Journal of Microbiology and Biotechnology Research

Scholars Research Library

J. Microbiol. Biotech. Res., 2013, 3 (1):61-64
(http://scholarsresearchlibrary.com/archive.html)

ISSN : 2231 3168
CODEN (USA) : JMBRB4

61
Available online at www.scholarsresearchlibrary.com
Isolation, media optimization and formulation of Trichoderma Harizanum in
agricultural soil

Subash*, N
1
., Viji, J
4
., Sasikumar, C
2
and Meenakshisundaram, M
3

1, 2, 3
PG & Research Department of Biotechnology, Nehru memorial College (Autonomous), Affiliated to
Bharathidasan University, puthanampatti, Tiruchirappalli, Tamilnadu.
4
Centre For Eco- Friendly Agro technologies (Vermibiotechnology), Research
______________________________________________________________________________

ABSTRACT

Trichoderma harizanum is a biocontrol agent, innovative, cost effective and eco-friendly approach for the control of
wide range of fungal disease in all types of crops. In order to overcome this problem, efforts have been to evaluate
the efficiency of the biocontrol agent Trichoderma harizanum was isolated from the garden soil and identified
through microscopic analysis. The T.harizanum optimized in different liquid media, the extreme growth and
sporulation was shown in mineral salts medium with whey followed by biogas slurry. Since PDB was quite
expensive, the mineral salts medium with whey was used in this study. The fungal mat formed in the mineral salts
medium with whey medium was used to the inoculate sorghum seeds and spore biomass was prepared in boiled
sorghum seeds. The fungal mycelium slowly colonized the sorghum seeds and after 7 days. The T. harizanum was
formulated with talcum powder 1:2 ratio. Hence we concluded that T. harizanum can be extreme growth occur in
mineral salts medium with whey medium. The investigation was carried out MCRC Taramani Chennai Tamil Nadu
In
______________________________________________________________________________

INTRODUCTION

Soil borne plant pathogenic fungi a major economic loss, which is a major problem among the agricultural
community. Now a days the diseases are managed with the application of chemical pesticides. Use of chemical
pesticides causes environmental problem, as they dont undergo biodegradation. So minimizing the application of
pesticides has become order of the day. To achieve this goal the biological control methods can be effectively used
along with other methods of disease control. Antagonistic interactions and cell free culture filtrate have been used to
demonstrate the role of antibiotics in biological control [1, 2, 3, 4] Knowledge on the modes of survival of
pathogens and the ways by which they could be suppressed are important especially in the control of plant diseases.
The pathogens, in the absence of their hosts, survive either as dormant propagules or actively as saprophytes on dead
organic substrates of the host in the soil. The survival structures of the pathogens in the soil are suppressed either
due to manipulation of the soil environment. The pathogen suppression in the soil is considered as important step in
the control of diseases as it involves the direct disinfestations of the soil. Fungi belonging to the genus Trichoderma
are important phytopathogen bioantagonists, various methods for controlling such diseases have been investigated
including the use of resistant varieties [5]. One of the major limitations to the application of biological control agents
(BCAs) such as Trichoderma species is the development of appropriately formulated products [6]. In recent years,
considerable success has been achieved by the use of fungal bioagent. But so far there is lack of feasible and
effective formulation of bioagents to exploit it commercially[7].

Subash, N

et al J. Microbiol. Biotech. Res., 2013, 3 (1):61-64
______________________________________________________________________________
62
Available online at www.scholarsresearchlibrary.com
MATERIALS AND METHODS

Isolation of Trichoderma harizanum
The soil samples were made from five cm depth near the root zone of chilli plants grown at Main Agricultural
Research Station Chennai. They were pooled and representative sample was drawn. The biocontrol agent
Trichoderma harizanum was isolated from the representative sample by following the serial dilution plate
technique.

Medium optimization of T. harizanum
In order to mass cultivate T.harizanum in a large scale using agricultural waste, initial inoculum preparation was
essential. To prepare the initial inoculum, T.harizanum was cultured in different media like Potato Dextrose broth
(PDB), Mineral salts medium with either whey or corn steep liquor or biogas slurry. The green conidial formation
and the time of conidial formation were checked and the medium in which the conidial formation was earlier was
selected for further studies.

Cultivation of T.harizanum
Preparation of Sorghum seeds and initial inoculum of T.harizanum
100 g of sorghum seeds were boiled up to 20 to 25 minutes to soften grains and cook about 25%, drain water and
spread sorghum seeds to cool down to decrease the moisture content. 2g of calcium carbonate was added per 100
gm of parboiled semi dried sorghum seeds to remove the excess moisture and transferred to auto clavable
polypropylene bags and autoclaved at 121c at 15 minutes. After cooling, the sorghum seeds were aseptically
inoculated with the T.harizanum mats grown in liquid culture and incubated at room temperature for 5-7 days.

Formulation of T.harizanum
Talc formulation
Talc formulation was made according to TNAU protocol. The sorghum seeds cultured with T.harizanum was mixed
with talc powder at 1:2 ratio. The mixture is air dried in shade and mixed with Tapioca starch @ 5 g / kg the
product and packed in polythene bags and it was used for pot culture experiments.

RESULTS AND DISCUSSION

Isolation of Trichoderma harizanum
A number of colonies were observed in PDA plate after 3-5 days when the serially diluted samples were plated on
PDA media. Colony that produced green colour conidia was picked, observed under microscope by staining with
lactophenol cotton blue stain. The microscopic analysis of the mycelium with spore revealed that the isolate was
Trichoderma harizanum (Fig 1.). The isolate was sub cultured and stored in PDA slants at -20C [8]

Fig 1. Colony and microscopic morphology of T.harizanum



Media Optimization
Large scale production and establishment of bio control agents in targeted environment determines the success of
biological control. Therefore, a study on cost effective large scale production of T. hazianum, their establishment in
Subash, N

et al
______________________________________________________________________________
Available online at
targeted niches and their consistency in disease control becomes the primary concern for augmentative biological
control. For this, development of easy to prepare and cost effective formulations for delivery is essential. For mass
multiplication through solid state fermentation technology an enormous quantity of spore biomass is required. In
order to prepare spore biomass from sorghum seeds, fungal mats are essential. The fungal mats of
were produced by culturing in different liquid media and a
Though T. harizanum grew in all the media tested, corn steep liquor media supported the growth of the mycelium
and not sporulation (Fig.2). The growth and sporulation was best in PDB followed by mineral
Whey / biogas slurry (Fig.2). Since PDB was quite expensive, the mineral salts medium with whey was used in this
study.


Preparation of fungal inoculum for large scale cultivation of T
The fungal mat formed in the mineral salts medium with whey medium was used to the inoculate sorghum seeds and
spore biomass was prepared in boiled sorghum seeds. The fungal mycelium slowly colonized th
after 7 days the entire bag was fully of green colour growth with mycelium and spores on the seeds.

Preparation of T.harizanum Formulation
[9]suggested different organic media like neem cake, coir pith, farmyard manure, and decomposed coffee pulp for
its multiplication. Addition of T. harzianum
decomposed coffee pulp causes an immediate increase in the population up to 3 day. Increase in the total population
density of T. harzianum in the media suggests that competition by native
limiting factor in colonization of the media by the antagonist. Soil amended with organic materials like neem cake,
coir compost, farmyard manure and
alone. The carrier materials like neem cake, coir pith, farmyard manure and decomposed coffee pulp serve as
J. Microbiol. Biotech. Res.
______________________________________________________________________________
Available online at www.scholarsresearchlibrary.com
targeted niches and their consistency in disease control becomes the primary concern for augmentative biological
control. For this, development of easy to prepare and cost effective formulations for delivery is essential. For mass
lid state fermentation technology an enormous quantity of spore biomass is required. In
order to prepare spore biomass from sorghum seeds, fungal mats are essential. The fungal mats of
were produced by culturing in different liquid media and a media was selected where quick sporulation was seen.
grew in all the media tested, corn steep liquor media supported the growth of the mycelium
and not sporulation (Fig.2). The growth and sporulation was best in PDB followed by mineral
Whey / biogas slurry (Fig.2). Since PDB was quite expensive, the mineral salts medium with whey was used in this
Fig 2. T.harizanum Culture in different liquid media

Mineral salts medium with whey
inoculum for large scale cultivation of T. harizanum
The fungal mat formed in the mineral salts medium with whey medium was used to the inoculate sorghum seeds and
spore biomass was prepared in boiled sorghum seeds. The fungal mycelium slowly colonized th
after 7 days the entire bag was fully of green colour growth with mycelium and spores on the seeds.


Fig. 3. Talc formulation of T. harizanum

Formulation
[9]suggested different organic media like neem cake, coir pith, farmyard manure, and decomposed coffee pulp for
T. harzianum into organic media like neem cake, coir pith, farmyard manure and
decomposed coffee pulp causes an immediate increase in the population up to 3 day. Increase in the total population
in the media suggests that competition by native fungi in non-sterile carrier media was not a
limiting factor in colonization of the media by the antagonist. Soil amended with organic materials like neem cake,
coir compost, farmyard manure and Gliricidia leaves showed better growth and survival of antago
alone. The carrier materials like neem cake, coir pith, farmyard manure and decomposed coffee pulp serve as
J. Microbiol. Biotech. Res., 2013, 3 (1):61-64
______________________________________________________________________________
63
www.scholarsresearchlibrary.com
targeted niches and their consistency in disease control becomes the primary concern for augmentative biological
control. For this, development of easy to prepare and cost effective formulations for delivery is essential. For mass
lid state fermentation technology an enormous quantity of spore biomass is required. In
order to prepare spore biomass from sorghum seeds, fungal mats are essential. The fungal mats of T. harizanum
media was selected where quick sporulation was seen.
grew in all the media tested, corn steep liquor media supported the growth of the mycelium
and not sporulation (Fig.2). The growth and sporulation was best in PDB followed by mineral salts medium with
Whey / biogas slurry (Fig.2). Since PDB was quite expensive, the mineral salts medium with whey was used in this

The fungal mat formed in the mineral salts medium with whey medium was used to the inoculate sorghum seeds and
spore biomass was prepared in boiled sorghum seeds. The fungal mycelium slowly colonized the sorghum seeds and
after 7 days the entire bag was fully of green colour growth with mycelium and spores on the seeds.
[9]suggested different organic media like neem cake, coir pith, farmyard manure, and decomposed coffee pulp for
into organic media like neem cake, coir pith, farmyard manure and
decomposed coffee pulp causes an immediate increase in the population up to 3 day. Increase in the total population
sterile carrier media was not a
limiting factor in colonization of the media by the antagonist. Soil amended with organic materials like neem cake,
leaves showed better growth and survival of antagonist than in soil
alone. The carrier materials like neem cake, coir pith, farmyard manure and decomposed coffee pulp serve as
Subash, N

et al J. Microbiol. Biotech. Res., 2013, 3 (1):61-64
______________________________________________________________________________
64
Available online at www.scholarsresearchlibrary.com
nutrient additives to the crop in addition to inoculum production they support. [10] Reported that the pre-boiled
sorghum grains, coir pith + neem cake (1:1), cow dung + neem cake (1:1) + wheat flour (10%) maintained high
populations of T. harzianum and T. viride within 10 days of inoculation.

In India, talc based formulations of T. viride was developed at Tamil Nadu Agricultural University, Coimbatore for
seed treatment of pulse crops and rice [11]. In this study, the T.harizanum grown in sorghum seeds was formulated
as per the TNAU protocol into wettable powder formulation using talc (Fig. 3 ). The talc formulations of
Trichoderma have shelf life of 3 - 4 months. It has become quite popular in India for management of several soil-
borne diseases of various crops through seed treatment at 4 - 5g/kg seed.

Acknowledgement
We boundless pleasure and heartfelt thanks to MCRC Chennai, and also thanks to Nehru Memorial College, P. G.
and Research Department of Biotechnology, Zoology Staff Members and Senior Research Scholars of our Research
Team.

REFERENCES

[1] Naik, N.K., and Sen, B., 1992. Biocontrol of disease caused by Fusarium sp. In Recent Development in
Biocontrol of Plant Diseases [K.G. Mukerji, J.P. Tewari, D.K. Arora and G.Saxena (eds.)]. New Delhi: Aditya
Books Pvt. Ltd., pp.37-51.
[2] Panneerselvam, A., and Saravanamuthu, R., 1999. Indian J. Geobios., 26: 123-126.
[3] Panneerselvam, A., and Saravanamuthu, R., 1994. Indian J. Bot. Soc., 73: 265-267.
[4] Tu, J.C., 1992. Biological control of Sclerotinia sclerotium. In Recent Development in Biocontrol of Plant
Diseases [K.G. Mukerjii, J.P. Tewari, D.K., Arara and G. Saxena (eds.)]. New Delhi: Aditya Books Pvt. Ltd., pp.24-
32.
[5] Brisa, R., Fernando, M.A., Asuncin, G.S., Noem, M.R., Arturo, P.E and Jos, M.D. 2007. Fungal genetics and
biology 44: 864-876.
[6] Fravel, D.R. 2005. Annu. Rev. phytopathol, 43:337359
[7] Mishra, B.K., Rohit Kumar Mishra., Mishra, R.C ., Amit Kumar Tiwari., and Ramesh Singh Yadav and
Anupam Dikshit (2011) Archives of Applied Science Research, 3 (2):361-369
[8] Harman GE (2006) Phytopathol. 96: 190- 194.
[9] Saju, K.A., Anandaraj, M and Sarma, Y.R. 2002. Indian Phytopath., 55: 277281.
[10] Rini, C.R and Sulochana, K.K. 2007. J.Tropical Agriculture, 45: 58-60.
[11] Jeyarajan, R., Ramakrishnan, G., Dinakaran, D and Sridar, R. 1994 Biotechnology in India (Ed Dwivedi B.K.)
Bioved Society, Allahabad