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TRICHODERMA HARZIANUM: MASS MULTIPLICATION AND ITS INTERACTION

WITH DIFFERENT FUNDICIDES

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 Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
TRICHODERMA HARZIANUM: MASS MULTIPLICATION AND ITS
INTERACTION WITH DIFFERENT FUNDICIDES
_________________________________
1* 2 The main objective of this study is to cultivate Trichoderma
Charan Singh Nevadita Sharma
Bhawna3 Roop singh4
1, 2, 3,4
Department of Biotechnology, Seth
G.L. Bihani S.D. PG College, Sri
Ganganagar- 335001 Rajasthan (India).
* Correspondence author email:
charan.singh201080@gmail.com
Keyword: Fungicides, Trichoderma harzianum, Soil borne diseases, biocontrol agent
INTRODUCTION
The art and science of plant disease control must
continue to move in the direction of biological control
of plant pathogens, including use of introduced
antagonists (Pal 2006). Chemical controls are necessary
with many diseases at present but are undesirable and
even inadequate as a long term solution to crop and
human health. During the past several years, some
notable successes of disease control were achieved
through introduction of microorganisms in the
laboratory, glass house and fields. It is now widely
recognized that biological control of plant pathogen is a
distinct possibility for future and can be successfully
exploited in the modern agriculture especially within
the frame work of integrated pest management system
(Bale et al., 2008).
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Research article
ABSTRACT
harizanum, formulated it and experiments was conducted on
different concentration combination of fungicides to prepare a mass
cultivation of the suitable bio-fungicides which use to manage
different of soil borne plant diseases in cost effective way.
Trichoderma harizanum was isolated from Pathology department of
Indian Agricultural Research Institute (IARI), Pusa, New Delhi and
subcultured, cultivated by solid and liquid fermentation technique,
formulated by talcum powder and followed by mass multiplication.
The effect on the mycelia growth of Trichoderma harzainumwas
tested with different concentration of fungicides namely Blitox,
Captan, Bavistin, Ridomil, Thiophenate methyl, Roxiltubecanezole.
The growth and sporulation of Trichoderma harizanum was faster
on Potato Dextrose Agar followed by talcum powder. Interaction
studies of bioagent (Trichoderma harzianum) with fungicides
indicated non compatibility of Thiophenatemethyl, Roxil and Blitox
withall the concentrations, whereas, Bavistin and Ridomil are
compatible with Trichoderma harzianum and Captan was the only
fungicide which showed full compatibility with all concentrations.
A simple and cost effective method to mass culture the biocontrol
agent Trichoderma harizanum by using liquid process and talcum
powder formulation.
Therefore, use of bio control agents like
Trichoderma spp. is a good option not only due to its
ability to control soil borne, seed borne and foliar
diseases but also due to its pronounced effect on yield
and growth enhancement. Increased growth of plants by
Trichoderma spp. has been reported as an added
advantage and is ascribed to control of many pathogens
and/or production of growth regulatory factors (Baker
1988; Howell 2003). The potential of T. harzianum to
induce growth of various floricultural and horticultural
crops were demonstrated. In raw soil containing the
fungus, pepper seed germinated 2 days earlier than
untreated controls. Steamed or raw soil infested with T.
harzianum hastened flowering of periwinkle, increased
the number of bloosoms per plant on Chrysanthemum
and increased the heights and weights of other plants
Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
(Chang et al., 1986). Therefore, our main objective of
this study is to cultivate Trichoderma harizanum,
formulated it and experiments was conducted on
different concentration combination of fungicides to
prepare a mass cultivation of the suitable bio-fungicides
which use to manage different of soil borne plant
diseases in cost effective way.
MATERIAL AND METHODS
Subculturing of Trichoderma harizanum
Trichoderma harizanum mother culture was isolated by
Department of Plant Pathology, IARI, Pusa, New delhi.
Transfer above mother culture dilution aseptically into a
petri plates containing the medium separately. The
plates were rotated manually for uniform distribution
and the suspension in the medium is allowed to solidify.
The plates were incubated at 27 ± 10 ºC for seven days
for the developments of fungal colonies. The colonies
with characteristic growth of Trichodermas spp were
observed under the microscope and growth from such
colonies was subcultured on agar slants.
CULTIVATION PROCESS OF
TRICHODERMA HARIZANUM
Solid state fermentation
Cultivation using cow dung + sorghum grain: 200gm
sorghum grains were boiled up to 20 to 30 minutes to
soften & cook about 25% drain water and spread grain
for cool down to decrees the moisture and transfer
10gm cow dung was added per 100gm grains.
Mixture transferred to autoclavable polythene bags and
autoclave 121C0 at 15 minute (Chahal 1985).
Cultivation using cow dung + wheat flour: 200gm
wheat flour + 10gm. cow dung per 100gm wheat
floursolidfermentation is very common method for
mass multiplication of Trichoderma harzianum these
combination are moistened sterilized and inoculated
with Trichoderma harzianum incubated for 10 days
when mixer is completely colonised by Trichoderma
harzianum and conida formed. It was used for the pot
culture experiments. Solid state fermentation technique
for commercial scale production of biocontrol fungi has
to work out in collaboration with industry (Chahal
1985).
Liquid fermentation
To prepare the initial inoculate Trichoderma
harzianum was culture in PDB (Hi media) at 250c for
10 days preparation of culture. Initial inoculum for
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inoculation of conical flask, T. harzianum grown on
PDA for 10 days than prepare a molasses media (3%
molasses, 0.5% yeast in 1000mL distilled water).
Molasses is sieve with 100 meshes, 50mL molasses
dispense into conical flak 250mL. Flakes are disinfected
in autoclave at 1210C for 30 min. under aseptic
condition in laminar air flow. Than four discs with 5
mm diameter from the 10 day culture colony of fungus
are pour into each 250mL flaks. Flaks are place inside
incubator at 270C for 10 days for fungal growth this
serves as mother culture (Papavizas et al., 1984).
Mass multiplication
10 day mother culture is added to the large flask
(1000, 2000, 2500mL) 750mL mother culture/ 2500mL.
The medium incubated at room temperature (22-300) for
10 days. The flaks biomass is formulated in talc
powder. This mixer ration is 1:2 and keeps for 3 day
under shade for drying after mixed with carboxy methyl
cellulose (CMC) 5gm/kg the product. Product contain
10% moisture is packed in polythene bags 500g/pack
then store at 300C for 5-10 months. Sample form these
packets are drown at 15 days intense and tested for
viability at the formulate product (Subash et al., 2014).
Talc based formulation
Trichoderma harzianum is grown in the liquid
medium is mixed with talc powder in the ratio at 1:2
and dried to 8% moisture under shade. The talc
formulation of Trichoderma harzianum has shelf life
of 3-4 months it has become quite popular in India
for management of several soil borne diseases of
various crops (Jeyarajan-2006).
Effect of different fungicides on the
mycelialgrowth of Trichodermaharzainum
These studies were conducted using poison
food technique (Singh et al., 2015). Different
fungicides in different concentration (ppm) i.e. 500,
1000, 1500 and 2000 were mixed with PDA before
sterilization to study the inhibitory effect against
mycelial growth of T. harzianum using poision food
technique (Dhingra and Sinclain, 1944). After cooling,
the plates were incubated with bit of Trichodrama in the
center. Mycelial growth of Trichoderma was then
recorded after 24 hr., 48 hr., 72 hr., 96 hr. of incubation
at 25 ± 2°C. For this interaction study following
fungicides were used: Blitox, Captan, Bavistin,
Ridomil, Thiophenate methyl, Roxiltubecanezole.
Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
Journal of Biotechnology and Biosafety

green or brown. Chlamydospores: Fairly abundant,

RESULTS
Table 1 showed the different substrate sorghum
grains + cow dung had highest growth of Trichoderma
harzainum. Maximum biomass production of
Trichoderma harzainum (150.3X10 8 cfug -1 ) at
10 days of inoculation. A population of 20.5X10 8
cfug-1 was recorded on this substrate 10 days after
incubation in third combination cow dung + wheat
flour for medium increase the growth rate and
population of Trichoderma harzainum. (90.7X10 8
cfug-1) at 10 days after incubation. The
Trichoderma harzainum. Inoculated with cultured
sorghum grain the growth and sporulation of
Trichoderma harzainum was faster and highest then
other combinations.
Morphology Character of
Trichodermaharzianum
Ten days old culture medium grown on Potato
Dextrose Agar was studied for morphological
characters. Colony characters: Sterile hyphae, grow
rapidly, green patches, cushioned aerial mycelium,
floccous septate mycelium. Conidiophores: Hyaline,
highly branched, primarily branching at regular
intervals, paired or in whorls at three. Conidia: 1-celled,
ovoid, born in small terminal cluster, rapid growth,
intercalary and terminal. Phialides: Ampuliform to
subglobose, rise mostly in crowded and diverse walls.
Interaction studies of bioagent with
fungicides
Interaction studies of bioagent were carried out with
fungicides having different concentration, the
observations of Blitoxare given in Table 2, Fig. 2 show
inhibited mycelia growth of T. harzianum at all
concentrations and showed full growth at 500ppm
concentration while at 1000 and 1500ppm it showed
lesser growth as compared to control. No growth
was observed at 2000ppm. The effect of Thiophenate
methyl on various concentrations was tabulated in Table
3, Fig.3 where this chemical inhibited mycelial growth
of T. harzianum. Table 4, Fig 4 indicates complete
inhibition of T. harzianumat all the concentration of
Roxiltabucanazole. Table 5, Fig 5 show the effect of all
the concentrations except 500ppm of Ridomilwhich is
lesser growth as compared to control. Bavistin,
observations with regard to the effect of various
concentrations, which show less growth as compared to
control, was tabulated in Table 6, Fig. 6. Full growth
of T. harzianum at all concentration was observed in
Captan was tabulated in Table 7, Fig. 7.

Table 1: Effect of different substrate on the population of Trichodermaharzianum

Substrate Mean population at 10 days inoculation

Cow dung + sorghum grains 150.3(12.25)
Cow dung + wheat flour 90.7 (9.52)

Table 2: Effect of various cones, of Blitoxon myeelial growth of T.harzianum

Cone, in ppm. Mycelial growth (cm) at different interval of time

24 48 72 96
Control 2.5 4.5 6.7 9
500 NG NG 0.2 0.5
1000 NG NG NG NG
1500 NG NG NG NG
2000 NG NG NG NG

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Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
Journal of Biotechnology and Biosafety

Table 3: Effect of various cones, of Thiophenatemethyl on mycelial growth of T.harzianum

Cone, in ppm. Mycelialgrowth (cm) at different interval of time

24 48 72 96
Control 3.0 5.0 7.2 9
500 NG NG NG NG
1000 NG NG NG NG
1500 NG NG NG NG
2000 NG NG NG NG

Table 4: Effect of various cones, of Roxiltabucanazole on mycelial growth of T. harzianum

Cone, in ppm. Mycelial growth (cm) at different interval of time
24 48 72 96
Control 2.7 5.2 7.1 9
500 NG NG NG NG
1000 NG NG NG NG
1500 NG NG NG NG
2000 NG NG NG NG

Table 5: Effect of various cones, of Ridomilon mycelial growth of T. harzianum

Cone, in ppm. Mycelial growth (cm) at different interval of time
24 48 72 96
Control 2 4.5 6.5 9
500 1.5 3.8 6.5 8.5
1000 2 4.1 6.6 8
1500 1.5 2.1 3.0 4.5
2000 NG NG 0.25 0.5

Table 6: Effect of various cones, of Bavistinon mycelial growth of T.harzianum

Cone, in ppm. Mycelial growth (cm) at different interval of time

24 48 72 96
Control 2 4 6 9
500 1.5 3.5 5.0 7
1000 5 1.5 2.0 5
1500 NG NG 0.5 1
2000 NG NG 0.5 0.5

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Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
Table 7: Effect of various cones, of Captanon mycelial growth of T.harzianum
Cone, in ppm. Mycelial growth (cm) at different interval of time
24
Control 2.7
500 2.5
1000 2.8
1500 2.3
2000 2.2
DISCUSSION
T. harzianum contain strains that could be of
importance in biological control of plant pathogens
(Harman 2006; Sanz et al., 2005; Viterbo and Chet
2006). Therefore, present investigation aim to scaling
up solid state production system with industry
collaboration, large scale demonstration of biocontrol
technologies in farmer field, development of liquid
formation suitable for foliar application and biomass,
sporulation was good in sorghum grain than other
substrates. The present investigation was carried out at
S.D. (P.G.) College, Sri Ganganagar. The pure culture
of bioagent Trichoderma harzianum was studied for
their morphological characters. Colonies of these
bioagent was green and greenish yellow in colour.
Canidia were single celled and canidophorus were
hyaline and branched. Phialides were ampuliform to
subglobose.
Interaction studies of bioagent (T.harzianum) with
fungicides indicated non-compatibility of Thiophenate
methyl with T.harzianum at all the concentrations used.
Similarly Roxil was not found compatible with
T.harzianum. Blitox showed incompatibility with
T.harzianum. Similarly Bavistin and Ridomil are
compatible with T.harzianum. Captan was the only
fungicide, which showed full compatibility with
T.harzianumat all the concentration, used i.e. 500, 1000,
1500 and 2000ppm. Fungicide resistance is a stable,
inheritable adjustment by a fungus to a fungicide,
resulting in reduced sensitivity of the fungus to the
fungicide (Chaparro et al., 2011). Resistant isolates are
less affected or not inhibited at all by application of a
fungicide (Ma and Michailides 2005). This study
suggest limitation or otherwise of these chemicals in
integrating with bioagents.
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48 72 96
4.8 6.9 9
4.5 6.5 9
4.9 7.0 9
4.7 7.1 9
4.6 6.9 9
CONCLUSION
There were 6 fungicide interacting with different
concentration can be treated with T. harizanum in which
Captan was the only fungicide which showed full
compatibility with all concentration. T. harizanum
grown by Liquid and solid fermentation process and
talcum powder treated pots. A simple and cost effective
method to mass culture the biocontrol agent T.
harizanum by using liquid process and talcum powder
formulation.
Acknowledgement
Authors are thankful to Dr. R.B Gaur, Associate
Professor of Pathology Department, ARS, Sri
Ganganagar (Raj) and Dr. M. K Kaul, Associate
Professor of Horticulture Department, ARS, Sri
Ganganagar(Raj) enlighten us with their valueable
supervision. Special thanks for Dr.Pratibha Sharma,
Department of Pathology from of Indian Agricultural
Research Institute (IARI), Pusa, New Delhi for
providing pure culture of Trichoderma harzianum.
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Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
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 Journal of Biotechnology and Biosafety
Volume 4, Issue 1, January-February 2016,332-338
ISSN 2322-0406
Journal of Biotechnology and Biosafety

(2) (3) (4)

(5) (6) (7)

Figure 2-7: Showing the effect of different concentration of fungicides (Balitox, Thiophenete methyl,Roxil, Ridomil,
Bavistin&Captan) on mycelial growth of T. harzianum

Citation of this article: Charan Singh, Nevadita Sharma, Bhawna Roop singh.
TRICHODERMA HARZIANUM: MASS MULTIPLICATION AND ITS INTERACTION
WITH DIFFERENT FUNDICIDES. Journal of Biotechnology and Biosafety. 4(1): 332-338.

Source of Support: Nil Conflict of Interest: None Declared

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