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BIOTECHNOLOGY
AND BIOLOGY OF
TRICHODERMA
Vijai K. Gupta, Monika Schmoll, Alfredo Herrera-Estrella,
R. S. Upadhyay, Irina Druzhinina, Maria G. Tuohy

AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD

PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO
Elsevier
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particular, independent verification of diagnoses and drug dosages should be made.

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Printed and bound in Poland

14 15 16 17 18 10 9 8 7 6 5 4 3 2 1
 Contents

Prefacexi Biodiversity Studies 45

Forewordxiii Identification of Industrial Trichoderma Strains 47
Identification of Biocontrol Trichoderma Strains 48
List of Contributors xv Identification of Trichoderma Isolates with Clinical Relevance 50
Identification of Mushroom Pathogenic Trichoderma Strains 51
Conclusions51
Acknowledgments52
A References52

BIOLOGY AND BIODIVERSITY 4 Understanding the Diversity and Versatility of

Trichoderma by Next-Generation Sequencing
1 Biodiversity of the Genus Hypocrea/Trichoderma in CHRISTIN ZACHOW AND GABRIELE BERG
Different Habitats
LSZL KREDICS, LRNT HATVANI, SHAHRAM NAEIMI, Introduction57
PTER KRMCZI, LSZL MANCZINGER, CSABA VGVLGYI, Access to Fungal and Trichoderma DiversityTaxonomic
IRINA DRUZHININA
Profiling58
Plants Life under Control of TrichodermaFunctional Profiling 62
Introduction3
Conclusion63
Methodology of Studying Trichoderma Biodiversity 3
Acknowledgments63
Trichoderma Diversity in Different Habitats 5
References63
Conclusions18
Acknowledgments18
References18 5 Molecular Evolution of Trichoderma Chitinases
VERENA SEIDL-SEIBOTH, KATARINA IHRMARK,
2 Ecophysiology of Trichoderma in Genomic Perspective IRINA DRUZHININA, MAGNUS KARLSSON
LEA ATANASOVA
Introduction67
Trichoderma in Its Ecological Niche 25 Phylogeny and Evolution of the GH Family 18 Gene Family in
From Diversity to Genomics 27 Trichoderma68
Mycotrophy of Trichoderma28 Subgroup A Chitinases 69
Saprotrophy of Trichoderma on Dead Wood 30 Subgroup B Chitinases 71
Trichoderma Growth in Soil 31 Subgroup C Chitinases 74
Rhizosphere Competence of Trichoderma32 Conclusions77
Trichoderma versus Mycorrhizae 32 Acknowledgments77
Trichoderma + Bacteria = ? 33 References77
Facultative Endophytism of Trichoderma33
Animal Nourishment of Trichoderma34
Most of the Famous Trichoderma Species are Environmental
Opportunists34 B
Versatile Carbon Utilization Patterns Reflect Ecological
Specialization of Trichoderma spp. 35
SECRETION AND PROTEIN
Acknowledgments37 PRODUCTION
References37
6 Protein ProductionQuality Control and Secretion
3 DNA Barcode for Species Identification in Trichoderma
Stress Responses in Trichoderma reesei
LRNT HATVANI, CSABA VGVLGYI, LSZL KREDICS,
IRINA DRUZHININA M. SALOHEIMO T. PAKULA N. ARO, J.J. JOENSUU

Introduction41 IntroductionMilestones of Trichoderma reesei81

The Tools 42 Protein Secretome of T. reesei82
Application of DNA Barcoding in Species-Level Identification ER Quality Control and Secretion Stress Responses 84
of Trichoderma43 Conclusion86
Taxonomic Studies 43 References86

v
vi CONTENTS

7 Heterologous Expression of Proteins in Trichoderma Diketopiperazine-Like Compounds 129

HELENA NEVALAINEN AND ROBYN PETERSON
Polyketides129
Pyrones130
Introduction89 Terpenes131
Promoter Options 92 Concluding Remarks and Future Directions 133
Fusion Partners 93 Acknowledgments134
Extracellular Proteases 94 References134
Secretion Stress in the Frame 95
Mass Production of Heterologous Protein by Fermentation 97 11 Recent Advancements on the Role and Analysis
N-glycosylation of Heterologous Proteins Produced in T. reesei97 of Volatile Compounds (VOCs) from Trichoderma
Conclusions98 SHAFIQUZZAMAN SIDDIQUEE
Acknowledgments99
References99 Introduction139
Detection Techniques of VOCs 140
8 Trichoderma Secretome: An Overview Types of Volatiles Compounds 142
SUNIL S. ADAV AND SIU KWAN SZE Application of VOCs in Agriculture 165
Conclusion168
Introduction103 References168
Proteomic Analysis of Secretory Proteins 105
Extraction of Extracellular Proteins for Proteomic Analysis 106
Extracellular Protein Secretion by T. reesei107
Polysaccharide Degradation Machinery of T. reesei108 D
New Candidates in Cellulose Degradation 109
Hemicellulose Hydrolyzing Enzymes 110 TOOLS
Lignin Degradation by T. reesei111
Industrial Applications of T. reesei Cellulolytic Enzymes 111 12 Molecular Tools for Strain Improvement of
Conclusion112 Trichoderma spp.
References112 ROBERT BISCHOF AND BERNHARD SEIBOTH

9 The Secretory Pathway in the Filamentous Introduction179

Fungus Trichoderma Genetic Transformation Techniques 180
MARCO J. HERNNDEZ-CHVEZ, ROBERTO J. GONZLEZ-HERNNDEZ, Auxotrophic and Dominant Selection Markers 181
JOS E. TRUJILLO-ESQUIVEL, ARTURO HERNNDEZ-CERVANTES, Marker Recycling Strategies and Marker Free Strains 182
HCTOR M. MORA-MONTES Advanced Methods for Gene Targeting 183
RNA Mediated Gene Silencing 184
Introduction115 Promoters for Recombinant Protein Expression and Targeting 185
Translocation115 Concluding Remarks 188
Cotranslational Translocation 116 References188
Post Translational Translocation 116
Protein Modifications in the ER 116
13 Genetic Transformation and Engineering of
Vesicle Transport from ER to Golgi Complex and Trafficking
within the Golgi Cisternae 118 Trichoderma reesei for Enhanced Enzyme Production
Transport after Trafficking within the Golgi Complex 119 ANLI GENG
Secreted Proteins in Trichoderma119
Concluding Remarks 120 Introduction193
Acknowledgments120 Engineering Cellulase and Hemicellulase Regulation 194
References120 Homologous and Heterologous Gene Expression and Gene
Disruption195
Protein Engineering 196
Engineering Promoters 197
C Conclusion198
References198
SECONDARY METABOLISM
10 Secondary Metabolism and Antimicrobial 14 Applications of RNA Interference for Enhanced
Metabolites of Trichoderma Cellulase Production in Trichoderma
SHAOWEN WANG AND GANG LIU
ROSA HERMOSA, ROSA ELENA CARDOZA, MARA BELN RUBIO,
SANTIAGO GUTIRREZ, ENRIQUE MONTE
Introduction201
Introduction125 RNA Interference in Fungus 202
Peptaibols126 Transcriptional Regulation of Cellulase Gene Expression 203
 CONTENTS vii
Application of Gene Downregulation Strategy for Enhanced Genetics of Industrial Trichoderma reesei Strains 263
Cellulase Production 204 The T. reesei Enzyme Cocktail 264
Combination of RNAi and Overexpression of the Regulating Hydrolysis of Cellulose 266
Genes208 Limitations in Lignocellulose Hydrolysis 267
Conclusions and Prospects 211 Improvement of Enzyme Cocktails by Optimization of
References211 Enzyme Ratios 269
Improvement by Supplementation of T. reesei Enzyme Cocktails 270
15 RNAi-Mediated Gene Silencing in Trichoderma: Adapting Cellulose Cocktails to Process Conditions 275
Principles and Applications Conclusions and Perspectives 275
XIAOYUN SU, LINA QIN, ZHIYANG DONG
References275

Introduction215 19 Beta-Glucosidase from Trichoderma to Improve

Molecular Mechanisms 216 the Activity of Cellulase Cocktails
Advantages and Disadvantages of Using RNAi-Mediated WARAWUT CHULALAKSANANUKUL
Gene Silencing as a Genetic Manipulation Tool in
Filamentous Fungi 218 Introduction281
Strategies of Applying RNAi for Gene Silencing in Trichoderma Cellulase Classification 282
and Other Filamentous Fungi 220 Trichoderma reesei Cellulases 282
Conclusions223 Trichoderma reesei BGLs 284
References224 BGLs from Aspergillus oryzae284
Synergism between Cellulases 286
Heterologous Expression of Cellulases 286
E Yarrowia Lipolytica Expression Platforms 286
Pichia pastoris Expression Platforms 287
CELLULASES -Glucosidase from Trichoderma to Improve the Activity of
Cellulase Cocktails 287
16 Cellulase Systems in Trichoderma: An Overview Acknowledgments288
LUIS H.F. DO VALE, EDIVALDO X.F. FILHO, ROBERT N.G. MILLER, References288
CARLOS A.O. RICART, MARCELO V. DE SOUSA
20 Regulation of Glycoside Hydrolase
Introduction229
Expression in Trichoderma
Degradation of Cellulose by Cellulase Systems 230
HODA BAZAFKAN, DORIS TISCH, MONIKA SCHMOLL
History of the Trichoderma Cellulase Research 232
Structural and Functional Diversity of Trichoderma Cellulases 232
Introduction291
Cellulase Systems and Complexes 240
Regulation by Environmental Parameters 292
Acknowledgments241
Regulatory Mechanisms 297
References241
Physiological Responses 302
17 Use of Cellulases from Trichoderma reesei in the References303
Twenty-First CenturyPart I: Current Industrial
21 Trichoderma Proteins with Disruption Activity on
Uses and Future Applications in the Production
Cellulosic Substrates
of Second Ethanol Generation
CHRISTIAN DERNTL, ASTRID R. MACH-AIGNER, ROBERT L. MACH
NICOLAS LOPES FERREIRA, ANTOINE MARGEOT,
SENTA BLANQUET, JEAN-GUY BERRIN
Structure and Occurrence of Cellulose in Nature 309
Overview of the Global Enzyme Market 245 General Aspects of Cellulose Degradation 310
Industrial Cellulases 246 Cellulose Degradation by T. reesei311
Current Applications 249 Cellulolytic Enzymes in Other Trichoderma Species 314
Perspectives253 Acknowledgments314
Application of Trichoderma Cellulases in the Bioethanol References314
Industry253
References258 22 Molecular Mechanism of Cellulase Production
Systems in Trichoderma
18 Use of Cellulases from Trichoderma reesei in the KATOCH MEENU, GURPREET SINGH, R. A. VISHWAKARMA
Twenty-First CenturyPart II: Optimization of
Cellulolytic Cocktails for Saccharification of Introduction319
Lignocellulosic Feedstocks Cellulase System of T. reesei319
JEAN-GUY BERRIN, ISABELLE HERPOEL-GIMBERT, Induction Mechanism of Cellulase Production 320
NICOLAS LOPES FERREIRA, ANTOINE MARGEOT, Promoter Involved in Cellulase Production 320
SENTA HEISS-BLANQUET Molecular Mechanism of Cellulase Production 320
viii CONTENTS

Approaches for Refining the Cellulases Production System Introduction363

in T. reesei321 Global Metabolism 364
References322 Carbohydrate Metabolism and Glycoside Hydrolases 366
Energy Metabolism 368
23 Trichoderma in Bioenergy Research: An Overview Secondary Metabolism 369
VIJAI K. GUPTA, ANTHONIA ODONOVAN, MARIA G. TUOHY, Metabolism and Transporters 372
GAURI DUTT SHARMA Acknowledgments374
References374
Introduction325
Fungal Enzyme Systems and Trichoderma Technology 326 28 Sequence Analysis of Industrially Important
Industrial Applications of Trichoderma327 Genes from Trichoderma
Trichoderma Enzyme Systems in Bioenergy Research 328 AHMED M.A. EL-BONDKLY
Conclusion332
References332 Introduction377
Gene Sequence Analysis Fundamentals 378
Genome Analysis of Trichoderma383
F Industrially Genes from Trichoderma384
Sequence Analysis of Industrially Genes from
INDUSTRIAL APPLICATIONS Trichoderma384
Conclusion389
24 Trichoderma Enzymes for Food Industries References390
ADINARAYANA KUNAMNENI, FRANCISCO J. PLOU,
ANTONIO BALLESTEROS 29 Biosynthesis of Silver Nano-Particles by
Trichoderma and Its Medical Applications
Introduction339 KHABAT VAHABI AND SEDIGHEH KARIMI DORCHEH
Fungus of Industrial Interest 340
Trichoderma Enzymes for Industries 340 Introduction393
Xylanases341 SNP Biosynthesis 395
Cellulases341 Mechanism397
Other Enzymes 342 Medical Application 399
Food Industry 342 References400
Perspectives for Biotechnological Production of Enzymes by
Trichoderma343
References343 30 Role of Trichoderma Species in
Bioremediation Process: Biosorption Studies
25 Trichoderma: A Dual Function Fungi and Their on Hexavalent Chromium
Use in the Wine and Beer Industries DHARA SHUKLA AND PADMA S. VANKAR
CARLOS ROBERTO FELIX, ELIANE FERREIRA NORONHA,
ROBERT N. G. MILLER Introduction405
Hexavalent Chromium Bioremediation will Be Discussed Here
Introduction345 with a Case Study Representing Chromium Biosorption by
Application in the Wine and Beer Industries 347 Trichoderma Species 407
Acknowledgments348 Conclusion411
References348 References412

26 Trichoderma Enzymes for Textile Industries

TERHI PURANEN, MARIKA ALAPURANEN, JARI VEHMAANPER
G
Substrate351 BIOCONTROL AND PLANT
Enzymes352 GROWTH PROMOTION
Textile Processes 353
Trichoderma Enzymes in Textile Finishing Processes 355
Trichoderma as a Production Host for Textile Enzymes 357 31 Applications of Trichoderma in Plant
Future Trends 359 Growth Promotion
Acknowledgments359 ALISON STEWART AND ROBERT HILL
References359
Introduction415
27 Metabolic Diversity of Trichoderma Trichoderma as a Plant Growth Promoter 416
ROBERTO NASCIMENTO SILVA, ANDREI STECCA STEINDORFF, Consistency of Growth Promotion 418
VALDIRENE NEVES MONTEIRO Commercialization419
 CONTENTS ix
Mechanisms of Growth Promotion 420 Signal Transduction Components and Pathways Affecting
Conclusions425 Vegetative Growth and Conidiation 469
References425 The Role of Signaling in Trichoderma Mycoparasitism and
Biocontrol471
32 Molecular Mechanisms of Biocontrol in Conclusions474
Acknowledgments474
Trichoderma spp. and Their Applications in References474
Agriculture
VIANEY OLMEDO MONFIL AND SERGIO CASAS-FLORES 35 Enhanced Resistance of Plants to Disease
Using Trichoderma spp.
Introduction429 M.G.B. SALDAJENO, H.A. NAZNIN, M.M. ELSHARKAWY,
Mycoparasitism430 M. SHIMIZU, M. HYAKUMACHI
Morphological Changes 430
Roll of Cell Wall Degrading Enzymes 431 Introduction477
Signal Transduction in Mycoparasitism 432 Induced Disease Resistance in Plants 478
ROS-Nox-Signal Transduction 433 Induced Resistance by Trichoderma spp. 481
Antibiosis (Secondary Metabolites Involved in Biocontrol) 435 Signaling Pathways of Trichoderma-Induced Resistance 482
Pyrones436 Trichoderma spp.-Secreted Elicitors of Plant Resistance 483
Polyketides437 Engineering Plants for Disease Resistance Using Trichoderma
Nonribosomal Peptides 437 Genes485
Mycotoxins Produced by Trichoderma spp. 438 Combination of Trichoderma with Other Beneficial
Synergism between Enzymes and Antibiotics 439 Microorganisms486
Competition for Nutrients 439 Other Effects of Trichoderma spp. Inoculation to the Plant 487
Plant Growth Promotion by Trichoderma440 Conclusion487
Plant Root Colonization 442 References488
Induction of Systemic Resistance to Plants by Trichoderma
spp.443 36 Enhanced Plant Immunity Using Trichoderma
Signal Transduction Pathways that Mediate Trichoderma-Plant HEXON ANGEL CONTRERAS-CORNEJO, LOURDES MACAS-RODRGUEZ,
Communication444 JESS SALVADOR LPEZ-BUCIO, JOS LPEZ-BUCIO
Trichoderma Elicitor of Systemic Resistance in Plants 446
Introduction495
Signal Transduction during PlantTrichoderma Interaction in
Mechanisms of Plant Protection by Microbes 495
Trichoderma448
Trichoderma-Induced Immunity 498
Transgenic Plants Expressing Trichoderma Genes 448
Plant Protection Conferred by Trichoderma500
Concluding Remarks 449
Conclusions501
Acknowledgments449
Acknowledgments501
References449
References501
33 Genome-Wide Approaches toward Understanding
37 Genes from Trichoderma as a Source
Mycotrophic Trichoderma Species
for Improving Plant Resistance to
ALFREDO HERRERA-ESTRELLA
Fungal Pathogen
Introduction455 BARBARA REITHNER AND ROBERT L. MACH

Lessons from the Genome Sequence 457

Introduction505
Transcriptome Analyses 458
Trichoderma Inducing Resistance in Plants 506
The Functional Genomics View of Mycoparasitism 458
Transgenic Plants Expressing Trichoderma Genes Develop
High-Throughput Analysis of the Trichoderma-Plant Interaction 459
Increased Resistance to Fungal Pathogens 506
Future Directions 461
Trichoderma Genes Involved in Elicitation of ISR 508
Concluding Remarks 462
Conclusion511
Acknowledgments462
Abbreviations511
References462
Acknowledgments511
References511
34 Insights into Signaling Pathways of Antagonistic
Trichoderma Species 38 Trichoderma Species as Abiotic Stress
SUSANNE ZEILINGER AND SABINE GRUBER Relievers in Plants
NAJAM W. ZAIDI, MANZOOR H. DAR,
Introduction465 SUDHANSHU SINGH, U.S. SINGH
G Protein Signaling 465
Effector Pathways of G Protein Signaling in Fungi 466 Introduction515
Signaling Pathways and Characterized Components in Microbes for the Management of Abiotic Stresses 516
Trichoderma Species 467 Alleviation of Abiotic Stress in Plants by Trichoderma516
x CONTENTS

Alleviation of Drought Stress in Plants by Trichoderma517 Conclusion and Future Prospects 530
Alleviation of Salinity Stress in Plants by Trichoderma518 References530
Alleviation of Heat Stress in Plants by Trichoderma519
Trichoderma Genes for Abiotic Stress Tolerance 520 40 Trichoderma: A Silent Worker of Plant Rhizosphere
Mechanism of Abiotic Stress Tolerance Using Trichoderma520 AKANKSHA SINGH, BIRINCHI K. SARMA, HARIKESH B. SINGH,
Host Gene: Stress Tolerant Varieties 521 R. S. UPADHYAY
Conclusion522
References523 Introduction533
Diverseness Amongst Trichoderma534
39 Advances in Formulation of Trichoderma Trichoderma as Inducer of Plant Defense Response 536
for Biocontrol Trichoderma as a Biofertilizer and Plant Growth Promoter 538
CHRISTIAN JOSEPH R. CUMAGUN
Commercialization538
Trichoderma Genes Responsible for Playing Big Games 539
Introduction527 Conclusion540
Types of Formulation 528 Acknowledgments540
Microencapsulation528 References540
Enhancement of Shelf Life and Application Efficiency 528
Compatibility with Other Biological Systems 529 Index543
 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 dioxide-
neutral 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 signifi-
cant 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 Tricho-
derma 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 pro-
teins, which facilitates complementation of this system
with efficient enzymes from other organisms, Tricho-
derma reesei has become one of the cornerstones for sec-
ond-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 pro-
duction are discussed. However, besides production of
second-generation biofuels from plant material, indus-
trial 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 expres-
sion of heterologous proteins and a broad array of tools
are available for modification of the genome of this fun-
gus for improvement of its production capacity. Chapters
on heterologous protein production with Trichoderma,
secretion and industrial strain improvement provide an
xi
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 physi-
ological 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 protec-
tion 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 ecophysiol-
ogy 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
 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 Guadal-
canal (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 research-
ers 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 com-
mon tropical ascomycete, Hypocrea jecorina. The inter-
est 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 culmi-
nating 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. Weindlings 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 commercializa-
tion of some of them. The cellulase and the biocontrol
researchers long formed two isolated communities with
little information exchange, but this improved once the
xiii
need for exchange of molecular genetics research tech-
niques 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 spe-
cies are among the most frequent mitosporic fungi com-
monly detected in cultivation-based surveys. They can
be isolated from an innumerable diversity of natural
and artificial substrata, particularly also from materi-
als infested with xenobiotics, demonstrating their high
opportunistic potential and adaptability to various eco-
logical conditions. Consequently, it has broad impacts
on mankind: one of the most stimulating recent find-
ings 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 immunocompro-
mised 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 Pleuro-
tus, 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 prog-
ress is needed.
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 Tech
nological University, Singapore
Marika Alapuranen Roal Oy, Rajamki, Finland
Miguel Alcalde Departamento de Biocatlisis, Instituto de
Catlisis y Petroleoqumica, CSIC, Madrid, Spain
N. Aro VTT Technical Research Centre of Finland, Espoo,
Finland
Lea Atanasova Research Area Biotechnology and Microbio
logy, Institute of Chemical Engineering, Vienna University
of Technology, Vienna, Austria
Antonio Ballesteros Departamento de Biocatlisis, Instituto
de Catlisis y Petroleoqumica, CSIC, Madrid, Spain
Hoda BazafkanHealth and Environment Department,
Austrian Institute of Technology GmbH (AIT), Tulln, Austria
Gabriele Berg Graz University of Technology, Environmen
tal Biotechnology, Graz, Austria
Jean-Guy Berrin Laboratoire de Biologie des Champignons
Filamenteux, INRA, Polytech Marseille, Aix Marseille Uni
versit, Marseille, France
Robert Bischof Institute of Chemical Engineering, Vienna
University of Technology and Austrian Centre of Industrial
Biotechnology (ACIB), Vienna, Austria
Senta Blanquet IFP Energies nouvelles, Biotechnology
Department, Rueil-Malmaison, France
Rosa Elena Cardoza Area of Microbiology, University School
of Agricultural Engineers, University of Len, Ponferrada,
Spain
Sergio Casas-Flores Divisin de Biologa Molecular, IPICyT,
San Luis Potos, Mxico
Warawut Chulalaksananukul Biofuels by Biocatalysts
Research Unit, Chulalongkorn University, Bangkok,
Thailand; Department of Botany, Chulalongkorn University,
Bangkok, Thailand
Hexon Angel Contreras-Cornejo Instituto de Investigacio
nes Qumico-Biolgicas, Universidad Michoacana de San
Nicols de Hidalgo, Morelia, Michoacn, Mxico
Christian Joseph R. Cumagun College of Agriculture, Uni
versity of the Philippines Los Baos, Los Baos, Laguna,
Philippines
Manzoor H. Dar International Rice Research Institute, IRRI,
New Delhi, India
Marcelo V. de Sousa Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Christian Derntl Research Area Gene Technology, Institute
of Chemical Engineering, Vienna University of Technology,
Vienna, Austria
xv
Luis H.F. Do Vale Department of Cell Biology, University of
Brasilia, Brasilia, Federal District, Brazil
Zhiyang Dong Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China
Sedigheh Karimi Dorcheh Institute for Genetic Microbiol
ogy, Friedrich-Schiller University Jena, Jena, Germany
Irina Druzhinina Institute of Chemical Engineering, Vienna
University of Technology, Research Area Biotechnology and
Microbiology, Vienna, Austria
Ahmed M.A. El-Bondkly National Research Centre, Dokki,
Giza, Egypt
M.M. Elsharkawy Laboratory of Plant Pathology, Faculty of
Applied Biological Sciences, Gifu University, Gifu City,
Japan
Carlos Roberto Felix Departamento de Biologia Celular,
Universidade de Brasilia, Brasilia, Federal District, Brasil
Nicolas Lopes Ferreira IFP Energies nouvelles, Biotechnol
ogy Department, Rueil-Malmaison, France
Edivaldo X.F. Filho Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Anli Geng School of Life Sciences and Chemical Technology,
Ngee Ann Polytechnic, Clementi, Singapore
Roberto J. Gonzlez-Hernndez Departamento de Biologa,
Universidad de Guanajuato, Guanajuato, Mxico
Sabine Gruber Research Area Biotechnology and Microbiol
ogy, Institute of Chemical Engineering, Vienna University of
Technology, Vienna, Austria
Vijai K. GuptaMolecular Glycobiotechnology Group,
Department of Biochemistry, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland
Santiago Gutirrez Area of Microbiology, University School
of Agricultural Engineers, University of Len, Ponferrada,
Spain
Lrnt Hatvani Department of Microbiology, University of
Szeged, Szeged, Hungary
Senta Heiss-Blanquet IFP Energies nouvelles, Biotechnology
Department, Rueil-Malmaison, France
Rosa Hermosa Centro Hispano-Luso de Investigaciones
Agrarias (CIALE), University of Salamanca, Salamanca, Spain
Arturo Hernndez-Cervantes Departamento de Biologa,
Universidad de Guanajuato, Guanajuato, Mxico
Marco J. Hernndez-Chvez Departamento de Biologa,
Universidad de Guanajuato, Guanajuato, Mxico
Isabelle Herpoel-GimbertLaboratoire de Biologie des
Champignons Filamenteux, INRA, Polytech Marseille, Aix
Marseille Universit, Marseille, France
xvi LIST OF CONTRIBUTORS
Alfredo Herrera-Estrella Laboratorio Nacional de Genmica
para la Biodiversidad, Cinvestav Sede Irapuato, Irapuato,
Guanajuato, Mexico
Robert Hill Bio-Protection Research Centre, Lincoln Univer
sity, 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 Agri
cultural Sciences, Uppsala, Sweden
J.J. Joensuu VTT Technical Research Centre of Finland,
Espoo, Finland
Magnus Karlsson Uppsala BioCenter, Department of Forest
Mycology and Plant Pathology, Swedish University of Agri
cultural Sciences, Uppsala, Sweden
Pter Krmczi Department of Microbiology, University of
Szeged, Szeged, Hungary
Lszl Kredics Department of Microbiology, University of
Szeged, Szeged, Hungary
Adinarayana Kunamneni Departamento de Biocatlisis,
Instituto de Catlisis y Petroleoqumica, CSIC, Madrid,
Spain
Gang Liu College of Life Science, Shenzhen University,
Shenzhen, China
Jess Salvador Lpez-Bucio Instituto de Biotecnologa,
Universidad Nacional Autnoma de Mxico, Cuernavaca,
Morelos, Mxico
Jos Lpez-Bucio Instituto de Investigaciones Qumico-
Biolgicas, Universidad Michoacana de San Nicols de
Hidalgo, Morelia, Michoacn, Mxico
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 Macas-RodrguezInstituto de Investigaciones
Qumico-Biolgicas, Universidad Michoacana de San
Nicols de Hidalgo, Morelia, Michoacn, Mxico
Lszl 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
Robert N.G. Miller Department of Cell Biology, University
of Brasilia, Brasilia, Federal District, Brazil
Vianey Olmedo Monfil Departamento de Biologa, Univer
sidad de Guanajuato, Guanajuato, Mxico
Enrique Monte Centro Hispano-Luso de Investigaciones
Agrarias (CIALE), University of Salamanca, Salamanca,
Spain
Valdirene Neves Monteiro Universidade Estadual de Gois,
Unidade Universitria de Cincias Exatas e Tecnolgicas da
Universidade Estadual de Gois-UnUCET, Anpolis, Gois,
Brazil
Hctor M. Mora-Montes Departamento de Biologa, Univer
sidad de Guanajuato, Guanajuato, Mxico
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
Helena Nevalainen Department of Chemistry and Biomo
lecular Sciences, Macquarie University, NSW, Australia;
Biomolecular Frontiers Research Centre, Macquarie Univer
sity, NSW, Australia
Eliane Ferreira Noronha Departamento de Biologia Celular,
Universidade de Brasilia, Brasilia, Federal District, Brasil
Anthonia ODonovan Molecular Glycobiotechnology
Group, Department of Biochemistry, School of Natural Sci
ences, National University of Ireland Galway, Galway,
Ireland
T. Pakula VTT Technical Research Centre of Finland, Espoo,
Finland
Robyn Peterson Department of Chemistry and Biomolecular
Sciences, Macquarie University, NSW, Australia; Biomolecu
lar Frontiers Research Centre, Macquarie University, NSW,
Australia
Francisco J. Plou Departamento de Biocatlisis, Instituto de
Catlisis y Petroleoqumica, CSIC, Madrid, Spain
Terhi Puranen Roal Oy, Rajamki, Finland
Lina Qin Institute of Microbiology, Chinese Academy of Sci
ences, 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
Mara Beln 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
Birinchi K. Sarma Department of Mycology and Plant
Pathology, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Monika Schmoll Health and Environment Department,
Austrian Institute of Technology GmbH (AIT), Tulln, Austria
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 Biotechnol
ogy and Microbiology, Vienna, Austria
Gauri Dutt Sharma Bilaspur University, Bilaspur, Chattis
garh, India
M. Shimizu Laboratory of Plant Pathology, Faculty of Applied
Biological Sciences, Gifu University, Gifu City, Japan
Dhara Shukla Facility for Ecological and Analytical Testing,
Indian Institute of Technology, Kanpur, Uttar Pradesh, India
Shafiquzzaman Siddiquee Biotechnology Research Institute,
University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia
Roberto Nascimento Silva Department of Biochemistry and
Immunology, School of Medicine, University of So Paulo,
Ribeiro Preto, So Paulo, Brazil
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
Gurpreet Singh Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India
Sudhanshu Singh International Rice Research Institute,
IRRI, New Delhi, India
U.S. Singh International Rice Research Institute, IRRI, New
Delhi, India
Andrei Stecca Steindorff Departamento de Biologia Celular,
Universidade de Braslia, Braslia, 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
xvii
Siu Kwan Sze School of Biological Sciences, Nanyang Tech
nological University, Singapore
Doris Tisch Health and Environment Department, Austrian
Institute of Technology GmbH (AIT), Tulln, Austria
Jos E. Trujillo-Esquivel Departamento de Biologa, Univer
sidad de Guanajuato, Guanajuato, Mxico
Maria G. Tuohy Molecular Glycobiotechnology Group,
Department of Biochemistry, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland
R.S. Upadhyay Department of Botany, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Csaba Vgvlgyi Department of Microbiology, University of
Szeged, Szeged, Hungary
Khabat Vahabi Institute of General Botany and Plant Physi
ology, Friedrich-Schiller University Jena, Jena, Germany
Padma S. Vankar Facility for Ecological and Analytical
Testing, Indian Institute of Technology, Kanpur, Uttar
Pradesh, India
Jari Vehmaanper Roal Oy, Rajamki, Finland
R.A. Vishwakarma Microbial Biotechnology Division, Indian
Institute of Integrative Medicine (CSIR), Jammu, Jammu and
Kashmir, India
Shaowen Wang College of Life Science, Shenzhen Univer
sity, Shenzhen, China
Christin Zachow Austrian Centre of Industrial Biotechnol
ogy (ACIB GmbH), Graz, Austria; Graz University of Tech
nology, Environmental Biotechnology, Graz, Austria
Najam W. Zaidi International Rice Research Institute, IRRI,
New Delhi, India
Susanne Zeilinger Research Area Biotechnology and Micro
biology, Institute of Chemical Engineering, Vienna Univer
sity 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
Lszl Kredics1, *, Lrnt Hatvani1, Shahram Naeimi2, Pter Krmczi1,
Lszl Manczinger1, Csaba Vgvlgyi1, Irina Druzhinina3
1Department
of Microbiology, University of Szeged, Szeged, Hungary,
2Department of Biological Control Research, Iranian Research Institute of Plant Protection, Amol, Mazandaran, Iran,
3Institute of Chemical Engineering, Vienna University of Technology, Research Area Biotechnology and Microbiology,

Vienna, Austria
*Corresponding author email: kredics@bio.u-szeged.hu

O U T L I N E
Introduction3 Living Plants (Endophytes) 11
Mushroom-Related Substrata 13
Methodology of Studying Trichoderma Biodiversity 3
Human Body 14
Methods for the Identification of Trichoderma Strains 3
Water-Related Environments 14
Evolution of the Approach: From the Culture-Based
Air and Settled Dust 17
Method to Metagenomics 4
Conclusions18
Trichoderma Diversity in Different Habitats 5
Natural Soils, Decaying Wood and Plant Material 5
Agricultural Habitats 9

INTRODUCTION METHODOLOGY OF STUDYING

Members of the genus Trichoderma are cosmopolitan
and prevalent components of different ecosystems in a
wide range of climatic zones (Kubicek etal. 2008). The
occurrence of Trichoderma species is modulated by sev-
eral 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 diver-
sity, high reproductive capacity and competitive capa-
bilities of Trichoderma strains in nature (Cardoso Lopes
etal. 2012). The aim of this chapter is to give an overview
about the studies aimed at the investigation of Tricho-
derma biodiversity in a wide variety of different ecologi-
cal habitats.
TRICHODERMA BIODIVERSITY
Methods for the Identification of Trichoderma
Strains
Formerly the species-level identification of Tricho-
derma/Hypocrea isolates was performed based on exclu-
sively their morphological characteristics (Danielson
and Davey, 1973; Summerbell, 2003; Gams and Bissett,
1998). Different media were used for culturing Tricho-
derma isolates for the analysis of their morphology and
culture characteristics, e.g. malt extract agar, which is
appropriate for conidium production and the observa-
tion of conidiophore branching, or potato dextrose agar,
which proved useful for observing pigment production

Biotechnology and Biology of Trichoderma 3

http://dx.doi.org/10.1016/B978-0-444-59576-8.00001-1 Copyright 2014 Elsevier B.V. All rights reserved.
4 1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
(Hoyos-Carvayal and Bissett, 2011). The preliminary
identification of species based on conidiophore struc-
ture, 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 etal., 2006b,
2012a,b). However, without professional expertise this
may often lead to incorrect diagnoses due to the difficul-
ties of morphology-based species identification, therefore
the results of early studies must be handled with special
care (Kubicek etal., 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 quanti-
tative measurements of growth and the assimilation of
different carbon and nitrogen sourcesproved to be a
useful tool to aid species identification and provide data
on the ecological roles of species (Kubicek etal., 2003;
Hoyos-Carvajal etal., 2009; Atanasova and Druzhinina,
2010).
A cellulose-acetate electrophoresis-based isoenzyme
analysiswith the involvement of glucose-6-phosphate
dehydrogenase, glucose-6-phosphate isomerase, 6-phos-
phogluconate dehydrogenase, peptidases A, B and D,
and phosphoglucomutase enzymes (Hebert and Beaton,
1993)was applied by Szekeres etal. (2006) and Kredics
etal. (2011a, 2012) for the identification of Trichoderma
strains deriving from clinical samples and winter wheat
fields, respectively.
Neuhof etal. (2007) suggested an alternative bio-
chemical technique for HypocreaTrichoderma 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 HypocreaTrichoderma
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-fingerprint-
ing (Arisan-Atac etal., 1995), the sequence analysis of
the ribosomal internal transcribed spacer (ITS) region
(ITS15.8S rDNAITS2) 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 etal., 2002; Druzhinina etal., 2008) about 12 species in arable soil. Later studies demon-
were found to be suitable for giving precise species iden-
tification of HypocreaTrichoderma strains.
The ITS-based online barcoding program TrichOKEY
(www.isth.info; Druzhinina etal., 2005; Druzhinina and
Kopchinskiy, 2006) provides another useful tool for
the identification of HypocreaTrichoderma species. The
A. BIOLOGY AND BIODIVERSITY
development and practical applications of ITS barcodes
are presented and discussed in chapter 3: DNA barcode
for species identification in Trichoderma. For the analy-
sis of tef1, ITS and rpb2 sequences the online programme
TrichoBLAST and its updated version, TrichoMARK are
recommended (www.isth.info; Kopchinskiy etal., 2005).
TrichoCHIT (www.isth.info), an online barcoding pro-
gramme for the screening and identification of excellent
chitinase producer strains of Hypocrea lixii/Trichoderma
harzianum was developed by Nagy etal. (2007).
The use of species-specific primers in polymerase
chain reaction can also lead to quick and precise diagno-
sis. For example, Chen etal. (1999a, b) developed a PCR-
based 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 etal. (2009) allows the rapid and
specific identification of Trichoderma pleurotum and Trich-
oderma pleuroticola, the causal agents of green mould in
the world-wide production of oyster mushroom (Pleu-
rotus osteatus) even directly from the growing substrate
without the need of cultivation.
Evolution of the Approach: From the Culture-
Based Method to Metagenomics
Most of the studies about Trichoderma biodiversity
applied the standard culture-based approach compris-
ing the collection of samples, isolation of Trichoderma
strains on one of the selective media described in the
literature (Elad etal., 1981; Papavizas and Lumsden,
1982; Askew and Laing, 1993; Williams etal., 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 habi-
tat. The application of the metagenomic approach pro-
vides 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 gath-
ering ground in Trichoderma biodiversity studies. In
the first metagenomic attempt, Hypocrea/Trichoderma-
specific primers were designed for the ITS1 fragment
of the rRNA gene cluster by Hagn etal. (2007). With
the application of this method, the authors found only
strated that ITS1 alone is not sufficiently diagnostic as
certain species share the same allele. Based on a master
alignment of ITS sequences, Meincke etal. (2010) devel-
oped a novel Trichoderma-specific primer pair for diver-
sity analysis, which amplifies an approximately 650bp
fragment of the ITS region suitable for identification
 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 fun-
gal 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 commu-
nities 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 30bp 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 etal., 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
sampleeach containing the same forward and one of
the reverse primersthe products are combined, puri-
fied 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 etal. (2010) applied this metage-
nomic 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) exam-
ined the Trichoderma propagules in a variety of forest soils
in the southeastern U.S. and Washington State and iden-
tified 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,
A. BIOLOGY AND BIODIVERSITY
5
T. koningii, T. pseudokoningii, and T. viride. Vajna (1983)
reported the isolation and morphology- as well as cul-
ture 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 bio-
diversity 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). Howeveras already mentionedthe results of
these early studies are hard to evaluate as no molecu-
lar 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. Nev-
ertheless, 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 rhizo-
plane of grasses of the subfamily Panicoideae in the Lak-
kavalli 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 etal.
(2000) studied 76 Trichoderma strains isolated from
Russiaincluding Siberiaand the Himalayas by ITS
sequence analysis, RAPD and DNA-fingerprinting and
reported the occurrence of T. asperellum, Trichoderma
atroviride, Trichoderma ghanense, T. hamatum, T. harzia-
num, T. koningii, Trichoderma oblongisporum, Trichoderma
virens as well as some previously undetected taxa, which
were later described based on morphological and physi-
ological characters as well as ITS1, 2 and tef1 sequences
as Trichoderma effusum, Trichoderma rossicum and Tricho-
derma velutinum (Bissett etal., 2003). The T. harzianum
species complex proved to be the most frequently occur-
ring and genetically most diverse taxon with six dif-
ferent ITS-genotypes (Kullnig etal., 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 iso-
lates (Kubicek etal., 2003), and revealed the occurrence
of T. asperellum, T. atroviride, T. ghanense, T. hamatum,
T. harzianum, Hypocrea jecorina/Trichoderma reesei, T. kon-
ingii, Trichoderma spirale, T. virens and T. viride. Based on
6 1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
the results of this study, the T. harzianum complex con-
tains 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 etal. (2000) could
also be isolated, along with additional four previously
undetected taxa, which were subsequently described by
Bissett etal. (2003) as Trichoderma cerinum, Trichoderma
erinaceum, Trichoderma helicum and Trichoderma sinense.
Zhang etal. (2005) examined the Trichoderma biodiver-
sity and biogeography on 135 isolates deriving from
four regions of China: provinces Hebei (North), Zheji-
ang (South-East), Yunnan (West) and the Himalayan
part (Tibet) and identified T. asperellum, T. atroviride, T.
cerinum, Trichoderma citrinoviride, T. harzianum, T. konin-
gii, 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 ori-
gin of a unique haplotype of T. harzianum. In a recent
study, Sun etal. (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, Tricho-
derma stromaticum, Trichoderma vermipilum and Hypocrea
virens/T. virens) from Chinese forest soils by ITS barcod-
ing. Tsurumi etal. (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. harzia-
num 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 etal. (2010) isolated Tricho-
derma 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 etal., 2010). Further species
known from Asia include Hypocrea catoptron/Tricho-
derma catoptron, Hypocrea cornea/T. sp., Hypocrea crassa/T.
crassum, Hypocrea rugulosa, T. spirale, Hypocrea tawa/
Trichoderma tawa, Hypocrea thailandica/Trichoderma thai-
landicum and Hypocrea thelephoricola/Trichoderma the-
lephoricola (Chaverri and Samuels, 2003), Trichoderma
capillare, Trichoderma gracile, Trichoderma parareesei and
Trichoderma pinnatum (Samuels etal., 2012a), Trichoderma
barbatum, Trichoderma caesareum/H. sp. and Trichoderma
A. BIOLOGY AND BIODIVERSITY
floccosum/H. sp. (Samuels etal., 2012b), as well as Trich-
oderma arundinaceum (Degenkolb etal., 2008).
Thanks to a series of biodiversity studies, plenty of
information is available about the biodiversity of the
genus in Europe. Wuczkowski etal. (2003) studied the
diversity of the genus Trichoderma in an original Euro-
pean 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 etal. (2012a) as T. capillare. Mysterud etal.
(2007) examined the plant litter-associated fungi from
the spring grazing corridor of a sheep herd in west-
ern Norway and detected a wide variety of fungi includ-
ing two Trichoderma isolates that the authors failed to
identify by NCBI BLAST search of their ITS sequences.
A search with TrichOKEY (Druzhinina etal., 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 biodiver-
sity of the Hypocrea/Trichoderma genus based on 620
specimens by examining their morphology, culture char-
acteristics 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 spe-
cies were detected including previously described species
(the holomorphs Hypocrea atroviridis/T. atroviride, Hypocrea
aureoviridis/T. aureoviride, Hypocrea citrina/Trichoderma
lacteum, Hypocrea epimyces/Trichoderma epimyces, Hypo-
crea gelatinosa/Trichoderma gelatinosum, H. lixii/T. harzia-
num, Hypocrea lutea/Trichoderma deliquescens, Hypocrea
koningii/T. koningii, Hypocrea minutispora/Tricho-
derma 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., Hypo-
crea rufa/T.viride, Hypocrea schweinitzii/T. citrinoviride,
Hypocrea sulphurea/T. sp. as well as Hypocrea species
without known anamorphs: Hypocrea argillacea, Hypo-
crea spinulosa, Hypocrea splendens, Hypocrea strobilina),
new taxa (the holomorphs Hypocrea aeruginea/Tricho-
derma aerugineum, Hypocrea albolutescens/Trichoderma
albolutescens, Hypocrea atlantica/Trichoderma atlanti
cum, Hypocrea auranteffusa/Trichoderma auranteffusum,
Hypocrea austriaca/Trichoderma austriacum, Hypocrea
bavarica/Trichoderma bavaricum, Hypocrea calamagrostidis/
 TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
Trichoderma calamagrostidis, Hypocrea fomiticola/Trichod
erma fomiticola, Hypocrea junci/Trichoderma junci, Hypocrea
luteffusa/Trichoderma luteffusum, Hypocrea luteocrystallina/
Trichoderma luteocrystallinum, Hypocrea margaretensis/
Trichoderma margaretense, Hypocrea neorufoides/Trichoderma
neorufoides, Hypocrea pachypallida/Trichoderma pachypal-
lidum, Hypocrea parepimyces/Trichoderma parepimyces,
Hypocrea parestonica/Trichoderma parestonicum, Hypo-
crea phellinicola/Trichoderma phellinicola, Hypocrea silvae-
virgineae/Trichoderma silvae-virgineae, Hypocrea subeffusa/
Trichoderma subeffusum and Hypocrea valdunensis/Tricho-
derma valdunense; the new teleomorphs Hypocrea danica,
Hypocrea rhododendri and Hypocrea sambuci; Hypocrea lon-
gipilosa described as the teleomorph state of Trichoderma
longipile; as well as Trichoderma alutaceum, Trichoderma
dacrymycellum, Trichoderma delicatulum, Trichoderma leu-
copus, 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 phyllo-
stachidis, Hypocrea sinuosa/Trichoderma sinuosum, Hypo-
crea strictipilosa/Trichoderma strictipile, H. thelephoricola
(Chaverry and Samuels, 2003), Hypocrea stilbohypoxyli
(Lu and Samuels, 2003), Hypocrea parapilulifera (Lu etal.,
2004), Hypocrea nybergiana (Chamberlain etal., 2004),
Hypocrea voglmayrii (Jaklitsch etal., 2005), Hypocrea
alcalifuscescens/T. sp. and Hypocrea parmastoi/T. sp. (Over-
ton etal., 2006), Hypocrea petersenii and Hypocrea rogerso-
nii (Samuels etal., 2006b), Hypocrea crystalligena (Jaklitsch
etal., 2006a), Hypocrea viridescens (Jaklitsch etal., 2006b),
Hypocrea alni and Hypocrea brunneoviridis (Jaklitsch etal.,
2008a), Hypocrea decipiens (Jaklitsch etal., 2008b), Hypo-
crea seppoi (Jaklitsch etal., 2008c) and Hypocrea rodmanii
(Degenkolb etal., 2008) also occur in Europe. From the
Longibrachiatum clade, Trichoderma saturnisporum is also
known from Sardinia (Samuels etal., 2012a). Results of
these studies demonstrated that not just a small num-
ber of Trichoderma species are capable of forming a tele-
omorph 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 sug-
gested 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 Vogl-
mayr, 2012), Hypocrea caerulescens, Hypocrea hispanica and
Trichoderma samuelsii (Jaklitsch etal. 2012). During an ITS
barcoding-based study on the species diversity of Tricho-
derma in Poland, Baszczyk etal. (2011) found that soil
and decaying wood were the most diverse among the
examined substrata. Species detected in forest soils were
A. BIOLOGY AND BIODIVERSITY
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. Identi-
fied taxa comprised H. alni, T. asperellum, H. atroviridis/T.
atroviride, T. brevicompactum, T. cerinum, T. harzianum
sensu stricto, H. pachybasioides, H. pachypallida, T. pleu-
roticola, 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 dis-
tribution 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, Tricho-
derma pubescens, H. strictipilosa/T. strictipile, Trichoderma
strigosum, Hypocrea surrotunda/Trichoderma surrotundum
and T. tomentosum with conidiophore elongations and
green conidia (Chaverri etal., 2003), the green asco-
spored species Hypocrea ceracea/Trichoderma ceraceum,
H. ceramica/Trichoderma ceramicum, Hypocrea chlorospora/
Trichoderma chlorosporum, Hypocrea chromosperma/Tricho-
derma chromospermum, Hypocrea cinnamomea/Trichoderma
cinnamomeum, H. crassa/T. crassum, H. cremea/Tricho-
derma cremeum, H. cuneispora/Trichoderma cuneisporum, H.
lixii/T. harzianum, H. sinuosa/T. sinuosum, H. strictipilosa/T.
strictipile and H. virens/T. virens (Chaverri and S amuels,
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
etal., 2012a), T. arundinaceum, T. brevicompactum and
H. rodmanii from the T. brevicompactum clade (Degenkolb
etal., 2008), H. alcalifuscescens/T. sp., Hypocrea farinosa/
T. sp. and H. sulphurea/T. sp. (Overton etal., 2006), T. bar-
batum (Samuels etal., 2012b) as well as T. capillare (Samu-
els etal., 2012a).
Central American species occurring in Costa
Rica comprise Hypocrea candida/Trichoderma candidum,
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
etal., 2003), T. brevicompactum and Trichoderma turri-
albense (Degenkolb etal., 2008), Hypocrea flaviconidia
(Druzhinina etal., 2004) as well as Hypocrea eucorticioides/
T. sp. (Overton etal., 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 etal.
(2009) to study the biodiversity of Trichoderma species
in habitats of neotropic regions in Peru, Mexico, Gua-
temala 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 pre-
dominant 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. asperel-
lum, Trichoderma asperelloides, T. atroviride, T. brevicom-
pactum, 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. longi-
brachiatum, Trichoderma neokoningii, T. ovalisporum, T. para-
reesei, 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 Ven-
ezuelan soils and found the T. harzianum species com-
plex to be the most frequently occurring taxon, followed
by T. virens, T. brevicompactum, T. theobromicola, T. kon-
ingiopsis, T. ovalisporum, T. asperellum, T. pleurotum and
T. koningiopsis. Other species known from South America
include T. strigosum and Hypocrea stromatica/Trichoderma
stromaticum (Chaverri etal., 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 andi-
nensis (Samuels etal., 2012a) and H. eucorticioides/T. sp.
(Overton etal., 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 investi-
gated by Okoth etal. (2009). Species isolated from indig-
enous forests of the Embu region in order of prevalence
were T. harzianum, T. viride, Trichoderma aggressivum and
A. BIOLOGY AND BIODIVERSITY
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 tech-
niques, therefore results like the reported occurrence of
the mushroom pathogenic species T. aggressivum in a nat-
ural habitat need to be handled critically. Sadfi-Zouaoui
etal. (2009) studied four different bioclimatic zones in
Tunisia for Trichoderma diversity. The T. harzianum spe-
cies complex proved to be the most prevalent taxon
identified. Trichoderma harzianum and T. longibrachia-
tum 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. har-
zianum 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 etal.,
2012a), H. catoptron/T. catoptron (Chaverri and Samuels,
2003), Hypocrea subcitrina (Overton etal., 2006) and T.
vermipilum (Samuels etal., 2012b) from South Africa,
T. arundinaceum from Namibia (Degenkolb etal., 2008),
Hypocrea subsulphurea (Overton etal., 2006), as well as
Trichoderma lanuginosum/H. sp., Trichoderma medusae/H.
sp. from Cameroon and Trichoderma ivoriense from Ivory
Coast (Samuels etal., 2012b).
Studying the biodiversity of the genus in islands
as geographically separated regions may reveal
important data about endemic taxa as well as inva-
sive ones arriving from the nearby continents. Spe-
cies reported from New Zealand include T. crassum,
Hypocrea semiorbis/Trichoderma sp. and H. tawa/T. tawa
(Chaverri etal., 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
etal., 2012a), as well as H. subcitrina (Overton etal.,
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
etal., 2006), as well as H. sulphurea/T. sp. and Hypo-
crea victoriensis/T. sp. (Overton etal., 2006). The
species Hypocrea melanomagna/Trichoderma melano-
magnum 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/
 TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
T. pseudokoningii are known from Sri Lanka (Samuels
etal., 2012a). The Trichoderma communities of the island of
Sardinia were studied by Migheli etal. (2009). Fifteen
soil samples from different habitats including undis-
turbed 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. spi-
rale, 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. hama-
tum, 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 etal., 2012a). Zachow etal. (2009) examined
the fungal biodiversity of soils at different vegetation
regions on Tenerife (Canary Islands). From the genus
Trichoderma/Hypocrea, the species isolated and identi-
fied by TrichOKEY were Trichoderma chionea, T. gamsii,
T. harzianum, H. rufa/T. viride, T. spirale and T. tomen-
tosum, 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 fertiliz-
ers as well as the geographical region.
Trichoderma spp. can be theoretically isolated from
almost all types of agricultural fields. They have sev-
eral 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 etal. 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 etal.
2011). Members of the genus were more frequently
A. BIOLOGY AND BIODIVERSITY
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 oth-
ers are limited to specific geographical areas (Harman
etal. 2004).
The majority of the research which involved the iso-
lation 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 num-
ber of studies deal with population, abundance and
diversity of the genus Trichoderma in specific crop fields
or agroecosystems.
Cereal crop fieldsTrichoderma spp. proved to be
among the dominating fungi in cereal (rye, triticale,
wheat) field soils in Poland (Pita etal. 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 etal. 2003). Tricho-
derma 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 iso-
lates. Diversity of Trichoderma spp. was very high in soil
samples of wheat fields of China (Liang etal. 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) com-
prising T. atroviride, T. brevicompactum, T. gamsii, T. har-
zianum, 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
etal. 2012). Trichoderma harzianum was the most abun-
dant species representing various ITS haplotypes includ-
ing 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 dif-
fered 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. harzia-
num and the anamorph of H. orientalis (Gherbawy etal.
2004). This low degree of diversity may occur due to the
alkalinity of the investigated soils (pH=7.38.4). Tricho-
derma 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
10 1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
the habitat. Corn field soils in Egypt (Gherbawy etal.
2004) and Mexico (Snchez-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. asperel-
lum, T. atroviride, T. erinaceum, T. harzianum, T. koningi-
opsis, 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 iso-
lates 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 etal. 2000). Similarly, T. harzianum was
the most common species in rice field soils in Bangla-
desh (Mostafa Kamal and Shahjahan, 1995), as well as in
upland and lowland rice fields of the Philippines (Naga-
mani 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 barcode-
based identification (Naeimi etal. 2010). Like the rhizo-
sphere of winter wheat in Hungary (Kredics etal. 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 inten-
sively 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 haplo-
types clustered into four groups. Trichoderma virens was
the only other species from this study that showed intra-
specific variation with three different genotypes in one
clade. Correlation of the genotypes with sampling site or
substrate (soil/phyllosphere) was not observed. In addi-
tion, the results suggested that different genotypes could
coexist in a single habitat (Naeimi etal. 2011).
Potato (Solanum tuberosum L.) rhizosphereSix species:
T. asperellum, T. atroviride, T. hamatum, T. harzianum, T. kon-
ingii 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). Tricho-
derma hamatum, T. harzianum and T. koningii appeared
to be the most frequent species. Moreover, biodiversity
analysis of Trichoderma communities in the rhizosphere
A. BIOLOGY AND BIODIVERSITY
soil of different potato cultivars grown in two fields
located in Southern Germany revealed that the popu-
lation of Trichoderma spp. and species diversity were
site-dependent, and high field heterogeneity of Tricho-
derma communities was revealed by DGGE fingerprints,
although differences among them were not statistically
significant (Meincke etal. 2010). A study undertaken in
Poland showed that Trichoderma spp. were predominant
in potato field soils (Pita etal. 2000).
Coffee (Coffea arabica) rhizosphereTrichoderma iso-
lates 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 fol-
lowing in order of abundance: T. harzianum and T. hama-
tum (the most predominant species in both habitats),
T. asperelloides, T. spirale, T. atroviride, T. koningiopsis, T.
gamsii and T. longibrachiatum (Mulaw etal. 2010). Cul-
tivated 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 Tricho-
derma 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. More-
over, strains of T. hamatum, T. atroviride and T. spirale rep-
resented 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.) rhizosphereOne hundred
and thirty five Trichoderma isolates collected from rhizo-
spheres in different locations across the Ivory Coast were
identified by ITS-barcoding as T. asperellum, T. harzianum,
T. virens and T. spirale (Mpika etal. 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.) rhizosphereSixteen
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 etal.
2010). Trichoderma velutinum and T. gamsii were the most
prevalent species.
Oilseed rape (Brassica napus L.) rhizosphereTrichoderma
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
(Fragariaananassa Duch.) in different locations of
Germany (Berg etal. 2005). Diversity and abundance of
Trichoderma in bulk soil was higher than in rhizosphere
soil and the occurring species showed more genotypic
 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 pro-
ductivity (Slezack-Deschaumes etal. 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 inter-
and intraspecific diversity in terms of metabolic pro-
files and assimilation of carbon sources was reported
(Cardoso Lopes etal. 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 unculti-
vated oil palm ecosystems (Sariah etal. 2005). Popula-
tions 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 cropsTrichoderma hamatum,
T. harzianum, T. koningii, T. pseudokoningii and T. viride
were detected from soybean (Glycine max (L.) Merr.) soils
in Poland (Pita 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 iden-
tified in Mexico from soils cultivated with Sorghum bicolor
based on morphological characteristics, enzymatic activ-
ity, macro- and microculture test results, rDNA restric-
tion patterns (AFLP), ITS15.8SITS2 rDNA sequences
and protein profiles (Larralde-Corona etal. 2008). In
Japan, T. hamatum, T. harzianum, T. koningii and T. viride
were identified from soils of a radish (Raphanus sativus
L.) field (Mghalu etal. 2007). Eleven species of Tricho-
derma 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 criteriawere the most dominant species
among 150 fungal species in cucumber rhizosphere soils
in Switzerland (Girlanda etal. 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 etal. 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
A. BIOLOGY AND BIODIVERSITY
11
(Shanmugam etal. 2008). Trichoderma spp. were report-
edly 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) rhi-
zosphere in South Korea (Hyun-Sung and Lee, 1986)
and arecanut palm (Areca catechu L.) rhizosphere in India
(Bopaiah and Bhat, 1981).
Sadfi-Zouaoui etal. (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 etal. (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, vegeta-
ble, farmland and pasture soils all over the country (Sun
etal. 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. longibra-
chiatum, T. stromaticum, T. velutinum), garden soils (eight
detected species: T. asperellum, T. erinaceum, T. hamatum,
T. harzianum, T. longibrachiatum, T. pleuroticola, T. tomento-
sum, 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 phyloge-
netic analysis of their ITS and tef1 sequences, T. harzia-
num 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 sym-
biotic 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 com-
mon in various tropical countries of the world. Certain
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 spe-
cies), witches' broom (Crinipellis perniciosa) and frosty
pod rot (Moniliophtora roreri) (Bailey etal., 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 exami-
nations proved that a series of Trichoderma species may
occur as endophytes of the cocoa plant, including mem-
bers of the former T. koningii species aggregate such as T.
ovalisporum and the new species T. caribbaeum var. aequa-
toriale, T. koningiopsis (Samuels etal., 2006a) and Tricho-
derma protrudens (Degenkolb etal., 2008). Trichoderma
theobromicola and T. paucisporum (Samuels etal., 2006a),
T. stromaticum (Samuels etal., 2012b) as well as Tricho-
derma martiale (Hanada etal., 2008) were also identified
as endophytic Trichoderma species of cocoa. Rubini etal.
(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 endo-
phytic microbiota of coffee seedlings was also reported
to contain Trichoderma species including T. hamatum and
T. harzianum identified by ITS sequence analysis (Posada
etal., 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 etal. (2011) described Trichoderma ama-
zonicum as a new species based on isolates from rubber
tree (Hevea spp.), Zhang etal. (2007) described Tricho-
derma taxi from Taxus mairei tree in China, while Samuels
etal. (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 etal.,
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 asperel-
lum and T. virens showed the highest frequencies in the
examined samples.
Dang etal. (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 well-
known 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 etal.,
2012). Moreover, in an independent examination, Chen
A. BIOLOGY AND BIODIVERSITY
etal. (2011) also identified T. atroviride as well as T. gam-
sii 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 Trich-
oderma 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 etal. (2012a,b). Souza etal. (2008) studied the
secondary metabolite production of a T. koningii iso-
late (identified by ITS sequence analysis) derived
from Strychnos cogens and described the production
of koninginins A, F and E. Two new octahydronaph-
thalene derivatives produced by a T. spirale strain iso-
lated from Aquilaria sinensis (Li etal., 2012), as well
as trichodermanin A produced by T. atroviride isolated
from Cephalotaxus fortunei (Sun etal., 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 rotun-
difolia 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 Den-
drobium nobile and Dendrobium chrysanthum belonging to
the Orchidaceae family (Chen etal., 2012). The presence
of Trichoderma species was proved among the 127 endo-
phytic fungal isolates. Moreover, Yuan etal. (2009) inves-
tigated 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 seri-
ous 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 endo-
phytic 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.
 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 (Pleuro-
tus ostreatus), causing serious losses for growers world-
wide. Overviews of the current status of A. bisporus and
P. ostreatus green mould were given by Kredics etal.
(2010) and Hatvani etal. (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 spe-
cies, causing aggressive compost colonization were
identified exclusively as the T. harzianum biotypes Th2
(Seaby, 1987, 1989; Doyle, 1991) and Th4 (Castle etal.
1998) in Great Britain/Ireland and United States/
Canada, respectively. The appearance of green mould
due to T. harzianum b iotype Th2 in other Western Euro-
pean countries was subsequently reported (Hermosa
etal. 1999; Mamoun etal. 2000). Based on morphologi-
cal characteristics as well as the phylogenetic analyses
of ITS1 and a fragment of the tef1 gene, Samuels etal.
(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. aggressi-
vum, respectively.
The cultivation of A. bisporus in Hungary was found
to be affected mostly by T. aggressivum f. europaeum, indi-
cating the spread of the Agaricus green mould epidemic
from Western to Central Europe. Besides T. aggressivum,
further five Trichoderma species, T. asperellum, T. atro-
viride, T. ghanense, T. harzianum and T. longibrachiatum
were detected in compost samples (Hatvani etal. 2007).
The holotype strain of the recently described new s pecies
T. capillare (Samuels etal., 2012a) was also recovered from
a Hungarian Agaricus-producing facility: it was isolated
from the wall of a mushroom growing cellar (Hatvani
etal., 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) (Baszczyk etal. 2011). This finding demon-
strates a change in the representation of species, as an
earlier study revealed the dominance of T. harzianum in
the country (Szczech etal., 2008). Green mould-affected
Agaricus compost in Croatia yielded exclusively T. har-
zianum, indicating a broadening spectrum of Trichoderma
species being able to cause green mould disease in but-
ton mushroom cultivation (Hatvani etal. 2012).
A. BIOLOGY AND BIODIVERSITY
13
Species found in green mould-affected oyster mush-
room substrate in Hungary were T. asperellum, T. a troviride,
T. longibrachiatum, and the yet undescribed species Tricho-
derma sp. DAOM 175924, which represented 90% of the
isolates (Hatvani etal. 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 corre-
sponded to Trichoderma sp. K1 and K2, the Pleurotus patho-
genic Trichoderma species observed in Korea (Park etal.
2004), which were subsequently described as the new
species T. pleurotum and T. pleuroticola (Park etal. 2006).
The results of the comprehensive study of Komo-
Zelazowska etal. (2007) confirmed that these two species
were responsible for green mould infections in Pleuro-
tus farms in various countries, such as Italy, Hungary,
Romania and the Netherlands. In Croatia the same spe-
cies were found to cause oyster mushroom green mould,
being the sole species recovered from infected Pleurotus
substrate samples (Hatvani etal. 2012). Pleurotus green
mould in Spain was shown to be caused exclusively
by T. pleurotum (Gea, 2009). Kredics etal. (2009) devel-
oped a PCR-based technique for the specific detection
of T. pleurotum and T. pleuroticola, the Trichoderma patho-
gens of cultivated oyster mushroom. Through the use of
the newly introduced method, the presence of T. pleu-
roticola 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 infec-
tion 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, fur-
ther investigations revealed that the natural substratum
of oyster mushroom is a habitat of this species as well
(Kredics etal. unpublished data). Recently, Kim etal.
(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 fre-
quent contaminants of shiitake (Lentinula edodes) cultiva-
tion 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 propor-
tions (Pukahuta etal. 2000). Turczi etal. (1996) reported
the isolation of T. hamatum from the fruiting bodies of
Lentinula edodes. The strains showed intermediate antag-
onistic properties towards phytopathogenic fungi.
Certain members of the genus Trichoderma (T. hama-
tum, T. harzianum, T. koningii, T. virens and T. viride) were
shown to be among the most abundant microfungi iso-
lated from the surroundings of wild Termitomyces species
in Thailand (Manoch etal., 2002), suggesting their poten-
tial 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
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 etal., 2008).
Rivera etal. (2010) reported the presence of Tricho-
derma species at 16% of the moulds isolated from the
ascocarps of the truffle Tuber aestivum. Wang etal. (2011)
examined the microbial communities of wild Chroogom-
phus rutilus, and found that T. koningiopsis strains repre-
sented 28.6% of the fungal isolates.
Human Body
The role of Trichoderma species as facultative patho-
gens of humans was firstly summarized by Kredics etal.
(2003) and later extensively reviewed and discussed
(Kredics etal., 2011b). Infections caused by Trichoderma
species known from the literature include peritonitis and
intra-abdominal abscess in patients undergoing continu-
ous ambulatory peritoneal dialysis (CAPD), liver infec-
tion, acute invasive sinusitis and disseminated infections
(e.g. abdominal, lung and skin disseminations) of trans-
plant recipients, brain abscess, skin infection, necrotiz-
ing stomatitis and pulmonary infections of patients with
hematological malignancies, fungemia by contaminated
saline, rhinosinusitis, pulmonary mycetoma and fibro-
sis, hypersensitivity pneumonitis, endocarditis, otitis
externa, cerebrospinal fluid infection and allergic reac-
tions (Kredics etal., 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 question-
able 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 morphol-
ogy-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 Tricho-
derma isolates.
As a biochemical solution, cellulose-acetate electro-
phoresis-based isoenzyme analysis according to Hebert
and Beaton (1993) was performed by Szekeres etal.
(2006) for the identification of clinical Trichoderma iso-
lates. The authors suggested this method as a cheap
and efficient alternative of molecular techniques for
the quick and specific identification of clinical T. lon-
gibrachiatum isolates. DNA-based molecular methods
applied to assess the genetic diversity of clinical Tricho-
derma isolates include RFLP of the mitochondrial DNA
A. BIOLOGY AND BIODIVERSITY
(Antal etal., 2006) as well as PCR-fingerprinting and ITS
sequence analysis (Kuhls etal. 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. orien-
talis 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
etal. (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 identi-
fied as an opportunistic human pathogen, and clinical T.
longibrachiatum isolates were shown not to form a par-
ticular subpopulation of the species. These findings sug-
gest that all strains of T. longibrachiatum and H. orientalis
might be able to cause infections in humans.
Besides T. longibrachiatum and H. orientalis, the involve-
ment of further four Trichoderma species was confirmed
with molecular tools: T. atroviride (Ranque etal., 2008),
T. citrinoviride (Kuhls etal. 1999), T. harzianum (Guarro
etal., 1999; Kantarciolu etal., 2009) and an unknown
Hypocreaceae species close to the genus Hypocrea/Tricho-
derma (Druzhinina etal., 2007), which shares identical
ITS and rpb2 sequences with Hypocrea peltata, a recently
described sexually reproducing Hypocrea species with-
out 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 nitro-
gen sources (Antal etal., 2005). Antifungal susceptibil-
ity 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 etal., 2011b).
Water-Related Environments
Trichoderma species were shown to be abundant in
ifferent water-related environments including marine
d
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 A gelas
 TRICHODERMA DIVERSITY IN DIFFERENT HABITATS
dispar collected from waters around the Island of Domi-
nica (Neumann etal., 2007), Latrunculia corticata from
the Red Sea at Sharm El-Sheikh, Egypt (El-Bondkly
etal., 2012), Geodia corticostylifera form the South Atlantic
Ocean, Brazil (Rocha etal., 2012), Mycale fibrexilis from
the South China Sea nearby Yongxing Island (Zhou
etal., 2011), Suberites zeteki from Rainbow Bay Marina
in Pearl Harbor and Gelliodes fibrosa from Coconut
Island in Kaneohe Bay on Oahu, Hawaii (Wang etal.
2008) and Psammocinia sp. from the Mediterranean Sea
at Sedot-Yam, Israel (Paz etal., 2010; Gal-Hemed etal.,
2011). Other sea animals from which the isolation of
Trichoderma strains was reported include the gorgonian
sea fan Annella sp. (Khamtong etal., 2012), the seastar
Acanthaster planci (Lan etal., 2012), the blue mussel Myti-
lus edulis (Ruiz etal., 2007a,b; Sallenave-Namont etal.,
2000) and the cockle Cerastoderma edule (Sallenave etal.,
1999; Sallenave-Namont etal., 2000), where the pres-
ence of Trichoderma was shown to contribute to shellfish
toxicity (Sallenave etal., 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 sedi-
ments on the root of mangrove Ceriops tagal collected in
the South Sea intertidal zone, China (Sun etal., 2009),
as well as from marine sediments collected at different
locations including the South China Sea (Burtseva etal.,
2003; Song etal., 2010), the Fujian province of China (Du
etal., 2009), the tideland of the Yellow Sea at Lianyun-
gang, China (Sun etal., 2008), various regions of the
Sea of Japan (Khudiakova etal., 2000), St. Helena Bay,
South Africa (Mouton etal., 2012), marine shellfish farm-
ing areas along the Western coast of France (Sallenave-
Namont etal., 2000) and the Antarctic Penguin Island
(Ren etal., 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
etal., 2008).
Marine-derived fungi including Trichoderma species
are attracting increasing interest as potential sources
of metabolites (Table 1.1) with a wide range of biologi-
cal activities including antibacterial activity against
methicillin-resistant S. aureus (Khamtong etal., 2012),
cytotoxicity against human colon carcinoma cells (Garo
etal., 2003), a melanoma cell line (Sun etal., 2006), other
cancer cell lines and bioactivities against HIV protease
(You etal., 2010). El-Bondkly etal. (2012) used a marine
Trichoderma strain for intergeneric protoplast fusion with
Penicillium and Aspergillus strains to improve cellulase
production. Activities of -1,3-glucanase (Burtseva etal.,
2003) and laminarinase enzymes (Burtseva etal., 2006)
A. BIOLOGY AND BIODIVERSITY
15
as well as a tyrosinase inhibitor (Tsuchiya etal., 2008)
were also studied in the case of marine-derived Tricho-
derma strains. A Trichoderma strain isolated from the
marine sponge G. corticostylifera was found to catalyze
the bioreduction of a-chloroacetophenone (Rocha etal.,
2009), the hydrolysis of benzyl glycidyl ether (Martins
etal., 2011) and the asymmetric bioconversion of iodo-
acetophenones to the corresponding iodophenyletha-
nols (Rocha etal., 2012). The potential application of
a strain (reported as T. viride) from sea water samples
collected from a heavy metal-polluted area in the Medi-
terranean Sea, Alexandria, Egypt was suggested for the
mycoremediation of Cr (VI) from water systems (El-
Kassas 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
etal., 2007a).
As the species level identification of marine Tricho-
derma isolates has not been performed in the majority of
these studies or it has been performed based on morpho-
logical 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 etal., 2012
identified an isolate as T. aureoviride while Song etal.
(2010) another one as T. koningii, however, a TrichOKEY
2.0 analysis of the respective sequences (GenBank acces-
sion numbers EU714396 and GU244589) reveals the
identity of the isolates as T. harzianum and T. koningiop-
sis/ovalisporum, respectively. An exact identification was
provided in the study of Mohamed-Benkada etal. (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 stud-
ies of Paz etal. (2010) and Gal-Hemed etal. (2011). Paz
etal. (2010) collected samples from the sponge Psammo-
cinia sp. at a depth of 26m, approximately 200m off-
shore 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 identifi-
cation. Trichoderma species with mycoparasitic potential
were also recorded. Species identifications of 29 Tricho-
derma strains from Psammocinia sp. were refined in the
subsequent study by Gal-Hemed etal. (2011). The great-
est 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
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 etal. (2009)
4-(4,5-Dimethyl-1,3-dioxolan-2-yl)methyl-phenol, Lu etal. (2012)
(3-hydroxybutan-2-yl)5-oxopyrrolidine-2-carboxylate,
atroviridetide

Trichoderma sp. Marine sediment, Fujian Sorbicillinoids: 6-demethylsorbicillin, sohirnones A, Du etal. (2009)
strain f-13 province, China sorbicillin, 2,3-dihydrosorbicillin
Bisorbicillinoids: bisvertinolone, 10,11-dihydrobisvertinolone,
trichodimerol, dihydrotrichodimerol, bisorbicillinol,
bisvertinoquinol, bisorbibutenolide

T. asperellum Sediment, Penguin island, Peptaibols: asperelines AF Ren etal. (2009)

Antarctica

T. koningii Marine mud of the South Koninginins A, D, E, and F Song etal. (2010)
China Sea Polyketide derivatives: 7-O-methylkoninginin D,
trichodermaketones AD

Trichoderma sp. Unidentified marine sponge Aminolipopeptides: trichoderins A, A1 and B Pruksakorn etal.
(2010)

T. reesei Marine mud, tideland of Cyclotetrapeptide: trichoderide A Sun etal. (2006)

Lianyungang, China
Polyketide derivatives: trichodermatides AD Sun etal. (2008)

T. aureoviride PSU-F95 Gorgonian sea fan (Annella sp.) Trichodermaquinone, trichodermaxanthone, Khamtong etal.
coniothranthraquinone 1, aloesone, 2-(2S-hydroxypropyl)- (2012)
5-methyl-7-hydroxychromone, isorhodoptilometrin,
pachybasin, 1-hydroxy-3-methoxyanthraquinone
2-methylquinizarin, -hydroxypachybasin, crysophanol,
-hydroxyemodin

Trichoderma sp. Seastar (Acanthaster planci) Sorbicillinoids: (4Z)-sorbicillin, (2S)-2,3-dihydro-7-hydroxy- Lan etal. (2012)
6-methyl-2-[(E)-prop-1-enyl]-chroman-4-one, (2S)-2,3-
dihydro-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 IIV and B IIV Mohamed-Benkada
etal. (2006)

T. longibrachiatum Blue mussels (Mytilus edulis), Peptaibols: 21 new trichobrachins Ruiz etal. (2007b)
shellfish-farming area from the trichobrachin C I and II
estuary of the Loire river trichobrachin A IIIX

T. virens Sea water Dipeptides: trichodermamides A and B Garo etal. (2003)

Trichoderma sp. Deep sea sediment, South China Cholesta-7,22-diene-3b,5a,6b-triol You etal. (2010)
Sea Cyclopentenone: trichoderone

T. viride Caribbean sponge (Agelas dispar), 2-Furancarboxylic acid Abdel-Lateff (2008),

island of Dominica Pyranone derivative: trichopyrone Abdel-Lateff etal.
Sorbicillinoid polyketide derivatives: trichodermanone AD (2009)
Hexaketide derivatives: rezishanone, vertinolide
Dodecaketides: trichodimerol, bislongiquinolide
(trichotetronine), bisvertinol

new species, Trichoderma sp. O.Y. 2407 from Strictipi-
losa 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 mycopara-
sitic abilities might be potential halotolerant biocontrol
agents in arid agricultural zones, where saline water
irrigation is becoming more common (Gal-Hemed
etal., 2011).
The above-mentioned Trichoderma species are consid-
ered as facultative marine fungi (true terrestrial fungi
capable of growing in the marine environment). How-
ever, a recent study suggests also the existence of obli-
gate water-inhabiting Trichoderma species (Yamaguchi
etal., 2012): two new aero-aquatic Trichoderma species
with Pseudaegerita-like propagules, Trichoderma matsu-
shimae 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 etal., 2012) or in bottled water (Ribeiro etal.,
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 etal., 2006). In a sub-
sequent study, a total of 123 Trichoderma strains were iso-
lated from Norwegian surface-sourced drinking water
(Hageskal etal., 2008). Examined samples included raw
water, treated water, and water from private homes and
hospital installations. Eleven known Trichoderma/Hypo-
crea species and a group of unidentified Trichoderma
strains representing a separate, strongly supported sub-
clade 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 iden-
tified strains, being present in 22% of the surface-derived
water samples.
Water-damaged buildings represent a further water-
related 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 dominat-
ing microfungi (Lbeck etal., 2000; Thrane etal., 2001;
Ebbehj etal. 2002). Release of spores by common indoor
fungi including T. harzianum from wet wallpapered gyp-
sum boards was examined by Kildes etal. (2003). The
authors concluded that these spores might be respon-
sible for certain negative health effects related to build-
ings contaminated with moulds. Andersen etal. (2011)
studied samples from water-damaged building materi-
als 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 etal. (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
A. BIOLOGY AND BIODIVERSITY
17
and the USA, hospitals in Austria, Iraq, Poland, Finland
and USA (from air filters) (Madsen etal., 2007). Tricho-
derma was also found in air ducts of houses in Finland
(Madsen etal., 2007), in air conditioners in Saudi Arabia
(Madsen etal., 2007), in the air of a library and archive
storage facilities in Poland (Zieliska-Jankiewicz etal.,
2008), homes in New Orleans after hurricanes Katrina
and Rita (Rao etal., 2007), an orchid greenhouse (Magyar
etal., 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 fil-
amentous 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, bed-
spread/furniture surfaces), hospitals in Iraq and schools
without water damage in Denmark (Madsen etal., 2007),
as well as on the walls of damp homes in Croatia and in
wall scrapes from basement in the USA (Madsen etal.,
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 bal-
conies in Taiwan, in a coastal area in Lithuania, in Pinus
nigra and Quercus forests in Turkey (Madsen etal., 2007),
in the Belgrade Forest area near Istanbul (olakoglu,
2003), in Trujillo, Peru (Requejo, 1975), Britain (Richards,
1956), Barcelona (Spain) (Calvo etal. 1980), Manhattan
(Kansas, USA) (Kramer etal., 1959, 1964; Kramer and
Pady, 1960), Sagamihara (Japan) (Takatori etal., 1994)
and Israel (Barkai-Golan, 1958; Barkai-Golan and Glazer,
1962; Barkai-Golan etal., 1977).
Trichoderma was also found to be present in settled
dust of roofs of houses and stationary cars in Egypt
(Madsen etal., 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 Eng-
land 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 etal., 2007), as well as
from spots visibly contaminated with fine plastic par-
ticles 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 identi-
fication has not been carried out in most of the cases.
Where species names were also provided in these ear-
lier studies, the occurrence of T. album, T. hamatum, T.
harzianum, T. koningii, T. lignorum, T. viride, as well as
18 1. BIODIVERSITY OF THE GENUS HYPOCREA/TRICHODERMA IN DIFFERENT HABITATS
Trichoderma inhamatuma taxon commonly regarded
a synonym of H. lixii, but also suggested as a sepa-
rate species within T. harzianum sensu lato (Druzhinina
etal., 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 metage-
nomic analyses like the one of Atanasova etal. (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 Wie-
nerwald, 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. stro-
maticum, 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. asperel-
lum, 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 etal. 2010). Interestingly, the most
frequent taxon of the genus, the T. harzianum species
complex could not be detected in the examined air sam-
ples, although it was abundant in soil samples from the
same area. Previously it was suggested that T. harzia-
num can rarely be found in the air, because its conidia
may not be released easily from growth materials to the
air (Madsen etal. 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
(olakoglu, 2003), and in the case of immunocompro-
mised 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 informa-
tion about the possible exposure of growers working
in such facilities to Trichoderma conidia. Hansen etal.
(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 etal., 2012), exposure to
Trichoderma could be observed for growers working in a
greenhouse with senescent cucumber plants, a c abbage
field and in a broccoli packing hall, however, PCR-
analysis revealed that the Trichoderma isolates respon-
sible for the exposure were different from the biocontrol
agents applied.
A. BIOLOGY AND BIODIVERSITY
CONCLUSIONS
The studies discussed above reflect that the genus
Trichoderma/Hypocrea can be characterized with high
adaptability to various ecological environments. How-
ever, it is important to mention that the results of any
study aimed at the examination of Trichoderma biodi-
versity 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 exami-
nations of a specific habitat may reveal higher biodi-
versity 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 dur-
ing 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 Vgvlgyi was realized in the frames of
TMOP 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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