Vol. 9(53), pp. 3838-3852, 31 December, 2014
DOI: 10.5897/AJAR2014. 9061
Article Number: CC4D79D50124
ISSN 1991-637X
African Journal of Agricultural
Copyright © 2014 Research
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJAR

Review

Trichoderma: Mass production, formulation, quality

control, delivery and its scope in commercialization in
India for the management of plant diseases
Sanjeev Kumar1*, Manibhushan Thakur2 and Archana Rani3
1
Department of Plant Pathology, College of Agriculture, Jawahar Lal Nehru Krishi Vishwavidyalaya-Jabalpur
(MP), 482004, India.
2
Department of Plant Pathology, B.K.S. College of Agriculture-Dumroan, Buxar (Bihar) 802136, India.
3
Department of Agricultural Biotechnology and Molecular Biology, (FBS & H), Rajendra Agricultural University,
Pusa-848125 (Bihar), India.
Received 11 August, 2014; Accepted 17 November, 2014

Popularization of biopesticides is very slow as compared to chemicals and only 2% biopesticides are
available. Among the different biopesticides, Trichoderma is most exploited and have many success
stories. Trichoderma viride and Trichoderma harzianum have curved a niche for themselves in India as
important biocontrol agents for management of various diseases. A number of successful products
based on different species of Trichoderma have been commercialized in India. The potential
Trichoderma isolates are formulated using different organic and inorganic carriers either through solid
or liquid fermentation technologies. They are delivered either through seed treatment, bio-priming,
seedling dip, soil application, and foliar spray. Application of Trichoderma formulations with strain
mixtures perform better than individual strains for the management of pest and diseases of crop plants,
in addition to plant growth promotion. Commercialization of the bioproducts is primarily hindered due
to the poor shelf life. Hence, research should be concentrated to increase the shelf life of the
formulation by developing superior strains that support the increased shelf life, or the organic
formulations that support the maximum shelf life with low level of contaminants must be standardized
for making biocontrol as a commercial venture. Whatever the limitations these Trichoderma products
may have, it can be addressed by enhancing biocontrol through manipulation of the environment,
accurate strain identification by molecular approach, using mixtures of beneficial organisms,
physiological and genetic enhancement of biocontrol mechanisms, and manipulation of formulations.
Of late, many small and large entrepreneurs have entered into the commercial production of bio control
agents resulting into the entry of various bio- control products into the world market.

Key words: Trichoderma, mass production, formulation, shelf-life, commercialization.

 
INTRODUCTION

The green revolution has led to intensified agriculture to globally. Nevertheless, this also damages the natural
meet the ever increasing demands for food and fiber ecosystems by polluting the ground water and food stuffs

*Corresponding author. Email: sanjeevcoa@gmail.com

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

besides degrading the environment. Plant diseases are
one of the major concerns in cultivation worldwide
consequential in loss of billions of dollars of farm
produce. There is a pressing need to manage diseases to
make sure a steady and constant supply of marketable
produce for the escalating world population. In disease
management, the amplified use of chemicals have
caused negative impact on environmental quality and
resulted in upward trend of many living forms which are
resistant to the chemicals (Kumar and Gupta, 2012).
Under the changing agriculture scenario, the only
technology that seems promising to manage the diseases
without disturbing the equilibrium of harmful and useful
composition of environment and ecosystem is the use of
more and more biological control agents. Trichoderma
spp. are biocontrol agents extensively used in
management of fungal diseases of crop plants exhibiting
mycoparasitism against a wide range of plant pathogens.
Their usage has been very much successful against soil
borne diseases for which no resistant sources have been
identified in the plants. Their biocontrol potential has also
been explored against several foliar diseases by seed or
soil application indicating their effectiveness in inducing
disease resistance in the crop plants. Interestingly,
similar to rhizobacteria, Trichoderma spp. has also been
exploited as a growth promoting agent. Thus, it has the
potential as a preferred input in Integrated Disease
Management (IDM) systems. They are also widely
exploited in industries as sources of enzymes. A large
number of research groups are working on various
aspects of Trichoderma viz., diversity, ecology and their
applications. In India, about 110 groups representing
various universities and research institutes are working
with about 15 different species and have published about
460 research papers. Trichoderma harzianum and
Trichoderma viride are the widely used species and have
been exploited on about 87 different crops and about 70
soil borne and 18 foliar pathogens, respectively (Sharma
et al., 2014).
Indian government is promoting research, production,
registration, and adoption of biopesticides with open
hand, through various rules, regulations, policies, and
schemes. The National Farmer Policy (2007) has strongly
recommended the promotion of biopesticides for
increasing agricultural production, sustaining the health of
farmers and environment. It also includes the clause that
biopesticides would be treated at par with chemical
pesticides in terms of support and promotion. In spite of
all these efforts the share of biopesticides in India is
merely 2% compared to 40% in USA and 20% each in
Europe and Oceanic countries (Sabalpara, 2014). A
major difficulty faced while translating the efficacious
biocontrol agents into merchandise was that the efficacy
of the developed strains was largely unstable under
diverse environmental conditions in which the finished
product of organismal origin was to tread before reaching
the farmer’s field. The proportion of registration of
Kumar et al. 3839
biocontrol agents for commercial availability is also very
slow. In addition, the present day bio-products needed to
be further improved to obtain greater levels of disease
reduction. Development of formulations with increased
shelf life and broad spectrum of action with consistent
performance under field conditions could pave the way
for commercialization of the technology at a faster rate. In
this context, the current review mainly focuses on
formulation development, mass production, quality
control, delivery system and its scope in commercial
lization in India for the management of plant diseases.
CHARACTERISTICS OF TRICHODERMA FOR
FORMULATION DEVELOPMENT
Our early work demonstrated that Trichoderma spp. did
indeed have the ability to control plant diseases.
However, the level of efficacy and the reliability of simple
approaches to biocontrol gave results that were
substantially less than that of commercial fungicides.
Over several years, we have concluded that
biocontrol systems must be developed if effective results
are to be obtained. To develop a successful Trichoderma
formulation, Trichoderma should possess
i) High rhizosphere competence.
ii) High competitive saprophytic ability.
iii) Enhanced plant growth.
iv) Ease for mass multiplication.
v) Broad spectrum of action.
vi) Excellent and reliable control.
vii) Safe to environment.
viii) Compatible with other bioagents.
ix) Should tolerate desiccation, heat, oxidizing agents and
UV radiations (Jeyarajan and Nakkeeran, 2000).
FORMULATION DEVELOPMENT
Major research on biocontrol is centered with the use of
spores of Trichoderma directly to seed. Technologies
become viable only when the research findings are
transferred from the lab to field. Though Trichoderma has
a very good potential in the management of diseases, it
could not be used as spore suspension under field
conditions. Thus, the culture of Trichoderma should be
immobilized in certain carriers and should be prepared as
formulations for easy application, storage, comer -
cialization, and field use.
Characteristics of an ideal formulation
i) Should have increased shelf life.
ii) Should not be phytotoxic to the crop plants.
iii) Should tolerate adverse environmental conditions.
3840 Afr. J. Agric. Res.
iv) Should be cost effective and should give reliable
control of plant diseases.
v) Should dissolve well in water.
vi) Carriers must be cheap and readily available for
formulation development.
vii) Should be compatible with other agrochemicals.
MODEL METHODS FOR THE MASS MULTIPLICATION
OF TRICHODERMA
Commercial application of Trichoderma either to increase
crop health or to manage plant diseases depend on the
development of commercial formulations with suitable
carriers that support the survival of Trichoderma for a
considerable length of time.
Talc based formulation
In India, talc based formulations of T. viride was
developed at Tamil Nadu Agricultural University,
Coimbatore for seed treatment of pulse crops and rice
(Jeyarajan et al., 1994). Trichoderma is grown in the
liquid medium is mixed with talc powder in the ratio of 1:2
and dried to 8% moisture under shade. The talc
formulations of Trichoderma has shelf life of 3 to 4
months. It has become quite popular in India for
management of several soil-borne diseases of various
crops through seed treatment at 4 to 5 g/kg seed. Several
private industries produce large quantities of talc
formulations in India for supply to the farmers. The
annual requirement of Trichoderma has been estimated
as 5,000 tones to cover 50 per cent area in India
(Jeyarajan, 2006).
Vermiculite-wheat bran based formulation
Trichoderma is multiplied in molasses-yeast medium for
10 days. 100 g vermiculite and 33 g wheat bran are
sterilized in an oven at 70°C for 3 days. Then, 20 g of
fermentor biomass, 0.05 N medium and concentrated or
entire biomass with HCl are added, mixed well and dried
in shade (Lewis, 1991).
Pesta granules based formulation
52 ml Fermentor biomass is added to wheat flour (100 g)
and mixed by gloved hands to form cohesive dough. The
dough is kneaded, pressed and folded by hand several
times. Then one mm thick sheets (pesta) is prepared and
air-dried till it breaks crisply. After drying, dough sheet
was ground and passed through an eighteen mesh and
granules were collected (Connick et al., 1991).
Alginate prills based formulation
Sodium alginate is dissolved in one portion and distilled
water (25 g/750 ml) and food base is suspended in
another portion (50 g/250 ml). These preparations are
autoclaved and when cool are blended together with
biomass. The mixture is added drop wise into CaCl2
solution to form spherical beads, which are air-dried and
stored at 5°C (Fravel et al., 1985).
Press mud based formulation
Press mud is available as a byproduct of the sugar
factory and this can be used as a substrate for mass
multiplication of Trichoderma. The method involved
uniformly mixing of 9 days old culture of T. viride
prepared in potato dextrose broth into 120 kg press mud.
Water was sprinkled intermittently in to keep it moisten.
This was covered by gunny bags to permit air movement
and trap moisture under shade. Within 25 days, nucleus
culture for further multiplication becomes ready. The
same was added to 8 tons of press mud, mixed
thoroughly and incubated for 8 days under shade
condition before being applied in the field. By this we
added 8000 times more inoculums in the soil than the
recommended doses of biopesticides which rapidly get
established showing rapid and visible effect. Similarly,
other substances could also be effectively used for the
multiplication of different bioagents at the mass level
(Sabalpara, 2014).
Coffee huskbased formulation
In Karnataka Sawant and Sawant (1996) developed a
Trichoderma formulation based on coffee husk which is a
waste from coffee curing industry. This product was very
effective in managing Phytophthora foot rot of black
pepper and is widely used in Karnataka and Kerala.
Oil-based formulations
They are prepared by mixing the conidia harvested from
the solid state/liquid state fermentation with a
combination of vegetable/mineral oils in stable emulsion
formulation. In such formulations, microbial agents are
suspended in a water immiscible solvent such as a
petroleum fraction (diesel, mineral oils), and vegetable
oils (groundnut etc.) with the aid of a surface active
agent. This can be dispersed in water to form a stable
emulsion. Emulsifiable concentrates require a high
concentration of an oil soluble emulsifying agent, to give
an instantaneous formation of a homogenous emulsion
on dilution in water. The oils used should not have toxicity
to the fungal spores, plants, humans and animals. Such

formulations of Trichoderma is now being used as foliar
sprays. Oil-based formulations are supposed to be
suitable for foliar sprays under dry weather and to have
prolonged shelf life. The spores can survive for longer
time in the plant surface even during the dry weather as
the spores are covered by oil that protects them 5°C from
drying. Batta (2005) developed an emulsion formulation
of T. harzianum for the control of post harvest decay of
apple caused by Botrytis cinerea. Invert-emulsion
formulation of T. harzianum with a shelf life of 8 months
has been developed using indigenous constituents at the
erstwhile Project Directorate of Biological Control
(PDBC), in India and this his formulation has been and
found to be effective against soil borne diseases of
groundnut.
Banana waste based formulations
The mass multiplication protocol of Trichoderma sp. in
banana waste -was proposed by Balasubramanian et al.
(2008). For the same banana waste, urea, rock
phosphate, culture of Bacillus polymixa, P. sajor caju and
T. viride area used. A pit of different banana waste viz.,
sheath pseudo stem and core is chopped in the length of
5 to 8 cm. A pit is prepared and different ingredients are
placed in five different layers. Each layer contains one
ton banana waste, 5 kg urea, 125 kg rock phosphate and
one litre broth culture of B. polymixa, P. sajor caju and T.
viride. Five different layers are prepared similarly and
mixed thoroughly Banana. Banana waste is decomposed
within 45 days and enriched culture is mass available for
field application.
SHELF LIFE OF TRICHODERMA FORMULATIONS
Shelf life of the formulated product of a biocontrol agent
plays a significant role in successful marketing. In
general, the antagonists multiplied in an organic food
base have longer shelf life than the inert or inorganic food
bases. Shelf life of Trichoderma in coffee husk was more
than 18 months. Talc, peat, lignite and kaolin based
formulation of Trichoderma, have a shelf life of 3 to 4
months. The viable propagules of Trichoderma in talc
formulation were reduced by 50% after 120 days of
storage (Sankar and Jeyarajan, 1996). Studies on the
storage of T. viride formulation in poly propylene bags of
various colours revealed that the population of T. viride
was maximum in milky white bags of 100 micron
thickness. At PDBC, Bangalore work on increasing shelf
life of talc formulations of Trichoderma using various
ingredients (chitin and glycerol) in production medium
and heat shock at the end of log phase of fermentation
was carried out which can extend the shelf of talc
formulation of successfully up to one year (Sriram et al.,
2010, 2011). Bhat et al. (2009) reported shelf life of talc
based formulation up to 180 days under ambient
Kumar et al. 3841
condition. The rate of desiccation was less up to the first
75 days of storage as compared to that observed from 75
to 180 days of storage. The formulation retained good
numbers of viable propagules (above 106 cfu/g) for more
than 150 days of storage.
IMPROVEMENT OF FORMULATION EFFICACY
In general, though biocontrol agents perform well in the
management of plant diseases, they are highly sensitive
to the fluctuations in environmental conditions and are
inconsistent in their performance. The consistency of
biocontrol agents could be enhanced through several
means.
Molecular approaches
The construction of superior biocontrol agents after
isolation and characterization of genes involved in
biocontrol properties is necessary for the successful
management of plant diseases. Moreover, molecular
techniques allow modification of wild type strains to
improve their ability to control diseases. The selected
biocontrol could be improved by genetic modification and
recombinations techniques (Raguchander et al., 2011). In
general, protoplast fusion (Harman et al., 1989; Minucci
et al., 1991). Transposon mutagenesis (Brown and
Holden, 1998) and transformation techniques are used
for the genetic manipulation of biocontrol agents.
Protoplast fusion
Protoplast fusion is a quick and easy method in strain
development for bringing genetic recombination and
developing hybrid strains in filamentous fungi
(Lalithakumar and Mathivanan, 2003). This technique is
mainly used to combine the advantageous properties of
distinct promising agents. Most frequently, the
introduction of exogenous DNA through protoplast fusion
is done in presence of polyethylene glycol and calcium
chloride (Penttilä et al., 1987). Isolation, fusion and
regeneration of protoplasts have been carried out in
Trichoderma strains mainly for improving the biocontrol
potential (Mrinalini and Lalithakumari, 1998). Ogama et
al. (1989) reported the enhanced cellulose production of
T. reesei by inter-specific protoplast fusion. Similarly, self
fusion of protoplasts from T. harzianum strain PTh18
showed two fold increases in chitinase and biocontrol
activity as compared to the parent strain against
Rhizoctonia solani (Prabavathy et al., 2006).
Genetic recombination
Integrating foreign DNA into the genome of a biocontrol
agent is powerful way to improve the biocontrol activity.
3842 Afr. J. Agric. Res.
To a certain extent, this involves the construction of
strains that produce increased levels of lytic enzymes
and antibiotics (Glick and Bashan, 1997). On the other
hand, the suppression or deletion of gene from the
biocontrol strains could also enhance the sustained
biocontrol activity of the strains. The biosynthesis of cell
wall degrading enzymes like glucanases, chitinase and
protease etc. produced by Trichoderma spp. which are
involved in mycoparasitism is controlled mainly at
transcriptional level and responsible genes are present
as single copy genes. To overproduce these enzymes,
their gene copy number has been increased by
transformation (Harmon and Bjoorman, 1998).
Transformants overproducing these enzymes are more
efficient as biocontrol agents. Biocontrol efficiency of
Trichoderma has been improved by transformation with
genes prbl (protease), egll (β-1,4-glucanase) and chit 33(
chitinase). Lorito et al. (1998) developed transformant of
T. harzianum strain 1295-22 by integrating β–
glucuronidase (GUS) and hygromycin B (hyg B)
phosphor-transferase genes that exhibited increased
biocontrol activity R. solani as compared with the wild
type.
Mutation
Mutation is employed to generate variability in
populations, which give an opportunity to select the
desirable type. Criteria defined for selecting a particular
phenotype from the population, N- methyl-N- nitro
nitrosoguanidine (NTG) has been the most widely used
chemical for inducing mutations in fungal Trichoderma.
By exposing the conidia of Trichoderma spp, to NTG,
Ahmad and Baker (1987) generated mutants that were
rhizosphere competent and superior to wild type in
respect of controlling Pythium ultimum. These mutants
were insensitive to up to 100 µg/ml of benomyl.
Mukhopadhyay et AL, (1993) developed seven stable
mutants of T. virens by exposing the cultures to 125 k rad
of gamma radiation. The mutants differed from the wild
type strains in phenotype, growth rate, sporulation and
antagonistic potential. One of the mutants (M-7) that
produced profuse chlamydospores but no conidia was
more aggressive than the wild type in antagonizing
Sclerotium rolfsii and Fusarium oxysporum f. sp. ciceri.
Selvakumar (2000) developed carboxin tolerant mutant T.
viride by exposing the culture to UV-light and ethyl
methane sulfonate, Triazole tolerant biotypes of T. viride
developed by UV-irradiation showed wide variation in
enzyme activity especially β-1,3-glucanase and
chitinases.
Development of compatible consortia
Apart from genetic manipulation of biocontrol strains to
improve their efficacy, the knowledge on mechanism of
action by biocontrol strains offers great insight for
combining various mode of action. For Trichoderma plant
beneficial purposes, strain mixtures and combinations
with other bacteria or fungi often provided more effective
disease control than the application of an individual
biocontrol of Trichoderma alone (de Boer, 2003). Another
approach to obtain a successful microbial biocontrol
consortium is to apply mixtures of biocontrol agents
display of different disease suppressive mechanisms that
are complementary to each other. Cocktail of various
Trichoderma strains provided enhanced protection than
the single organism (Raguhchander et al., 2011; Kumar,
2013). Kumar and Upadhyay (2003) reported that
integration of seed treatment with T. viride with
Rhizobium significantly improved root and shoot length,
number of nodules per plant, their fresh and dry weight in
chickpea. Development of strain mixtures with non-
competitive nature of these fungal and bacterial strains
will have an additive effect in increasing the yield and
growth. Consortia of T. viride in combinations with T.
harzianum were found effective than the application of
individual organisms for management of maydis leaf
blight of maize (Jha et al., 2004). Singh and Singh (2012)
studied the effect of individual and consortium of T.
harzianum isolates BHU-51 and BHU-105 against
Sclerotinia sclerotiorum rot of brinjal. He observed
maximum percent disease reduction and yield in the
seedlings treated with consortium. The seeds and
seedlings treatment with consortium of Trichoderma spp
and Pseudomonas spp. results in plant growth promotion,
yield and simultaneously reduce the disease severity of
sclerotium rolfsii rot in tomato in contrast to application of
individual bioagent (Singh et al., 2013). In another,
biocontrol agent combination study by Rao et al. (2013),
T. harzianum and Pseudomonas fluorescens were found
effective against brown leaf spot disease of paddy.
Manjunatha et al. (2013) reported combination of T. viride
and P. fluorescens to be effective against dry root rot of
chickpea caused by M. phaseolina. Manoranitham et al.
(2000) reported that the application of T. viride and P.
fluorescens effectively checked the pre-emergence and
post-emergence damping off of tomato caused by P.
aphanidermatum under pot culture experiments.
Thankamani et al. (2005) observed the biocontrol effect
of a P. fluorescens isolate IISR-6 and T. harzianum
isolate P-26 on the growth of black pepper. Shanmugam
et al. (2012) designed effective strain mixtures of
antagonistic T. harzianum isolate S17TH and a
Burkholderia cepacia isolate TEPF Sungaln for rhizome
rot control in polyhouse and field conditions. A systematic
study was carried out in mustard by Gaur et al. (2010)
where seed and foliar spray treatments of mixed
formulation of T. hamatum (HP-20) and T. viride (Tv-1)
were found superior in field trials. Hence, the application
of Trichoderma, especially in the consortium form, are
more useful than the individual because of two or more
than two compatible isolates of same or different species

with same or different quality work synergistically and
give the high impact of their use.
Adjuvants, spreaders and stickers
Performance of Trichoderma in the formulations can be
increased by the incorporation of water-soluble
adjuvants, oils, stickers and emulsions. It increases the
efficacy of biocontrol agents by supplying nutrients and
by protecting the microbes from desiccation and death
(Connick et al., 1991; Bateman et al., 1993; Barnes and
Moore, 1997; Green et al., 1998; Ibrahim et al., 1999).
Incorporation of carboxy methyl cellulose (CMC) in
formulations serves as stickers in uniform seed coating of
microbes. Though adjuvants and stickers increase the
efficacy of bio-products it has its own demerits.
Adjuvants/stickers in the formulations will be diluted when
exposed to rain or heavy dew. It would alter the efficacy
of formulations by reducing the establishment or
colonization of Trichoderma onto the infection court.
Sometimes spray application of emulsions or oil-based
formulations may be toxic to plants. Consequently, a
comprehensive knowledge on the usage of adjuvants,
stickers is crucial for increasing the efficacy of
formulations.
DELIVERY OF TRICHODERMA FOR DISEASE
MANAGEMENT
For successful diseases control, delivery and
establishment of Trichoderma to the site of action is very
important. The most common methods of application of
Trichoderma are seed treatment, seed biopriming,
seedling dip, soil application and wound dressing.
Seed treatment
Seed coating with Trichoderma is one of the easy and
effective methods of delivering the antagonist for the
management of seed/soil-borne diseases. Seed is coated
with dry powder/dusts of Trichoderma just before sowing.
For commercial purpose, dry powder of antagonist is
used at 3 to 10 g per kg seed based on seed size
(Mukhopadhyay et al., 1992). Propagules of biocontrol
agents germinate on the seed surface and colonize roots
of germinated seedlings and rhizosphere (Tewari, 1996;
Kumar et al., 2009). T. harzianum, T. virens and T. viride
were found to be effective seed protectants against
Pythium spp. and R. solani (Mukherjee and
Mukhopadhyay 1995). The rice seed treated with two
antagonistic fungi, viz., T. viride and T. harzianum were
found effective to control sheath blight of rice and
increase the yield of the crop (Das and Hazarika, 2000).
In another study T. viride was found an efficient agent to
Kumar et al. 3843
control the R. solani toxin activity against the same
disease (Sriram et al., 2000). Singh and Maheshwari
(2001) reported seed treatment with bioagents like T.
viride, T. harzianum, and G. virens found it useful in
combating loose smut of wheat. Trichoderma, being a
growth promoting agent also helps in increasing yield of
crops which has been demonstrated by application of T.
harzianum (Th3) in irrigated and dry areas of Kota and
Jaipur districts of Rajasthan which is also ecologically
competent (Sharma et al., 2012). Seed treatment with T.
harzianum, A. sativum and A. indica on par with the foliar
spray of mancozeb showed results against Alternaria
blight disease mustard caused by A. brassicae and A.
brassicicola and increasing the yield (Jagana et al.,
2013). Seed treatment with Trichoderma species
inhibited the growth of oilseed-borne fungi like Aspergillus
flavus, Alternaria alternata, Curvularia lunata, Fusarium
moniliforme, F. oxysporum, Rhizopus nigricans,
Penicillium notatum and Penicillium chrysogenum which
affects oil seed crops like soybean, sesame and
sunflower (Jat and Agalave, 2013).
Seed biopriming
Treating of seeds with biocontrol agents and then
incubating under warm and moist conditions until just
prior to emergence of radical is referred to as
bioprimming. This technique has potential advantages
over simple coating of seeds as it results in rapid and
uniform seedling emergence. Trichoderma conidia
germinate on the seed surface and form a layer around
bioprimed seeds. Such seeds tolerate adverse various
soil conditions better. Biopriming could also reduce the
amount of biocontrol agents that is applied to the seed.
Seed biopriming is successfully used in tomato, brinjal,
soybean and chickpea in Tarai region of Uttaranchal.
(Mishra et al., 2001). Three rhizosphere competent
microbial strains, viz., P. fluorescens OKC, Trichoderma
asperellum T42 and Rhizobium sp. RH4, individually and
in combination in bioprimed seeds of chickpea and rajma
in pots and fields showed higher germination percentage,
and better plant growth in both the crops compared to
non-bioprimed control plants. It was also observed that
the combined application of the microbes enhanced seed
germination and plant growth better than their individual
application. Among the combinations, all combinations
comprising Trichoderma showed better results compared
to the others and the triple microbial combination
demonstrated best results in terms of seed germination
and seedling growth in both chickpea and rajma (Yadav
et al., 2013).
Soil treatment
Soil is the repertoire of both beneficial and pathogenic
3844 Afr. J. Agric. Res.
microbes. Delivering of Trichoderma spp. to soil will
increase the population dynamics of augmented fungal
antagonists and thereby would suppress the
establishment of pathogenic microbes onto the infection
court. There are several reports on the application of
biocontrol agents to the soil either before or at the time of
planting for control of a wide range of soil-borne fungal
pathogens (Baby and Manibhushanrao, 1996; Kumar,
2010a; Kumar et al., 2009). Soil application of T. viride
either alone and in combination with other treatments
significantly reduced red rot caused by C. falcatum
(Reddy et al., 2009). Srivastava et al. (2010) suggested
that the soil application of T. viride was found to be best
in controlling seedling blight, color rot, stem rot and root
rot disease of Jute. Soil application of organic preparation
of Trichoderma was effective in managing seed borne
pathogenic fungi F. oxysporum, F. moniliforme, F. solani,
B. theobromae, A. alternata and R. solani and in seedling
establishment of Dalbergia sissoo Roxb (Mustafa et al.,
2009). Trichoderma is capable of colonizing farm yard
manure (FYM) and therefore application of colonized
FYM to the soil is more appropriate and beneficial. This is
the most effective method of application of Trichoderma
particularly for the management of soil-borne diseases.
Root treatment
Seedling roots can be treated with spore or cell
suspension of antagonists either by drenching the
Trichoderma in nursery beds or by dipping roots in
Trichoderma suspension before transplanting. Root
dipping in antagonist's suspension not only reduces
disease severity but also enhances seedling growth in
rice, tomato, brinjal, chili and capsicum (Singh and Zaidi,
2002). There are also reports on the reduction of sheath
blight disease of rice by root dip of seedlings before
transplantation (Vasudevan et al. 2002).
Foliar spraying/Wound dressing
The efficacy of biocontrol agents for foliar diseases is
greatly affected by fluctuation of microclimate.
Phyllosphere is subjected to diurnal and nocturnal, cyclic
and non-cyclic variation in temperature, relative humidity,
dew, rain, wind and radiation. Hence water potential of
phylloplane microbes will be varying constantly. It will
also vary between leaves or the periphery of the canopy
and on sheltered leaves. Higher relative humidity could
be observed in the shaded, dense region of the plant
than that of peripheral leaves. The dew formation is
greater in centre and periphery. The concentration of
nutrients like amino acid, organic acids and sugars
exuded through stomata, lenticels, hydathodes and
wounds varies highly. It affects the efficacy and survival
of antatognist in phylloplane (Andrews, 1992). The liquid
suspension of Trichoderma has been successfully
applied to the aerial plant parts for the biocontrol of
Alternaria leaf spot of Vicia faba (Kumar et al., 2005).
Khan and Sinha (2005, 2007) emphasized on the
usefulness of T. harzianum and T. virens in foliar sprays
and talc-based formulations for reducing disease
incidence of sheath blight of rice. Sharma et al. (2012)
carried out field trails in Rajasthan on the groundnut root
rot disease caused by multiple pathogen complex mainly
A. niger, A. flavus, S. rolfsii, Thievaliopsis basicola, R.
solani and P. aphanidermatum by the application of T.
harzianum in the form of powder and liquid bio-
formulation and found it effective in controlling disease in
field. Singh et al. (2000) managed citrus scab caused by
Elsinoe fawcettii. They found that T. harzianum and E.
purpurascens reduced the disease incidence in the field
on spraying by 17.8 and 10%, respectively. Though foliar
application of Trichoderma reduce the severity of
diseases under field conditions, it is not technically
feasible due to increased dosage and economy realized
from the crop. Hence, dosage and frequency of
application has to be standardized based on the crop
value, which could be as a reliable and practical
approach.
Multiple delivery systems
Plant pathogens establish host parasite relationships by
entering through infection court such as spermosphere,
rhizosphere and phyllosphere. Hence, protection of sites
vulnerable for the entry and infection of pathogens would
offer a better means for disease management.
Jeyalakshmi et al. (2013) revealed that seed treatment
and soil application (2.5 kg/ha) of T. viride followed by
foliar spray of azadirachtin @ 3 mL/L on 30 and 45 DAS
was found to be significantly effective by recording
minimum disease incidence coupled with maximum seed
yield in Sesame. Seed and foliar application of T. viride
reduced Alternaria blight in linseed under field conditions
(Singh et al., 2013). A systematic study was carried out
by Gaur et al. (2010) where seed and foliar spray
treatments of mixed formulation of T. hamatum (HP-20)
and T. viride (Tv-1) were found superior in field trials for
management of Sclerotinia rot of mustard. Recently,
several isolates of Trichoderma species were
characterized and evaluated against different fungal
pathogens of pulse crops (Dubey et al., 2007). One of
each isolate of T. viride, T. harzianum and T. viriens were
selected for development of formulations and out of the
several formulations developed, Pusa 5SD for seed
treatment and Pusa biopellet for soil application were
found to be the best in respect of their shelf life as well as
efficacy (Dubey et al., 2009). These formulations alone
and in combinations were proved highly effective against
several diseases of pulse crops, namely, dry root rot of
mungbean (Dubey et al., 2009), dry root rot of chickpea

Table 1. Some commercial products of Trichoderma spp. available in India.
Product Species/strain of Trichoderma
Ecofit Trichoderma viride
Funginil Trichoderma viride
Trichogourd Trichoderma viride
Defense SF Trichoderma viride
Tricho-X Trichoderma viride
Biogourd Trichoderma viride
Biocon Trichoderma viride
Bip T Trichoderma viride
Pant biocontrol agent-1 Trichoderma harzianum
Bioderma Trichoderma viride+ Trichoderma harzianum
Ecoderma Trichoderma viride+ Trichoderma harzianum
(Dubey et al., 2011), wet root rot of chickpea (Dubey et
al., 2012) and wilt of chickpea (Dubey et al., 2013).
Delivering of Trichoderma through combined application
of different delivery systems will increase the population
load of Trichoderma and thereby might suppress the
pathogenic propagules.
COMMERCIAL PRODUCTS OF BIO-CONTROL
AGENTS
Unlike the bio control of insects, bio control of plant
diseases is relatively new. The first bacterium called
agrobacterium radiobacter strain K 84 was registered
with the United States Environmental Protection agency
(EPA) for the control of crown gall in 1979. Ten years
later the first fungus T. harzianumi ATCC 20476 was
registered with the EPA for the control of plant diseases.
Currently a total of 12 biological organisms have been
registered in India for the control of plant diseases
(Sabalpara, 2014). Most of these are sold commercially
as one or more products. The technology of
commercialization is still in its initial phase. Many
technological problems were overcome and shifts in
tinking occurred for these products to reach the shelves.
Some of the commercially available bio control products
available in the market are shown in Table 1.
COMMERCIALIZATION
Once an effective biocontrol system has been identified,
it can and should be commercialized. As a biocontrol
researcher, it has become clear that solving the
technological problems is perhaps the easiest part of
developing biocontrol agents and systems. Legal and
commercial considerations are also extremely important.
A total of 246 pesticides have been registered in India
under the Insecticide act till now (Anonymous, 2013). Out
Kumar et al. 3845
Agency/Company
Hoechest and Schering Agro. Evo. Ltd., Mumbai, India
Crop Helath Bioproduct Research Centre, Gaziabad (UP), India
Anu Biotech international Ltd. Bangalore, India
Wockhardt Life Science Ltd., Mumbai India
Excel Industries Ltd., Mumbai,India
Krishi Rasayan Export Pvt. Ltd., Solan (HP), India
Tocklai Experimental Station Tea Research Association, Jorhat (Assam), India
Poland
Department of Plant Pathology, GB Plant University of Agriculture and
Technology, Panatnagar, Uttarakhand, India
Biotech International Ltd., New Delhi, India
Morgo Biocontrol Pvt. Ltd., Bangalore, India
of these 12 are biological organisms, and only five
(Ampelomyces quisqualis, Beauveria bassiana, P.
fluorescens, T. harzianum and T. viride) are used for the
management of plant diseases. Popularization of
biopesticides is very slow as compared to chemicals and
only 2% biopesticides are available. Among the different
biopesticides, Trichoderma is most exploited and have
many success stories. In recent years many small and
large entrepreneurs have entered into the commercial
production of bio control agents resulting into the entry of
various bio-control products into the world market.
Commercialization of bio-control products is a multi-step
process involving a wide range of activities:
Isolation of antagonist
Isolation of an effective strain plays a prime role in
disease management. It is done from the pathogen
suppressive soils either by dilution plate technique or by
baiting the soil with fungal structures like sclerotia of
pathogen. Consortium of biocontrol agents could be
established by isolating the location specific and crop
specific isolates. It could be used for the development of
mixtures of biocontrol agents suited for different
ecological niche.
Screening of antagonist
All the strains isolated from the different cropping system
have to be ascertained for its virulence and broad
spectrum of action against different pathogens causing
serious economic threat to cultivation. Selection of an
effective strain decides the viability of the technology.
Hence a proper yardstick should be developed to screen
the antagonistic potentiality of the biocontrol agents.
Once active strains have been identified with the in vitro
assays, a further selection must be done by studying
3846 Afr. J. Agric. Res.
other factors such as:
1) Should have the ability to secrete increased level of
cell wall lytic enzymes (glucanses, chitinases, cellulases),
antibiotics and plant growth promotion.
2) Activity in vivo using experimentally induced diseases
on plants.
3) Tolerance of high or low temperatures (necessary to
survive other IPM treatments).
4) Suitability for formulation as foliar sprays and/or soil
enhancements (e.g. high sporulation levels, rapid growth
in bulk conditions).
5) Specificity (strains should be inactive against beneficial
organisms and plant crops).
6) Long-term survival in field conditions.
7) Interactions with other Trichoderma strains already
present in the cropping systems.
8) Compatibility with agrochemicals used in the crop, or
9) Shelf life and inoculum efficacy under commercial
conditions.
Hence the yardstick should be developed, comprising of
above-mentioned components. Each component should
be given weightage depending upon their role in disease
management. This type of rigorous and meticulous
screening will lead to identification of an effective
biocontrol strain suited for commercialization (Nakkeeran
et al., 2005).
Pot test and field efficacy
The plant, pathogen and antagonists are co exposed to
controlled environmental conditions. Exposure of the host
to the heavy inoculums pressure of the pathogen along
with the antagonist will provide ecological data on the
performance of the antagonist under controlled
conditions. Promising antagonists from controlled
environment are tested for its efficacy under field
conditions along with the standard recommended
fungicides. Since the variation in the environment under
field condition influence the performance of biocontrol
agent, trials on the field efficacy should be conducted for
at least 15 to 20 locations under different environmental
conditions to promote the best candidate for mass
multiplication and formulation development (Jeyarajan
and Nakkeeran, 2000).
Mass production and formulation development
The first major concern in commercial production
systems involves the achievement of adequate growth of
the biocontrol agent. In many cases biomass production
of the antagonist is not easy owing to the specific
requirement of nutritional and environmental conditions
for the growth of organism. Mass production is achieved
through solid and liquid fermentation techniques. The
commercial success of biocontrol agents requires
i) Consistent and broad spectrum action.
ii) Safety and stability.
iii) Longer shelf life.
iv) Low capital costs.
v) Easy availability of career materials.
vi) Economical and viable market demand.
Fermentation
Liquid and solid fermentation methods are used for the
mass production of Trichoderma.
Liquid fermentation
Term liquid state fermentation (LSF) is applied for the
processes in which soluble materials in water is used for
the microbial growth. In fermentative processes of this
type, the quantity of water should exceed. Water is
essential both for microbial growth, in LSF, is present in
thick layers and on occasions absorbs inside the
substrates.
This fermentation system has been adopted for the
mass multiplication of fungal biocontrol agents. For mass
multiplication the selected medium should be inexpensive
and readily available with appropriate nutrient balance.
Potato dextrose broth, V-8 juice, Molasses-yeast medium
and Wheat bran are generally used for the mass
production of Trichoderma spp., through liquid
fermentation technology (Prasad and Rangeshwaran,
1998, 2000; Prasad et al., 2002).
Solid fermentation
Term solid state fermentation (SSF) is applied for the
processes in which insoluble materials in water is used
for the microbial growth. In fermentative processes of this
type, the quantity of water should not exceed the capacity
of saturation of the solid bed in which the microorganisms
(fungus) grow. Water is essential for the microbial growth,
in SSF, present in thin layers and on occasions absorbs
inside the substrates.
Solid-state fermentation (SSF) is an effective method
for the mass production of fungal biopesticides since it
provides micropropagules with higher conidia content.
Various cheap cereal grains like, sorghum, millets, ragi
are used as substrates (Lewis, 1991; Jeyarajan, 2006).
The grains are moistened, sterilized and inoculated with
Trichoderma and incubated for 10 to 15 days for
production of Trichoderma through liquid Trichoderma
which produces dark green spore coating on the grains.
These grains can be powdered finely and used as seed

treatment or the grains can be used as it is for enriching
FYM for soil application. SSF processes, which involve
the growth of microorganisms (typically fungi) on moist
solid substrates in the absence of free-flowing water,
have considerable economic potential in producing
products for the food, feed, pharmaceutical, and
agricultural industries (Mienda et al., 2011). SSF has
gained renewed interest because it exhibits certain
product recovery, and reduced energy requirements. Two
types of SSF systems can be distinguished depending on
the nature of the solid phase used. The first and the most
commonly used system involve cultivation on natural
substrates. The second system, which is not frequently
used, involves cultivation on an inert support impregnated
with a liquid medium. This technique is suitable for small-
scale production in cottage industries or at individual
farmer level. Solid state fermentation technique for
commercial/industrial scale has to worked out in
collaboration with industry.
Formulation
Formulation is blending of active ingredients such as
fungal spores with the inert material such as diluents and
surfactants in order to alter the physical characteristics of
to a more desirable form. A final formulation must:
i) Be easy to handle.
ii) Be stable over a range of -5 to 35°C.
iii) Have a minimum shelf-life of two years at room
temperature.
One of the critical obstacles in the commercialization of
bio agents is the loss of viability of the propagulues over
time. Shelf life of the bio control product is dependent on
the storage temperature and the carriers used in the
formulation of bio control product. A talc based
preparation of the T. virens conidia retain 82% viability at
5ºC in refrigerator after 6 months while at room
temperature same level of viability was observed for a
period of 3 months (Chaube et al., 2002). Shelf life of the
formulations decides the commercialization of biocontrol
agents. Formulations should sustain the viable nature of
the product for the increased period of storage. Bio
control product should have the minimum shelf life of 8 to
12 months for industrialization. Carrier material should
not influence the viable nature of the biocontrol agent.
Commercialization of the bio products is primarily
hindered due to the poor shelf life. Hence research
should be concentrated to increase the shelf life of the
formulation by developing superior strains that support
the increased shelf life, or the organic formulations that
support the maximum shelf life with low level of
contaminants must be standardized for making biocontrol
as a commercial venture.
Kumar et al. 3847
Toxicology
Safety and environmental considerations could not be
taken for granted and it is vital that biopesticides are
regulated in an appropriate way to confirm the
international standards. The regulatory environment is
usually favorable for the bio-pesticides than the chemical
pesticides.
However the cost of carrying out the toxicological study
for registration is still too expensive. Toxicology includes
information of antagonist on safety to men, plants,
animals and soil microflora. Cost incurred for the
toxicological studies is high. These studies have to be
done separately for each and every biocontrol organism
separately. The huge investment on toxicological studies
warrants for the linkages between research organizations
and stakeholders (Jeyarajan and Nakkeeran, 2000;
Sabitha et al., 2001).
Industrial linkages
The research institutes carry out the initial discovery of an
effective organism, genetic manipulation of organisms to
develop superior strains, and studies on mechanisms,
field efficacy and protocols for the development of
formulations. But to take this technology to the entire
country depends on the partnership between the
institutes and stakeholders. Corporate resources are
required for the large-scale production, toxicology, wide
scale field-testing, registration and marketing.
Entrepreneurship may be defined as the exchange of
intellectual property for research grants, and a royalty
stream, with the establishment of University–Industry
partnership for the benefit of both. The first requirement
for the entrepreneurship requires a patent application on
the strain and the related technology, especially on the
then efficacy data, identity of the organism, toxicological
data and delivery system. Ideally, the process of
entrepreneurship will result in an academic corporate
research team working towards a common goal.
Quality control
This is very much obligatory to retain the confidence of
the farmers on the efficacy of biocontrol agents. Being
the living organisms, their population in a product
influences the shelf life. The population load of the
antagonists decides the minimum level of requirement for
bringing the effective biological control of the plant
diseases.
Standards for Trichoderma formulations
i. Colony Forming Units (CFUs) of Trichoderma spp.
3848 Afr. J. Agric. Res.
should be a minimum of 2x106 CFU per ml or gm on
selective medium.
ii. Pathogenic contaminants such as Salmonella, Shigella
or Vibrio should not be present. Other microbial
contaminants need not exceed 1x104 count ml/gm.
iii. Maximum moisture content should not be more than
8% for dry formulation of fungi.
Quality control parameters set by CIB are inadequate for
knowing potentiality of a bioagent. It is time now for
Ministry of Agriculture to identify a Central Agency for
quality testing not only in terms of amount of live
propagules in the formulation but also their bio efficacy
against plant pathogens. Quality of the products in these
lines should be periodically checked by the identified
agency and that will ensure availability of better products
to farmers.
Registration of bioagents
Two important factors in the registration of bio agents are
the toxicity and environmental fate. Under Section 9(3) of
Pesticide Act of India 1968, information required for the
registration of any bio pesticide are:
i) Systemic name and common name of the bio control
agent
ii) Natural occurrence
iii) Morphological description of the of the bio agent
iv) Details of manufacturing process
v) Mammalian toxicity
vi) Environmental toxicity
vii) Residual analysis
CONSTRAINTS TO COMMERCIALIZATION
The success of biopesticides to suppress pests and
diseases depends on the availability of microbes as a
product or formulation, which facilitate the technology to
transfer from lab to land. The constraints to biopesticides
development and utilization mirror some of those factors
that limit the development worldwide. Constraints include
i) Lack of right screening protocol for the selection of
promising candidate of Trichoderma.
ii) Lack of sufficient knowledge on the microbial ecology
of Trichoderma and plant pathogens.
iii) Optimization of fermentation technology and mass
production of Trihcoderma.
iv) Inconsistent performance and poor shelf life.
v) Lack of patent protection.
vi) Prohibitive registration cost.
vii) Awareness, training and education shortfalls.
viii) Lack of multi disciplinary approach.
ix) Technology constraints.
Screening and selection of potential Trichoderma
strains
Success of commercialization of Trichoderma depends
on the selection of effective strains after adopting precise
screening strategies as any error during strain selection
will be a costly mistake in product development (Schisler
and Slininger, 1997). The potentiality of the Trichoderma
in the suppression of plant pathogen should be carried
out at both lab and field conditions in different soil types
with diversified microbial communities and climatic
conditions (Roberts and Lohrke, 2003). It would lead to
the development of a viable Trichoderma spp.
Microbial ecology and interaction
Suppression of plant disease is a four-way interaction of
biocontrol agents, plants, pathogens and the
environment. Hence, understanding of interaction
between all these components is essential for developing
a suitable biocontrol agent in disease management
(Handelsman and Stabb, 1996; Larkin et al., 1998). Root
colonization by Trichoderma leads to a modification in the
plant transcriptome that limit the progress of the fungus to
the epidermal and cortex intercellular spaces. Effective
Trichoderma strains elicit plant beneficial responses by
the production of proteins or small molecules (several
enzymes, hydrophobins, cerato- platanins, expansin—
like proteins, avirulence–like proteins, secondary
metabolites with antimicrobial activity, phytohormones,
etc.) that alter host-cell structure and function (Hermosa
et al., 2012). As a result Trichoderma induces systemic
defense responses in the plant that are primed to
respond faster and to a greater extent to potential
pathogen attack (Kumar, 2013b, 2014c). However,
knowledge on the influence of biotic and abiotic
environment on Trichoderma strains to express its
antimicrobial action has to be studied in depth under in
vivo to improve the efficacy of Trichoderma spp. This will
facilitate identification of Trichoderma spp. that could
perform well under diverse environmental conditions
around the court of infection.
Fermentation technology and shelf life of
formulations
Optimization of liquid or solid fermentation technology
with suitable medium (synthetic or semi-synthetic) for
mass multiplication and identification of suitable carrier
material for formulation development with increased shelf
life is a barrier in the commercial success of formulation
development. Commercial biomass production of fungal
antagonists requires large-scale fermenters. The biomass
production and efficacy of biocontrol agents to suppress
plant pathogens varies depending on the nutrient

composition of the medium (Schisler et al., 1997). Hence,
the medium selected for biomass production should
support the growth and efficacy of antagonist and the
cost of medium should be economical so that the
technology remains viable.
Patent protection and prohibitive registration cost
The environmental protection agency in India should
relax the formalities and registration cost to promote
registration of biocontrol agents either by universities or
private companies. The patent protection rights for the
effective products should be strengthened to encourage
the organizations involved in identification and
development of commercial biocontrol agents.
Awareness, training and education shortfalls
The common level of awareness among stakeholders
and farmers about the potential value of biopesticides is
lacking. Thus there is a need for
i) Awareness level among the policy makers of the
potential for biopesticides, their efficacy and their effect in
reducing the health and environmental problems.
ii) The opportunities offered by the commercialization in
terms of generation of wealth and employment are to be
promoted.
iii) Government extension workers have to be trained in
biopesticides and the communication between research
and extension sectors have to be intensified.
iv) The nature and mode of action of biopesticides have
to be explained to farmers who are used to chemical
pesticides, which are often fast acting and are visibly
effective.
v) Entrepreneurs and investors need to be informed
about the opportunities that exist for establishing
commercial companies to manufacture market and sell
biopesticides.
Lack of multidisciplinary approach
The process of biopesticides development to complete
product requires research in areas of screening,
formulation, field application, production, storage,
toxicology as well as the steps necessary for
commercialization, such as scale up production,
registration and regulatory matters. Most of the research
efforts undertaken with the use of biopesticides are
confined only to the collection, isolation and identification
of biocontrol agents combined with laboratory based
bioassays. But in the process of product development the
above research aspects shares only a fraction of work
required to develop a complete product. Product
development requires a multidisciplinary approach to
Kumar et al. 3849
biopesticides research and development. Rarely, a
complete range of expertise exists in a single institute or
organization.
Technology constraints
Delivery system
Success in biocontrol depends on understanding and use
of delivery system. The research on delivery system is
well below that of chemical pesticides. The awareness on
the application technology can improve biopesticides
performance.
Biopesticides quality
The major predicament in the field of biopesticides
production is the product quality and stability. In small-
scale production, contamination of inoculum is a common
problem. The long -term shelf life of the product is highly
essential to attract the multinational companies to invest
on a large scale.
STRATEGIES TO PROMOTE COMMERCIALIZATION
In the current crop production scenario, biocontrol is of
utmost importance, but its potential is yet to be fully
exploited primarily because the research in this area is
still confined to the laboratory and very little attention has
been paid to producing the commercial formulations of
bio agents. Moreover, whatsoever has been
commercially produced has not been used professionally
by the farmers due to the lack of information regarding its
use. So commercialization of biocontrol could be
promoted by
1) Popularization of biocontrol agents.
2) Industrial linkages.
Popularization of biocontrol agents
Motivating the growers through
1) Publicity
2) Field demonstrations
3) Farmers days
4) Bio village adoption
5) Conducting periodical trainings for commercial
producers and farmers to increase / improve the supply.
Industrial linkages
1) Technical support should be made available to
entrepreneurs on quality control and registration.
3850 Afr. J. Agric. Res.
2) Regular monitoring is essential to maintain the quality.
3) Constant research support should be extended to
standardize the dosage, storage, and delivery systems.
Positive policy support from government to use more of
biocontrol agents in crop protection.
FUTURE RESEARCH
The future research should focus on the following
aspects for better utilization of Trichoderma as a
biocontrol agent for plant disease management.
i) Suitability of Trichoderma for control of the foliar/aerial
pathogens.
ii) Development of liquid/oil formulations suitable for foliar
applications.
iii) Formulations with prolonged shelf life, field
persistence and suitable for dry weather conditions.
iv) Scaling up of solid state production systems with
Industry collaboration.
v) Large scale demonstration of biocontrol technologies
in farmers’ fields.
vi) Fast track registration.
REFERENCES
Ahmad JS, Baker R (1987). Thizosphere competence of Trichoderma
harzianum. Phytopathology 77:182.
Andrews JH (1992). Biological control in the phyllosphere. Ann. Rev.
Phytopathol. 30:603-635.
http://dx.doi.org/10.1146/annurev.py.30.090192.003131
Anonymous (2013). India Statistics collected from
http://www.indiastat.com/agriculture/2/consumption of
pesticides/206872/stats.aspx
Baby UI, Manibhushanrao K (1996). Fungal antagonists and VA
mycorrhizal fungi for biocontrol of Rhizoctonia solani, the rice sheath
blight pathogen, pp 1-9. In Recent Developments in Biocontrol of
Plant Pathogens (Eds Manibhushanrao K Mahadevan A) Today and
Tomorrow's Printers and Publishers, New Society, Allahabad. Delhi
P. 160.
Balasubramanian C, Udaysoorian P, Prabhu C, Kumar GS (2008).
Enriched compost for yield and quality enhancement in sugarcane. J.
Ecobiol, 22:173-176.
Barnes S, Moore D (1997). The effect of fatty, organic, or phenolic acids
on the germination of conidia of Metarhizium flavoviride. Mycol. Res
10:662-666. http://dx.doi.org/10.1017/S0953756296003152
Bateman R, Carey M, Morre D, Prior C (1993). The enhanced infectivity
of Metarhizum flavoviridae in oil formulations to desert locust at low
humidities. Ann. Appl. Biol. 122:145-152.
Batta YA (2005). Postharvest biological control of apple gray mold by
Trichoderma harzianum formulated in an invert emulsion. Crop
Protect. 23(1):19-26.
Bhat KA, Anwar Ali, Lone GM. Hussain K, Nazir G (2009) .Shelf life of
liquid fermented product of Trichoderma harzianum in talc J. Mycol.
Pl. Pathol. 39(2):263-265.
Brown JS, Holden DW (1998). Insertional mutagenesis of pathogenic
fungi. Curr. Opin. Microbial. 1(4):390-394.
Connick W, Daigle D, Quimby P (1991). An improved invert emulsion
with high water retention for mycoherbicide delivery. Weed Technol.
5:442-444.
Chaube HS, Mishra DS, Varshney S, Singh US (2002). Biological
control of plant pathogens by fungal antagonists: Historical
background, present status and future prospect, Annu. Rev. Plant
Pathol. P. 2.
Das BC, Hazarika DK (2000). Biological management of sheath blight of
rice. Indian Phytopathol. 53(4):433-435.
de Boer M, Bom P, Kindt F, Keurentjes JJB, van dex Sluism I, van Loon
LC, Bakker PAHM (2003). Control of Fusarium wilt of radishby
combining Pseudomonas putida strains that have different disease
suppressive mechanisms. Phytopathology 93:626-632.
Dubey SC Suresh M, Singh B (2007). Evalaution of Trichoderma
species agasint Fusarium oxysporum f.sp. ciceris for integrated
management of chickpea wilt. Biol. Control 40:118-127.
http://dx.doi.org/10.1016/j.biocontrol.2006.06.006
Dubey SC, Bhavani R, Singh B (2009). Development of Pusa 5SD for
seed dressing and Pusa Biopellet 10G for soil application
formulations of Trichoderma harzianum and their evaluation for
integrated management of dry root rot of mungbean (Vigna radiata).
Biol. Control 50:231-242.
http://dx.doi.org/10.1016/j.biocontrol.2009.04.008
Dubey SC, Bhavani R, Singh B (2011). Integration of soil application
and seed treatment formulations of Trichoderma species for
management of wet root rot of mungbean caused by Rhizoctonia
solani. Pest Manage. Sci. 67:1163-1168.
Dubey SC, Tripathi A, Singh B (2012).Combination of soil application
and seed treatment formulations of Trichoderma species for
integrated management of wet root rot caused by Rhizoctonia solani
in chickpea. Indian J. Agric. Sci. 82:357-364.
Dubey SC, Tripathi A, Singh B (2013). Integrated management of
Fusarium wilt by combined application of soil and seed dressing
formulations of Trichoderma species to increase grain yield of
chickpea. Int. J. Pest Manage. 59:47-54.
http://dx.doi.org/10.1080/09670874.2012.744494
Fravel DR, Rhodes DJ, Larkin RP (1999). Production and
commercialization of biocontrol products. In: Integrated pest and
disease management in greenhouse crops (Albajes, R., Lodovica
Gullino, M., Van Lenteren, J. C and Elad, Y. eds.,), Kluwer Academic
Publishers, Boston pp. 365–376.
Glick GR, Bashan Y (1997). Genetic manipulation of plant growth
promoting bacteria to enhance biocontrol of phytopathogens.
Biotechnol. Adv. 15:353-378. http://dx.doi.org/10.1016/S0734-
9750(97)00004-9
Gaur RB, Sharma RN, Gautam VS, Dangi RP (2010). Management of
Sclerotinia rot of mustard through bioagents. Indian Phytopathol.
63(4):392-397.
Green S, Wade-Stewart S, Boland G, Teshler M, Liu S (1998).
Formulating microorganisms for biological control of weeds. In: Plant-
/Microbe Interactions and Biological Control (Boland, G. and
Kuykendall, L., eds.). Marcel Dekker, Inc., New York pp. 249-281.
Handelsman J, Stabb EV (1996) Biocontrol of soilborne plant
pathogens. Plant Cell 8:1855–1869.
http://dx.doi.org/10.2307/3870235
Harmon GE, Bjoorman T (1998), Potential and existing uses of
Trichoderma and Gliocladium for plant disease control and plant
growth enhancement . In : Trichoderma and Gliocladium , vol. II. G.E.
Harman and C.K. Kubicek, (Eds), 229, London: Taylor and Francis
Ltd. http://dx.doi.org/10.1099/mic.0.052274-0
Hermosa R, Viterbo A, Chet I, Monte E (2012). Plant-beneficial effects
of Trichoderma and of its genes. Microbiology 158(1):17-25.
Ibrahim L, Butt T, Beckett A, Clark S (1999). The germination of oil-
formulated conidia of the insect pathogen, Metarhizium anisolpliae.
Mycol. Res. 103:901-907.
http://dx.doi.org/10.1017/S0953756298007849
Jagana M, Zacharia S, Basayya A (2013). Management of Alternaria
blight in Mustard. An. Plant Prot. Sci. 21(2):441-442.
Jat JG, Agalave HR (2013). Antagonistic properties of Trichoderma
species against oilseed-borne fungi. Sci. Res. Reporter 3(2):171-174.
Jeyalakshmi C, Rettinassababady C, Nema S (2013). Integrated
management of sesame diseases. J. Biopestic. 6(1):68-70.
Jeyarajan R, Nakkeeran S (2000). Exploitation of microorganisms and
viruses as biocontrol agents for crop disease mangement. In:
Biocontrol Potential and their Exploitation in Sustainable agriculture,
(Ed. Upadhyay et al.,) Kluwer Academic/ Plenum Publishers, USA
pp. 95-116. http://dx.doi.org/10.1007/978-1-4615-4209-4_8
Jeyarajan R, Ramakrishnan G, Dinakaran D, Sridar R (1994).
Development of products of Trichoderma viride and Bacillus subtilis
3852 Afr. J. Agric. Res.
for biocontrol of root rot diseases. In “Biotechnology in India” (Ed
Dwivedi B.K.) Bioved Research Society, Allahabad. pp. 25-36.
Jeyarajan R (2006). Prospects of indigenous mass production and
formulation of Trichroderma, In Current Status of Biological Control of
Plant diseases using antagonistic organisms in India (Eds Rabindra
RJ Ramanujam B), Project Directorate of Biological Control,
Bangalore, pp. 74-80, 445.
Jha MM, Kumar S, Saleh H (2004). Response of bioagents against
maydis leaf blight of maize, Ann. Biol. 20(2):177-179.
Khan AA, Sinha AP (2005). Influence of different factors on the
effectivity of fungal bioagents to manage rice sheath blight in nursery.
Indian Phytopathol. 58(3):289-293.
Khan AA, Sinha AP (2007). Screening of Trichoderma spp. Against
Rhizoctonia solani the causal agent of rice sheath blight. Indian
Phytopathol. 60(4):450-456.
Kumar S (2010a). Integrated management of maydis leaf blight of
maize Annals of plant Protect. Sci. 18(2):536-537.
Kumar S (2013b). Trichoderma ; A Biological Weapon for Managing
Plant Diseases and Promoting Sustainability. Int. J. Agric. Sci. Vet.
Med. 1(3):1-16.
Kumar S (2014c). Plant disease management: advances and
challenges. Afr. J. Agric. Sci. 9(15):1207-1217.
http://dx.doi.org/10.5897/AJAR2014.7311
Kumar S, Gupta OM (2012). Expanding dimension of Plant Pathology.
JNKVV Res. J. 46(3):286-293.
Jha AMM (2009). Potential of Trichoderma spp. as biocontrol agents
against pathogens causing maydis leaf blight of maize. J. Biol.
Control 23(1):89-91.
Kumar S, Upadhyay JP, Rani A (2009). Evaluation of Trichoderma
species against Fusarium udum Butler causing wilt of Pigeon pea. J.
Biol. Control 23(3):329-332.
Kumar Sanjeet , Upadhyay JP, Kumar S (2002). Biocontrol of Alternaria
leaf spot of Vicia faba using antogonistic fungi J. Biol. Control
20(2):247-251.
Kumar S, Upadhyay JP (2003). Compatibility of Rhizobium and
Trichoderma in vitro and in vivo. RAU J. Res. 13(1-2):61-64.
Lalithakumari D, Mathivanan N (2003). Strain improvement in
filamentous fungi by protoplast fusion. In: Mathivanan N, Prabavathy
VR, Gomathinayagam S (Eds). Innovative Methods and Techinques
for integrated Pest and Disease Management. Centre for Advanced
Studies in Botany, University of Madras, Chennai, India. pp. 76-97.
Kumar R, Kumar S, Upadhyay JP (2012). Effect of different solid and
liquid media on growth and sporulation of T. viride. Adv. Appl. Res.
4(2):131-132.
Larkin RP, Roberts DP, Gracia-Garza J A (1998). Biological control of
fungal diseases. In: Fungicidal activity, chemical and biological
approaches (Hutson, D and Miyamoto, Y eds.), Jhon Wiley and Sons,
New York, pp.149-191.
Lewis JA (1991). Formulation and delivery system of biocontrol agents
with emphasis on fungi Beltsville symposia. In: The rhizosphere and
plant growth (Keister, D. L. and Cregan, P. B. eds.,). Agric. Res.
14:279-287.
Lorito M, Woo sl, García Fernández I, Colucci G, Harman Ge, Pintor-
Toro JA, Filippone E Mucciflora S, Lawrence C, Zoina A, Tuzun S,
Scala F (1998). Genes from mycoparasitic fungi as a source for
improving plant resistance to fungal pathogens. Proc. Natl. Acad. Sci.
USA 95:7860-7865. http://dx.doi.org/10.1073/pnas.95.14.7860
Manjunatha SV, Naik MK, Khan MFR, Goswami RS (2013). Evaluation
of bio-control agents for management of dry root rots of chickpea
caused by Macrophomina phaseolina. Crop Protect. 45:147-150.
http://dx.doi.org/10.1016/j.cropro.2012.09.003
Manoranitham SK, Prakasam V, Rajappan K, Amutha G (2000). Effect
of two antagonists on damping off disease of tomato. Indian
Phytopathol. 53(4):441-443.
Mienda BS, Idi A Umar A (2011). Microbiological Features of Solid State
Fermentation and its Applications - An overview, Res. Biotechnol.
2(6):21-26.
Minucci C, Migheli Q, Gullino ML (1991). Protoplast fusion for the
production of superior strains of antagonistic Fusarium spp., In:
Beemster ABR, Bollen GJ, Gerlagh, M.A. Ruissen, B. Schippers, A.
Tempel, eds.)., Elsevier, Amsterdam pp. 316-321.
Mishra DS, Singh US, Dwivedi TS (2001). Comparative efficacy of
Kumar et al. 3851
normal seed treatment and seed biopriming with commercial
formulations of Trichoderma spp. In 53rd Annual meeting of Indian
Phytopathological Society and National symposium on Ecofriendly
approaches for Trichoderma Chennai, India 21-23.
Mrinalini C, Lalithakumari D (1998). Integration of enhanced biocontrol
efficacy and fungicide tolerance in Trichoderma spp. by electrofusion.
J. Plant Dis. Protect. 105:34-40.
Mukhopadhyay AN, Shrestha SM, Mukherjee PK (1992). Biological
seed treatment for control of soilborne plant pathogens. FAO Plant
Protect. Bull. 40:21-30.
Mukherjee PK, Mukhopadhyay AN (1995). In situ, mycoparasitism of
Gliocladium virens on Rhizoctonia solani. Indian Phytopathol.
48(1):101-102.
Mustafa A, Khan MA, Inam-ul-Haq M, Khan SH, Pervez MA (2009).
Mass multiplication of Trichoderma spp. on organic substrate and
their effect in management of seed borne fungi.  Pak. J. Phytopathol.
21(2):108-114.
Ogama K, Ohara H, Koide T Toyama N (1989). Interspecific
hybridization of Trichoderma reeseai by protoplast fusion. J. Ferment.
Bio-eng. 67:207-209. http://dx.doi.org/10.1016/0922-338X(89)90124-
4
Nakkeeran S, Fernando D, Siddiqui ZA (2005). Plant growth promoting
rhizobacteria formulations and its scope in commercialization for the
management of pests and diseases. PGPR: Biocontrol and
Biofertilization, Springer, Dordrecht, The Netherlands pp. 257-296.
Penttilä M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987). A
versatile transformation system for the cellulytic filamentous fungus
Trichoderma reesei. Gene 61:155-164.
http://dx.doi.org/10.1016/0378-1119(87)90110-7
Prabavathy VR, Mathivanan N, Sagadevan E, Murugesan K,
Lalithakumari D (2006). Intra-strain protoplast usion enhances
carboxymethyl cellulase activity in Trichoderma reesei. Enzyme
Microb. Technol. 38:719–723.
Prasad RD, Rangeshwaran R, Anuroop CP, Phanikumar PR (2002).
Bioefficacy and shelf life of conidial and chlamydospore formulation
of Trichoderma harzianum. J. Biol. Control 16:145-48.
Prasad RD, Rangeshwaran R (1998). Amodified liquid medium for mass
production of Trichoderma by fermentation process, P. 26. In
Abstracts of National symposium on Eco friendly approaches in the
management of plant diseases. December 22-24, Shimoga
Karnataka, Inida.
Prasad RD, Rangeshwaran R (2000). Shelf life and bioefficacy of
Trichoderma harzianum formulated in various carrier materials. Plant
Dis. Res. 15:38-42.
Raguhchander T, Saravankumar S, Balasubramanian P (2011).
Molecular approaches to improvement of biocontrol agents of plant
diseases. J. Biol. Control. 25(2):71-84
Rao Y, Lal AA, Simon S, Singh L (2013). Management of brown spot
(Drechslera oryzae) of Rice. Ann. Plant Protect. Sci. 21(2):450-452.
Reddy K, Krishnamma, Narayana P (2009). Efficacy of Trichoderma
viride against Colletotrichum falcatum in Sugarcane. Indian J. Plant
Prot. 37:111-115.
Roberts DP, Lohrke SM (2003). United States Department of
Agriculture -Agricultural Research Service research programs in
biological control of plant diseases. Pest Manage. Sci. 59(6-7):654-
664.
Sabalpara AN (2014). Mass multiplication of biopesticides at farm level.
J. Mycol. Plant Pathol. 44(1):1-5.
Sabitha D, Nakkeeran S, Chandrasekar G (2001). Trichoderma –
bioarsenal in plant disease management and its scope for
commercialization. In proceedings of Indian Phytopathological
Society, Southern Zone Meeting, 10-12 December, Indian Institute of
Spice Research, Calicut, kerala. pp.43-55
Sawant IS, Sawant SD (1996). A simple method for achieving high cfu
of Trichoderma harzianum on organic wastes for field applications.
Indian Phytopathol. 9:185-87.
Schisler DA, Slininger PJ (1997). Effects of antagonist cell
concentration and two strain mixtures on biological control of
Fusarium dry rot of potatoes. Phytopathology 87:177-183.
http://dx.doi.org/10.1094/PHYTO.1997.87.2.177
Shanmugam V, Gupta S, Dohroo NP (2012). Selection of compatible
biocontrol strain mixture based on co-cultivation for control of rhizome
 rot of ginger. Crop Prot. 43:119–127.
Singh US, Zaidi NW (2002). Current Status of formulation and delivery
of fungal and bacterial antagonists for disease management in India,
pp 168-179. In Microbial Biopesticide Formulations and Application
(Eds Rabindra RJ Hussaini SS Ramanujam B) Project Directorate of
Biological Control, Bangalore P. 269.
Srivastava, Singh RK, Kumar RKN, Singh S (2010). Management of
Macrophomia disease complex in jute(Corchorus olitorius) by
Trichoderma viride. J. Biol. Control, 24(1):77–79.
Sankar P, Jeyarajan R (1996). Biological control ofsesamum root rot by
seed treatment withTrichoderma spp. and Bacillus subtilis. Indian J.
Mycol. Plant Pathol. 26:147-53.
Singh D Maheshwari VK (2001). Biological seed treatment for the
control of loose smut of wheat. Indian Phytopathol. 54(4):457-460.
Singh SP, Singh HB (2012). Effect of consortium of Trichoderma
harzianum isolates on growth attributes and Sclerotinia sclerotiorum
rot of brinjal. Veg. Sci. 39(2):144-148.
http://dx.doi.org/10.1007/s12088-012-0335-2
Sharma P, Sharma M, Raja M, Shanmugam V (2014). Status of
Trichoderma research in India: A review. Indian Phytopathol. 67(1):1-
19.
Sharma V, Bhandari P, Singh B, Bhatacharya A, Shanmugam V (2012).
Chitinase expression due to reduction in fusaric acid level in an
antagonistic Trichoderma harzianum S17TH. Indian J. Microbiol.
53:214-220.
Sharma P, Patel AN, Saini MK, Deep S (2012). Field demonstration of
Trichoderma harzianum as a plant growth promoter in wheat
(Triticum aestivum L). J. Agric. Sci. 4(8):65-73.
Singh D, Kapur SP, Singh K (2000). Management of citrus scab caused
by Elsinoe fawcettii. Indian Phytopathol. 53(4):461-467.
Singh R, Parmar H, Arpita (2013). Integrated management of Alternaria
blight in linseed. Biol. Sci. 83(3):465-469.
Sriram S, Raguchander T, Babu S, Nandakumar R, Shanmugam V,
Vidhysekaran P, Balasubramanian P, Samiyappan R (2000).
Inactivation of phytotoxin produced by the rice sheath blight pathogen
Rhizoctonia solani. Canadian J. Microbiol. 46:520-524.
http://dx.doi.org/10.1139/cjm-46-6-520
Sriram S, Palanna KB, Ramanujam B (2010). Effect of chitin on the
shelf life of Trichoderma harzianum in talc formulation. Indian J.
Agric. Sci. 80:930-932.
Sriram S, Roopa KP, Savitha MJ (2011). Extended shelf life of liquid
fermentation derived talc formulations of Trichoderma harzianum with
the addition of glycerol. Crop Protect. 30:1334-1339.
http://dx.doi.org/10.1016/j.cropro.2011.06.003
Tewari AK (1996). Biological Control of chickpea wilt using different
formulations of Gliocladium virens through seed treatment. Ph,D
Thesis, GB Pant University of Agriculture Karnataka, India, and
Technology, Pantnagar, India P. 167.
Thankamani CK, Sreekala K, Anandaraj M (2005). Effect of
Pseudomonas fluorescens (IISR-6) and Trichoderma harzianum (P-
26) on growth of black pepper (Piper nigrum L.) in the nursery. J.
Spices Arom. Crops 14(2):112-116.
Vasudevan P, Kavitha S, Priyadarisini VB, Babujee L
Gnanamanickamm SS (2002). Biological control of rice diseases. In
Biological control of crop diseases. (Ed Gnanamanickam SS) Marcel
Decker, Newyork, pp. 11-32. 480 p.
Yadav SK, Dave A, Sarkar A, Singh HB, Sharma BK (2013). Co-
inoculated biopriming with Trichoderma, Pseudomonas and
Rhizobium improves crop growth inCicer arietinum and Phaseolus
vulgari. Int. J. Agric. Biol. 6(2):255-259.