification of Trichoderma strains on one of the selective media described in the
strains deriving from clinical samples and winter wheat literature (Elad et al., 1981; Papavizas and Lumsden,
fields, respectively. 1982; Askew and Laing, 1993; Williams et al., 2003) and
Neuhof et al. (2007) suggested an alternative bio- their maintenance in culture, which can be followed by
chemical technique for Hypocrea⁄Trichoderma species and the application of the above-mentioned species-level
strains, which was developed based on intact-cell mass identification methods. The problem of this approach
spectrometry for the direct detection of hydrophobins in is that certain Trichoderma species may be easier, while
the mycelia as well as spores of 32 Hypocrea⁄Trichoderma others harder to isolate, therefore the diversity detected
strains representing 29 species. The hydrophobin in the culture-based studies does not necessarily reflect
patterns were shown to be characteristic to species and the actual diversity of the genus in the examined habi-
isolates, and the method is proposed to enable the rapid tat. The application of the metagenomic approach pro-
and direct detection of class II hydrophobins. vides a solution to this problem, as it is examining the
Among the molecular methods, DNA-fingerprint- habitats in situ, without the isolation and culturing of
ing (Arisan-Atac et al., 1995), the sequence analysis of the Trichoderma strains. This approach is recently gath-
the ribosomal internal transcribed spacer (ITS) region ering ground in Trichoderma biodiversity studies. In
(ITS1–5.8S rDNA–ITS2) and of segments from genes the first metagenomic attempt, Hypocrea/Trichoderma-
encoding for the translation elongation factor 1-alpha specific primers were designed for the ITS1 fragment
(tef1), endochitinase (chi18-5, formerly known as ech42), of the rRNA gene cluster by Hagn et al. (2007). With
RNA polymerase II subunit (rpb2) and calmodulin (cal1) the application of this method, the authors found only
( Kullnig-Gradinger et al., 2002; Druzhinina et al., 2008) about 12 species in arable soil. Later studies demon-
were found to be suitable for giving precise species iden- strated that ITS1 alone is not sufficiently diagnostic as
tification of Hypocrea⁄Trichoderma strains. certain species share the same allele. Based on a master
The ITS-based online barcoding program TrichOKEY alignment of ITS sequences, Meincke et al. (2010) devel-
(www.isth.info; Druzhinina et al., 2005; Druzhinina and oped a novel Trichoderma-specific primer pair for diver-
Kopchinskiy, 2006) provides another useful tool for sity analysis, which amplifies an approximately 650 bp
the identification of Hypocrea⁄Trichoderma species. The fragment of the ITS region suitable for identification
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS 5
by TrichOKEY and TrichoBLAST from all taxonomic T. koningii, T. pseudokoningii, and T. viride. Vajna (1983)
clades of the genus Trichoderma The authors applied a reported the isolation and morphology- as well as cul-
seminested strategy for DNA amplification from soil: ture characteristics-based identification of Trichoderma
the first PCR-a mplification was performed with a fun- aureoviride, T. harzianum, T. koningii, T. longibrachiatum
gal specific forward primer and the Trichoderma-specific and T. viride from dead wood of apple twigs, oak wood
reverse primer, while the Trichoderma-specific forward and cork wood samples collected in Hungary. In the
and reverse primers were used together in the second 1990s, broad studies on Trichoderma taxonomy and bio-
reaction. ITS amplicons were subjected to denaturing diversity were performed by Bissett (1991a,b,c, 1992) in
gradient gel electrophoresis (DGGE) analysis or cloned North America and some European regions. Trichoderma
to pGEM-T Easy vector and sequenced. The designed harzianum, T. polysporum and T. viride were the three taxa
primer system was applied to study Trichoderma commu- reported from the Hubbard Brook Experimental Forest in
nities in the rhizosphere of potatoes. However, several New Hampshire (USA), which were examined for their
species are undetectable by the use of this method as the potential to degrade organochlorine xenobiotics (Smith,
reverse primer of this system is located 30 bp upstream 1995). However—as already mentioned—the results of
of the last genus-specific TrichOKEY hallmark in a still these early studies are hard to evaluate as no molecu-
polymorphic and indel-rich area of ITS2. In a more lar tools were available for species identification and
recent study, Friedl and Druzhinina (2012) designed six the taxonomy of the genus Trichoderma has also changed
reverse primers and demonstrated their high specificity substantially since the publication of these reports. The
and selectivity. Applied along with the forward primer advent of molecular techniques resulted in a new era
ITS5 (White et al., 1990), this set of reverse primers is also in the field of Trichoderma biodiversity studies. Nev-
able to amplify the entire diagnostic region of ITS1 and ertheless, the results of certain recent studies should still
2 of all members of the genus. The strategy is that after be handled with care due to the lack of the application
six separate PCR amplifications from the tested soil of molecular techniques for species identification. For
sample—each containing the same forward and one of instance Vasanthakumari and Shivanna (2011) reported
the reverse primers—the products are combined, puri- the occurrence of Trichoderma asperellum, T. harzianum,
fied and subcloned to pGEM-T Easy vector resulting in T. koningii and T. viride from the rhizosphere and rhizo-
a clone library. The sequences of the individual clones plane of grasses of the subfamily Panicoideae in the Lak-
are determined and analyzed with TrichOKEY 2.0 and kavalli Region of Karnataka, India, however, the isolates
TrichoBLAST. Atanasova et al. (2010) applied this metage- were identified based on morphological and cultural
nomic strategy to study the diversity of the Trichoderma characteristics only.
genus in air samples. Several studies addressed the biodiversity of the
genus Hypocrea and Trichoderma in Asia. Kullnig et al.
(2000) studied 76 Trichoderma strains isolated from
TRICHODERMA DIVERSITY Russia—including Siberia—and the Himalayas by ITS
IN DIFFERENT HABITATS sequence analysis, RAPD and DNA-fingerprinting and
reported the occurrence of T. asperellum, Trichoderma
atroviride, Trichoderma ghanense, T. hamatum, T. harzia-
Natural Soils, Decaying Wood and Plant
num, T. koningii, Trichoderma oblongisporum, Trichoderma
Material
virens as well as some previously undetected taxa, which
In an early study, Danielson and Davey (1973) exam- were later described based on morphological and physi-
ined the Trichoderma propagules in a variety of forest soils ological characters as well as ITS1, 2 and tef1 sequences
in the southeastern U.S. and Washington State and iden- as Trichoderma effusum, Trichoderma rossicum and Tricho-
tified the isolates as Trichoderma hamatum, T. harzianum, derma velutinum (Bissett et al., 2003). The T. harzianum
Trichoderma koningii, Trichoderma polysporum, Trichoderma species complex proved to be the most frequently occur-
pseudokoningii and Trichoderma viride. T. koningii and ring and genetically most diverse taxon with six dif-
T. hamatum were reported as the most widely distributed ferent ITS-genotypes (Kullnig et al., 2000). A follow-up
species aggregates. Trichoderma polysporum and T. viride study on the biodiversity of Trichoderma in Southeast
were found to be largely restricted to cool temperate Asia including Burma, Cambodia, Malaysia, Singapore,
regions, T. harzianum was reported to be characteristic of Taiwan, Thailand and Western Indonesia applied the
warm climates, while T. hamatum and T. pseudokoningii sequence analysis of the ITS region as well as Biolog
were the dominant forms under conditions of excessive phenotype microarrays to examine 96 Trichoderma iso-
moisture. Widden and Abitbol (1980) studied the seasonal lates (Kubicek et al., 2003), and revealed the occurrence
distribution of Trichoderma species in a spruce-forest soil of T. asperellum, T. atroviride, T. ghanense, T. hamatum,
in Canada and reported the occurrence of T. hamatum, T. harzianum, Hypocrea jecorina/Trichoderma reesei, T. kon-
T. harzianum, Trichoderma longibrachiatum, T. polysporum, ingii, Trichoderma spirale, T. virens and T. viride. Based on
A. BIOLOGY AND BIODIVERSITY