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Biopesticides and Bioagents Novel Tools for Pest
Management 1st Edition Md. Arshad Anwer Digital
Instant Download
Author(s): Md. Arshad Anwer
ISBN(s): 9781771885195, 177188519X
Edition: 1
File Details: PDF, 38.58 MB
Year: 2017
Language: english
BIOPESTICIDES AND BIOAGENTS
Novel Tools for Pest Management
BIOPESTICIDES AND BIOAGENTS
Novel Tools for Pest Management

Edited by
Md. Arshad Anwer
Apple Academic Press Inc. Apple Academic Press Inc.
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© 2018 by Apple Academic Press, Inc.
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Biopesticides and bioagents : novel tools for pest management / edited by Md. Arshad Anwer.
Includes bibliographical references and index.
Issued in print and electronic formats.
ISBN 978-1-77188-519-5 (hardcover).--ISBN 978-1-315-36555-8 (PDF)
1. Natural pesticides. 2. Biological pest control agents. 3. Phytopathogenic microorganisms--Control. 4.
Weeds--Control. I. Anwer, Md. Arshad, editor
SB951.145.N37B56 2017 632’.95 C2017-903060-4 C2017-903061-2

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Names: Anwer, Md. Arshad, editor.

Title: Biopesticides and bioagents : novel tools for pest management / editor: Md. Arshad Anwer.
Description: Waretown, NJ : Apple Academic Press, 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2017019826 (print) | LCCN 2017022123 (ebook) | ISBN 9781315365558 (ebook) |
ISBN 9781771885195 (hardcover : alk. paper)
Subjects: LCSH: Natural pesticides. | Biological pest control agents. | Phytopathogenic microorganisms-
-Control. | Weeds--Control.
Classification: LCC SB951.145.N37 (ebook) | LCC SB951.145.N37 B53 2017 (print) | DDC 632/.96--
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ABOUT THE EDITOR

Md. Arshad Anwer, PhD

Md. Arshad Anwer, PhD, is currently Assistant Professor-cum-Jr. Scien-
tist in the Department of Plant Pathology at Bihar Agricultural Univer-
sity (BAU), Sabour-Bhagalpur, Bihar, India. He is engaged in developing
low-cost biopesticides that have increased shelf life. His areas of interest
include the development of preliminary information of economically
important pathogens associated with maize and the ability to recognize
key diseases and their hot spots through survey and surveillance of major
crops under agro-ecological condition. He is also working on Panama wilt
of banana and host specificity of plant pathogen, evaluation of biocon-
trol agents against wilts of several crops, and establishment of sick-plots
for studies on several soil borne plant pathogens. He is associated with
more than eight research groups at BAU, Sabour, including maize research
team, viz. organic farming, host parasite research groups, bacteriology,
mycology research group, biopesticides and bio-fertilizer research groups.
He teaches undergraduate as well as postgraduate students and has
published 24 peer-reviewed research papers as well as one book and seven
book chapters, mostly in publications from the USA, UK and Italy.
 Dedicated to
my wife Aishatul Bushra, my son Md. Huzaifa Anwer,
and my daughter Aamna Anwer
CONTENTS

List of Contributors....................................................................................... ix
List of Abbreviations................................................................................... xiii
Preface..........................................................................................................xv

1. Status of Biopesticides and Biocontrol Agents in Agriculture:

An Overview................................................................................................. 1
Md. Arshad Anwer

Part I: Plant Health Promoting Biocontrol Agents............................. 15

2. Trichoderma—An Impeccable Plant Health Booster.............................. 17
Najam Waris Zaidi and Uma Shankar Singh

3. Aspergillus niger: A Phosphate Solubilizing Fungus as

Biocontrol Agent......................................................................................... 43
Md. Arshad Anwer, Kundan Singh, and Raj Narain Singh

4. Flourescent Pseudomonads: A Potential Bio-Control Agents

Against Plant Diseases............................................................................... 77
Kahkashan Arzoo, Erayya, and Nishant Prakash

Part II: Entomopathogens..................................................................... 99

5. Entomopathogenic Fungi: Introduction, History, Classification,
Infection Mechanism, Enzymes, and Toxins......................................... 101
Waheed Anwar, Ahmad Ali Shahid, and Muhammad Saleem Haider

6. Entomopathogens and Their Mass Production and Application......... 153

Kalmesh Managanvi and Ramanuj Vishwakarma

7. Entomopathogenic Nematodes: An Emerging Biocontrol

Agent for Insect Pests Management....................................................... 181
Rashid Pervez and Santhosh J. Eapen

8. Role of Insect Viruses in the Management of Insect Pests................... 209

Chandra Shekhar Prabhakar, Amit Choudhary, Jaipal Singh Choudhary, Pankaj Sood,
and Pawan Kumar Mehta
viii Contents

Part III: Parasitoids and Predators.................................................... 239

9. Trichogramma: An Egg Parasitoid in Insect Pest Management.......... 241
Tarak Nath Goswami, Anil, Tarak Brambha Maji, Pranab Barma, and
Shree Niwas Ray

10. Coccinellids in Insect Pest Management: Problems and Prospects.... 273

Pranab Barma, Suprakash Pal, and Tarak Nath Goswami

11. Chrysopid: A Potential Biocontrol Agent.............................................. 289

Nithya Chandran and Tamoghna Saha

Part IV: Genetically Modified Crops, Bacillus thuringiensis and

Phytochemicals in Bioconrol........................................................ 305
12. Genetically Modified Crops for Insect Pests and
Disease Resistance.................................................................................... 307
Tushar Ranjan, Sangita Sahni, Bishun Deo Prasad, and Vijay Kumar Jha

13. Bacillus thuringiensis and Insect Pest Management............................. 331

Anil, Lokender Kashyap, Tarak Nath Goswami, Vikas Kumar Patel, and
Ramesh Kumar Sharma

14. Role of Phytochemicals in Insect Pest Management............................. 371

Tamoghna Saha, Nithya Chandran, and Shyambabu Sha

Index.................................................................................................................. 393
LIST OF CONTRIBUTORS

Anil
Department of Entomology, Bihar Agricultural University, Sabour, India

Waheed Anwar
Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore 54000, Pakistan

Md. Arshad Anwer

Department of Plant Pathology, Bihar Agricultural University, Sabour 813210, India. E-mail: arshad_
[email protected]
Kahkashan Arzoo
Department of Plant Pathology, GBPUA&T, Pantnagar, Uttara Khand, India

Pranab Barma
Darjeeling Krishi Vigyan Kendra, Uttar Banga Krishi Viswavidyalaya, Kalimpong, Darjeeling
734301, West Bengal 734301, India. E-mail: [email protected]
Nithya Chandran
Division of Entomology, Indian Agricultural Research Institute, New Delhi 110012, India. E-mail:
[email protected]
Amit Choudhary
Department of Entomology, Punjab Agricultural University, Ludhiana 141004, Punjab, India

Jaipal Singh Choudhary

Research Centre, ICAR-Research Complex for Eastern Region, Research Centre, Plandu, Ranchi
834010, Jharkhand, India

Santhosh J. Eapen
Division of Crop Protection, ICAR-Indian Institute of Spices Research, Kozhikode 673012, Kerala,
India

Erayya
Department of Plant Pathology, Bihar Agricultural University, Sabour, Bhagalpur, Bihar, India.
E-mail: [email protected]

Tarak Nath Goswami

Department of Entomology, Bihar Agricultural University, Sabour, India. [email protected]

Muhammad Saleem Haider

Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore 54000, Pakistan

Vijay Kumar Jha

Department of Botany, Patna University, Patna, Bihar, India

Lokender Kashyap
Department of Plant Protection, Lovely Professional University, Jalandhar 144410, Punjab, India
x List of Contributors

Tarak Brambha Maji

Department of Agricultural Entomology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur 741252,
Nadia, West Bengal, India

Kalmesh Managanvi
Department of Entomology, Bihar Agricultural University, Sabour, Bhagalpur 813 210, India. E-mail:
[email protected]

Pawan Kumar Mehta

Department of Entomology, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Kangra
176062, Himachal Pradesh, India
Suprakash Pal
Directorate of Research (RRS-TZ), Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar,
West Bengal 736165, India
Vikas Kumar Patel
Department of Entomology, Bihar Agricultural University, Sabour 813210, Bhagalpur, Bihar, India

Chandra Shekhar Prabhakar

Department of Entomology, Veer Kunwar Singh College of Agriculture, Bihar Agricultural University,
Dumroan, Buxar 802136, Bihar, India
Nishant Prakash
Department of Plant Pathology, Krishi Vigyan Kendra, Arwal, BAU, Sabour, Bhagalpur, Bihar, India

Bishun Deo Prasad

Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Sabour,
Bhagalpur, Bihar, India. E-mail: [email protected]

Rashid Pervez
Division of Crop Protection, ICAR-Indian Institute of Spices Research, Kozhikode 673 012, Kerala,
India. E-mail: [email protected]
Tushar Ranjan
Department of Basic Science Humanities & Genetics, Bihar Agricultural University, Sabour,
Bhagalpur, Bihar, India
Shree Niwas Ray
Department of Entomology, Bihar Agricultural University, Sabour, India

Tamoghna Saha
Department of Entomology, Bihar Agricultural University, Sabour, Bhagalpur 813210, India. E-mail:
[email protected]

Sangita Sahni
Department of Plant Pathology, Tirhut College of Agriculture, Dholi, RAU, Pusa, Bihar, India

Shyambabu Sha
Department of Entomology, Bihar Agricultural University, Sabour, Bhagalpur 813210, India

Ahmad Ali Shahid

Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore 54000, Pakistan.
E-mail: [email protected]

Ramesh Kumar Sharma

Department of Horticulture (Veg. & Flori.), Bihar Agricultural University, Sabour 813210, Bhagalpur,
Bihar, India
List of Contributors xi

Uma Shankar Singh

International Rice Research Institute, IRRI India office, 1st Floor, NASC Complex, DPS Marg, New
Delhi 110012, India

Kundan Singh
Department of Plant Pathology, Bihar Agricultural University, Sabour 813210, India

Raj Narain Singh

Directorate of Extension Education, Bihar Agricultural University, Sabour 813210, India

Pankaj Sood
Krishi Vigyan Kendra, CSK Himachal Pradesh Krishi Vishvavidyalaya, Sundernagar, Mandi, 175019,
Himachal Pradesh, India
Ramanuj Vishwakarma
Department of Entomology, Bihar Agricultural University, Sabour, Bhagalpur 813 210, India. E-mail:
[email protected]
Najam Waris Zaidi
International Rice Research Institute, IRRI India office, 1st Floor, NASC Complex, DPS Marg, New
Delhi 110012, India. E-mail: [email protected]
LIST OF ABBREVIATIONS

ABC ATP-binding cassette

ACC 1-aminocyclopropane-1-carboxylic acid
APX ascorbate peroxidase
CAT catalase
CWDEs cell wall degrading enzymes
CWPOX cell wall bound peroxidises
DAPG diacetylphloroglucinol
DJ dauer juvenile
EBPM ecologically based pest management
EPA Environmental Protection Agency
EPNs entomopathogenic nematodes
FYM farm yard manure
GA gibberelic acid
GMCs genetically modified crops
GSP gossyplure and organic pesticides
GV granulosis virus
IAA indole acetic acid
ICM integrated crop management
ICTV International Committee on Taxonomy of Viruses
IJs infective juveniles
IPM integrated pest management
ISR induced systemic resistance
KCZ ketoconazole
LOX lipoxygenase
MAMPs microbe-associated molecular patterns
MAPK mitogen-activated protein kinase
MCZ miconazole
MIC minimum inhibitory concentrations
PAL phenylalanine ammonia lyase
PCNB pentachloronitrobenzene
PCR polymerase chain reaction
PDB potato dextrose broth
PO peroxidase
xiv List of Abbreviations

PPO polyphenol oxidase

PR pathogenesis-related
PSM phosphate-solubilizing microorganisms
Pvd pyoverdin
ROS reactive oxygen species
SAR systemic acquired resistance
SOD superoxide dismutase
SPOX soluble proxidases
PREFACE

Swift changes in the agro ecosystem leave a problem in the establishment

of harmony in the discord of a disturbed agro ecosystem. This distur-
bance is caused by extension of cultivated areas, monoculture, intensive
cultivation, faulty cropping pattern, irrational crop husbandry by way of
unbalanced fertilizer application, tapping of subsoil water, and indiscrimi-
nate application of chemical pesticides, etc. The pesticide consumption
has increased 20% per annum although agricultural production increased
nominally throughout the globe. The insects, diseases, and weeds cause
almost 30% yield loss per annum. This situation has come up because of
killing of natural enemies, resistance problem among insects and patho-
gens to chemicals, residue hazards of used chemicals, disturbing the
balance of nature, resurgence of treated insect populations, and emergence
of new pathogen races.
When we headed for the green revolution in the past, we had used
all kinds of affluent situations like good land, water resources, etc. Even
fertilizer and pesticide response was much better than what we can expect
today. As a matter of fact, we are now getting some kind of decline in total
food productivity. That is the reason why we are saying that there is a need
of a different kind of agriculture and different kind of technology. This has
to be a major shift and we have to move to a very high level of precision
agricultural technology. This technology will enhance the production and
the productivity per unit of resource.
Considering the drawbacks of our faulty cropping pattern, it becomes
necessary to lay special emphasis on integrated pest management (IPM).
To achieve our goal of the next green revolution, we have to phase out the
unnecessary consumption of poisonous chemicals and integrated multidis-
ciplinary methodologies in developing agro ecosystem management strat-
egies that are practical, economical, and protective of both human health
and environment.
Integrated control or pest management does imply the use of the best
combination of controls in organized ways that are designed to avoid harm
to anything but pests. The use of biopesticides and bioagents is on the
increase to control our crop pests by artificial introduction or increase
xvi Preface

of their natural enemies such as antagonists, parasites predators, insect

pathogens, and plant products.
A number of pathogenic bacteria viz., Bacillus thuringiensis, B.
popillae, fungi viz., Trichoderma spp., Aspergillus niger, Beauveria
bassiana, Metarrhizium anisoplae. Entomophthora spp. Verticillium
lecanii, and viruses viz., nuclear polyherosis, cytoplasmic polydedrosis,
Ganulosis, the nematode viz., Steinernema carpocapsae. Rhabditis spp.
Parasitorhadbitis, and protozoans viz., Nosema spp., Tetrahymena spp.,
are under use to control crop pests.
Besides, a number of important parasites viz., Trichogramma spp.,
Grniozus spp., Tetrastichus spp. Telonomus spp., and predators viz., Cryp-
tolaemus spp., Chrysoperla spp., Coccinella spp., Mallada boninensis, and
Chilochorus nigritus, are being used in farmers’ field for pest management.
This book is a standard reference work, offering available basic facts,
re-evaluating and reviewing the past research, and providing the new and
current discoveries on the subject and up-to-date information on biopesti-
cides and bioagents.
The main features—(1) scope of the biopesticides and bioagents as
a tool in integrated pest management, (2) emerging technologies of the
subject, (3) sustainability of the technology, and (4) future directions in
effective pest management to ensure safety of the environment—have
been comprehensively covered in various chapters of the book.
CHAPTER 1

STATUS OF BIOPESTICIDES
AND BIOCONTROL AGENTS IN
AGRICULTURE: AN OVERVIEW
MD. ARSHAD ANWER
Department of Plant Pathology, Bihar Agricultural University,
Sabour 813210, India
E-mail: [email protected]

CONTENTS

Abstract...................................................................................................... 2
1.1 Introduction....................................................................................... 2
1.2 Definition and Types of Biopesticides.............................................. 3
1.3 Mass Production of Fungal Biological Control Agents.................... 3
1.4 Immobilization.................................................................................. 5
1.5 Shelf-Life Test.................................................................................. 8
1.6 Evaluation for Effectiveness............................................................. 8
1.7 Biosafety Analysis............................................................................ 9
1.8 Registration..................................................................................... 10
1.9 Conclusion and Future Prospects.................................................... 11
Keywords................................................................................................. 11
References................................................................................................ 11
2 Biopesticides and Bioagents: Novel Tools for Pest Management

ABSTRACT

To achieve sustainable development of agriculture it is a need for advance

research and development in the field of biopesticides applications
which greatly reduce the environmental pollution caused by the chem-
ical synthetic insecticides residues. Since the advent of biopesticides, a
large number of products have been registered and released; some of them
have played a leading role in the agriculture market. The development
of biopesticide has encouraged replacing the chemical synthetic pesticide
in pest management in some extent. Biopesticides and biocontrol agents
have proven to be highly effective, species specific and eco-friendly in
nature, leading to their adoption in pest management strategies around the
world. The microbial biopesticides market constitutes about 92% of total
biopesticides and there is sufficient scope for further development in agri-
culture, although there are challenges as well. This chapter reviews the
various microbial especially fungal biopesticides, the different approaches
for their production and development, field evaluation, registration and
recent technological advances, and the challenges faced by the microbial
biopesticide field in the future.

1.1 INTRODUCTION

Biopesticides are the formulations of biocontrol agents in a form which

keep the organism at higher count and viable for their introduction or
application in the field. Hence, for huge-scale or business use of biocon-
trol agents, their biopesticides are necessarily produced. Among the total
pesticides used in India, more than 60% of the chemical pesticides are
used in the agriculture sector. The use of chemical pesticides is highest
in Andhra Pradesh with 20%, followed by Punjab with 10%, Tamil Nadu
with 9%, and Karnataka and Gujarat with 6%. Cotton with 40–50%, rice
with 17–18% and vegetables with 14–16% use maximum quantity of
pesticide in the country. In India, among the chemical pesticides, insec-
ticides are used to a large extent of about 60% followed by fungicides
and bactericides (20%) herbicides (17%) and other chemicals (3%). While
in Western countries, herbicide use is the highest. The world average for
herbicide use is about (45%) followed by insecticides (36%), fungicides
(17%), and other chemicals (2%) (Wahab, 2003, 2005, 2009).
Status of Biopesticides and Biocontrol Agents in Agriculture: An Overview 3

Till now, 452 biocontrol agents are used in the production of 2000
commercial products worldwide. It includes 149 micro-organisms; 89
natural products; 140 macro-organisms; and 74 semiochemicals. In India,
about 16 commercial preparations of Bacillus thuringiensis, 38 fungal
formulations based on Trichoderma, Metarhizium, Beauveria and about
45 baculovirus-based formulations of Helicoverpa and Spodoptera are
available. Microbials are expected to replace at least 20% of the chem-
ical pesticides. Biotic agents are being supplied by about 128 units in the
country (80 private companies). Besides, ICAR institutes (8), State Agri-
cultural Universities (10) and Central Integrated Pest Management centers
(30), and four parasitoid-producing laboratories are also supplying natural
enemies (Wahab, 2003, 2004, 2009).

1.2 DEFINITION AND TYPES OF BIOPESTICIDES

In the European Union, biopesticides have been defined as “a form of

pesticide based on micro-organisms or natural products” (Anonymous,
2008). According to the United States Environmental Protection Agency
(EPA), they “include naturally occurring substances that control pests
(biochemical pesticides), microorganisms that control pests (microbial
pesticides), and pesticidal substances produced by plants containing added
genetic material (plant-incorporated protectants) or PIPs” (Anonymous,
2012). Biopesticides belong to mainly three categories: (a) living organ-
isms, which include predatory insects, nematodes and micro-organisms,
(b) naturally occurring substances which include plant extracts and semio-
chemicals, for example, insect pheromones, and (c) genetically modified
plants that express introduced genes that confer protection against pests or
diseases (plant-incorporated products).

1.3 MASS PRODUCTION OF FUNGAL BIOLOGICAL CONTROL

AGENTS

Fermentation methods are important for mass production of microorgan-

isms and to harvest a much better yield quantitatively as well as quali-
tatively. Three methods of fermentation are described by Lewis and
Papavizas (1991).
4 Biopesticides and Bioagents: Novel Tools for Pest Management

1.3.1 LIQUID FERMENTATION

This technology has been adopted to produce bacterial and fungal biomass.
A suitable medium should consist of inexpensive, readily available agri-
cultural byproducts with appropriate nutrient balance. Acceptable mate-
rials include molasses, brewer’s yeast, corn steep liquor, sulfate waste
liquor, cotton seed, and soya flours (Lisansky, 1985). Alegre et al. (2003)
proposed liquid fermentation method consisting of molasses, wheat bran,
and yeast for large-scale production of T. harzianum. A higher produce
of Trichoderma chlamydospores was harvested through liquid fermenta-
tion technology. The preparation based on chlamydospores prevented the
disease more effectively than a preparation that contained conidia only
(Lewis et al., 1990; Papavizas & Lewis, 1989). Small-scale fermentation
in molasses-brewers yeast medium has also resulted in abundant chlamyd-
ospore production of Trichoderma (Papavizas et al., 1984).

1.3.2 SOLID FERMENTATION

Mass production of antagonists on solid substrates for the production of

inoculum of various biocontrol fungi includes straws, wheat bran, and
sawdust; bagasse moistened with water or nutrient solutions through
fermentation technology is referred to as solid fermentation (Papavizas,
1985). This technology is also effective especially for those organisms,
which can multiply on dry substrates.

1.3.3 SEMISOLID FERMENTATION

Semisolid fermentation is done for the fungi which do not sporulate in

liquid culture. Diatomaceous earth granules impregnated with molasses
(Backman & Rodriguez-kabana, 1975), wheat bran, and vermiculite-wheat
bran (Lewis et al., 1989) yield good produce of bioagents. This method,
however, requires more area and is labor intensive, and the chances of
contamination are high when compared to liquid fermentation.
For commercial production of biological control agents, different tech-
nologies have been adopted on industrial scale. Fermented Trichoderma
consisted mainly of chlamydospores and conidia with some amount of
mycelial fragments. Air-dried mats have been grounded and mixed with a
Status of Biopesticides and Biocontrol Agents in Agriculture: An Overview 5

commercially available carrier, the formulations thus developed, contained

108–109 propagules/g (Table 1.1; Chaube et al., 2002; Chaube & Pundhir,
2010).

TABLE 1.1 Base Material/Carriers Used for Mass Production of Fungus Biocontrol
Agents.
Antagonists Base material(s) Form of
formulation
Trichoderma Black gram shell, shelled maize cob, coir-pith, Powder, pellets
harzianum peat, gypsum, coffee fruit skin + biogas slurry,
coffee husk, coffee-cherry husk, fruit skin and
berry mucilage, molasses-yeast, molasses-soy,
molasses-NaNO3, mushroom-grown waste,
sugarcane straw, wheat bran + biogas manure
(1:1), wheat-bran + kaolin.
T. viride Barley grains, black gram shell, shelled maize Pellets
cob, coir-pith, peat, gypsum, coffee husk,
coffee-cherry husk, fruit skin and berry muci-
lage, mushroom grown waste, mustard oil cake,
neem cake + cow dung, poultry manure, spent
tea leaf waste, sugarcane straw, talc, vermiculite
+ wheat bran + HCL
T. virens Barley grains, coffee husk, coffee-cherry husk, Pellets
fruit skin and berry mucilage, mushroom-grown
waste, neem cake + cow dung, poultry manure,
soil, sorghum grains, talc, wheat bran sawdust.
T. longibrachiatum Talc, wheat bran + saw dust Powder
Aspergillus niger Citrus pomace (waste from canning industry), Pellet, powder
talc + cmc
A. terreus Maize-meal + sand Powder

1.4 IMMOBILIZATION

For field application of a biocontrol agent, an efficient substrate for mass

production and an inert immobilizing material are required, which could
carry the maximum number of propagules of the organism with minimum
volume and necessarily maintain its survival and integrity. An excel-
lent bioinoculant is one that is introduced to an ecosystem, and subse-
quently survives, proliferates, becomes active, and establishes itself in a
6 Biopesticides and Bioagents: Novel Tools for Pest Management

new environment (Khan, 2005). For preparing a commercial formulation,

these attributes must be considered. In addition, the bioinoculant should
be mass cultured on an inexpensive substrate in a short time. Easy applica-
tion, effectiveness, and consistent results under a variety of environmental
conditions are other desirable features required for production of bioin-
oculant formulations.
Different techniques of cell immobilization have been developed to
devise efficient carrier systems to produce commercial formulations of
bioinoculants. A number of carriers for immobilization of microorganisms
have been used to develop commercial formulations of biocontrol agents
viz., peat, seeds, meals, kernels, husks, bran, bagasse, farmyard manure,
cow dung cake, compost, oil cakes, wood bark, vermiculite, sand, clay,
and liquid carriers. Three types of formulations viz., pellet, granular, and
liquid, are widely produced (Mukhopadhyay, 1987; Kousalya & Jeyarajan,
1990; Bhai et al., 1994; Khan & Anwer, 2011). Tiwari et al. (2004) evalu-
ated grains of sorghum, wheat, bajra, wheat bran, rice bran, and sugarcane
bagasse for mass multiplication of T. viride. Grains of sorghum (cv. swanki)
were found the best substrate that provided maximum spore concentration
(8 × 109 spores) and spore viability (92.5%) after 15 days of incubation at
27 ± 1 °C. Spores remained viable for 6 months at 5 °C. Wheat bran and
sawdust mixture have been used as a carrier media for the mass multipli-
cation of T. harzianum (Elad et al., 1980; Mukhopadhyay et al., 1986).
Khan et al. (2001) evaluated various agricultural and industrial wastes for
mass multiplication of T. harzianum, T. virens, and P. chlamydosporia, and
found highest CFU, 1.2 × 106 on bagasse-soil-molasses for T. harzianum,
1.0 × 106 for T. virens and 1.1 × 106 on corn meal-sucrose mixture for P.
chlamydosporia. Vidhyasekaran et al. (1997) developed powder formula-
tions of P. fluorescens using talc powder, peat, vermiculite, lignite, and
kaolinite. All freshly prepared powdered formulations were effective in
controlling pigeon pea wilt, but their efficacy varied depending upon the
length of storage. Talc formulation was effective even after 6 months of
storage. Bhai et al. (1994) evaluated a number of agricultural wastes which
could be used as carrier and multiplication media at the same time. They
reported that sterilized tea waste, coffee husk, or a mixture of coffee husk
and cattle manure were ideal combinations for the fast growth and multi-
plication of T. harzianum and T. virens. Angappan (1992) used molasses
yeast medium for growing T. viride and mixed it with talc powder to
develop commercial formulation. The initial population in the produce
Status of Biopesticides and Biocontrol Agents in Agriculture: An Overview 7

was 3 × 108 CFU/g, whereas the product should contain 2 × 106 CFU/g
at the time of use. The shelf life of this product was 4 months. Seed treat-
ment of chickpea with this product maintained the rhizosphere population
of the bioagent at 11–13 × 103 CFU/g soil throughout crop. Ranganathan
et al. (1995) found that gypsum is a good and cheap substitute for talc.
Nakkeeran and Jayrajan (1996) tested two industrial wastes—precipitated
silica and calcium silicate as carriers for Trichoderma in the place of talc.
The material gave a population of 0.99 and 1.04 × 108 CFU/g, respec-
tively compared to 1.4 × 108 CFU/g in talc substrate after 4 months of
storage. Both the substrates were much cheaper than talc. Backman and
Rodriguez-Kabana (1975) used diatomaceous earth granules impregnated
with 10% molasses solution for rearing T. harzianum. It was applied to
peanut at 140 kg/h on 70 and 100 days after sowing to control Sclerotium
rolfsii. The disease was reduced by 42% over control and yield increased
by 13.5%.
Several researchers have used combination of two or more agricultural
materials. Elad et al. (1986) used wheat bran: sawdust: tap water mixture
(3:1:4 v/v) for T. harzianum. It was applied at the time of sowing and
mixed with the soil to a depth of 7–10 cm with a rotatory hoe. It increased
yield of beans (15 q/h), tomato (3 q/h), cotton (5 q/h) and potato (4–6 q/h)
and controlled Sclerotium rolfsii and Rhizoctonia solani. Vidhya (1995)
applied the formulation of T. harzianum based on vermiculate-wheat bran
(@ 250 kg/ha) to mungbean and found 41% reduction in root-rot (Macro-
phomina phaseolina) and 91% increase in yield. Papavizas and Lewis
(1989) prepared T. virens on alginate-bran-fermenter biomass pellets and
pyrax–fermenter biomass mixture. Soil application of the product checked
the damping off caused by R. solani. Several other substrates such as
farm yard manure (FYM), biogas plant slurry, press mud, paddy chaff,
rice bran, groundnut shell (Kousalya & Jeyarajan, 1988), FYM, FYM +
sand, sawdust, wheat bran, pigeon pea leaves, wheat straw, and urdbean
straw (Chaudhary & Prajapati, 2004) have been tested to grow T. viride
and T. harzianum. The enumeration of viable CFUs revealed that pigeon
pea leaves and urdbean straw were the best substrates showing 3.4 and
3.4 × 105 propagules at 4 months, 1.2 and 1.1 × 105 at 8 months, and
1.5 and 3.0 × 104 at 12 months of storage at room temperature, whereas
sorghum seed showed 11.4, 3.8, and 0.6 × 104 propagules at the same
intervals, respectively. Next suitable substrates were wheat straw and saw
dust. Cabanillas and Bakar (1989) tested some carriers like wheat grains,
8 Biopesticides and Bioagents: Novel Tools for Pest Management

alginate pellets, and diatomaceous earth granules for soil application of P.

lilacinus. Kerry et al. (1984) used oat seeds to rear P. chlamydosporia for
field application. Soil application of the colonized oat kernels @ 0.5 and
1.0% (w/w soil:seed) considerably reduced the population of root-knot
and cyst nematodes (Godoy et al., 1983; Rodriguez-kabana et al., 1984).
De Leij and Kerry (1991) did encapsulation of liquid suspension of spores
and hyphae of P. chlamydosporia with sodium alginate containing 10%
(w/v) kaolin or wheat bran. On soil application, the fungus proliferated
in soil from only those granules which contained wheat bran as energy
source. In other study, Kerry (1988) estimated approximately 9 × 104 and
4 × 104 CFUs of the fungus/g soil after 1 and 12 weeks of application of
granules, respectively.

1.5 SHELF-LIFE TEST

The limiting factor in commercialization of a biocontrol agent prepara-

tion is its loss of viability of the biocontrol agents over time. Considerable
efforts have been made in India itself to determine the viability of biocon-
trol agents in their preparations when stored at room temperature and in
refrigerator. Most of the results are variable and therefore it appears that
shelf life is also dependent upon species/isolate/strain.

1.6 EVALUATION FOR EFFECTIVENESS

In general, the antagonist multiplied in an organic food base has greater

shelf life than that on an inert or inorganic food base (Jeyarajan &
Nakkeeran, 2000). A talc-based preparation of T. virens conidia retained
82% viability at 5 °C in refrigerator after 6 months, while at room tempera-
ture (25–35 °C), same level of viability was observed only up to 3 months.
Shelf life was same, when T. virens treated chickpea or soybean seeds
were stored at room temperature (Tiwari et al., 2004). Seed coating with
biocontrol agents has emerged as a feasible was of delivering the antago-
nists, that is, supplying the coated seeds to the farmers directly by the
seed companies/agencies. The time gap between coating seeds and sowing
such seeds by farmers is critical. Mukherjee (1991) quantitatively assessed
the viability of T. virens on coated chickpea seeds when the seeds were
Status of Biopesticides and Biocontrol Agents in Agriculture: An Overview 9

stored at low temperature (5 °C) and at room temperature (25–35 °C),

88% of the propagules remained viable for up to 4 months. Conidia of
Trichoderma in pyrophyllite survived better than fermenter biomass prop-
agules alone at −5 to 30 °C. The most suitable temperature to prolong
shelf life of conidia and fermenter biomass propagules in pyrophyllite
were −5 to 5 °C (Mukherjee, 1991). Storage at 5 °C increased the shelf
life of T. virens and T. hamatum in granular formulations of pre gelati-
nizing starch flour up to 6 months. Chlamydospore-based formulations of
T. virens and T. harzianum exhibited longer shelf life (80% viability for 9
months) than conidia-based formulations (80% viability after 4 months)
at room temperature and a preparation of T. virens (mainly in the form
of chlamydospores was from peat moss Czapek broth culture) stored at
25 °C for 6 months without loss of viability (Mishra, 2002). Lewis et al.
(1995) reported that among the different carriers tested; the shelf life of B.
subtilis in soybean flour was increased up to three months. Ranganathan
et al. (1995) also reported 4-month shelf life of T. viride in gypsum-based
formulations. Talc-based formulations of T. harzianum can retain more
than 106 viable propagules per gram up to 90 days (Prasad & Rangesh-
waran, 2000). Jeyarajan et al. (1994) developed talc-, peat-, lignite-, and
kaolin-based formulations of T. viride, which had a shelf life of 4 months.
Studies on storage temperature revealed that 20–30°C was optimum to
store vermiculite fermenter biomass of Trichoderma up to 75 days without
losing the viability (Nakkeeran et al., 1997). Commercial formulations of
Aspergillus niger AN27 showed an extraordinary long shelf life of more
than two years at room temperature (25–35 °C) when packed in polyeth-
ylene bags and stored under less than 80% relative humidity (Sen, 2000).

1.7 BIOSAFETY ANALYSIS

Quality control is the most essential aspect of biopesticide production. A

good quality of the preparation is necessarily required to retain the confi-
dence of farmers on the efficacy of biocontrol formulation. Being living
agents their population in a product may be influenced by storage. The
other contaminating microorganisms in the product should also be within
permissible limits.
10 Biopesticides and Bioagents: Novel Tools for Pest Management

1.8 REGISTRATION

Current legislation demands that new products are subjected to detailed

study of their environment impact and toxicological effects and they are
registered. As current legislation stands, there are certain categories of
biocontrol agents that have an easier and quicker passage for registra-
tion. Indigenous microorganisms that are specific to a defined group of
targets have a comparatively straightforward progress. Under Section
9(3) of Pesticide Act of India (1968), information required for registra-
tion of any biopesticides includes: systematic name and common name;
natural occurrence and morphological descriptions; details of manufac-
turing process (active and inert ingredients of formulation); test methods
(dual culture of pathogenicity); quantitative analysis (CFU on selec-
tive medium, absence of Gram negative bacteria contaminants); mois-
ture content; shelf life; mammalian toxicity; bioefficacy; environmental
toxicity and residue analysis. Because of less awareness of growers
toward biocontrol programs, the Indian Biopesticide Industry involves
more than 15–20% expenditure on marketing compared to only 1–2%
marketing expenses in the case of conventional pesticides (Singhal &
Sharma, 2003). Therefore, there is a need for simplification of registra-
tion requirements and government subsidies should be granted to farmers
to promote biopesticide use.
The registration policies may vary with the country. In the United
States, registration of microbial pesticide requires toxicological tests for
oral, dermal, eye, and other health hazards using test animals or fish. If these
tests show no adverse effects and the biocontrol agent is not a pathogen,
it is registered and can be sold. The cost required for research and devel-
opment for biopesticide is only US$ 0.8–1.6 million as against US$ 20
million for chemical pesticides. The toxicological tests for a biocontrol
agent cost US$ 0.5 million as against US$ 10 million for chemical pesti-
cides. The number of candidates to be tested to develop one biocontrol
product will be in 100s as against 20,000 for a chemical pesticide. It was
estimated that the market size required for profit for a biocontrol agent is
US$ 1.6 million per year as against US$ 4 million per year for a chemical
pesticide (Cook, 1993).
Status of Biopesticides and Biocontrol Agents in Agriculture: An Overview 11

1.9 CONCLUSION AND FUTURE PROSPECTS

It would be impractical to expect that biocontrol products will replace

chemical pesticides in the near future. There will always be a need for
chemical pesticides, given that the world population continues to increase
at a swift rate and worldwide food production must keep balance with it.
However, there is likely to be some decline in the use of synthetic pesti-
cides, especially in developed countries and this will provide an opportu-
nity for microbial products to satisfy markets.
Developing a safe, easy to use, cost-effective formulation that will keep
the microorganism alive is one of the most important steps in developing a
biological product. Formulation is the blending of active ingredients such
as fungal spores with inert carriers such as diluents and surfactants in order
to improve the physical characteristics. A final formulation must have a
long shelf life at room temperature, be easy to handle, be insensitive to
abuse, and must be stable over a range of −5 to 35 °C. The most needed
technique is drying techniques, which allows retention of maximum
number of viable propagules in dried product. Most important steps of
production of biopesticides are mass production of biocontrol agents,
their immobilization, shelf-life test, evaluation for effectiveness, biosafety
analysis, registration, etc.

KEYWORDS

•• biopesticides
•• biocontrol agents
•• chemical pesticides
•• bacterial and fungal biomass
•• liquid fermentation

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 PART I
Plant Health Promoting
Biocontrol Agents
CHAPTER 2

TRICHODERMA—AN IMPECCABLE
PLANT HEALTH BOOSTER
NAJAM WARIS ZAIDI* and UMA SHANKAR SINGH
Department of Plant Breeding, International Rice Research Institute,
IRRI India Office, 1st Floor, NASC Complex, DPS Marg,
New Delhi 110012, India
Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract.................................................................................................... 18
2.1 Introduction..................................................................................... 19
2.2 Trichoderma General Physiognomies............................................. 21
2.3 Trichoderma as an Endophyte and Defense Inducer...................... 25
2.4 Trichoderma as a Plant Growth Stimulant...................................... 27
2.5 Trichoderma as an Abiotic Stress Reliever of Crop Plants............. 28
2.6 Delivery of Trichoderma................................................................ 30
2.7 Trichoderma as an Efficient Colonizer of Compost....................... 31
2.8 Limitations...................................................................................... 32
2.9 Conclusion...................................................................................... 32
Keywords................................................................................................. 33
References................................................................................................ 33
18 Biopesticides and Bioagents: Novel Tools for Pest Management

ABSTRACT

Species of Trichoderma belong to one of the most useful group of microbes

to have had an impact on human welfare in recent times. Trichoderma
(teleomorph Hypocrea; Hypocreaceae, Hypocreales, and Ascomycota)
spp. have had a major influence on agriculture too. Trichoderma spp. are
one of the most plentiful fungal genera found in many ecosystems. They
are most widespread and popular research tools as microbial inoculants
that are largely used against several plant pathogenic fungi causing soil
borne, foliar, and post-harvest diseases of plants. This is well reflected by
the completion of whole genome sequencing of as many as seven species
of Trichoderma by the Joint Genomic Institute (JGI) (Mukharjee et al.,
2013). Having highly antagonistic and mycoparasitic potential in labora-
tory conditions, they have shown to reduce the severity of plant diseases by
inhibiting plant pathogens in the soil. Trichoderma can have direct effects
on plants, increasing their biomass and nutrient uptake, fertilizer use effi-
ciency, seed germination, and stimulation of plant defenses by enhanced
photosynthetic ability, against biotic and abiotic stresses and hence is a
famous plant growth promoter. Some species can kill plant parasitic nema-
todes and have the potential to be used as bio-nematicides.
Most Trichoderma are endophytic plant symbionts. These changes alter
plant physiology and may result in the improvement of nitrogen uptake,
resistance to pathogen, photosynthetic efficiency, as well as abiotic stress
tolerance. According to an estimate, about 60% of all the registered biofun-
gicides worldwide are Trichoderma based with more than 250 Tricho-
derma-based formulations being commercially sold in India alone (Verma
et al., 2007). Trichoderma strains interact with the plant by colonizing
roots, establishing chemical communication, and systemically altering the
expression of numerous plant genes.
So far major bottleneck with the use of Trichoderma is their uneven
field performance. This is due to the fact that they are biotic agents.
Their field performance depends upon a number of factors like, strain/
host selectivity, environmental conditions, inoculum density, method of
delivery, and so forth. By far, application of Trichoderma both through
seed and colonized compost is the most effective delivery system for the
management of seedling and root diseases in different crops. Also they can
help alleviate abiotic stresses in plants, including water deficit (drought),
salinity, and temperature.
Trichoderma—An Impeccable Plant Health Booster 19

2.1 INTRODUCTION

The taxonomy of Trichoderma is relatively young compared with that of

Hypocrea. Trichoderma spp. are free-living fungi that are common in soil,
root, and foliar ecosystems throughout the world and are highly interac-
tive. They are present in nearly all types of soils and other natural habitats
especially those containing high organic matter. Being secondary colo-
nizers, they are often isolated from well-decomposed organic matter like
decaying barks and from sclerotia and fruiting bodies of other fungi. Trich-
oderma generally exhibit a preference for wet soils but individual species
exhibit different preferences for soil temperature, moisture, and pH. While
species like Trichoderma viride and Trichoderma polysporum are gener-
ally found in areas with low temperature, Trichoderma harzianum most
commonly occurs in warm climatic regions. Trichoderma hamatum and
Trichoderma koningii can occur in diverse climatic conditions (Zaidi &
Singh, 2013). Trichoderma spp. have the ability to utilize a wide range
of compounds as sole carbon and nitrogen source and can utilize mono-
saccharides, disaccharides, and polysaccharides for carbon with ammonia
being the most preferred source of nitrogen.
The presence of carbon dioxide has been reported to favor growth of
Trichoderma. Apart from these factors, the iron content of the soil, HCO3−,
salt and organic matter content, and presence or absence of other microbes
are also important determinants of microsite preference by Trichoderma
spp. (Papavizas, 1985).
Trichoderma spp. are adapted to various ecological niches as fungal
antagonists, opportunistic, avirulent plant symbionts, and endophytes
and saprophytes on bark and dead wood (Harman et al., 2004). Benefi-
cial actions of Trichoderma/plant interactions include direct antagonism
against plant pathogens mainly fungi and nematodes (Singh et al., 2003),
increased plant growth, especially of roots, and particularly under stress
(Harman, 2000; Shoresh et al., 2010), systemic resistance to diseases
(Lorito et al., 2010; Bae et al., 2011), enhanced tolerance to abiotic
plant stresses, including water deficit (drought), salinity, and tempera-
ture, enhancement of the vigor of poor-quality seeds (Mastouri et al.,
2010; Shoresh et al., 2010), decomposition of organic matter resulting in
increased concentration of humic acid (Singh et al., 2003), solubilization
of phosphorus and increased availability of micronutrients (Altomare et
20 Biopesticides and Bioagents: Novel Tools for Pest Management

al., 1999; Singh et al., 2003; Harman, 2011), and improved nitrogen use
efficiency (NUE) by plants (Harman, 2006, 2011; Shoresh et al., 2010).
Trichoderma spp. also produce many extra-cellular enzymes like cellu-
lases, chitinases, glucanases, proteases, and so forth. They are used in foods
and textiles and also in poultry feed. Trichoderma chitinases are used in
generating disease resistant transgenic plants, in plant disease control and
improvement of plant growth.
Species of Trichoderma have been reported to be excellent colonizers
of cow dung compost/farm yard manure (FYM). Reports also suggest that
it helps in faster decomposition of cattle dung to convert it into compost
(Zaidi & Singh, 2004a). The antifungal action which is driven by mecha-
nisms that comprise both enzymatic and antibiotic activities, allows them
to switch between biotrophic and saprophytic lifestyles. Current knowl-
edge suggests that all species are fungicolous (Jacklitsch, 2009; Druzhinina
& Kubieck, 2013).
Trichoderma spp. have the ability to reduce the severity of plant
diseases by inhibiting plant pathogens, mainly in the soil or on plant roots,
through their high antagonistic and mycoparasitic potential (Viterbo &
Horwitz, 2010). Studies by Advanced Biological Marketing, Inc. (ABM),
Van Wert, Ohio and Cornell University showed that Trichoderma strains
induce changes in the microbial composition on roots, enhance nutrient
uptake, stabilize soil nutrients, promote root development, and increase
root hair formation (Harman, 2006). Since Trichoderma is a living entity
and can be affected by the surroundings, it will not be justifiable enough to
compare its efficacy with the chemical pesticides and fertilizers. Success
of Trichoderma spp. as a biopesticide under field condition depends not on
its antagonistic activity alone but a blend of several other characteristics
and soil and climatic conditions and plant host. The consistency in perfor-
mance is still considered as a constraint to the widespread adaptation with
high variability across sites and seasons, most likely as a result of the
differential impact of fluctuating biotic and abiotic factors. T. harzianum
and T. viride are the widely used species and have been exploited on about
87 different crops and about 70 soil borne and 18 foliar pathogens, respec-
tively (Sharma et al., 2014).
Trichoderma—An Impeccable Plant Health Booster 21

2.2 TRICHODERMA GENERAL PHYSIOGNOMIES

2.2.1 TRICHODERMA AS A MYCOPARASITE

Trichoderma has been known as a successful biofungicide on a commer-

cial scale. This success is mainly attributed to its ability to parasitize and
kill other fungi (Mukherjee et al., 2012a). Several Trichoderma spp. have
been long known for their potent necrotrophic mycoparasitic abilities
(Elad 1995; Druzhinina et al., 2011; Benítez et al., 2004) and are there-
fore used as model systems to study the mechanisms of mycoparasitism.
Mycoparasitism in itself is a complicated process that involves sequen-
tial events, including recognition, attack and subsequent penetration, and
killing of the host.
Trichoderma can detect its host from a distance and branches atypically
toward the host fungus (Chet et al., 1997). The coiling of Trichoderma is
not merely a contact stimulus (Dennis & Webster, 1971). Inbar and Chet
(1992, 1994) provided direct evidence for the role of lectins in myco-
parasitism. Once Trichoderma is attacked, it coils around the pathogen
and forms the appresoria-like structures. The following step consists of
the production of cell wall degrading enzymes (CWDEs) and secondary
metabolites (Howell, 2003), which facilitate both the entry of Trichoderma
hypha into the lumen of the parasitized fungus and the assimilation of
the cell wall content. The significance of lytic enzymes has been demon-
strated by overexpression and deletion of the respective genes (Viterbo et
al., 2002; Mukherjee et al., 2012a).
Mitogen-activated protein kinase (MAPK) cascades and G-protein
α-subunits transduce a large variety of signals, including those associ-
ated with pathogenesis. Signal transduction pathways triggering the
genes involved in biocontrol and mycoparasitism have been studied
in considerable depth and include heterotrimeric G-protein signaling,
MAPK cascades, and the cAMP pathway (Zeilinger & Omann, 2007).
Especially the MAP-kinase TVK1/TmkA, characterized in Trichoderma
virens (Mendoza-Mendoza et al., 2003; Mukherjee et al., 2003; Mendoza-
Mendoza et al., 2007) as well as its orthologs in Trichoderma asperellum
(Viterbo et al., 2005) and Trichoderma atroviride (Tmk1; Reithner et
al., 2007), is important in regulation of signaling mechanisms targeting
output pathways relevant for efficient biocontrol. The seven-transmem-
brane G-protein coupled receptor, Gpr1 is involved in sensing the fungal
22 Biopesticides and Bioagents: Novel Tools for Pest Management

prey; silencing of the Gpr1 gene in T. atroviride rendered the mycoparasite

unable to respond to the presence of the host fungus (Omann et al., 2012).
Binding of a ligand to such receptors leads to downstream signaling events
via activation of G-protein cascades. Indeed, deletion of the Tga3 G alpha
protein-encoding gene affected the mycoparasitic abilities of T. atroviride
in a similar way to loss of Gpr1 (Zeilinger et al., 2005). Deletion of the
adenylate cyclase gene tac1 severely impaired growth and mycoparasitic
abilities of T. virens (Mukherjee et al., 2007). Like most other filamen-
tous fungi, Trichoderma spp. have three MAPK cascades comprising
MAPKKK, MAPKK, and MAPK (Schmoll, 2008) and MAPK pathways
may act in mycoparasitism and biocontrol (Reithner et al., 2007; Kumar
et al., 2010).
Cell wall degradation during mycoparasitism is mediated by a set
of hydrolytic enzymes secreted by Trichoderma including chitinases,
β-(1,4)-, β-(1,3)- and β-(1,6)-glucanases, and proteases (Benítez et al.,
2004). Genome analysis enabled assessment of the total numbers of
CWDEs encoded in the genomes of Trichoderma spp. and unraveled even
more complex enzymatic degradation machinery for fungal cell walls than
previously anticipated (Kubicek et al., 2011).
Diverse DNA array experiments have determined that an expansin-like
protein, aspartyl proteases, and hydrophobins, among others, are involved
in the biocontrol and mycoparasitism of Trichoderma spp. (Brotman et al.,
2008; Samolski et al., 2009).
Trichoderma genes can be expressed functionally in plants to control
of plant diseases. High expression levels of the T. harzianum endochi-
tinase gene, ech42 were obtained in different plant tissues, with no visible
effect on plant growth and development (Lorito et al., 1998). Trichoderma
chitinase genes are used to generate transgenic plants resistant to fungal
diseases. Transgenic cotton plants expressing the T. virens endochitinase
gene Tv-ech42 showed significant resistance to Alternaria alternata and
Rhizoctonia solani (Emani et al., 2003; Kumar et al., 2009). The expres-
sion of ech42 in lemon enhanced resistance to Phoma tracheiphila and
Botrytis cinerea (Gentile et al., 2007; Distefano et al., 2008). The homolo-
gous ech42 gene from T. virens can enhance resistance against R. solani
when it was expressed in rice, tobacco, and tomato (Shah et al., 2009,
2010). Ech42 expression enhanced resistance to Venturia inaequalis,
but reduced plant growth in apple (Bolar et al., 2000) and significantly
increased the resistance of broccoli to Alternaria brassicicola (Mora &
Trichoderma—An Impeccable Plant Health Booster 23

Earle, 2001). Liu et al. (2004) reported the multiple expression of rice
transgenes encoding two chitinases (ech42 and nag70) and one β-1,3-
glucanase (gluc78) of T. atroviride resulted in resistance to R. solani and
Magnaporthe grisea in rice. The expression of the endochitinase chit36
gene of T. harzianum in carrot significantly enhanced tolerance to Alter-
naria radicina and B. cinerea (Baranski et al., 2008).
Although no quantitative studies have been performed previously,
Trichoderma spp. is a suitable genus and has been recognized for their
extreme facility in producing a large variety of extra-cellular enzymes and
the degradation of lignocellulose (Kirk & Farrell, 1987). Furthermore, T.
viride and Trichoderma reesei are the most extensively studied fungi in the
field of cellulosic material degradation (Cullen & Kersten, 1992).

2.2.2 TRICHODERMA AS A PRODUCER OF SECONDARY

METABOLITES

Trichoderma is known to produce volatile and non-volatile toxic

metabolites that impede colonization by competing microorganisms.
Some of them are harzianic acid, tricholin, peptaibols, 6-penthyl-alpha-
pyrone, massoilactone, viridin, gliovirin, glisoprenins, and heptelidic
acid (Lumsden et al., 1992; Mukherjee et al., 2012b). One of the first
characterized peptide antibiotics of Trichoderma spp. was Paracelsin
(Bruckner et al., 1984). Mycotoxins and more than 100 metabolites
derived from amino acids, with antibiotic activity including antibiotics
like gliotoxin and glioviridin from T. virens, viridin, pyrones, alkyl
pyrones, isonitriles, terpenes, polyketides, peptaibols, diketopiperazines,
sesquiterpenes polypeptides (Sivasithamparam & Ghisalberti, 1998), and
some steroids from other Trichoderma species have been suggested to be
used for chemotaxonomy of these species.

2.2.3 COMPETITION AND RHIZOSPHERE COMPETENCY OF

TRICHODERMA

Trichoderma is known to be a good competitor that involves competi-

tion between antagonist and plant pathogen for space and nutrients (Chet,
1987). Usually competition is thought to occur when there is absence of
24 Biopesticides and Bioagents: Novel Tools for Pest Management

any evidences that mycoparasitism or antibiosis has occurred during a

particular Trichoderma–host fungus interaction.
Howell (2003) used ultraviolet light irradiation to produce mutants
of T. virens, deficient for both mycoparasitism and antibiotic production.
However, the mutants still retained biocontrol efficacy equal to that of the
parent strain against both Pythium ultimum and R. solani causing cotton
seedling diseases. This indicated that neither mycoparasitism nor anti-
biosis is the principal mechanism involved in the biocontrol of seedling
diseases in cotton.
Most fungi excrete siderophores that are low-molecular-weight ferric
iron-specific chelators to mobilize environmental iron (Eisendle et al.,
2004). Some Trichoderma spp. produce highly efficient siderophores
that chelate iron and stop the growth of other fungi (Chet & Inbar, 1994).
Siderophores also help in improving antagonistic activities, rhizospheric
competence, and plant growth. T. virens produces three types of hydroxy-
mate siderophores: a monohydroxamate (cis- and trans-fusarinines), a
dipeptide of trans-fusarinine (dimerum acid), and a trimer disdepsipeptide
(coprogen) (Mukherjee et al., 2012b).
Trichoderma spp. are also highly efficient in mobilizing and taking
up soil nutrients. The efficient use of available nutrients is based on the
ability of Trichoderma to obtain ATP from the metabolism of different
sugars, such as those derived from polymers widespread in environments:
cellulose, glucan, and chitin (Chet et al., 1997).

2.2.4 TRICHODERMA AS A HYPER PARASITE OF NEMATODES

Trichoderma has been proved to parasitize nematodes and inactive

pathogen enzymes and therefore can be exploited as a bio nematicide to
control diseases caused by plant parasitic nematodes (Sharon, 2011).
T. harzianum colonized egg masses, eggs, and second stage juveniles
of Meloidogyne incognita. It formed loops and trapped second stage juve-
niles of M. incognita. Trichoderma penetrated nematode body by forming
haustoria like structures and colonized internally replacing all internal
organs with fungal mycelia resulting in death of the nematode. Culture
filtrate of T. harzianum and T. virens suppressed hatching and release of
second stage juveniles of Meloidogyne.
Reduced root galling was reported by Sharon et al. (2001) under green-
house experiments on the potential of T. harzianum to control root knot
Trichoderma—An Impeccable Plant Health Booster 25

nematode, Meloidogyne javanica associated with protease production

by T. harzianum. Prasad and Anes (2008) reported that ethyl acetate and
methanol extracts of T. viride and T. harzianum significantly reduced the
total number of galls and M. incognita population in Okra. Windham et al.
(1989) reported the reduction of egg production by the root knot nematodes
M. incognita following soil treatment with a Trichoderma conidial suspen-
sion. Saifullah and Thomas (1996) have also reported direct interactions
between T. harzianum and potato cyst nematode Globodera rostochiensis.
However, a lot of experimentations under field conditions need to be
done to fully harness the potential of Trichoderma for the control of plant
parasitic nematodes.

2.3 TRICHODERMA AS AN ENDOPHYTE AND DEFENSE

INDUCER

Trichoderma isolates form intimate relationships with plants including the

colonization of internal plant tissues. It is the internal colonization of plant
tissues by Trichoderma without causing harm to the plant that supports
the consideration of Trichoderma as an endophyte. In recent years it has
become apparent that Trichoderma isolates not only penetrate and survive
inside root tissues but can also internally colonize above ground plant
tissues, including woody tissues. The colonization of below and above
ground plant tissues have similarities, being influenced by factors such as
nutrient availability, water availability, host tissue structural characteris-
tics, host plant genetics, and the Trichoderma isolates involved (Bailey &
Melnick, 2013).
In addition to direct mycoparasitism of soil-borne fungal pathogens,
Trichoderma spp. interact with roots. This interaction induces systemic
resistance (ISR), which reduces disease in above ground parts of the plant.
In the molecular dialog between fungus and plant leading to ISR, proteins
secreted by T. virens provide signals (Lamdan et al., 2015). Trichoderma
spp. and other beneficial microorganisms do not cause disease, but result
in a stronger immune response upon subsequent challenge by a pathogen
(Van Vees et al., 1999; Prime et al., 2006; Conrath, 2011).
Recent discoveries indicate Trichoderma spp. can also induce
systemic and localized resistance to a variety of plant pathogens and act
as opportunistic, avirulent plant symbiont. Trichoderma spp. produce or
26 Biopesticides and Bioagents: Novel Tools for Pest Management

release a variety of compounds that induce localized or systemic resis-

tance responses. Few strains can establish robust and long-lasting colo-
nization of root surfaces, penetrate into the epidermis and a few cortical
cells below (Harman et al., 2004). This restricts further advance of the
Trichoderma and make the plants resistant to other diseases. Induced
resistance as a result of activated plant defense in response to Tricho-
derma colonization has been reported by several workers (Alfano et al.,
2007; Djonovic et al., 2006; Harman et al., 2004; Korolev et al., 2007;
Meyer et al., 1998; Segarra et al., 2009; Shoresh & Harman, 2008;
Shoresh et al., 2005). Root colonization by Trichoderma spp. frequently
enhances root growth and development, crop productivity, resistance to
abiotic stresses, and the uptake and use of nutrients (Harman et al., 2005).
Trichoderma strains become endophytic in roots, but the greatest changes
in gene expression occur in shoots. These changes alter plant physiology
and may result in the improvement of abiotic stress resistance, nitrogen
fertilizer uptake, resistance to pathogens, and photosynthetic efficiency.
Typically, the net result of these effects is an increase in plant growth and
productivity (Hermosa et al., 2012). The induction of defense response
in plants by Trichoderma spp. is mostly associated with accumulation of
various antimicrobial compounds like phytoalexins, pathogenesis-related
(PR) proteins along with the strengthening of cell walls and other barriers
in the plant cells.
Various groups of compounds secreted by Trichoderma spp. may act
as elicitors for the induction of defense responses in plants. Xylanase from
Trichoderma spp. induce systemic resistance in cotton, tobacco, grapevine,
and so forth (Anderson et al., 1993; Bailey & Lumsden 1998; Calderpn et
al., 1993; Elad 2000; Howell 2003). Cellulase produced by T. harzianum
acts as elicitor for systemic acquired resistance (SAR) by triggering perox-
idase and chitinase activity (Elad, 2003; Elad & Kapat, 1999). Fusarium
oxysporum f. sp. melonis and powdery mildew infections on greenhouse
melon plants were reduced in plants treated with cellulases.
Recently chitin oligosaccharides were found to act as elicitors of
defense response in plants, and the scavenging of such oligomers is funda-
mental to the lifestyle of fungal pathogens upon colonization of their hosts
(de Jonge et al., 2010). Effective Trichoderma strains produce a variety
of microbe-associated molecular patterns (MAMPs). The proteome and
transcriptome of plant leaves are observed to be systemically affected as a
result of Trichoderma root colonization and MAMP interactions (Shoresh
et al., 2010).
Trichoderma—An Impeccable Plant Health Booster 27

Xylanase and peptaibols (peptaibiotics with high content of

α-aminoisobutyric acid) like alamethicin and trichovirin II which are
produced by Trichoderma spp. can elicit an immune response in plants
(Leitgeb et al., 2007; Druzhinina et al., 2011). Recently, a PKS/NRPS
hybrid enzyme involved in defense responses in maize was identified
(Mukharjee et al., 2012a). The best characterized elicitor produced by
Trichoderma spp. is Sm1/Epl1, an abundantly secreted, small cysteine-rich
hydrophobin-like protein of the cerato-platanin (CP) family (Djonovic et
al., 2006; Seidl et al., 2006). Deletion of this Trichoderma gene impairs
elicitation of ISR in maize (Djonovic et al., 2006).
Trichoderma can colonize root intercellular spaces by recognizing and
adhering to the plant roots. Once inside the plant, it can tolerate the toxic
metabolites produced by the plant as a defense in response to fungal inva-
sion. Plant cell-wall-degrading enzymes are involved in active root coloni-
zation. Hermosa et al. (2012) reported that adherence of Trichoderma to the
root surface is facilitated by hydrophobins, which are small hydrophobic
proteins of the outermost cell wall layer that coat the fungal cell surface,
and expansin-like proteins related to cell wall development. T. asperellum
produces the class I hydrophobin TasHyd1, which support the coloniza-
tion of plant roots, possibly by enhancing its attachment to the root surface
and protecting the hyphal tips from plant defense compounds (Viterbo &
Chet, 2006), and the swollenin TasSwo, an expansin-like protein with a
cellulose-binding domain was able to recognize cellulose and modify the
plant cell wall architecture, to help in root colonization (Brotman et al.,
2008). In Trichoderma, this resistance has been associated with the pres-
ence of ATP-binding cassette (ABC) transport systems, which are key
factors in the multiple interactions established by Trichoderma biocon-
trol strains with other microbes in a potentially toxic or antagonistic envi-
ronment with rapid degradation of the phenolic compounds exuded from
plants (Chen et al., 2011), and with the suppression of phytoalexin produc-
tion, as detected in Lotus japonica during colonization with T. koningii
(Masunaka et al., 2011).

2.4 TRICHODERMA AS A PLANT GROWTH STIMULANT

Trichoderma has recently emerged out as a successful plant growth

promotor and is reported to enhance plant growth (Hermosa et al., 2012;
Harman et al., 2004; Mc Lean et al., 2005; Bae et al., 2009; Joshi et al.,
28 Biopesticides and Bioagents: Novel Tools for Pest Management

2010; Rojan et al., 2010; Singh, 2010; Masunaka et al., 2011; Sharma et
al., 2012). Seed treatment with Trichoderma spp. usually improves seed-
ling vigor. The growth promotion effect is dependent not only on isolate
of Trichoderma but also on plant species and/or cultivar involved (Singh
et al., 2011).
Trichoderma spp. have a positive effect on plant growth and yield in
some vegetable crops by promoting efficiency of basal organic fertilizer
application. The increased growth response is mainly due to hydrolysis
of cellulose in soil, mineral solubilization and uptake of minor and other
minerals as well as improvement in the root morphology enabling the
roots to explore a large volume of soil (Maral et al., 2012).
The colonization of cucumber roots by T. asperellum (asperelloides)
colonization in cucumber roots enhances the availability of P and Fe to
plants resulting in significant increases in dry weight, shoot length, and
leaf area (Yedidia et al., 2001). The uses of T. harzianum improves the
growth and development of maize plants (Akladious & Abbas, 2012)
causing an increase in all measured parameters which include growth,
chlorophyll content, starch content, nucleic acids content, total protein
content, and phytohormones content of maize plants when applied to the
soil or the seeds but the magnitude of these increases was much more
pronounced in case of plants developed from seeds treated with various
concentrations of metabolic solution of T. harzianum. Even if planted
in poor nitrogen soil, corn seeds pre-treated with T. harzianum (T-22)
were greener and larger initially and gave increased yields at maturity
(Harman, 2000).
Trichoderma is also reported to increase the production of humic acid.
According to an unpublished report, almost six-fold increase in water-
soluble humic matter content was reported in colonized FYM as compared
to non-colonized FYM that led to a significant increase in growth of tomato
and okra plants as compared to non-colonized FYM and/or control. This
report suggests an additive effect of microorganism with humic acid in
better plant growth.

2.5 TRICHODERMA AS AN ABIOTIC STRESS RELIEVER OF

CROP PLANTS

Apart from biocontrol of plant diseases, one of the major attribute of the
fungus is its ability to enhance abiotic stress tolerance of plants, including
Exploring the Variety of Random
Documents with Different Content
Montcontour, 177.
Montlhery, 179.
Montmarthe, 78. — Montmartre.
Mores, 36, 37.
Motin, 8, 30.
Mun (Jan de), 164.
Naples, 164.
Narcis, 178.
Neptune, 159.
Nonne (tour de), 48. — Contraction de Torre dell’ annona. Tour de
Rome, qui, après avoir servi de grenier à blé, devint une prison.
Normans (les), 29.
Oger, 164. — Oger dit le Danois, l’un des compagnons de Roland.
Osse, 79. — Le mont Ossa en Thessalie.
Othomans (les), 166.
Ovide, 78, 143.
Oye (mere l’), 124.
Oyse, 122.
Palais (le), 61, 64, 69, 137.
Palatin (le mont), 44.
Pape (le), 69.
Paris, 49, 63, 108, 119, 122, 161.
Parnasse, 13, 30, 34.
Passerat, 149.
Patisson, 34. — Célèbre imprimeur français du XVIe siècle, à qui l’on
doit un grand nombre d’ouvrages, modèles de typographie & de
correction.
Patrasse, 83. — Le golfe de Patras & celui de Lépante ne forment
qu’un long golfe resserré à son milieu par un détroit de chaque
 côté duquel se trouvent, au nord, Lépante en Phocide, & au sud,
Patras en Achaïe.
Pedre (Domp), 76. — Don Pedro de Tolede, connétable de Castille,
général des galères de Naples & parent de Marie de Médicis. Il
arriva à Paris le 22 juillet 1608. M. de Fréville a fait paraître dans
la Bibliothèque de l’École des Chartes (1844, p. 344) le pamphlet
publié au sujet de son entrée & l’on trouve dans le Registre
journal de l’Estoile des détails piquants sur ses entrevues avec
Henri IV.
Pelion, 79.
Perou, 27.
Perron (du), 241, 242.
Perse, 37.
Pescheurs (la Guide des), 106. — Ouvrage de Fr. Luis de Granada,
dont on ne connaît pas moins de cinq traductions françaises
publiées en 1574 à Douai, en 1577 à Reims, en 1585 & 1674 à
Lyon, & en 1658 à Paris.
Petrarque, 88. — Le remede de Petrarque est le traité de ce poëte,
de remediis utriusque fortunæ. Cremonæ, 1492, in-fo.
Phœbus, 11, 15, 34, 45, 67, 78, 87, 122, 150.
Pin (la Pomme de), 79. — Cabaret déjà célèbre du temps de Villon. Il
était situé dans la Cité, rue de la Juiverie, vis-à-vis de la
Madeleine.
Pinde (le), 149.
Platon, 20, 28, 73.
Pline, 81.
Plutus, 201.
Polyenne, 95. — Héros d’une aventure amoureuse décrite dans
Pétrone.
Pontalais (Janin du), 206. — Le vrai nom de ce farceur, qui débitait
ses bons mots à la pointe Saint-Eustache, était Jehan de l’Espine
du Pont-Allez, & son surnom Songe-creux. On trouve dans la
 Bibliothèque de du Verdier, 1773, III, 503, des indications à
consulter. Il est à peu près certain aujourd’hui que les Contredits
de Songe-creux, attribués à Gringore, sont de Pontalais. V. à ce
sujet une curieuse note des Var. hist. & litt. de M. Fournier, X,
356.
Pont-Neuf (le), 63.
Pontoise, 161.
Poytou (le), 160.
Priape, 150.
Prothée, 24.
Provence, 160.
Provins (le sieur de), 118.
Puis (Pierre du), 46. — Fou qui courait les rues, un pied chaussé d’un
chapeau. V. Bruscambille, Paradoxes, 1622, p. 45.
Quinze-Vingts (les), 86. — Hôpital fondé en 1254 par saint Louis,
pour 300 gentilshommes auxquels les Sarrasins avaient crevé les
yeux. Sauval rapporte dans ses Antiquités de Paris que, vers la
mi-carême, les quinze-vingts étaient donnés en spectacle. Cette
comédie d’un nouveau genre, à laquelle Charles IX & Henri III
assistèrent plus d’une fois, consistait dans une course au cochon.
L’animal, poursuivi par les quinze-vingts armés de bâtons,
devenait le prix de son vainqueur, c’est-à-dire de l’aveugle qui
parvenait à le rouer de coups.
Rapin, 66, 69, 70, 73, 175.
Rhain, 160.
Rhée, 48.
Rochelle (La), 26.
Roland, 164.
Romains, 12.
Rome, 27, 41, 59, 78, 106, 179.
Ronsard, 18, 22, 33, 38, 73.
Rosete, 60, 119. — Coquette chansonnée par Desportes.
Rousset, 125.
Royaumont, 122. — Abbaye de l’ordre de Cîteaux, fondée par saint
Louis en 1228, entre Beaumont-sur-Oise & la forêt du Lys, en un
lieu appelé Cuimont qui fut nommé depuis Royaumont. V.
l’histoire de cette abbaye par l’abbé Duclos. Paris, Douniol, 1867.
Sardaigne, 45.
Saturne, 150.
Savoye, 22, 80.
Savoye (l’Escu de), 177. — Taverne meritoire. V. Rab., II, 6.
Scaures, 38.
Scipion, 78.
Sées, 36.
Seine, 205.
Sicille, 164.
Socrate, 17, 74.
Sydon, 122. — Aujourd’hui Saïda, l’une des échelles du Levant. Cette
ville a été prise en 1110, par Baudouin, premier roi de Jérusalem.
C’est par erreur que Regnier en attribue deux fois la conquête à
saint Louis. Ce dernier roi n’a en effet séjourné en Palestine
qu’après sa captivité à Mansourah en 1251. Avant de revenir en
France, il passa trois ans à réparer les forteresses restées en
possession des chrétiens, Césarée, Jaffa, Saint-Jean-d’Acre &
Sidon.
Symonide, 84.
Tantale, 119.
Tasse (le), 73.
Thebaide (la), 202.
Therese (la mere), 106. — Sainte Thérèse, morte en 1582, canonisée
en 1621. Regnier a ici en vue le livre des Méditations sur la
communion, l’un des ouvrages de la célèbre carmélite.
Terence, 81.
Thespean (antre), 34. — Thespies, ville de Béotie située au pied de
l’Hélicon & consacrée aux Muses.
Tibre, 44.
Toscane, 22.
Trace, 32, 83.
Troyen (le), 180. — Pâris.
Tuileries, 63.
Turc, 55.
Turpin, 80.
Tyrtée, 6.
Urgande, 124.
Vandôme, 161.
Vanves, 20.
Venise, 50.
Venus, 53, 56, 168, 197.
Verrès, 38.
Vialard, 221.
Virgille, 11, 20, 73, 177.
Zephire, 121.
FAC SIMILE DE LA PROFESSION DE FOI DE MATHURIN REGNIER
D’après le livre de profession des chanoines de Chartres, conservé
aux Archives du Dépt d’Eure & Loir.
 TABLE DES MATIERES

Pages.
Avertissement I
Notice V

PREMIERES ŒVVRES DE M. REGNIER

Epitre liminéaire au Roy 3

Ode à Regnier 5
Satyre I. Discours au Roy 9
— II. A M. le Comte de Caramain 14
— III. A M. le Marquis de Cœuures 22
— IIII. A M. Motin 30
— V. A M. Bertault, Euesque de Sées 36
— VI. A M. de Bethune 44
— VII. A M. le Marquis de Cœuures 52
— VIII. A M. l’Abé de Beaulieu 58
— IX. A M. Rapin 66
— X. Ce mouuement de temps 74
— XI. Suitte. Voyez que c’est du monde 88
— XII. A M. Freminet 100
— XIII. Macette 105
 — XIIII. I’ay pris cent & cent fois 114
— XV. Ouy i’escry rarement 120
— XVI. A M. de Forqueuaus 129
— XVII. Non non i’ay trop de cœur 131
Élegie zelotipique 135
Autre. Aymant comme i’aymois 141
Impuissance. Imitation d’Ouide 143
Sur le trespas de M. Passerat 149
Stanses. Le tout puissant Iupiter 150
La C. P. Infame bastard 151
Sur le portraict d’vn Poëte couronné 154
Contre vn amoureux transy 155
Quatrains. Si des maux qui 157
— Ie n’ay peu rien voir qui me plaise »
— Ie croy que vous auez faict vœu »
— Le Dieu d’Amour se deuoit peindre »
— Ceste femme à couleur de bois 158
Discours au Roy 159
Plainte. En quel obscur seiour 167
Ode. Iamais ne pourray-ie bannir 173
Sonnet sur la mort de M. Rapin 175
Discours d’vne maquerelle 176
Épitaphe de Regnier 182

ŒVVRES POSTHVMES

Dialogue. Cloris & Phylis 185

Satyre. N’avoir crainte de rien 199
— Perclus d’vne jambe & des bras 203
Élegie. L’homme s’oppose en vain 207
Vers spirituels. Stances. Quand sur moy 211
Sur la Nativité de Nostre Seigneur 215
Sonnet I. O Dieu, si mes pechez 217
— II. Quand devot vers le ciel 218
— III. Cependant qu’en la croix »
Commencement d’vn poëme sacré 220
Épigramme. Vialard, plein d’hypocrisie 221
Ode sur une vieille maquerelle 222
Stances. Ma foy, ie fus bien de la feste 225
Épigramme I. Amour est vne affection 226
— II. Madelon n’est point difficile »
— III. Hier la langue me fourcha »
— IV. Lorsque i’estois comme inutile 227
— V. Dans vn chemin vn pays »
— VI. Lizette à qui l’on faisoit tort 228
Stances. Si vostre œil tout ardant 229
Complainte. Vous qui violentez 231
Stances pour la belle Cloris 236
Épigramme I. Faut auoir le cerueau 238
— II. Le violet tant estimé »
— III. L’argent, tes beaux iours »
— IV. Quelque moine 239
— V. Vn homme gist »
Appendice 241
Variantes & notes 243
Glossaire 281
Index 315
 Achevé d’imprimer
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PAR J. CLAYE
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