BIOTECHNOLOGY
AND BIOLOGY OF
TRICHODERMA
VIJAI K. GUPTA, MONIKA SCHMOLL, ALFREDO HERRERA-ESTRELLA,
R. S. UPADHYAY, IRINA DRUZHININA, MARIA G. TUOHY
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14 15 16 17 18 10 9 8 7 6 5 4 3 2 1
Contents
Biodiversity Studies 45
Identification of Industrial Trichoderma Strains 47
Identification of Biocontrol Trichoderma Strains 48
Identification of Trichoderma Isolates with Clinical Relevance 50
Identification of Mushroom Pathogenic Trichoderma Strains 51
Conclusions 51
Acknowledgments 52
References 52
Preface xi
Foreword xiii
List of Contributors xv
A
BIOLOGY AND BIODIVERSITY
4 Understanding the Diversity and Versatility of
Trichoderma by Next-Generation Sequencing
1 Biodiversity of the Genus Hypocrea/Trichoderma in
Different Habitats
CHRISTIN ZACHOW AND GABRIELE BERG
Introduction 57
Access to Fungal and Trichoderma Diversity—Taxonomic
Profiling 58
Plants Life under Control of Trichoderma—Functional Profiling 62
Conclusion 63
Acknowledgments 63
References 63
LÁSZLÓ KREDICS, LÓRÁNT HATVANI, SHAHRAM NAEIMI,
PÉTER KÖRMÖCZI, LÁSZLÓ MANCZINGER, CSABA VÁGVÖLGYI,
IRINA DRUZHININA
Introduction 3
Methodology of Studying Trichoderma Biodiversity
Trichoderma Diversity in Different Habitats 5
Conclusions 18
Acknowledgments 18
References 18
3
5 Molecular Evolution of Trichoderma Chitinases
VERENA SEIDL-SEIBOTH, KATARINA IHRMARK,
IRINA DRUZHININA, MAGNUS KARLSSON
2 Ecophysiology of Trichoderma in Genomic Perspective
LEA ATANASOVA
Introduction 67
Phylogeny and Evolution of the GH Family 18 Gene Family in
Trichoderma 68
Subgroup A Chitinases 69
Subgroup B Chitinases 71
Subgroup C Chitinases 74
Conclusions 77
Acknowledgments 77
References 77
Trichoderma in Its Ecological Niche 25
From Diversity to Genomics 27
Mycotrophy of Trichoderma 28
Saprotrophy of Trichoderma on Dead Wood 30
Trichoderma Growth in Soil 31
Rhizosphere Competence of Trichoderma 32
Trichoderma versus Mycorrhizae 32
Trichoderma + Bacteria = ? 33
Facultative Endophytism of Trichoderma 33
Animal Nourishment of Trichoderma 34
Most of the Famous Trichoderma Species are Environmental
Opportunists 34
Versatile Carbon Utilization Patterns Reflect Ecological
Specialization of Trichoderma spp. 35
Acknowledgments 37
References 37
B
SECRETION AND PROTEIN
PRODUCTION
6 Protein Production—Quality Control and Secretion
Stress Responses in Trichoderma reesei
3 DNA Barcode for Species Identification in Trichoderma
LÓRÁNT HATVANI, CSABA VÁGVÖLGYI, LÁSZLÓ KREDICS,
IRINA DRUZHININA
M. SALOHEIMO T. PAKULA N. ARO, J.J. JOENSUU
Introduction—Milestones of Trichoderma reesei 81
Protein Secretome of T. reesei 82
ER Quality Control and Secretion Stress Responses 84
Conclusion 86
References 86
Introduction 41
The Tools 42
Application of DNA Barcoding in Species-Level Identification
of Trichoderma 43
Taxonomic Studies 43
v
vi
CONTENTS
7 Heterologous Expression of Proteins in Trichoderma
HELENA NEVALAINEN AND ROBYN PETERSON
Introduction 89
Promoter Options 92
Fusion Partners 93
Extracellular Proteases 94
Secretion Stress in the Frame 95
Mass Production of Heterologous Protein by Fermentation 97
N-glycosylation of Heterologous Proteins Produced in T. reesei 97
Conclusions 98
Acknowledgments 99
References 99
8 Trichoderma Secretome: An Overview
SUNIL S. ADAV AND SIU KWAN SZE
Introduction 103
Proteomic Analysis of Secretory Proteins 105
Extraction of Extracellular Proteins for Proteomic Analysis 106
Extracellular Protein Secretion by T. reesei 107
Polysaccharide Degradation Machinery of T. reesei 108
New Candidates in Cellulose Degradation 109
Hemicellulose Hydrolyzing Enzymes 110
Lignin Degradation by T. reesei 111
Industrial Applications of T. reesei Cellulolytic Enzymes 111
Conclusion 112
References 112
9 The Secretory Pathway in the Filamentous
Fungus Trichoderma
MARCO J. HERNÁNDEZ-CHÁVEZ, ROBERTO J. GONZÁLEZ-HERNÁNDEZ,
JOSÉ E. TRUJILLO-ESQUIVEL, ARTURO HERNÁNDEZ-CERVANTES,
HÉCTOR M. MORA-MONTES
Introduction 115
Translocation 115
Cotranslational Translocation 116
Post Translational Translocation 116
Protein Modifications in the ER 116
Vesicle Transport from ER to Golgi Complex and Trafficking
within the Golgi Cisternae 118
Transport after Trafficking within the Golgi Complex 119
Secreted Proteins in Trichoderma 119
Concluding Remarks 120
Acknowledgments 120
References 120
C
SECONDARY METABOLISM
10 Secondary Metabolism and Antimicrobial
Metabolites of Trichoderma
ROSA HERMOSA, ROSA ELENA CARDOZA, MARÍA BELÉN RUBIO,
SANTIAGO GUTIÉRREZ, ENRIQUE MONTE
Introduction 125
Peptaibols 126
Diketopiperazine-Like Compounds 129
Polyketides 129
Pyrones 130
Terpenes 131
Concluding Remarks and Future Directions 133
Acknowledgments 134
References 134
11 Recent Advancements on the Role and Analysis
of Volatile Compounds (VOCs) from Trichoderma
SHAFIQUZZAMAN SIDDIQUEE
Introduction 139
Detection Techniques of VOCs 140
Types of Volatiles Compounds 142
Application of VOCs in Agriculture 165
Conclusion 168
References 168
D
TOOLS
12 Molecular Tools for Strain Improvement of
Trichoderma spp.
ROBERT BISCHOF AND BERNHARD SEIBOTH
Introduction 179
Genetic Transformation Techniques 180
Auxotrophic and Dominant Selection Markers 181
Marker Recycling Strategies and Marker Free Strains 182
Advanced Methods for Gene Targeting 183
RNA Mediated Gene Silencing 184
Promoters for Recombinant Protein Expression and Targeting 185
Concluding Remarks 188
References 188
13 Genetic Transformation and Engineering of
Trichoderma reesei for Enhanced Enzyme Production
ANLI GENG
Introduction 193
Engineering Cellulase and Hemicellulase Regulation 194
Homologous and Heterologous Gene Expression and Gene
Disruption 195
Protein Engineering 196
Engineering Promoters 197
Conclusion 198
References 198
14 Applications of RNA Interference for Enhanced
Cellulase Production in Trichoderma
SHAOWEN WANG AND GANG LIU
Introduction 201
RNA Interference in Fungus 202
Transcriptional Regulation of Cellulase Gene Expression
203
vii
CONTENTS
Application of Gene Downregulation Strategy for Enhanced
Cellulase Production 204
Combination of RNAi and Overexpression of the Regulating
Genes 208
Conclusions and Prospects 211
References 211
15 RNAi-Mediated Gene Silencing in Trichoderma:
Principles and Applications
XIAOYUN SU, LINA QIN, ZHIYANG DONG
Introduction 215
Molecular Mechanisms 216
Advantages and Disadvantages of Using RNAi-Mediated
Gene Silencing as a Genetic Manipulation Tool in
Filamentous Fungi 218
Strategies of Applying RNAi for Gene Silencing in Trichoderma
and Other Filamentous Fungi 220
Conclusions 223
References 224
E
CELLULASES
16 Cellulase Systems in Trichoderma: An Overview
LUIS H.F. DO VALE, EDIVALDO X.F. FILHO, ROBERT N.G. MILLER,
CARLOS A.O. RICART, MARCELO V. DE SOUSA
Introduction 229
Degradation of Cellulose by Cellulase Systems 230
History of the Trichoderma Cellulase Research 232
Structural and Functional Diversity of Trichoderma Cellulases
Cellulase Systems and Complexes 240
Acknowledgments 241
References 241
19 Beta-Glucosidase from Trichoderma to Improve
the Activity of Cellulase Cocktails
WARAWUT CHULALAKSANANUKUL
Introduction 281
Cellulase Classification 282
Trichoderma reesei Cellulases 282
Trichoderma reesei BGLs 284
BGLs from Aspergillus oryzae 284
Synergism between Cellulases 286
Heterologous Expression of Cellulases 286
Yarrowia Lipolytica Expression Platforms 286
Pichia pastoris Expression Platforms 287
β-Glucosidase from Trichoderma to Improve the Activity of
Cellulase Cocktails 287
Acknowledgments 288
References 288
20 Regulation of Glycoside Hydrolase
Expression in Trichoderma
HODA BAZAFKAN, DORIS TISCH, MONIKA SCHMOLL
232
17 Use of Cellulases from Trichoderma reesei in the
Twenty-First Century—Part I: Current Industrial
Uses and Future Applications in the Production
of Second Ethanol Generation
NICOLAS LOPES FERREIRA, ANTOINE MARGEOT,
SENTA BLANQUET, JEAN-GUY BERRIN
Overview of the Global Enzyme Market 245
Industrial Cellulases 246
Current Applications 249
Perspectives 253
Application of Trichoderma Cellulases in the Bioethanol
Industry 253
References 258
18 Use of Cellulases from Trichoderma reesei in the
Twenty-First Century—Part II: Optimization of
Cellulolytic Cocktails for Saccharification of
Lignocellulosic Feedstocks
JEAN-GUY BERRIN, ISABELLE HERPOEL-GIMBERT,
NICOLAS LOPES FERREIRA, ANTOINE MARGEOT,
SENTA HEISS-BLANQUET
Genetics of Industrial Trichoderma reesei Strains 263
The T. reesei Enzyme Cocktail 264
Hydrolysis of Cellulose 266
Limitations in Lignocellulose Hydrolysis 267
Improvement of Enzyme Cocktails by Optimization of
Enzyme Ratios 269
Improvement by Supplementation of T. reesei Enzyme Cocktails 270
Adapting Cellulose Cocktails to Process Conditions 275
Conclusions and Perspectives 275
References 275
Introduction 291
Regulation by Environmental Parameters
Regulatory Mechanisms 297
Physiological Responses 302
References 303
292
21 Trichoderma Proteins with Disruption Activity on
Cellulosic Substrates
CHRISTIAN DERNTL, ASTRID R. MACH-AIGNER, ROBERT L. MACH
Structure and Occurrence of Cellulose in Nature 309
General Aspects of Cellulose Degradation 310
Cellulose Degradation by T. reesei 311
Cellulolytic Enzymes in Other Trichoderma Species 314
Acknowledgments 314
References 314
22 Molecular Mechanism of Cellulase Production
Systems in Trichoderma
KATOCH MEENU, GURPREET SINGH, R. A. VISHWAKARMA
Introduction 319
Cellulase System of T. reesei 319
Induction Mechanism of Cellulase Production 320
Promoter Involved in Cellulase Production 320
Molecular Mechanism of Cellulase Production 320
viii
CONTENTS
Approaches for Refining the Cellulases Production System
in T. reesei 321
References 322
23 Trichoderma in Bioenergy Research: An Overview
VIJAI K. GUPTA, ANTHONIA O’DONOVAN, MARIA G. TUOHY,
GAURI DUTT SHARMA
Introduction 325
Fungal Enzyme Systems and Trichoderma Technology 326
Industrial Applications of Trichoderma 327
Trichoderma Enzyme Systems in Bioenergy Research 328
Conclusion 332
References 332
F
INDUSTRIAL APPLICATIONS
24 Trichoderma Enzymes for Food Industries
ADINARAYANA KUNAMNENI, FRANCISCO J. PLOU,
ANTONIO BALLESTEROS
Introduction 339
Fungus of Industrial Interest 340
Trichoderma Enzymes for Industries 340
Xylanases 341
Cellulases 341
Other Enzymes 342
Food Industry 342
Perspectives for Biotechnological Production of Enzymes by
Trichoderma 343
References 343
25 Trichoderma: A Dual Function Fungi and Their
Use in the Wine and Beer Industries
CARLOS ROBERTO FELIX, ELIANE FERREIRA NORONHA,
ROBERT N. G. MILLER
Introduction 345
Application in the Wine and Beer Industries
Acknowledgments 348
References 348
347
Introduction 363
Global Metabolism 364
Carbohydrate Metabolism and Glycoside Hydrolases
Energy Metabolism 368
Secondary Metabolism 369
Metabolism and Transporters 372
Acknowledgments 374
References 374
366
28 Sequence Analysis of Industrially Important
Genes from Trichoderma
AHMED M.A. EL-BONDKLY
Introduction 377
Gene Sequence Analysis Fundamentals 378
Genome Analysis of Trichoderma 383
Industrially Genes from Trichoderma 384
Sequence Analysis of Industrially Genes from
Trichoderma 384
Conclusion 389
References 390
29 Biosynthesis of Silver Nano-Particles by
Trichoderma and Its Medical Applications
KHABAT VAHABI AND SEDIGHEH KARIMI DORCHEH
Introduction 393
SNP Biosynthesis 395
Mechanism 397
Medical Application 399
References 400
30 Role of Trichoderma Species in
Bioremediation Process: Biosorption Studies
on Hexavalent Chromium
DHARA SHUKLA AND PADMA S. VANKAR
Introduction 405
Hexavalent Chromium Bioremediation will Be Discussed Here
with a Case Study Representing Chromium Biosorption by
Trichoderma Species 407
Conclusion 411
References 412
26 Trichoderma Enzymes for Textile Industries
TERHI PURANEN, MARIKA ALAPURANEN, JARI VEHMAANPERÄ
Substrate 351
Enzymes 352
Textile Processes 353
Trichoderma Enzymes in Textile Finishing Processes 355
Trichoderma as a Production Host for Textile Enzymes 357
Future Trends 359
Acknowledgments 359
References 359
27 Metabolic Diversity of Trichoderma
ROBERTO NASCIMENTO SILVA, ANDREI STECCA STEINDORFF,
VALDIRENE NEVES MONTEIRO
G
BIOCONTROL AND PLANT
GROWTH PROMOTION
31 Applications of Trichoderma in Plant
Growth Promotion
ALISON STEWART AND ROBERT HILL
Introduction 415
Trichoderma as a Plant Growth Promoter 416
Consistency of Growth Promotion 418
Commercialization 419
ix
CONTENTS
Mechanisms of Growth Promotion 420
Conclusions 425
References 425
32 Molecular Mechanisms of Biocontrol in
Trichoderma spp. and Their Applications in
Agriculture
VIANEY OLMEDO MONFIL AND SERGIO CASAS-FLORES
Introduction 429
Mycoparasitism 430
Morphological Changes 430
Roll of Cell Wall Degrading Enzymes 431
Signal Transduction in Mycoparasitism 432
ROS-Nox-Signal Transduction 433
Antibiosis (Secondary Metabolites Involved in Biocontrol) 435
Pyrones 436
Polyketides 437
Nonribosomal Peptides 437
Mycotoxins Produced by Trichoderma spp. 438
Synergism between Enzymes and Antibiotics 439
Competition for Nutrients 439
Plant Growth Promotion by Trichoderma 440
Plant Root Colonization 442
Induction of Systemic Resistance to Plants by Trichoderma
spp. 443
Signal Transduction Pathways that Mediate Trichoderma-Plant
Communication 444
Trichoderma Elicitor of Systemic Resistance in Plants 446
Signal Transduction during Plant–Trichoderma Interaction in
Trichoderma 448
Transgenic Plants Expressing Trichoderma Genes 448
Concluding Remarks 449
Acknowledgments 449
References 449
33 Genome-Wide Approaches toward Understanding
Mycotrophic Trichoderma Species
ALFREDO HERRERA-ESTRELLA
Introduction 455
Lessons from the Genome Sequence 457
Transcriptome Analyses 458
The Functional Genomics View of Mycoparasitism 458
High-Throughput Analysis of the Trichoderma-Plant Interaction 459
Future Directions 461
Concluding Remarks 462
Acknowledgments 462
References 462
34 Insights into Signaling Pathways of Antagonistic
Trichoderma Species
SUSANNE ZEILINGER AND SABINE GRUBER
Introduction 465
G Protein Signaling 465
Effector Pathways of G Protein Signaling in Fungi 466
Signaling Pathways and Characterized Components in
Trichoderma Species 467
Signal Transduction Components and Pathways Affecting
Vegetative Growth and Conidiation 469
The Role of Signaling in Trichoderma Mycoparasitism and
Biocontrol 471
Conclusions 474
Acknowledgments 474
References 474
35 Enhanced Resistance of Plants to Disease
Using Trichoderma spp.
M.G.B. SALDAJENO, H.A. NAZNIN, M.M. ELSHARKAWY,
M. SHIMIZU, M. HYAKUMACHI
Introduction 477
Induced Disease Resistance in Plants 478
Induced Resistance by Trichoderma spp. 481
Signaling Pathways of Trichoderma-Induced Resistance 482
Trichoderma spp.-Secreted Elicitors of Plant Resistance 483
Engineering Plants for Disease Resistance Using Trichoderma
Genes 485
Combination of Trichoderma with Other Beneficial
Microorganisms 486
Other Effects of Trichoderma spp. Inoculation to the Plant 487
Conclusion 487
References 488
36 Enhanced Plant Immunity Using Trichoderma
HEXON ANGEL CONTRERAS-CORNEJO, LOURDES MACÍAS-RODRÍGUEZ,
JESÚS SALVADOR LÓPEZ-BUCIO, JOSÉ LÓPEZ-BUCIO
Introduction 495
Mechanisms of Plant Protection by Microbes 495
Trichoderma-Induced Immunity 498
Plant Protection Conferred by Trichoderma 500
Conclusions 501
Acknowledgments 501
References 501
37 Genes from Trichoderma as a Source
for Improving Plant Resistance to
Fungal Pathogen
BARBARA REITHNER AND ROBERT L. MACH
Introduction 505
Trichoderma Inducing Resistance in Plants 506
Transgenic Plants Expressing Trichoderma Genes Develop
Increased Resistance to Fungal Pathogens 506
Trichoderma Genes Involved in Elicitation of ISR 508
Conclusion 511
Abbreviations 511
Acknowledgments 511
References 511
38 Trichoderma Species as Abiotic Stress
Relievers in Plants
NAJAM W. ZAIDI, MANZOOR H. DAR,
SUDHANSHU SINGH, U.S. SINGH
Introduction 515
Microbes for the Management of Abiotic Stresses 516
Alleviation of Abiotic Stress in Plants by Trichoderma 516
x
CONTENTS
Alleviation of Drought Stress in Plants by Trichoderma 517
Alleviation of Salinity Stress in Plants by Trichoderma 518
Alleviation of Heat Stress in Plants by Trichoderma 519
Trichoderma Genes for Abiotic Stress Tolerance 520
Mechanism of Abiotic Stress Tolerance Using Trichoderma 520
Host Gene: Stress Tolerant Varieties 521
Conclusion 522
References 523
39 Advances in Formulation of Trichoderma
for Biocontrol
CHRISTIAN JOSEPH R. CUMAGUN
Introduction 527
Types of Formulation 528
Microencapsulation 528
Enhancement of Shelf Life and Application Efficiency 528
Compatibility with Other Biological Systems 529
Conclusion and Future Prospects
References 530
530
40 Trichoderma: A Silent Worker of Plant Rhizosphere
AKANKSHA SINGH, BIRINCHI K. SARMA, HARIKESH B. SINGH,
R. S. UPADHYAY
Introduction 533
Diverseness Amongst Trichoderma 534
Trichoderma as Inducer of Plant Defense Response 536
Trichoderma as a Biofertilizer and Plant Growth Promoter 538
Commercialization 538
Trichoderma Genes Responsible for Playing “Big Games” 539
Conclusion 540
Acknowledgments 540
References 540
Index
543
Preface
A growing world population and the increased
energy consumption caused by a higher standard of
living pose a challenge on current efforts to sustain a
healthy environment and counteract climate change in
the future. Replacing the limited resource of fossil oil
and related products with renewable, carbon dioxideneutral resources requires a considerate strategy, as also
renewable biomass is not an unlimited resource. In order
to achieve a sustainable economy, the delicate balance
between use of biomass/land for food production and
for use in industry and as an energy resource has to be
kept. Species of the genus Trichoderma can play a significant role in the strategy for a sustainable future and this
book summarizes the capabilities these fungi offer.
On the one hand, the metabolic capacities of Trichoderma are of central importance for breakdown of plant
cell walls into small compounds that can be utilized by
yeast not only for bioethanol production, but also as
building blocks for chemical synthesis. With its potent
cellulase system and its versatility for heterologous proteins, which facilitates complementation of this system
with efficient enzymes from other organisms, Trichoderma reesei has become one of the cornerstones for second-generation biofuel production. Several chapters of
this book provide an overview of the enzyme system of
Trichoderma and its optimization for efficient utilization
and conversion of lignocellulosic material. Additionally,
novel and established tools for enhancing cellulase production are discussed. However, besides production of
second-generation biofuels from plant material, industrial use of Trichoderma also extends to production of
silver nanoparticles and applications in beer and wine
industry as well as in textile industry.
Trichoderma also serves as a versatile host for expression of heterologous proteins and a broad array of tools
are available for modification of the genome of this fungus for improvement of its production capacity. Chapters
on heterologous protein production with Trichoderma,
secretion and industrial strain improvement provide an
overview of the use of this fungus as a cell factory in
biotechnology. The enzyme systems of Trichoderma have
even been used for bioremediation, which is a further
important contribution to environmental sustainability.
Other products of potential relevance for industry are
the secondary metabolites produced by Trichoderma spp.
as well as metabolic byproducts with interesting physiological or chemical functions.
While T. reesei serves as a workhorse for industrial
enzyme production, other species of the genus are used
for plant protection in agriculture. Thereby, these fungi
play an important role in establishing this important and
delicate balance between food production and the use
of biomass for energy production and chemical industry.
Efficient and sustainable use of biomass requires protection of energy plants and food crops from pathogens in
order to guarantee that biomass as a limited resource can
fulfill the need of both society and industry. Different
species of Trichoderma act positively on plant growth and
resistance of plants against disease. The chapters of this
book include a thorough summary on mechanisms and
application of biocontrol, the enhancing effect on plant
immunity and mycoparasitism.
Considering the huge potential of Trichoderma for
use in agriculture and industry, exploration of natural
isolates of the genus is warranted to further increase
the genomic resources to be exploited. Screening the
biodiversity of different habitats and the ecophysiology of Trichoderma in a genomic perspective as well as
analysis of this diversity delivers important insights
into the promises the genus Trichoderma holds for the
future.
In summary, this book gives a detailed overview of
the field of industrial and agricultural use as well as
the research with Trichoderma from industrial enzyme
production to strain improvement to biocontrol and
diversity.
Editors
xi
Foreword
Trichoderma exists probably since at least 100 millions
of years, but it entered the scientific spotlight only in the
late seventies of the last century, when the first oil shock
prompted governments to look for alternatives for fossil
fuel. Researchers at the US military laboratories at Natick,
Massachusetts, then remembered to possess a culture of
a green fungus that had been destroying all the cotton
material (tents, belts, clothes) of the US soldiers during
the Second World War in the South Pacific at Guadalcanal (Solomon Islands), and who was subsequently
demonstrated to have exceptional cellulolytic abilities.
This fungus, like any other Trichoderma isolate at that
time, was then named “T. viride” because the genus was
believed to consist only of a single species. It was later
re-identified as “T. reesei” (in honor of one of the researchers exploring its cellulolytic properties, Elwyn T. Reese),
for a few years misnamed as T. longibrachiatum, and
finally found out to be the asexual form of a very common tropical ascomycete, Hypocrea jecorina. The interest in this organism was of outmost importance to the
Trichoderma community in general, because it challenged
researchers to develop a whole toolbox of molecular
genetics techniques for its manipulation, finally culminating in the sequencing of the genome of the original
isolate QM6a and several of its cellulase-producing
mutants, which comprise an invaluable aid to study this
organism.
While Trichoderma is consequently known to many
people only as the organism that makes cellulases, a
parallel world of Trichoderma started to develop in 1932
when R. Weindling published the mycoparasitic abilities
of Trichoderma “lignorum” (an illegitimate name) on plant
pathogenic fungi. This biocontrol ability is due to the
profound ability of Trichoderma to parasitize or even prey
on other fungi, which today is known to be the innate
nature of the whole genus. Weindling’s findings formed
the basis for a multitude of studies on the potential use
of various Trichoderma spp. for the biological control of
plant pathogenic fungi, resulting in the commercialization of some of them. The cellulase and the biocontrol
researchers long formed two isolated communities with
little information exchange, but this improved once the
need for exchange of molecular genetics research techniques became apparent. Most recently the genomes
of several Trichoderma biocontrol species have been
sequenced, and two of them (T. atroviride, T. virens) have
been published.
Yet Trichoderma offers much more to science: its species are among the most frequent mitosporic fungi commonly detected in cultivation-based surveys. They can
be isolated from an innumerable diversity of natural
and artificial substrata, particularly also from materials infested with xenobiotics, demonstrating their high
opportunistic potential and adaptability to various ecological conditions. Consequently, it has broad impacts
on mankind: one of the most stimulating recent findings is that some Trichoderma spp. occur or can occur as
symptomless associates of plant-endophytes, thereby
stimulating plant growth, delaying the onset of drought
stress and preventing attacks of pathogens. Yet, there
are also negative impacts of Trichoderma on mankind:
in a clinical context, a pair of genetically related species
(T. longibrachiatum and T. orientale) have been shown to
occur as opportunistic pathogens of immunocompromised humans, and several Trichoderma spp. can occur as
indoor molds, although their effect on human health is
less severe than that of other fungal species. Finally, some
species like T. aggressivum, T. pleuroticola, T. pleurotum,
and T. mienum have turned their mycoparasitic abilities
against commercial mushrooms like Agaricus and Pleurotus, thereby causing large economic losses.
All these properties make Trichoderma one of the most
versatile and intriguing fungal genus, which still offers
numerous aspects to be dealt with in more detail. This
book has been initiated to describe the current stage of
knowledge on Trichoderma from various perspectives,
thereby outlining also those areas where further progress is needed.
xiii
Christian P. Kubicek
Professor for Biotechnology and Microbiology
Department of Chemical Engineering
Vienna University of Technology
Vienna, Austria
List of Contributors
Sunil S. Adav School of Biological Sciences, Nanyang Technological University, Singapore
Luis H.F. Do Vale Department of Cell Biology, University of
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 Microbiology, Friedrich-Schiller 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
Microbiology, Vienna, Austria
Lea Atanasova Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University
of Technology, Vienna, Austria
Ahmed M.A. El-Bondkly
Giza, Egypt
Antonio Ballesteros Departamento de Biocatálisis, Instituto
de Catálisis y Petroleoquímica, CSIC, Madrid, Spain
National Research Centre, Dokki,
M.M. Elsharkawy Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Japan
Hoda Bazafkan Health and Environment Department,
Austrian Institute of Technology GmbH (AIT), Tulln, Austria
Carlos Roberto Felix Departamento de Biologia Celular,
Universidade de Brasilia, Brasilia, Federal District, Brasil
Gabriele Berg Graz University of Technology, Environmental Biotechnology, Graz, Austria
Nicolas Lopes Ferreira IFP Energies nouvelles, Biotechnology Department, Rueil-Malmaison, France
Jean-Guy Berrin Laboratoire de Biologie des Champignons
Filamenteux, INRA, Polytech Marseille, Aix Marseille Université, 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
Biotechnology (ACIB), Vienna, Austria
Anli Geng School of Life Sciences and Chemical Technology,
Ngee Ann Polytechnic, Clementi, Singapore
Senta Blanquet IFP Energies nouvelles, Biotechnology
Department, Rueil-Malmaison, France
Roberto J. González-Hernández Departamento de Biología,
Universidad de Guanajuato, Guanajuato, México
Rosa Elena Cardoza Area of Microbiology, University School
of Agricultural Engineers, University of León, Ponferrada,
Spain
Sabine Gruber Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of
Technology, Vienna, Austria
Sergio Casas-Flores División de Biología Molecular, IPICyT,
San Luis Potosí, México
Vijai K. Gupta Molecular Glycobiotechnology Group,
Department of Biochemistry, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland
Warawut Chulalaksananukul Biofuels by Biocatalysts
Research Unit, Chulalongkorn University, Bangkok,
Thailand; Department of Botany, Chulalongkorn University,
Bangkok, Thailand
Santiago Gutiérrez Area of Microbiology, University School
of Agricultural Engineers, University of León, Ponferrada,
Spain
Lóránt Hatvani Department of Microbiology, University of
Szeged, Szeged, Hungary
Hexon Angel Contreras-Cornejo Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San
Nicolás de Hidalgo, Morelia, Michoacán, México
Senta Heiss-Blanquet IFP Energies nouvelles, Biotechnology
Department, Rueil-Malmaison, France
Christian Joseph R. Cumagun College of Agriculture, University of the Philippines Los Baños, Los Baños, Laguna,
Philippines
Rosa Hermosa Centro Hispano-Luso de Investigaciones
Agrarias (CIALE), University of Salamanca, Salamanca, Spain
Manzoor H. Dar International Rice Research Institute, IRRI,
New Delhi, India
Arturo Hernández-Cervantes Departamento de Biología,
Universidad de Guanajuato, Guanajuato, México
Marcelo V. de Sousa Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Marco J. Hernández-Chávez Departamento de Biología,
Universidad de Guanajuato, Guanajuato, México
Christian Derntl Research Area Gene Technology, Institute
of Chemical Engineering, Vienna University of Technology,
Vienna, Austria
Isabelle Herpoel-Gimbert Laboratoire de Biologie des
Champignons Filamenteux, INRA, Polytech Marseille, Aix
Marseille Université, Marseille, France
xv
xvi
LIST OF CONTRIBUTORS
Alfredo Herrera-Estrella Laboratorio Nacional de Genómica
para la Biodiversidad, Cinvestav Sede Irapuato, Irapuato,
Guanajuato, Mexico
Robert Hill Bio-Protection Research Centre, Lincoln University, Canterbury, New Zealand
M. Hyakumachi Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Japan
Katarina Ihrmark Uppsala BioCenter, Department of Forest
Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
J.J. Joensuu VTT Technical Research Centre of Finland,
Espoo, Finland
Valdirene Neves Monteiro Universidade Estadual de Goiás,
Unidade Universitária de Ciências Exatas e Tecnológicas da
Universidade Estadual de Goiás-UnUCET, Anápolis, Goiás,
Brazil
Héctor M. Mora-Montes Departamento de Biología, Universidad de Guanajuato, Guanajuato, México
Shahram Naeimi Department of Biological Control Research,
Iranian Research Institute of Plant Protection, Amol,
Mazandaran, Iran
H.A. Naznin Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Japan
Magnus Karlsson Uppsala BioCenter, Department of Forest
Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Helena Nevalainen Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW, Australia;
Biomolecular Frontiers Research Centre, Macquarie University, NSW, Australia
Péter Körmöczi Department of Microbiology, University of
Szeged, Szeged, Hungary
Eliane Ferreira Noronha Departamento de Biologia Celular,
Universidade de Brasilia, Brasilia, Federal District, Brasil
László Kredics Department of Microbiology, University of
Szeged, Szeged, Hungary
Anthonia
O’Donovan Molecular
Glycobiotechnology
Group, Department of Biochemistry, School of Natural Sciences, National University of Ireland Galway, Galway,
Ireland
Adinarayana Kunamneni Departamento de Biocatálisis,
Instituto de Catálisis y Petroleoquímica, CSIC, Madrid,
Spain
Gang Liu College of Life Science, Shenzhen University,
Shenzhen, China
Jesús Salvador López-Bucio Instituto de Biotecnología,
Universidad Nacional Autónoma de México, Cuernavaca,
Morelos, México
José López-Bucio Instituto de Investigaciones QuímicoBiológicas, Universidad Michoacana de San Nicolás de
Hidalgo, Morelia, Michoacán, México
Robert L. Mach Research Area Gene Technology, Institute of
Chemical Engineering, Vienna University of Technology,
Vienna, Austria
Astrid R. Mach-Aigner Research Area Gene Technology,
Institute of Chemical Engineering, Vienna University of
Technology, Vienna, Austria
Lourdes Macías-Rodríguez Instituto de Investigaciones
Químico-Biológicas, Universidad Michoacana de San
Nicolás de Hidalgo, Morelia, Michoacán, México
László Manczinger Department of Microbiology, University
of Szeged, Szeged, Hungary
Antoine Margeot IFP Energies nouvelles, Biotechnology
Department, Rueil-Malmaison, France
Katoch Meenu Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India
T. Pakula VTT Technical Research Centre of Finland, Espoo,
Finland
Robyn Peterson Department of Chemistry and Biomolecular
Sciences, Macquarie University, NSW, Australia; Biomolecular Frontiers Research Centre, Macquarie University, NSW,
Australia
Francisco J. Plou Departamento de Biocatálisis, Instituto de
Catálisis y Petroleoquímica, CSIC, Madrid, Spain
Terhi Puranen Roal Oy, Rajamäki, Finland
Lina Qin Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Barbara Reithner Research Area Gene Technology, Institute
of Chemical Engineering, Vienna University of Technology,
Vienna, Austria
Carlos A.O. Ricart Department of Cell Biology, University of
Brasilia, Brasilia, Federal District, Brazil
María Belén Rubio Centro Hispano-Luso de Investigaciones
Agrarias (CIALE), University of Salamanca, Salamanca,
Spain
M.G.B. Saldajeno Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Japan
M. Saloheimo VTT Technical Research Centre of Finland,
Espoo, Finland
Robert N.G. Miller Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Birinchi K. Sarma Department of Mycology and Plant
Pathology, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Vianey Olmedo Monfil Departamento de Biología, Universidad de Guanajuato, Guanajuato, México
Monika Schmoll Health and Environment Department,
Austrian Institute of Technology GmbH (AIT), Tulln, Austria
Enrique Monte Centro Hispano-Luso de Investigaciones
Agrarias (CIALE), University of Salamanca, Salamanca,
Spain
Bernhard Seiboth Institute of Chemical Engineering, Vienna
University of Technology and Austrian Centre of Industrial
Biotechnology (ACIB), Vienna, Austria
LIST OF CONTRIBUTORS
Verena Seidl-Seiboth Institute of Chemical Engineering,
Vienna University of Technology, Research Area Biotechnology and Microbiology, Vienna, Austria
Gauri Dutt Sharma Bilaspur University, Bilaspur, Chattisgarh, India
M. Shimizu Laboratory of Plant Pathology, Faculty of Applied
Biological Sciences, Gifu University, Gifu City, Japan
xvii
Siu Kwan Sze School of Biological Sciences, Nanyang Technological University, Singapore
Doris Tisch Health and Environment Department, Austrian
Institute of Technology GmbH (AIT), Tulln, Austria
José E. Trujillo-Esquivel Departamento de Biología, Universidad de Guanajuato, Guanajuato, México
Dhara Shukla Facility for Ecological and Analytical Testing,
Indian Institute of Technology, Kanpur, Uttar Pradesh, India
Maria G. Tuohy Molecular Glycobiotechnology Group,
Department of Biochemistry, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland
Shafiquzzaman Siddiquee Biotechnology Research Institute,
University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia
R.S. Upadhyay Department of Botany, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Roberto Nascimento Silva Department of Biochemistry and
Immunology, School of Medicine, University of São Paulo,
Ribeirão Preto, São Paulo, Brazil
Csaba Vágvölgyi Department of Microbiology, University of
Szeged, Szeged, Hungary
Akanksha Singh Department of Botany, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Harikesh B. Singh Department of Mycology and Plant
Pathology, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Khabat Vahabi Institute of General Botany and Plant Physiology, Friedrich-Schiller University Jena, Jena, Germany
Padma S. Vankar Facility for Ecological and Analytical
Testing, Indian Institute of Technology, Kanpur, Uttar
Pradesh, India
Jari Vehmaanperä Roal Oy, Rajamäki, Finland
Gurpreet Singh Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India
R.A. Vishwakarma Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India
Sudhanshu Singh International Rice Research Institute,
IRRI, New Delhi, India
Shaowen Wang College of Life Science, Shenzhen University, Shenzhen, China
U.S. Singh International Rice Research Institute, IRRI, New
Delhi, India
Christin Zachow Austrian Centre of Industrial Biotechnology (ACIB GmbH), Graz, Austria; Graz University of Technology, Environmental Biotechnology, Graz, Austria
Andrei Stecca Steindorff Departamento de Biologia Celular,
Universidade de Brasília, Brasília, Distrito Federal, Brazil
Alison Stewart Bio-Protection Research Centre, Lincoln
University, Canterbury, New Zealand
Xiaoyun Su Institute of Microbiology, Chinese Academy of
Sciences, Beijing, China
Najam W. Zaidi International Rice Research Institute, IRRI,
New Delhi, India
Susanne Zeilinger Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria
S E C T I O N
A
BIOLOGY AND BIODIVERSITY
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,
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: kredics@bio.u-szeged.hu
2Department
O U T L I N E
Introduction
3
Methodology of Studying Trichoderma Biodiversity
Methods for the Identification of Trichoderma Strains
Evolution of the Approach: From the Culture-Based
Method to Metagenomics
3
3
Trichoderma Diversity in Different Habitats
Natural Soils, Decaying Wood and Plant Material
Agricultural Habitats
5
5
9
Living Plants (Endophytes)
Mushroom-Related Substrata
Human Body
Water-Related Environments
Air and Settled Dust
4
Conclusions
INTRODUCTION
18
METHODOLOGY OF STUDYING
TRICHODERMA BIODIVERSITY
Members of the genus Trichoderma are cosmopolitan
and prevalent components of different ecosystems in a
wide range of climatic zones (Kubicek et al. 2008). The
occurrence of Trichoderma species is modulated by several factors including microclimate, the availability of
substrates as well as complex ecological interactions
(Hoyos-Carvajal and Bissett, 2011). Survival in different
geographical habitats can be related to metabolic diversity, high reproductive capacity and competitive capabilities of Trichoderma strains in nature (Cardoso Lopes
et al. 2012). The aim of this chapter is to give an overview
about the studies aimed at the investigation of Trichoderma biodiversity in a wide variety of different ecological habitats.
Biotechnology and Biology of Trichoderma
http://dx.doi.org/10.1016/B978-0-444-59576-8.00001-1
11
13
14
14
17
Methods for the Identification of Trichoderma
Strains
Formerly the species-level identification of Trichoderma/Hypocrea isolates was performed based on exclusively their morphological characteristics (Danielson
and Davey, 1973; Summerbell, 2003; Gams and Bissett,
1998). Different media were used for culturing Trichoderma isolates for the analysis of their morphology and
culture characteristics, e.g. malt extract agar, which is
appropriate for conidium production and the observation of conidiophore branching, or potato dextrose agar,
which proved useful for observing pigment production
3
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
identification of species based on conidiophore structure, morphology as well as the size and morphology of
conidia can be performed with the aid of taxonomic keys
and descriptions available in the literature (Bissett, 1984,
1991a, b, c, 1992; Gams and Bissett, 1998; Chaverri and
Samuels, 2003; Jaklitsch, 2009, 2011; Samuels et al., 2006b,
2012a,b). However, without professional expertise this
may often lead to incorrect diagnoses due to the difficulties of morphology-based species identification, therefore
the results of early studies must be handled with special
care (Kubicek et al., 2008). In order to get around such
problems and give precise species-level diagnoses, the use
of biochemical and molecular methods is recommended.
Among the biochemical methods, the metabolic
profiling technique of Biolog Incorporated (Hayward,
California)—which provides the possibility of quantitative measurements of growth and the assimilation of
different carbon and nitrogen sources—proved to be a
useful tool to aid species identification and provide data
on the ecological roles of species (Kubicek et al., 2003;
Hoyos-Carvajal et al., 2009; Atanasova and Druzhinina,
2010).
A cellulose-acetate electrophoresis-based isoenzyme
analysis—with the involvement of glucose-6-phosphate
dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidases A, B and D,
and phosphoglucomutase enzymes (Hebert and Beaton,
1993)—was applied by Szekeres et al. (2006) and Kredics
et al. (2011a, 2012) for the identification of Trichoderma
strains deriving from clinical samples and winter wheat
fields, respectively.
Neuhof et al. (2007) suggested an alternative biochemical technique for Hypocrea⁄Trichoderma species and
strains, which was developed based on intact-cell mass
spectrometry for the direct detection of hydrophobins in
the mycelia as well as spores of 32 Hypocrea⁄Trichoderma
strains representing 29 species. The hydrophobin
patterns were shown to be characteristic to species and
isolates, and the method is proposed to enable the rapid
and direct detection of class II hydrophobins.
Among the molecular methods, DNA-fingerprinting (Arisan-Atac et al., 1995), the sequence analysis of
the ribosomal internal transcribed spacer (ITS) region
(ITS1–5.8S rDNA–ITS2) and of segments from genes
encoding for the translation elongation factor 1-alpha
(tef1), endochitinase (chi18-5, formerly known as ech42),
RNA polymerase II subunit (rpb2) and calmodulin (cal1)
(Kullnig-Gradinger et al., 2002; Druzhinina et al., 2008)
were found to be suitable for giving precise species identification of Hypocrea⁄Trichoderma strains.
The ITS-based online barcoding program TrichOKEY
(www.isth.info; Druzhinina et al., 2005; Druzhinina and
Kopchinskiy, 2006) provides another useful tool for
the identification of Hypocrea⁄Trichoderma species. The
development and practical applications of ITS barcodes
are presented and discussed in chapter 3: DNA barcode
for species identification in Trichoderma. For the analysis of tef1, ITS and rpb2 sequences the online programme
TrichoBLAST and its updated version, TrichoMARK are
recommended (www.isth.info; Kopchinskiy et al., 2005).
TrichoCHIT (www.isth.info), an online barcoding programme for the screening and identification of excellent
chitinase producer strains of Hypocrea lixii/Trichoderma
harzianum was developed by Nagy et al. (2007).
The use of species-specific primers in polymerase
chain reaction can also lead to quick and precise diagnosis. For example, Chen et al. (1999a, b) developed a PCRbased assay for the fast and specific detection of Th2 and
Th4, the aggressive Trichoderma biotypes causing green
mould disease of Agaricus bisporus, while the method
developed by Kredics et al. (2009) allows the rapid and
specific identification of Trichoderma pleurotum and Trichoderma pleuroticola, the causal agents of green mould in
the world-wide production of oyster mushroom (Pleurotus osteatus) even directly from the growing substrate
without the need of cultivation.
Evolution of the Approach: From the CultureBased Method to Metagenomics
Most of the studies about Trichoderma biodiversity
applied the standard culture-based approach comprising the collection of samples, isolation of Trichoderma
strains on one of the selective media described in the
literature (Elad et al., 1981; Papavizas and Lumsden,
1982; Askew and Laing, 1993; Williams et al., 2003) and
their maintenance in culture, which can be followed by
the application of the above-mentioned species-level
identification methods. The problem of this approach
is that certain Trichoderma species may be easier, while
others harder to isolate, therefore the diversity detected
in the culture-based studies does not necessarily reflect
the actual diversity of the genus in the examined habitat. The application of the metagenomic approach provides a solution to this problem, as it is examining the
habitats in situ, without the isolation and culturing of
the Trichoderma strains. This approach is recently gathering ground in Trichoderma biodiversity studies. In
the first metagenomic attempt, Hypocrea/Trichodermaspecific primers were designed for the ITS1 fragment
of the rRNA gene cluster by Hagn et al. (2007). With
the application of this method, the authors found only
about 12 species in arable soil. Later studies demonstrated that ITS1 alone is not sufficiently diagnostic as
certain species share the same allele. Based on a master
alignment of ITS sequences, Meincke et al. (2010) developed a novel Trichoderma-specific primer pair for diversity analysis, which amplifies an approximately 650 bp
fragment of the ITS region suitable for identification
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
by TrichOKEY and TrichoBLAST from all taxonomic
clades of the genus Trichoderma The authors applied a
seminested strategy for DNA amplification from soil:
the first PCR-amplification was performed with a fungal specific forward primer and the Trichoderma-specific
reverse primer, while the Trichoderma-specific forward
and reverse primers were used together in the second
reaction. ITS amplicons were subjected to denaturing
gradient gel electrophoresis (DGGE) analysis or cloned
to pGEM-T Easy vector and sequenced. The designed
primer system was applied to study Trichoderma communities in the rhizosphere of potatoes. However, several
species are undetectable by the use of this method as the
reverse primer of this system is located 30 bp upstream
of the last genus-specific TrichOKEY hallmark in a still
polymorphic and indel-rich area of ITS2. In a more
recent study, Friedl and Druzhinina (2012) designed six
reverse primers and demonstrated their high specificity
and selectivity. Applied along with the forward primer
ITS5 (White et al., 1990), this set of reverse primers is
able to amplify the entire diagnostic region of ITS1 and
2 of all members of the genus. The strategy is that after
six separate PCR amplifications from the tested soil
sample—each containing the same forward and one of
the reverse primers—the products are combined, purified and subcloned to pGEM-T Easy vector resulting in
a clone library. The sequences of the individual clones
are determined and analyzed with TrichOKEY 2.0 and
TrichoBLAST. Atanasova et al. (2010) applied this metagenomic strategy to study the diversity of the Trichoderma
genus in air samples.
TRICHODERMA DIVERSITY
IN DIFFERENT HABITATS
Natural Soils, Decaying Wood and Plant
Material
In an early study, Danielson and Davey (1973) examined the Trichoderma propagules in a variety of forest soils
in the southeastern U.S. and Washington State and identified the isolates as Trichoderma hamatum, T. harzianum,
Trichoderma koningii, Trichoderma polysporum, Trichoderma
pseudokoningii and Trichoderma viride. T. koningii and
T. hamatum were reported as the most widely distributed
species aggregates. Trichoderma polysporum and T. viride
were found to be largely restricted to cool temperate
regions, T. harzianum was reported to be characteristic of
warm climates, while T. hamatum and T. pseudokoningii
were the dominant forms under conditions of excessive
moisture. Widden and Abitbol (1980) studied the seasonal
distribution of Trichoderma species in a spruce-forest soil
in Canada and reported the occurrence of T. hamatum,
T. harzianum, Trichoderma longibrachiatum, T. polysporum,
5
T. koningii, T. pseudokoningii, and T. viride. Vajna (1983)
reported the isolation and morphology- as well as culture characteristics-based identification of Trichoderma
aureoviride, T. harzianum, T. koningii, T. longibrachiatum
and T. viride from dead wood of apple twigs, oak wood
and cork wood samples collected in Hungary. In the
1990s, broad studies on Trichoderma taxonomy and biodiversity were performed by Bissett (1991a,b,c, 1992) in
North America and some European regions. Trichoderma
harzianum, T. polysporum and T. viride were the three taxa
reported from the Hubbard Brook Experimental Forest in
New Hampshire (USA), which were examined for their
potential to degrade organochlorine xenobiotics (Smith,
1995). However—as already mentioned—the results of
these early studies are hard to evaluate as no molecular tools were available for species identification and
the taxonomy of the genus Trichoderma has also changed
substantially since the publication of these reports. The
advent of molecular techniques resulted in a new era
also in the field of Trichoderma biodiversity studies. Nevertheless, the results of certain recent studies should still
be handled with care due to the lack of the application
of molecular techniques for species identification. For
instance Vasanthakumari and Shivanna (2011) reported
the occurrence of Trichoderma asperellum, T. harzianum,
T. koningii and T. viride from the rhizosphere and rhizoplane of grasses of the subfamily Panicoideae in the Lakkavalli Region of Karnataka, India, however, the isolates
were identified based on morphological and cultural
characteristics only.
Several studies addressed the biodiversity of the
genus Hypocrea and Trichoderma in Asia. Kullnig et al.
(2000) studied 76 Trichoderma strains isolated from
Russia—including Siberia—and the Himalayas by ITS
sequence analysis, RAPD and DNA-fingerprinting and
reported the occurrence of T. asperellum, Trichoderma
atroviride, Trichoderma ghanense, T. hamatum, T. harzianum, T. koningii, Trichoderma oblongisporum, Trichoderma
virens as well as some previously undetected taxa, which
were later described based on morphological and physiological characters as well as ITS1, 2 and tef1 sequences
as Trichoderma effusum, Trichoderma rossicum and Trichoderma velutinum (Bissett et al., 2003). The T. harzianum
species complex proved to be the most frequently occurring and genetically most diverse taxon with six different ITS-genotypes (Kullnig et al., 2000). A follow-up
study on the biodiversity of Trichoderma in Southeast
Asia including Burma, Cambodia, Malaysia, Singapore,
Taiwan, Thailand and Western Indonesia applied the
sequence analysis of the ITS region as well as Biolog
phenotype microarrays to examine 96 Trichoderma isolates (Kubicek et al., 2003), and revealed the occurrence
of T. asperellum, T. atroviride, T. ghanense, T. hamatum,
T. harzianum, Hypocrea jecorina/Trichoderma reesei, T. koningii, Trichoderma spirale, T. virens and T. viride. Based on
A. BIOLOGY AND BIODIVERSITY
6
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
the results of this study, the T. harzianum complex contains species with high metabolic diversity and partially
unique metabolic characteristics, which may explain its
wide distribution over different habitats. The three new
species previously detected by Kullnig et al. (2000) could
also be isolated, along with additional four previously
undetected taxa, which were subsequently described by
Bissett et al. (2003) as Trichoderma cerinum, Trichoderma
erinaceum, Trichoderma helicum and Trichoderma sinense.
Zhang et al. (2005) examined the Trichoderma biodiversity and biogeography on 135 isolates deriving from
four regions of China: provinces Hebei (North), Zhejiang (South-East), Yunnan (West) and the Himalayan
part (Tibet) and identified T. asperellum, T. atroviride, T.
cerinum, Trichoderma citrinoviride, T. harzianum, T. koningii, T. longibrachiatum, T. sinense, T. velutinum, T. virens, T.
viride, as well as two putative new species. The results
of the study provided evidence for a North to South
distribution of Trichoderma species in East Asia and
identified Northern China as a potential center of origin of a unique haplotype of T. harzianum. In a recent
study, Sun et al. (2012) identified 12 taxa (T. asperellum,
T. atroviride, Trichoderma brevicompactum, T. citrinoviride,
T. erinaceum, T. hamatum, Trichoderma koningiopsis, H.
lixii/T. harzianum, T. reesei/H. jecorina, T. spirale, Trichoderma stromaticum, Trichoderma vermipilum and Hypocrea
virens/T. virens) from Chinese forest soils by ITS barcoding. Tsurumi et al. (2010) explored the distribution of
Trichoderma species in four countries of Asia: Indonesia,
Japan, Mongolia and Vietnam through the examination
of 332 isolates. Trichoderma crassum, T. hamatum, T. harzianum and T. virens occurred in most habitats. Trichoderma
atroviride, T. koningiopsis and Trichoderma stramineum
were also frequent but not in cooler regions, where the
occurrence of T. polysporum and Trichoderma viridescens
was reported. Trichoderma brevicompactum, T. erinaceum
and T. ghanense were prevalent in tropical areas. In
addition to these species, potentially new taxa were
also detected. Abd-Elsalam et al. (2010) isolated Trichoderma strains from soil collected from protected areas
(Rawdet Khuraim) in Saudi Arabia. Identification of the
isolates by M13-microsatellite PCR and ITS barcoding
revealed the presence of T. harzianum/H. lixii and the
T. longibrachiatum/Hypocrea orientalis species duplet,
suggesting them as pan-global taxa of Trichoderma/
Hypocrea (Abd-Elsalam et al., 2010). Further species
known from Asia include Hypocrea catoptron/Trichoderma catoptron, Hypocrea cornea/T. sp., Hypocrea crassa/T.
crassum, Hypocrea rugulosa, T. spirale, Hypocrea tawa/
Trichoderma tawa, Hypocrea thailandica/Trichoderma thailandicum and Hypocrea thelephoricola/Trichoderma thelephoricola (Chaverri and Samuels, 2003), Trichoderma
capillare, Trichoderma gracile, Trichoderma parareesei and
Trichoderma pinnatum (Samuels et al., 2012a), Trichoderma
barbatum, Trichoderma caesareum/H. sp. and Trichoderma
floccosum/H. sp. (Samuels et al., 2012b), as well as Trichoderma arundinaceum (Degenkolb et al., 2008).
Thanks to a series of biodiversity studies, plenty of
information is available about the biodiversity of the
genus in Europe. Wuczkowski et al. (2003) studied the
diversity of the genus Trichoderma in an original European river-floodplain habitat, the Danube national
park, which is a primeval, riparian forest area located
south-east from Vienna, Austria. Besides morphological
examinations, sequence analysis of the ITS region and a
fragment of the tef1 gene as well as RAPD analysis were
applied for the identification of the isolated Trichoderma
strains. In the order of abundance, the species identified
were T. harzianum, T. rossicum, T. cerinum, T. hamatum,
T. atroviride, T. koningii (recognized now by TrichOKEY
as T. koningiopsis) and Trichoderma sp. MA3642 from
section Longibrachiatum, which was recently described
by Samuels et al. (2012a) as T. capillare. Mysterud et al.
(2007) examined the plant litter-associated fungi from
the spring “grazing corridor” of a sheep herd in western Norway and detected a wide variety of fungi including two Trichoderma isolates that the authors failed to
identify by NCBI BLAST search of their ITS sequences.
A search with TrichOKEY (Druzhinina et al., 2005)
reveals that one of these isolates is T. hamatum while
the other one belongs to the T. koningiopsis/Trichoderma
ovalisporum/T. asperellum species triplet. Jaklitsch (2009,
2011) performed a wide-scale survey over 14 European
countries with temperate climate to study the biodiversity of the Hypocrea/Trichoderma genus based on 620
specimens by examining their morphology, culture characteristics as well as ITS, rpb2 and tef1 sequences. Far
exceeding the previous estimations about the number of
Hypocrea/Trichoderma species in Europe, a total of 75 species were detected including previously described species
(the holomorphs Hypocrea atroviridis/T. atroviride, Hypocrea
aureoviridis/T. aureoviride, Hypocrea citrina/Trichoderma
lacteum, Hypocrea epimyces/Trichoderma epimyces, Hypocrea gelatinosa/Trichoderma gelatinosum, H. lixii/T. harzianum, Hypocrea lutea/Trichoderma deliquescens, Hypocrea
koningii/T. koningii, Hypocrea minutispora/Trichoderma minutisporum, Hypocrea neorufa/T. sp., Hypocrea
ochroleuca/T. sp., Hypocrea pachybasioides/T. polysporum,
Hypocrea pilulifera/Trichoderma piluliferum, Hypocrea
protopulvinata/T. sp., Hypocrea pulvinata/T. sp., Hypocrea rufa/T.viride, Hypocrea schweinitzii/T. citrinoviride,
Hypocrea sulphurea/T. sp. as well as Hypocrea species
without known anamorphs: Hypocrea argillacea, Hypocrea spinulosa, Hypocrea splendens, Hypocrea strobilina),
new taxa (the holomorphs Hypocrea aeruginea/Trichoderma aerugineum, Hypocrea albolutescens/Trichoderma
albolutescens, Hypocrea atlantica/Trichoderma atlanticum, Hypocrea auranteffusa/Trichoderma auranteffusum,
Hypocrea austriaca/Trichoderma austriacum, Hypocrea
bavarica/Trichoderma bavaricum, Hypocrea calamagrostidis/
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
Trichoderma calamagrostidis, Hypocrea fomiticola/Trichoderma fomiticola, Hypocrea junci/Trichoderma junci, Hypocrea
luteffusa/Trichoderma luteffusum, Hypocrea luteocrystallina/
Trichoderma luteocrystallinum, Hypocrea margaretensis/
Trichoderma margaretense, Hypocrea neorufoides/Trichoderma
neorufoides, Hypocrea pachypallida/Trichoderma pachypallidum, Hypocrea parepimyces/Trichoderma parepimyces,
Hypocrea parestonica/Trichoderma parestonicum, Hypocrea phellinicola/Trichoderma phellinicola, Hypocrea silvaevirgineae/Trichoderma silvae-virgineae, Hypocrea subeffusa/
Trichoderma subeffusum and Hypocrea valdunensis/Trichoderma valdunense; the new teleomorphs Hypocrea danica,
Hypocrea rhododendri and Hypocrea sambuci; Hypocrea longipilosa described as the teleomorph state of Trichoderma
longipile; as well as Trichoderma alutaceum, Trichoderma
dacrymycellum, Trichoderma delicatulum, Trichoderma leucopus, Trichoderma moravicum, Trichoderma placentula,
Trichoderma psychrophilum, Trichoderma subalpinum and
Trichoderma tremelloides, which are the anamorphs of
previously described sexually reproducing species).
Among the new Hypocrea species described during the
past decade, Hypocrea estonica/T. sp., Hypocrea phyllostachidis, Hypocrea sinuosa/Trichoderma sinuosum, Hypocrea strictipilosa/Trichoderma strictipile, H. thelephoricola
(Chaverry and Samuels, 2003), Hypocrea stilbohypoxyli
(Lu and Samuels, 2003), Hypocrea parapilulifera (Lu et al.,
2004), Hypocrea nybergiana (Chamberlain et al., 2004),
Hypocrea voglmayrii (Jaklitsch et al., 2005), Hypocrea
alcalifuscescens/T. sp. and Hypocrea parmastoi/T. sp. (Overton et al., 2006), Hypocrea petersenii and Hypocrea rogersonii (Samuels et al., 2006b), Hypocrea crystalligena (Jaklitsch
et al., 2006a), Hypocrea viridescens (Jaklitsch et al., 2006b),
Hypocrea alni and Hypocrea brunneoviridis (Jaklitsch et al.,
2008a), Hypocrea decipiens (Jaklitsch et al., 2008b), Hypocrea seppoi (Jaklitsch et al., 2008c) and Hypocrea rodmanii
(Degenkolb et al., 2008) also occur in Europe. From the
Longibrachiatum clade, Trichoderma saturnisporum is also
known from Sardinia (Samuels et al., 2012a). Results of
these studies demonstrated that not just a small number of Trichoderma species are capable of forming a teleomorph and suggest that the biodiversity of the genus
is higher on and above the litter layer than inside the
soil. Although specific associations with host fungi or
trees were found, the majority of the species were suggested to be necrotrophic on diverse fungi on wood and
bark. Since the publication of the papers of Jaklitsch
(2009, 2011), the above list of European species was
supplemented with additional, recently described taxa
Hypocrea britdaniae, Hypocrea foliicola (Jaklitsch and Voglmayr, 2012), Hypocrea caerulescens, Hypocrea hispanica and
Trichoderma samuelsii (Jaklitsch et al. 2012). During an ITS
barcoding-based study on the species diversity of Trichoderma in Poland, Błaszczyk et al. (2011) found that soil
and decaying wood were the most diverse among the
examined substrata. Species detected in forest soils were
7
T. atroviride, Trichoderma gamsii, T. hamatum, T. harzianum,
Trichoderma tomentosum, T. viride and T. viridescens. Except
for T. tomentosum, decaying wood samples also harbored
these species as well as T. citrinoviride, T. koningii and T.
koningiopsis. Following T. harzianum, the most abundant
Trichoderma species collected from forest soil and forest
wood were T. atroviride and T. viridescens, respectively.
A taxon-specific metagenomic approach was applied
by Friedl and Druzhinina (2012) for the assessment of
the Trichoderma diversity in situ in soil samples of aspen
and beech forests along the Danube floodplain. Identified taxa comprised H. alni, T. asperellum, H. atroviridis/T.
atroviride, T. brevicompactum, T. cerinum, T. harzianum
sensu stricto, H. pachybasioides, H. pachypallida, T. pleuroticola, Hypocrea pseudoharzianum, the species duplet H.
orientalis/T. longibrachiatum, T. rossicum, H. schweinitzii,
Trichoderma sp. C.P.K. 2974 and H. virens/T. virens, with
the highest abundance of T. asperellum in both habitats.
Two presumably new taxa, Trichoderma sp. MOTU 2B
48 from section Trichoderma and Trichoderma sp. MOTU
1A 64 from section Longibrachiatum were also detected
in aspen and beech forests, respectively. The species distribution proved to be uneven in the vertical profiles of
the examined soils. The authors concluded that only a
relatively small number of Hypocrea/Trichoderma species
adapted to soil as a habitat.
Members of the genus Hypocrea/Trichoderma occurring
in natural habitats of the North-American subcontinent
include Hypocrea ceramica/T. sp., T. crassum, Hypocrea
cremea/T. sp., Hypocrea cuneispora/T. sp., Trichoderma
fertile, T. hamatum, T. longipile, T. oblongisporum, Trichoderma pubescens, H. strictipilosa/T. strictipile, Trichoderma
strigosum, Hypocrea surrotunda/Trichoderma surrotundum
and T. tomentosum with conidiophore elongations and
green conidia (Chaverri et al., 2003), the green ascospored species Hypocrea ceracea/Trichoderma ceraceum,
H. ceramica/Trichoderma ceramicum, Hypocrea chlorospora/
Trichoderma chlorosporum, Hypocrea chromosperma/Trichoderma chromospermum, Hypocrea cinnamomea/Trichoderma
cinnamomeum, H. crassa/T. crassum, H. cremea/Trichoderma cremeum, H. cuneispora/Trichoderma cuneisporum, H.
lixii/T. harzianum, H. sinuosa/T. sinuosum, H. strictipilosa/T.
strictipile and H. virens/T. virens (Chaverri and Samuels,
2003), T. ghanense, T. longibrachiatum, T. parareesei,
Hypocrea pseudokoningii/T. pseudokoningii, Trichoderma
saturnisporopsis, T. saturnisporum and H. schweinitzii/
T. citrinoviride from the Longibrachiatum clade (Samuels
et al., 2012a), T. arundinaceum, T. brevicompactum and
H. rodmanii from the T. brevicompactum clade (Degenkolb
et al., 2008), H. alcalifuscescens/T. sp., Hypocrea farinosa/
T. sp. and H. sulphurea/T. sp. (Overton et al., 2006), T. barbatum (Samuels et al., 2012b) as well as T. capillare (Samuels et al., 2012a).
Central American species occurring in Costa
Rica comprise Hypocrea candida/Trichoderma candidum,
A. BIOLOGY AND BIODIVERSITY
8
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
H. chlorospora/T. chlorosporum, Hypocrea costaricensis/T.
sp., Hypocrea nigrovirens/Trichoderma nigrovirens, Hypocrea
substipitata, Hypocrea tuberosa, Hypocrea virescentiflava/T.
sp. (Chaverri and Samuels, 2003) and T. spirale (Chaverri
et al., 2003), T. brevicompactum and Trichoderma turrialbense (Degenkolb et al., 2008), Hypocrea flaviconidia
(Druzhinina et al., 2004) as well as Hypocrea eucorticioides/
T. sp. (Overton et al., 2006).
In South America, a polyphasic method based on the
analysis of ITS1, 2 and tef1 sequences as well as Biolog
metabolic profiling was used by Hoyos-Carvajal et al.
(2009) to study the biodiversity of Trichoderma species
in habitats of neotropic regions in Peru, Mexico, Guatemala and Colombia including rainforest soils, river
sand, humus and wood. A total of 182 isolates were
identified from 18 species (T. asperellum, T. atroviride,
T. brevicompactum, T. crassum, T. erinaceum, T. gamsii, T.
hamatum, T. harzianum, H. jecorina/T. reesei, T. koningiopsis,
T. longibrachiatum, T. ovalisporum, T. pubescens, T. rossicum,
T. spirale, T. tomentosum, T. virens and T. viridescens) and
11 undescribed species were also discovered. The predominant species were T. asperellum and T. harzianum. In
a subsequent paper, Hoyos-Carvajal and Bissett (2011)
reviewed the biodiversity of the genus Trichoderma in
tropical American regions. The occurrence of T. asperellum, Trichoderma asperelloides, T. atroviride, T. brevicompactum, Trichoderma caribbaeum, Trichoderma caribbaeum
var. aequatoriale, T. crassum, T. erinaceum, Trichoderma
evansii, T. gamsii, T. hamatum, T. harzianum, H. jecorina/
T. reesei, T. koningiopsis, Trichoderma lieckfeldtiae, T. longibrachiatum, Trichoderma neokoningii, T. ovalisporum, T. parareesei, Trichoderma paucisporum, T. pleurotum, T. pubescens,
T. rossicum, Trichoderma scalesiae, T. spirale, Trichoderma
stilbohypoxyli, Trichoderma theobromicola, T. tomentosum,
T. virens and T. viridescens was reported, providing a
wide repertoire for the selection of biocontrol agents of
crop diseases. Rivas and Pavone (2010) examined Venezuelan soils and found the T. harzianum species complex to be the most frequently occurring taxon, followed
by T. virens, T. brevicompactum, T. theobromicola, T. koningiopsis, T. ovalisporum, T. asperellum, T. pleurotum and
T. koningiopsis. Other species known from South America
include T. strigosum and Hypocrea stromatica/Trichoderma
stromaticum (Chaverri et al., 2003), Hypocrea gyrosa and
H. virescentiflava/T. sp. (Chaverri and Samuels, 2003)
from Brazil, Hypocrea clusiae/T. sp. from French Guyana
(Chaverri and Samuels, 2003), as well as Hypocrea andinensis (Samuels et al., 2012a) and H. eucorticioides/T. sp.
(Overton et al., 2006) from Venezuela.
Less information is available about the biodiversity of
the genus in natural habitats of Africa. The distribution
of Trichoderma species in soils of Embu and Taita regions
in Kenya with relation to land use practices was investigated by Okoth et al. (2009). Species isolated from indigenous forests of the Embu region in order of prevalence
were T. harzianum, T. viride, Trichoderma aggressivum and
T. atroviride, while in the case of indigenous forests of
the Taita region the detected species were T. harzianum,
T. atroviride, T. koningii, T. aggressivum, T. viride and T.
asperellum. The identities of the isolates were determined
based on a morphological key, but unfortunately the
identifications were not confirmed by molecular techniques, therefore results like the reported occurrence of
the mushroom pathogenic species T. aggressivum in a natural habitat need to be handled critically. Sadfi-Zouaoui
et al. (2009) studied four different bioclimatic zones in
Tunisia for Trichoderma diversity. The T. harzianum species complex proved to be the most prevalent taxon
identified. Trichoderma harzianum and T. longibrachiatum proved to be predominant in North-Tunisian forest
soils. Trichoderma harzianum, T. saturnisporum and a yet
unidentified Trichoderma species were detected in forest
soils from central Tunisia while T. hamatum and T. harzianum could be isolated from oasis soils in the Southern
part of the country. Further species reported from Africa
include Trichoderma aethiopicum, Trichoderma flagellatum
and T. parareesei from Ethiopia, Trichoderma konilangbra
from Uganda, H. orientalis from Zambia (Samuels et al.,
2012a), H. catoptron/T. catoptron (Chaverri and Samuels,
2003), Hypocrea subcitrina (Overton et al., 2006) and T.
vermipilum (Samuels et al., 2012b) from South Africa,
T. arundinaceum from Namibia (Degenkolb et al., 2008),
Hypocrea subsulphurea (Overton et al., 2006), as well as
Trichoderma lanuginosum/H. sp., Trichoderma medusae/H.
sp. from Cameroon and Trichoderma ivoriense from Ivory
Coast (Samuels et al., 2012b).
Studying the biodiversity of the genus in islands
as geographically separated regions may reveal
important data about endemic taxa as well as invasive ones arriving from the nearby continents. Species reported from New Zealand include T. crassum,
Hypocrea semiorbis/Trichoderma sp. and H. tawa/T. tawa
(Chaverri et al., 2003), Hypocrea atrogelatinosa/T. sp., H.
cremea/T. cremeum and Hypocrea macrospora (Chaverri
and Samuels, 2003), Hypocrea novae-zelandiae/T. sp.
and H. pseudokoningii/T. pseudokoningii (Samuels
et al., 2012a), as well as H. subcitrina (Overton et al.,
2006). Members of the genus occurring in Japan
comprise Hypocrea aureoviridis f. macrospora and H.
ceramica (Chaverri and Samuels, 2003), Hypocrea
albocornea/T. sp., Hypocrea centristerilis/T. sp. and H.
strictipilosa/T. strictipile (Chaverri and Samuels, 2003),
H. farinosa/T. sp. and H. subsulphurea/T. sp. (Overton
et al., 2006), as well as H. sulphurea/T. sp. and Hypocrea victoriensis/T. sp. (Overton et al., 2006). The
species Hypocrea melanomagna/Trichoderma melanomagnum and Hypocrea sulawesensis/T. sp. are known
from Australia and Indonesia, respectively (Chaverri
and Samuels, 2003). Hypocrea catoptron/T. catoptron,
H. cornea/T. sp., H. rugulosa and Hypocrea straminea/
T. stramineum (Chaverri and Samuels, 2003), as well
as T. parareesei, T. pinnatum/H. sp. and H. pseudokoningii/
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
T. pseudokoningii are known from Sri Lanka (Samuels
et al., 2012a). The Trichoderma communities of the island of
Sardinia were studied by Migheli et al. (2009). Fifteen
soil samples from different habitats including undisturbed or extensively grazed grass steppes, forests and
shrub lands were examined and the widely distributed
species T. asperellum, H. atroviridis/T. atroviride, T. gamsii,
T. hamatum, H. koningii/T. koningii, Hypocrea koningiopsis/
T. koningiopsis, H. lixii/T. harzianum, H. semiorbis, T. spirale, T. tomentosum, H. virens/T. virens, H. viridescens/
T. viridescens and T. velutinum could be identified by
ITS barcoding. Only a single, potentially endemic
ITS1 allele could be detected in the case of T. hamatum, suggesting a significant reduction in the diversity
of native species from the genus in Sardinia and an
invasion of nonendemic species from Eurasia, Africa
and the Pacific Basin. From the Longibrachiatum clade,
T. saturnisporopsis is also known from Sardinia
(Samuels et al., 2012a). Zachow et al. (2009) examined
the fungal biodiversity of soils at different vegetation
regions on Tenerife (Canary Islands). From the genus
Trichoderma/Hypocrea, the species isolated and identified by TrichOKEY were Trichoderma chionea, T. gamsii,
T. harzianum, H. rufa/T. viride, T. spirale and T. tomentosum, with a clear dominance of T. harzianum. The
majority of the isolates could be characterized with
excellent mycoparasitic activities against the fungal
plant pathogens Botrytis cinerea, Guignardia bidwellii,
Rhizoctonia solani, Sclerotium rolfsii and Verticillium
dahliae, suggesting the colonization of the island
Tenerife by highly competitive Trichoderma species
from the continent.
Agricultural Habitats
Several biotic and abiotic factors affect populations
and diversity of microbial communities in agricultural
ecosystems including plant species and their growth
stage, total microbial competition, soil physical and
chemical properties, application of pesticides or fertilizers as well as the geographical region.
Trichoderma spp. can be theoretically isolated from
almost all types of agricultural fields. They have several positive impacts on cultivated plants including
biological control of plant diseases, inducing systemic
resistance, increasing nutrient availability and uptake,
promotion of plant growth, improving crop yields and
degrading xenobiotic pesticides (Harman, 2006). For the
reasons mentioned above, these fungi have been widely
studied and commercially marketed as biofungicides,
biofertilizers and soil amendments (Vinale et al. 2008).
The rhizosphere is among the common ecological
niches for Trichoderma spp., which attracts them by the
presence of different soil borne fungi as their prey and
by rich plant root- derived nutrients (Druzhinina et al.
2011). Members of the genus were more frequently
9
isolated from rhizosphere and non-rhizosphere soils
than from phyllospheres. Numerous Trichoderma species
have been collected from different crop fields in diverse
climatic zones of all continents. Members of the genus
Trichoderma are among the most frequently isolated soil
fungi. However, some species are ubiquitous while others are limited to specific geographical areas (Harman
et al. 2004).
The majority of the research which involved the isolation and identification of Trichoderma strains from
various agricultural and horticultural crop fields in
different agro-climatic zones was undertaken in order
to evaluate them for biological control potential against
various phytopathogens. Therefore, only a limited number of studies deal with population, abundance and
diversity of the genus Trichoderma in specific crop fields
or agroecosystems.
Cereal crop fields—Trichoderma spp. proved to be
among the dominating fungi in cereal (rye, triticale,
wheat) field soils in Poland (Pięta et al. 2000), and
reported to be the most prevalent taxa among the fungal
communities in winter wheat (Triticum aestivum L.) soils
of Germany, where the most frequently isolated species
were T. atroviride and T. viride (Hagn et al. 2003). Trichoderma piluliferum was also isolated but surprisingly, the
cosmopolitan species T. harzianum has not been obtained
in this study. Season, soil type and farming management
practice influenced only the distribution of T. viride isolates. Diversity of Trichoderma spp. was very high in soil
samples of wheat fields of China (Liang et al. 2004). In
another study, 11 Trichoderma species were identified
by ITS-barcoding from rhizosphere soils of five winter
wheat fields in the Pannonian Plain (Hungary) comprising T. atroviride, T. brevicompactum, T. gamsii, T. harzianum, T. koningiopsis/T. ovalisporum, the species duplet
T. longibrachiatum/H. orientalis, T. pleuroticola, T. rossicum,
T. spirale, T. tomentosum/T. cerinum and T. virens (Kredics
et al. 2012). Trichoderma harzianum was the most abundant species representing various ITS haplotypes including two yet unknown ones. Other frequent species were
T. virens, T. rossicum and T. atroviride, each of which
grouped into two ITS-genotypes. Agricultural fields differed in species composition as well as the abundance
of individual Trichoderma species. Trichoderma spp. in
rhizospheres of winter wheat in the Pannonian Plain
were found to be common and very diverse. In contrast,
Trichoderma biodiversity in agricultural soils (cultivated
with various crops including wheat) of the Nile valley
in Egypt was very low and contained only T. harzianum and the anamorph of H. orientalis (Gherbawy et al.
2004). This low degree of diversity may occur due to the
alkalinity of the investigated soils (pH = 7.3–8.4). Trichoderma harzianum isolates were genetically more diverse
and displayed three different ITS haplotypes and three
RAPD genotypes. Furthermore, enzymatic activities and
RAPD fingerprints of the isolates did not correlate with
A. BIOLOGY AND BIODIVERSITY
10
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
the habitat. Corn field soils in Egypt (Gherbawy et al.
2004) and Mexico (Sánchez-Perez, 2009) revealed only
two (T. harzianum and the anamorph of H. orientalis) and
three (T. harzianum, T. koningiopsis and T. virens) species,
respectively, while nine Trichoderma species (T. asperellum, T. atroviride, T. erinaceum, T. harzianum, T. koningiopsis, T. pleurotum, T. reesei, T. spirale and T. virens) were
identified from the soils of the same crop in Venezuela
(Pavone and Domenico, 2012). Trichoderma harzianum
was the most prevalent species in all three cases. Based
on UP-PCR and rDNA-ITS1 analysis, 42 Trichoderma isolates obtained from rice (Oryza sativa L.) field soils in four
provinces of the Philippines belonged only to T. viride and
T. harzianum, the latter comprised the majority of isolates
(93%) (Cumagun et al. 2000). Similarly, T. harzianum was
the most common species in rice field soils in Bangladesh (Mostafa Kamal and Shahjahan, 1995), as well as in
upland and lowland rice fields of the Philippines (Nagamani and Mew, 1987) however, molecular identifications
were not carried out in these studies. Two hundred and
two Trichoderma isolates were collected from soil and
phyllosphere of rice in paddy fields located at different
geographical areas at the southern coast of the Caspian
Sea, Iran, which belonged to six species: T. asperellum,
T. atroviride, T. brevicompactum T. hamatum, T. harzianum
and T. virens according to the results of ITS barcodebased identification (Naeimi et al. 2010). Like the rhizosphere of winter wheat in Hungary (Kredics et al. 2011a,
2012), rice paddy field habitats in Northern Iran are rich
sources of potential biocontrol isolates belonging to T.
atroviride, T. harzianum and T. virens, taxa that are intensively studied and applied in biological control of plant
diseases. Trichoderma harzianum and T. virens were two
most dominant species (>90%) in this region and only
these two taxa were isolated from rice phyllosphere.
Phylogenetic analysis revealed that T. harzianum was the
most diverse species representing 14 different ITS haplotypes clustered into four groups. Trichoderma virens was
the only other species from this study that showed intraspecific variation with three different genotypes in one
clade. Correlation of the genotypes with sampling site or
substrate (soil/phyllosphere) was not observed. In addition, the results suggested that different genotypes could
coexist in a single habitat (Naeimi et al. 2011).
Potato (Solanum tuberosum L.) rhizosphere—Six species:
T. asperellum, T. atroviride, T. hamatum, T. harzianum, T. koningii and T. tomentosum were identified by ITS barcoding
and restriction fragment length polymorphism (RFLP)
analysis from the rhizosphere, rhizoplane and bulk soil
of potato (S. tuberosum L.) as well as onion (Allium cepa
L.) in New Zealand, and similar species diversity was
reported in these habitats (Bourguignon, 2008). Trichoderma hamatum, T. harzianum and T. koningii appeared
to be the most frequent species. Moreover, biodiversity
analysis of Trichoderma communities in the rhizosphere
soil of different potato cultivars grown in two fields
located in Southern Germany revealed that the population of Trichoderma spp. and species diversity were
site-dependent, and high field heterogeneity of Trichoderma communities was revealed by DGGE fingerprints,
although differences among them were not statistically
significant (Meincke et al. 2010). A study undertaken in
Poland showed that Trichoderma spp. were predominant
in potato field soils (Pięta et al. 2000).
Coffee (Coffea arabica) rhizosphere—Trichoderma isolates were recovered from the rhizosphere soils of coffee
plants in forests and disturbed semiforests of Ethiopia
and 134 isolates belonging to eight common species
were identified by ITS-barcoding, which were the following in order of abundance: T. harzianum and T. hamatum (the most predominant species in both habitats),
T. asperelloides, T. spirale, T. atroviride, T. koningiopsis, T.
gamsii and T. longibrachiatum (Mulaw et al. 2010). Cultivated and uncultivated coffee regions were rich in
Trichoderma populations and showed relatively high
diversity, but interestingly the biodiversity indices and
evenness were the same for both habitats. In addition,
correlation analysis of the existence of individual Trichoderma species to altitude and some chemical properties
of sampling site soils revealed that Trichoderma spp. did
not have an ecological preference. Intraspecific variation
detected by phylogenetic analysis based on tef1 revealed
that T. harzianum was the most diverse species. Moreover, strains of T. hamatum, T. atroviride and T. spirale represented new genotypes. It was concluded that the high
genetic diversity of Trichoderma from coffee plantation
soil and the establishment of new taxa were influenced
by the variability of the host plant.
Cocoa (Theobroma cacao L.) rhizosphere—One hundred
and thirty five Trichoderma isolates collected from rhizospheres in different locations across the Ivory Coast were
identified by ITS-barcoding as T. asperellum, T. harzianum,
T. virens and T. spirale (Mpika et al. 2009). The first two
species were obtained from all cocoa fields and proved
to be the most abundant in this habitat.
Sugar beet (Beta vulgaris L.) rhizosphere—Sixteen
isolates of Trichoderma were obtained from soils of a sugar
beet field in France and identified based on morphology
as well as ITS and tef1 sequence analysis as T. gamsii, T.
harzianum, T. tomentosum and T. velutinum (Anees et al.
2010). Trichoderma velutinum and T. gamsii were the most
prevalent species.
Oilseed rape (Brassica napus L.) rhizosphere—Trichoderma
spp. were among the prevalent fungi and showed high
biodiversity and plant specificity in the rhizosphere
and bulk soil of oilseed rape as well as strawberry
(Fragaria × ananassa Duch.) in different locations of
Germany (Berg et al. 2005). Diversity and abundance of
Trichoderma in bulk soil was higher than in rhizosphere
soil and the occurring species showed more genotypic
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
diversity by BOX-PCR compared to other fungal genera.
Another study in France showed that Trichoderma was
among the dominant fungal genera in rape rhizosphere
and contributed to the mineralization of organic sulfur,
which is an essential element for plant growth and productivity (Slezack-Deschaumes et al. 2012).
Common bean (Phaseolus vulgaris L.) rhizosphere—
Trichoderma asperellum, T. erinaceum, T. harzianum,
T. koningiopsis and T. tomentosum were identified by
ITS barcoding in the rhizosphere soils of common bean
fields in different areas of Brazil and high level of interand intraspecific diversity in terms of metabolic profiles and assimilation of carbon sources was reported
(Cardoso Lopes et al. 2012). Trichoderma asperellum and
T. harzianum were the most frequent and diverse species
detected.
Oil palm (Elaeis guineensis Jacq.) rhizosphere—
Trichoderma harzianum, T. virens and T. koningii were
the most prevalent Trichoderma species recovered from
oil palm soils in Malaysia and the population of these
fungi was almost the same in cultivated and uncultivated oil palm ecosystems (Sariah et al. 2005). Populations of Trichoderma spp. increased by adding empty fruit
bunches to the fields, while soil pH and moisture did not
affect their distribution and frequency.
Rhizosphere of other crops—Trichoderma hamatum,
T. harzianum, T. koningii, T. pseudokoningii and T. viride
were detected from soybean (Glycine max (L.) Merr.) soils
in Poland (Pięta and Patkowska, 2003), but identities of
the isolates were not confirmed by molecular techniques.
Six species, (T. atroviride, T. citrinoviride, T. harzianum,
T. longibrachiatum, T. koningiopsis and T. reesei) were identified in Mexico from soils cultivated with Sorghum bicolor
based on morphological characteristics, enzymatic activity, macro- and microculture test results, rDNA restriction patterns (AFLP), ITS1–5.8S–ITS2 rDNA sequences
and protein profiles (Larralde-Corona et al. 2008). In
Japan, T. hamatum, T. harzianum, T. koningii and T. viride
were identified from soils of a radish (Raphanus sativus
L.) field (Mghalu et al. 2007). Eleven species of Trichoderma were obtained from tobacco (Nicotiana tabacum
L.) fields in China, among which T. harzianum, T. viride
and T. hamatum were the most dominant species (Yu and
Zhang, 2004). Trichoderma harzianum and T. hamatum—
identified solely on the basis of classical macro- and
microscopic criteria—were the most dominant species
among 150 fungal species in cucumber rhizosphere soils
in Switzerland (Girlanda et al. 2001). From greenhouse
soils in China, T. atroviride, T. aureoviride, T. citrinoviride,
T. fertile, T. harzianum, T. longibrachiatum, Trichoderma
parceramosum, T. reesei, T. virens and T. viride were
reported (Zhao et al. 2004). Trichoderma spp. (mostly
T. harzianum based on RAPD-analysis) were isolated
from rhizosphere soils of various flowers (e.g. carnation,
gladiolus and lilium) and vegetables (e.g. tomato) in India
11
(Shanmugam et al. 2008). Trichoderma spp. were reportedly common or even the most abundant fungi obtained
from various crop fields worldwide such as undisturbed
cotton (Gossypium hirsutum L.) roots in USA (Baird and
Carling, 1998), ginseng (Panax ginseng C.A. Meyer) rhizosphere in South Korea (Hyun-Sung and Lee, 1986)
and arecanut palm (Areca catechu L.) rhizosphere in India
(Bopaiah and Bhat, 1981).
Sadfi-Zouaoui et al. (2009) isolated Trichoderma strains
from the soils of cultivated fields in North-East Tunisia
and identified them as T. atroviride and T. hamatum.
In the study of Hoyos-Carvajal et al. (2009), 10 out of 29
Trichoderma species originated from agricultural related
habitats in Colombia and Mexico. Trichoderma harzianum
and T. asperellum were the most dominant species in this
region. Distribution of the species was related to the soil
and substrate type as well as to the climatic zone. Recent
investigations of Trichoderma diversity in China by ITS
barcoding and tef1 sequence analysis resulted in the
identification of 23 species from different garden, vegetable, farmland and pasture soils all over the country (Sun
et al. 2012). The diversity of the genus was the highest in
vegetable soils with 13 detected species (T. asperellum, T.
atroviride, T. brevicompactum, T. citrinoviride, T. harzianum,
T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. sinense,
T. stromaticum, T. velutinum, T. virens, T. viride), followed
by pasture soils (eight detected species: T. asperellum, T.
atroviride, T. erinaceum, T. hamatum, T. koningii, T. longibrachiatum, T. stromaticum, T. velutinum), garden soils (eight
detected species: T. asperellum, T. erinaceum, T. hamatum,
T. harzianum, T. longibrachiatum, T. pleuroticola, T. tomentosum, T. virens) and farmland soils (6 detected species: T.
asperellum, T. atroviride, T. aureoviride, T. brevicompactum,
T. erinaceum, T. harzianum). The distribution, proposition
and frequency of the species were associated with the
geographical area. Trichoderma harzianum was the most
abundant and widely distributed species followed by
T. asperellum and T. hamatum. According to the phylogenetic analysis of their ITS and tef1 sequences, T. harzianum was the most variable species in China representing
12 different ITS and 17 tef1 genotypes.
Living Plants (Endophytes)
The studies discussed above demonstrate that the
occurrence of Trichoderma is general in the rhizosphere
of a wide variety of soils. Certain Trichoderma strains
can also colonize the plant roots and take part in symbiotic relationships. In recent times, numerous studies
were carried out to prove that some Trichoderma species
can reach the inner tissues of the plants and develop an
endophytic relationship.
The cocoa plant (Theobroma cacao) was in the focus
of several studies, as growing this plant is very common in various tropical countries of the world. Certain
A. BIOLOGY AND BIODIVERSITY
12
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
plant pathogenic fungi can cause serious crop losses. In
Central- and South America, the three most common
diseases of cocoa plants are black pod (Phytophtora species), witches' broom (Crinipellis perniciosa) and frosty
pod rot (Moniliophtora roreri) (Bailey et al., 2006). In
order to find an efficient biocontrol agent against these
pathogens, the endophytic microbial community of the
cocoa plant is being studied intensively. These examinations proved that a series of Trichoderma species may
occur as endophytes of the cocoa plant, including members of the former T. koningii species aggregate such as T.
ovalisporum and the new species T. caribbaeum var. aequatoriale, T. koningiopsis (Samuels et al., 2006a) and Trichoderma protrudens (Degenkolb et al., 2008). Trichoderma
theobromicola and T. paucisporum (Samuels et al., 2006a),
T. stromaticum (Samuels et al., 2012b) as well as Trichoderma martiale (Hanada et al., 2008) were also identified
as endophytic Trichoderma species of cocoa. Rubini et al.
(2005) studied the diversity of endophytic fungi of the
cocoa plant and successfully identified a range of fungal
species, however, the prevalence of Trichoderma species
was very low among the strains isolated. The endophytic microbiota of coffee seedlings was also reported
to contain Trichoderma species including T. hamatum and
T. harzianum identified by ITS sequence analysis (Posada
et al., 2007).
The identification of endophytic fungi is an intensively
investigated field in the case of other plants as well. Such
studies may result in the description of new Trichoderma
species. Chaverry et al. (2011) described Trichoderma amazonicum as a new species based on isolates from rubber
tree (Hevea spp.), Zhang et al. (2007) described Trichoderma taxi from Taxus mairei tree in China, while Samuels
et al. (2012) described T. solani as an endophyte in tubers
of Solanum hintonii in Mexico.
Six different Trichoderma species were identified by
TrichOKEY as endophytic fungi of banana root (Xia et al.,
2011), among which four species: T. asperellum, T. virens,
T. brevicompactum and H. lixii could be found inside the
roots while two species: T. atroviride and T. koningiopsis
were detected only on root surface. Trichoderma asperellum and T. virens showed the highest frequencies in the
examined samples.
Dang et al. (2010) examined the endophytic fungi of
Panax notoginseng, a traditional Chinese medicinal plant.
According to ITS-based identifications, a T. ovalisporum
strain with antibacterial activity against Escherichia coli,
Bacillus cereus, Staphylococcus aureus and Micrococcus
luteus could be isolated during this study. Other wellknown medicinal plants such as Salvia miltiorrhiza and
Huperzia serrata were also examined. A T. atroviride
strain identified by its morphology and ITS sequence
analysis was isolated as an endophytic fungus, which
produced tanshinone I and tanshinone IIA (Ming et al.,
2012). Moreover, in an independent examination, Chen
et al. (2011) also identified T. atroviride as well as T. gamsii strains based on their ITS sequences from Huperzia
serrata.
A series of studies were aimed at the examination
of the production of secondary metabolites by Trichoderma strains. Trichoderma gamsii identified by ITS
sequencing from P. notoginseng, was further examined
and as a result, four new cytochalasins: trichoderones
A and B and trichalasins C and D were identified by
Ding et al. (2012a,b). Souza et al. (2008) studied the
secondary metabolite production of a T. koningii isolate (identified by ITS sequence analysis) derived
from Strychnos cogens and described the production
of koninginins A, F and E. Two new octahydronaphthalene derivatives produced by a T. spirale strain isolated from Aquilaria sinensis (Li et al., 2012), as well
as trichodermanin A produced by T. atroviride isolated
from Cephalotaxus fortunei (Sun et al., 2011) were also
reported (both producer strains were identified by ITS
barcoding).
Studies on endophytic fungi of carnivorous plants
also resulted in the detection of endophytic Trichoderma
strains. Quilliam and Jones (2010) studied Drosera rotundifolia plants during spring and autumn and observed
seasonal distribution of endophytic fungi, however,
T. viride (confirmed by ITS) could be isolated from all
of the samples. Later the same authors carried out the
investigation of endophytic fungi from the carnivorous
plant Pinguicula vulgaris's (Quilliam and Jones, 2012).
Although differences could be observed between the
endophytes of the two plants, Trichoderma species were
detected in both cases.
The endophytic and mycorrhizal fungi were studied
also in the case of seeds and roots originated from Dendrobium nobile and Dendrobium chrysanthum belonging to
the Orchidaceae family (Chen et al., 2012). The presence
of Trichoderma species was proved among the 127 endophytic fungal isolates. Moreover, Yuan et al. (2009) investigated further 288 samples from D. nobile and identified
T. chlorosporum based on ITS sequence analysis among
the detected species.
One of the most harmful pathogenic fungi of the
potato plant is R. solani. It is capable of causing serious quality and quantity damages in the potato tuber.
Therefore a lot of laboratory work is aimed worldwide
at finding an effective biocontrol agent against this
dangerous pathogen. The biocontrol ability of endophytic fungi isolated from potato plants was examined
in agar confrontation tests (Lahlali and Hijri, 2010). On
the basis of the results of these tests it was concluded
that the isolates of Epicoccum nigrum and T. atroviride
(confirmed by ITS) showed the highest inhibition of
R. solani. In the case of T. atroviride the mycoparasitic
phenomenon was found to be determinative in the
inhibition process.
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
Mushroom-Related Substrata
The association of Trichoderma species with wild as
well as cultivated mushrooms has been reported from
various countries. The Trichoderma-caused green mould
disease severely affects the production of both button
mushroom (A. bisporus) and oyster mushroom (Pleurotus ostreatus), causing serious losses for growers worldwide. Overviews of the current status of A. bisporus and
P. ostreatus green mould were given by Kredics et al.
(2010) and Hatvani et al. (2008), respectively.
During the early appearance of mushroom green
mould disease, various Trichoderma species such as T.
atroviride, T. citrinoviride, T. harzianum, T. koningii and
T. longibrachiatum were found to be associated with
cultivated A. bisporus. However, the predominant species, causing aggressive compost colonization were
identified exclusively as the T. harzianum biotypes Th2
(Seaby, 1987, 1989; Doyle, 1991) and Th4 (Castle et al.
1998) in Great Britain/Ireland and United States/
Canada, respectively. The appearance of green mould
due to T. harzianum biotype Th2 in other Western European countries was subsequently reported (Hermosa
et al. 1999; Mamoun et al. 2000). Based on morphological characteristics as well as the phylogenetic analyses
of ITS1 and a fragment of the tef1 gene, Samuels et al.
(2002) redescribed the T. harzianum biotypes Th2 and
Th4, the causal agents of Agaricus green mould disease
in Europe and North America, as the new species T.
aggressivum f. europaeum and T. aggressivum f. aggressivum, respectively.
The cultivation of A. bisporus in Hungary was found
to be affected mostly by T. aggressivum f. europaeum, indicating the spread of the Agaricus green mould epidemic
from Western to Central Europe. Besides T. aggressivum,
further five Trichoderma species, T. asperellum, T. atroviride, T. ghanense, T. harzianum and T. longibrachiatum
were detected in compost samples (Hatvani et al. 2007).
The holotype strain of the recently described new species
T. capillare (Samuels et al., 2012a) was also recovered from
a Hungarian Agaricus-producing facility: it was isolated
from the wall of a mushroom growing cellar (Hatvani
et al., 2006).
In Poland T. aggressivum, T. atroviride, T. citrinoviride,
T. harzianum, T. longibrachiatum, T. virens and T. viride
were identified in association with mushrooms, with
T. aggressivum being the most abundant species (60% of
the isolates) (Błaszczyk et al. 2011). This finding demonstrates a change in the representation of species, as an
earlier study revealed the dominance of T. harzianum in
the country (Szczech et al., 2008). Green mould-affected
Agaricus compost in Croatia yielded exclusively T. harzianum, indicating a broadening spectrum of Trichoderma
species being able to cause green mould disease in button mushroom cultivation (Hatvani et al. 2012).
13
Species found in green mould-affected oyster mushroom substrate in Hungary were T. asperellum, T. atroviride,
T. longibrachiatum, and the yet undescribed species Trichoderma sp. DAOM 175924, which represented 90% of the
isolates (Hatvani et al. 2007). Strains belonging to this
taxon could be divided into two groups based on an A/C
transversion at position 447 of the ITS2 region and corresponded to Trichoderma sp. K1 and K2, the Pleurotus pathogenic Trichoderma species observed in Korea (Park et al.
2004), which were subsequently described as the new
species T. pleurotum and T. pleuroticola (Park et al. 2006).
The results of the comprehensive study of KomońZelazowska et al. (2007) confirmed that these two species
were responsible for green mould infections in Pleurotus farms in various countries, such as Italy, Hungary,
Romania and the Netherlands. In Croatia the same species were found to cause oyster mushroom green mould,
being the sole species recovered from infected Pleurotus
substrate samples (Hatvani et al. 2012). Pleurotus green
mould in Spain was shown to be caused exclusively
by T. pleurotum (Gea, 2009). Kredics et al. (2009) developed a PCR-based technique for the specific detection
of T. pleurotum and T. pleuroticola, the Trichoderma pathogens of cultivated oyster mushroom. Through the use of
the newly introduced method, the presence of T. pleuroticola was detected in high proportions in the growing
substrate and on the fruiting bodies of wild Pleurotus
species, which might act as potential sources of infection of mushroom farms. Further Trichoderma species
found in these habitats were T. atroviride, T. harzianum
and T. longibrachiatum. Trichoderma pleurotum could not
be detected in any of the samples tested, however, further investigations revealed that the natural substratum
of oyster mushroom is a habitat of this species as well
(Kredics et al. unpublished data). Recently, Kim et al.
(2012) described Trichoderma mienum as a new species of
the Semiorbis clade isolated from oyster mushroom and
shiitake bed logs in Japan.
Trichoderma species were found to be the most frequent contaminants of shiitake (Lentinula edodes) cultivation in Thailand. The majority of the isolates belonged to
T. harzianum, but T. aureoviride, T. koningii, T. piluliferum
and T. pseudokoningii were also detected in small proportions (Pukahuta et al. 2000). Turóczi et al. (1996) reported
the isolation of T. hamatum from the fruiting bodies of
Lentinula edodes. The strains showed intermediate antagonistic properties towards phytopathogenic fungi.
Certain members of the genus Trichoderma (T. hamatum, T. harzianum, T. koningii, T. virens and T. viride) were
shown to be among the most abundant microfungi isolated from the surroundings of wild Termitomyces species
in Thailand (Manoch et al., 2002), suggesting their potential role in the stimulation of the occurrence of termite
fungi. The ITS barcoding-based detection of T. hamatum,
T. harzianum, T. spirale, T. virens and an unidentified
A. BIOLOGY AND BIODIVERSITY
14
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
Trichoderma sp. was reported from the nests of leafcutter
ants cultivating basidiomycetous fungi from Agaricales
for nutritional purposes (Rodrigues et al., 2008).
Rivera et al. (2010) reported the presence of Trichoderma species at 16% of the moulds isolated from the
ascocarps of the truffle Tuber aestivum. Wang et al. (2011)
examined the microbial communities of wild Chroogomphus rutilus, and found that T. koningiopsis strains represented 28.6% of the fungal isolates.
Human Body
The role of Trichoderma species as facultative pathogens of humans was firstly summarized by Kredics et al.
(2003) and later extensively reviewed and discussed
(Kredics et al., 2011b). Infections caused by Trichoderma
species known from the literature include peritonitis and
intra-abdominal abscess in patients undergoing continuous ambulatory peritoneal dialysis (CAPD), liver infection, acute invasive sinusitis and disseminated infections
(e.g. abdominal, lung and skin disseminations) of transplant recipients, brain abscess, skin infection, necrotizing stomatitis and pulmonary infections of patients with
hematological malignancies, fungemia by contaminated
saline, rhinosinusitis, pulmonary mycetoma and fibrosis, hypersensitivity pneumonitis, endocarditis, otitis
externa, cerebrospinal fluid infection and allergic reactions (Kredics et al., 2011b). Trichoderma species reported
in case descriptions of human infections in the literature
are T. atroviride, T. citrinoviride, T. harzianum, T. koningii,
T. longibrachiatum, H. orientalis, T. pseudokoningii, T. reesei,
T. viride and a Hypocreaceae sp. However, it is questionable whether all of these Trichoderma species are in fact
able to cause human infections, as many Trichoderma
isolates recovered from clinical samples were identified
based on their morphological characters only, which is
frequently problematic. Although a key for the morphology-based identification of clinical Trichoderma isolates
was introduced by Summerbell (2003) and the use of the
morphological key of Gams and Bissett (1998) was also
proposed, this may still result in incorrect identifications
due to the lack of expertise. Therefore the application
of biochemical and molecular techniques is suggested
to confirm the species-level diagnosis of clinical Trichoderma isolates.
As a biochemical solution, cellulose-acetate electrophoresis-based isoenzyme analysis according to Hebert
and Beaton (1993) was performed by Szekeres et al.
(2006) for the identification of clinical Trichoderma isolates. The authors suggested this method as a cheap
and efficient alternative of molecular techniques for
the quick and specific identification of clinical T. longibrachiatum isolates. DNA-based molecular methods
applied to assess the genetic diversity of clinical Trichoderma isolates include RFLP of the mitochondrial DNA
(Antal et al., 2006) as well as PCR-fingerprinting and ITS
sequence analysis (Kuhls et al. 1999), which revealed
that the reported identities of clinical Trichoderma strains
were incorrect in several cases and that T. longibrachiatum
was the most frequent, almost exclusive causal agent of
opportunistic infections within the genus Trichoderma.
However, as the species T. longibrachiatum and H. orientalis are not distinguishable based on their ITS sequences
alone, the analysis of further phylogenetic markers is
needed in the cases of the clinical involvement of this
species pair. For the examination of a Trichoderma strain
collection of 15 clinical and 36 environmental isolates
belonging to T. longibrachiatum/H. orientalis, Druzhinina
et al. (2008) applied multilocus phylogenetic analysis
involving the ITS region along with fragments of tef1,
calmodulin (cal1) and endochitinase (chit18-5) genes.
The results of this study have reinforced that H. orientalis
is a sexually recombining, while T. longibrachiatum is a
strictly clonal species. Hypocrea orientalis was also identified as an opportunistic human pathogen, and clinical T.
longibrachiatum isolates were shown not to form a particular subpopulation of the species. These findings suggest that all strains of T. longibrachiatum and H. orientalis
might be able to cause infections in humans.
Besides T. longibrachiatum and H. orientalis, the involvement of further four Trichoderma species was confirmed
with molecular tools: T. atroviride (Ranque et al., 2008),
T. citrinoviride (Kuhls et al. 1999), T. harzianum (Guarro
et al., 1999; Kantarcioğlu et al., 2009) and an unknown
Hypocreaceae species close to the genus Hypocrea/Trichoderma (Druzhinina et al., 2007), which shares identical
ITS and rpb2 sequences with Hypocrea peltata, a recently
described sexually reproducing Hypocrea species without a Trichoderma anamorph (Samuels and Ismaiel, 2011).
Potential virulence factors of Trichoderma species as
opportunistic pathogens of humans are suggested to
include the ability to grow at elevated temperatures and
neutral pH, the production of extracellular proteases
and the ability to utilize amino acids as carbon and nitrogen sources (Antal et al., 2005). Antifungal susceptibility studies on clinical Trichoderma strains revealed high
resistance of numerous isolates to a series of widely used
antimycotics, but e.g. voriconazole can be suggested for
the treatment of patients (Kredics et al., 2011b).
Water-Related Environments
Trichoderma species were shown to be abundant in
different water-related environments including marine
and sweet water habitats as well as water-damaged
building materials.
The marine occurrence of Trichoderma species was
firstly mentioned by Kohlmeyer (1974). Since that time,
Trichoderma has frequently been reported in association
with different marine sponge species, including Agelas
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
dispar collected from waters around the Island of Dominica (Neumann et al., 2007), Latrunculia corticata from
the Red Sea at Sharm El-Sheikh, Egypt (El-Bondkly
et al., 2012), Geodia corticostylifera form the South Atlantic
Ocean, Brazil (Rocha et al., 2012), Mycale fibrexilis from
the South China Sea nearby Yongxing Island (Zhou
et al., 2011), Suberites zeteki from Rainbow Bay Marina
in Pearl Harbor and Gelliodes fibrosa from Coconut
Island in Kaneohe Bay on Oahu, Hawaii (Wang et al.
2008) and Psammocinia sp. from the Mediterranean Sea
at Sedot-Yam, Israel (Paz et al., 2010; Gal-Hemed et al.,
2011). Other sea animals from which the isolation of
Trichoderma strains was reported include the gorgonian
sea fan Annella sp. (Khamtong et al., 2012), the seastar
Acanthaster planci (Lan et al., 2012), the blue mussel Mytilus edulis (Ruiz et al., 2007a,b; Sallenave-Namont et al.,
2000) and the cockle Cerastoderma edule (Sallenave et al.,
1999; Sallenave-Namont et al., 2000), where the presence of Trichoderma was shown to contribute to shellfish
toxicity (Sallenave et al., 1999). Besides sea animals, a
Trichoderma strain identified as T. harzianum was isolated
from the green alga Chaetomorpha linum collected from
the North Sea around the Island of Helgoland, Germany
(Neumann, 2008).
Trichoderma species could be isolated from sediments on the root of mangrove Ceriops tagal collected in
the South Sea intertidal zone, China (Sun et al., 2009),
as well as from marine sediments collected at different
locations including the South China Sea (Burtseva et al.,
2003; Song et al., 2010), the Fujian province of China (Du
et al., 2009), the tideland of the Yellow Sea at Lianyungang, China (Sun et al., 2008), various regions of the
Sea of Japan (Khudiakova et al., 2000), St. Helena Bay,
South Africa (Mouton et al., 2012), marine shellfish farming areas along the Western coast of France (SallenaveNamont et al., 2000) and the Antarctic Penguin Island
(Ren et al., 2009). In the Alimini Grande brackish lake
in Italy, Trichoderma proved to be the dominant genus in
sediment samples in a marshy area, where it is supposed
to be involved in the decomposition of allochthonous
plant material (mainly Phragmites australis) (De Donno
et al., 2008).
Marine-derived fungi including Trichoderma species
are attracting increasing interest as potential sources
of metabolites (Table 1.1) with a wide range of biological activities including antibacterial activity against
methicillin-resistant S. aureus (Khamtong et al., 2012),
cytotoxicity against human colon carcinoma cells (Garo
et al., 2003), a melanoma cell line (Sun et al., 2006), other
cancer cell lines and bioactivities against HIV protease
(You et al., 2010). El-Bondkly et al. (2012) used a marine
Trichoderma strain for intergeneric protoplast fusion with
Penicillium and Aspergillus strains to improve cellulase
production. Activities of β-1,3-glucanase (Burtseva et al.,
2003) and laminarinase enzymes (Burtseva et al., 2006)
15
as well as a tyrosinase inhibitor (Tsuchiya et al., 2008)
were also studied in the case of marine-derived Trichoderma strains. A Trichoderma strain isolated from the
marine sponge G. corticostylifera was found to catalyze
the bioreduction of a-chloroacetophenone (Rocha et al.,
2009), the hydrolysis of benzyl glycidyl ether (Martins
et al., 2011) and the asymmetric bioconversion of iodoacetophenones to the corresponding iodophenylethanols (Rocha et al., 2012). The potential application of
a strain (reported as T. viride) from sea water samples
collected from a heavy metal-polluted area in the Mediterranean Sea, Alexandria, Egypt was suggested for the
mycoremediation of Cr (VI) from water systems (ElKassas and El-Taher, 2009). A T. longibrachiatum strain
isolated from mussels in a shellfish farming area was
investigated for total lipid production, total lipid fatty
acids, and phospholipid fatty acids and did not found
marked differences when compared to lipid class and
fatty acid profiles of terrestrial Trichoderma species (Ruiz
et al., 2007a).
As the species level identification of marine Trichoderma isolates has not been performed in the majority of
these studies or it has been performed based on morphological characters only, the marine occurrence of certain
species reported in these articles (e.g. T. reesei, T. viride)
lacks molecular evidence. In certain cases, ITS sequences
were determined but their NCBI BLAST analysis resulted
in an incorrect identification, e.g. Khamtong et al., 2012
identified an isolate as T. aureoviride while Song et al.
(2010) another one as T. koningii, however, a TrichOKEY
2.0 analysis of the respective sequences (GenBank accession numbers EU714396 and GU244589) reveals the
identity of the isolates as T. harzianum and T. koningiopsis/ovalisporum, respectively. An exact identification was
provided in the study of Mohamed-Benkada et al. (2006),
who identified the studied trichobrachin-producing
strain as T. longibrachiatum using metabolic profiles on
Biolog FF MicroPlatesTM and sequence analysis of the
ITS region and the intron-rich region of the tef1 gene.
The most detailed data about Trichoderma population
structure in a marine habitat were provided by the studies of Paz et al. (2010) and Gal-Hemed et al. (2011). Paz
et al. (2010) collected samples from the sponge Psammocinia sp. at a depth of 2–6 m, approximately 200 m offshore at Sedot-Yam, Israel in the winter and summer of
2007 and isolated 400 fungal strains, among which 220
were subjected to ITS and tef1-based molecular identification. Trichoderma species with mycoparasitic potential
were also recorded. Species identifications of 29 Trichoderma strains from Psammocinia sp. were refined in the
subsequent study by Gal-Hemed et al. (2011). The greatest number of isolates proved to belong to T. atroviride
(9), the T. harzianum species complex (7), as well as to T.
longibrachiatum (5) and the closely related H. orientalis
(4). A single isolate of T. asperelloides and two putative
A. BIOLOGY AND BIODIVERSITY
16
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
TABLE 1.1 Metabolites Isolated from Marine Trichoderma Strains
Trichoderma Strain
Isolation Source
Detected Metabolites
References
T. atroviride G20-12
Root of mangrove (Ceriops tagal),
South Chinese Sea
Methyl 3-(3-oxocyclopent-1-enyl) propionate
Sun et al. (2009)
4′-(4,5-Dimethyl-1,3-dioxolan-2-yl)methyl-phenol,
(3′-hydroxybutan-2′-yl)5-oxopyrrolidine-2-carboxylate,
atroviridetide
Lu et al. (2012)
Trichoderma sp.
strain f-13
Marine sediment, Fujian
province, China
Sorbicillinoids: 6-demethylsorbicillin, sohirnones A,
Du et al. (2009)
sorbicillin, 2,3-dihydrosorbicillin
Bisorbicillinoids: bisvertinolone, 10,11-dihydrobisvertinolone,
trichodimerol, dihydrotrichodimerol, bisorbicillinol,
bisvertinoquinol, bisorbibutenolide
T. asperellum
Sediment, Penguin island,
Antarctica
Peptaibols: asperelines A−F
Ren et al. (2009)
T. koningii
Marine mud of the South
China Sea
Koninginins A, D, E, and F
Polyketide derivatives: 7-O-methylkoninginin D,
trichodermaketones A–D
Song et al. (2010)
Trichoderma sp.
Unidentified marine sponge
Aminolipopeptides: trichoderins A, A1 and B
Pruksakorn et al.
(2010)
T. reesei
Marine mud, tideland of
Lianyungang, China
Cyclotetrapeptide: trichoderide A
Sun et al. (2006)
Polyketide derivatives: trichodermatides A–D
Sun et al. (2008)
T. aureoviride PSU-F95
Gorgonian sea fan (Annella sp.)
Trichodermaquinone, trichodermaxanthone,
coniothranthraquinone 1, aloesone, 2-(2′S-hydroxypropyl)5-methyl-7-hydroxychromone, isorhodoptilometrin,
pachybasin, 1-hydroxy-3-methoxyanthraquinone
2-methylquinizarin, ω-hydroxypachybasin, crysophanol,
ω-hydroxyemodin
Khamtong et al.
(2012)
Trichoderma sp.
Seastar (Acanthaster planci)
Sorbicillinoids: (4′Z)-sorbicillin, (2S)-2,3-dihydro-7-hydroxy- Lan et al. (2012)
6-methyl-2-[(E)-prop-1-enyl]-chroman-4-one, (2S)-2,3dihydro-7-hydroxy-6,8-dimethyl-2-[(E)-prop-1-enyl]chroman-4-one, sorbicillin, 2′,3′-dihydrosorbicillin
T. longibrachiatum
Mytilus edulis, Tharon, France
Peptaibols: trichobrachins A I–IV and B I–IV
Mohamed-Benkada
et al. (2006)
T. longibrachiatum
Blue mussels (Mytilus edulis),
shellfish-farming area from the
estuary of the Loire river
Peptaibols: 21 new trichobrachins
trichobrachin C I and II
trichobrachin A II–IX
Ruiz et al. (2007b)
T. virens
Sea water
Dipeptides: trichodermamides A and B
Garo et al. (2003)
Trichoderma sp.
Deep sea sediment, South China
Sea
Cholesta-7,22-diene-3b,5a,6b-triol
Cyclopentenone: trichoderone
You et al. (2010)
T. viride
Caribbean sponge (Agelas dispar),
island of Dominica
2-Furancarboxylic acid
Abdel-Lateff (2008),
Pyranone derivative: trichopyrone
Abdel-Lateff et al.
Sorbicillinoid polyketide derivatives: trichodermanone A–D (2009)
Hexaketide derivatives: rezishanone, vertinolide
Dodecaketides: trichodimerol, bislongiquinolide
(trichotetronine), bisvertinol
new species, Trichoderma sp. O.Y. 2407 from Strictipilosa clade and O.Y. 14707 from Longibrachiatum clade
were also identified based on sequences of cal1, chi18-5
and rpb2 gene fragments. The authors suggested that
marine-derived Trichoderma strains showing mycoparasitic abilities might be potential halotolerant biocontrol
agents in arid agricultural zones, where saline water
irrigation is becoming more common (Gal-Hemed
et al., 2011).
The above-mentioned Trichoderma species are considered as facultative marine fungi (true terrestrial fungi
capable of growing in the marine environment). However, a recent study suggests also the existence of obligate water-inhabiting Trichoderma species (Yamaguchi
et al., 2012): two new aero-aquatic Trichoderma species
with Pseudaegerita-like propagules, Trichoderma matsushimae and Trichoderma aeroaquaticum were described
from Thailand and Japan. The authors hypothesized
A. BIOLOGY AND BIODIVERSITY
TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
that these fungi evolved from soil-inhabiting species
of Trichoderma by adaptation to aquatic environments
via the formation of bulbil-like propagules floating on
water.
The abundance of Trichoderma species is also known
in natural and artificial sweet water environments, e.g.
in an acid mine drainage lake in Anhui Province, China
(Zhang et al., 2012) or in bottled water (Ribeiro et al.,
2006). In a study about the diversity and significance
of mold species in Norwegian drinking water, besides
Penicillium and Aspergillus, Trichoderma was also among
the fungi with the highest maximum number of CFU in
drinking water samples (Hageskal et al., 2006). In a subsequent study, a total of 123 Trichoderma strains were isolated from Norwegian surface-sourced drinking water
(Hageskal et al., 2008). Examined samples included raw
water, treated water, and water from private homes and
hospital installations. Eleven known Trichoderma/Hypocrea species and a group of unidentified Trichoderma
strains representing a separate, strongly supported subclade within the Pachybasium A/Hamatum clade could
be detected based on ITS and tef1 sequences. Trichoderma
viride was the predominant species with 49% of the identified strains, being present in 22% of the surface-derived
water samples.
Water-damaged buildings represent a further waterrelated habitat from which Trichoderma species (T.
atroviride, T. viride, T. hamatum as well as the clinically
relevant species T. longibrachiatum, T. citrinoviride and
T. harzianum) are frequently reported as the dominating microfungi (Lübeck et al., 2000; Thrane et al., 2001;
Ebbehøj et al. 2002). Release of spores by common indoor
fungi including T. harzianum from wet wallpapered gypsum boards was examined by Kildesø et al. (2003). The
authors concluded that these spores might be responsible for certain negative health effects related to buildings contaminated with moulds. Andersen et al. (2011)
studied samples from water-damaged building materials with visible fungal growth from private residences
(houses, apartments and holiday cottages) and private
businesses (shops and offices) as well as from public
buildings (kindergartens, schools and offices) from all
parts of Denmark and Greenland. Concrete, glass fiber,
gypsum, plaster, plywood, wallpaper and wood samples
were all positive for Trichoderma.
Air and Settled Dust
Air can play an important role in the dispersal of
fungal spores and conidia. Based on a comprehensive
review by Madsen et al. (2007), Trichoderma species could
be isolated from the air of a series of different indoor and
outdoor environments.
Indoor air samples positive for Trichoderma included
buildings heated by wooden chips in Sweden, flats in
Lithuania, homes in Germany, Poland, Taiwan, Turkey
17
and the USA, hospitals in Austria, Iraq, Poland, Finland
and USA (from air filters) (Madsen et al., 2007). Trichoderma was also found in air ducts of houses in Finland
(Madsen et al., 2007), in air conditioners in Saudi Arabia
(Madsen et al., 2007), in the air of a library and archive
storage facilities in Poland (Zielińska-Jankiewicz et al.,
2008), homes in New Orleans after hurricanes Katrina
and Rita (Rao et al., 2007), an orchid greenhouse (Magyar
et al., 2011), a greenhouse with ornamental plants (Li and
La-Mondia, 2010), child care centers in Turkey (Aydogdu
and Asan, 2008) and intensive care units in Brazil, where
it was reported among the most frequently occurring filamentous fungi (Mobin and Salmito, 2006). Trichoderma
was found to be present also in the settled dust of homes
in Saudi Arabia and the USA (low-traffic carpets, bedspread/furniture surfaces), hospitals in Iraq and schools
without water damage in Denmark (Madsen et al., 2007),
as well as on the walls of damp homes in Croatia and in
wall scrapes from basement in the USA (Madsen et al.,
2007).
Outdoor isolations of Trichoderma from air samples
could be realized at roofs of houses in Saudi Arabia, at
rooftop of a hospital in the Netherlands, in urban areas of
Kuwait including a balcony of Kuwait University, on balconies in Taiwan, in a coastal area in Lithuania, in Pinus
nigra and Quercus forests in Turkey (Madsen et al., 2007),
in the Belgrade Forest area near Istanbul (Çolakoglu,
2003), in Trujillo, Peru (Requejo, 1975), Britain (Richards,
1956), Barcelona (Spain) (Calvo et al. 1980), Manhattan
(Kansas, USA) (Kramer et al., 1959, 1964; Kramer and
Pady, 1960), Sagamihara (Japan) (Takatori et al., 1994)
and Israel (Barkai-Golan, 1958; Barkai-Golan and Glazer,
1962; Barkai-Golan et al., 1977).
Trichoderma was also found to be present in settled
dust of roofs of houses and stationary cars in Egypt
(Madsen et al., 2007).
Regarding environments related with agriculture
or industry, Trichoderma could be isolated from the air
of hop farms and herb processing plants in Poland,
wood chip terminals in Sweden, different parts of
swine farms in Finland, combine harvesters in England and storey buildings undergoing renovation in
Egypt, from the air and settled dust of carpentries in
Italy, from corn dust in the USA, from settled sawdust
at sawmills in England and the USA, from dust blown
from hay in England (Madsen et al., 2007), as well as
from spots visibly contaminated with fine plastic particles in a manufacturing factory for plastic caps for
soft drinks (Sato, 2010).
The studies above indicate that Trichoderma conidia
are commonly occurring in air samples and settled
dust, however, unfortunately a species-level identification has not been carried out in most of the cases.
Where species names were also provided in these earlier studies, the occurrence of T. album, T. hamatum, T.
harzianum, T. koningii, T. lignorum, T. viride, as well as
A. BIOLOGY AND BIODIVERSITY
18
1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
Trichoderma inhamatum—a taxon commonly regarded
a synonym of H. lixii, but also suggested as a separate species within T. harzianum sensu lato (Druzhinina
et al., 2010). However, as a sequence-based molecular
identification has not been carried out in these studies,
they do not provide data about the real diversity of the
genus and abundance of its different members in air
samples. The actual Trichoderma diversity and species
abundance in air samples could be revealed by metagenomic analyses like the one of Atanasova et al. (2010),
who applied the metagenomic approach to study the
occurrence and diversity of members of the genus in air
samples taken in the Viennese suburban area of Wienerwald, Austria. Sequence analysis of a total of 159
molecular operational taxonomic units (MOTUs) by
TrichOKEY 2.0 revealed 15 known species with T. virens
being the most abundant (52% of all MOTUs). The first
European detection of tropical species (T. reesei, T. stromaticum, T. taxi) and rare temperate species known from
North-America (T. fertile, T. ceramicum) was reported in
this study, supporting the possibility of long-distance
spore dispersal. Other species detected were T. asperellum, T. citrinoviride, T. gamsii, the T. longibrachiatum/H.
orientalis species duplet and T. minutisporum that all
occur frequently in European ecosystems, as well as
H. neorufa and Hypocrea psychrophila which are rare in
Europe (Atanasova et al. 2010). Interestingly, the most
frequent taxon of the genus, the T. harzianum species
complex could not be detected in the examined air samples, although it was abundant in soil samples from the
same area. Previously it was suggested that T. harzianum can rarely be found in the air, because its conidia
may not be released easily from growth materials to the
air (Madsen et al. 2007).
The prevalence of conidia in the air has a significance
also from the clinical point of view: allergic diseases
may be associated with airborne Trichoderma conidia
(Çolakoglu, 2003), and in the case of immunocompromised patients, air may also be a potential source of
opportunistic Trichoderma infections like sinusitis or
pneumonia. As Trichoderma species are frequently used
as biocontrol agents against plant pathogenic fungi in
both field agriculture and closed production systems
like greenhouses, it is very important to gain information about the possible exposure of growers working
in such facilities to Trichoderma conidia. Hansen et al.
(2010) revealed that T. harzianum from the biocontrol
product Supresivit could be detected in the air of the
examined greenhouse only on the day of treatment. In
a subsequent study (Hansen et al., 2012), exposure to
Trichoderma could be observed for growers working in a
greenhouse with senescent cucumber plants, a cabbage
field and in a broccoli packing hall, however, PCRanalysis revealed that the Trichoderma isolates responsible for the exposure were different from the biocontrol
agents applied.
CONCLUSIONS
The studies discussed above reflect that the genus
Trichoderma/Hypocrea can be characterized with high
adaptability to various ecological environments. However, it is important to mention that the results of any
study aimed at the examination of Trichoderma biodiversity should always be evaluated in the context of
the developmental stage of Trichoderma taxonomy and
the species identification methods available at the time
of the publication of the respective paper. Due to the
constant development of the taxonomy of the genus
and the description of new species, more recent examinations of a specific habitat may reveal higher biodiversity of the genus and refine the results of previous
studies.
Thanks to the introduction of new methods and the
evolution of the approaches in biodiversity studies during the past two decades, the amount of information
available about the distribution of Trichoderma species
is constantly growing, therefore it can be expected that
the biogeography of the genus will be understood more
deeply in the near future.
Acknowledgments
The contribution of Csaba Vágvölgyi was realized in the frames of
TÁMOP 4.2.4. A/2-11-1-2012-0001 „National Excellence Program –
Elaborating and operating an inland student and researcher personal
support system”. The project was subsidized by the European Union
and co-financed by the European Social Fund.
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