Use of locally available agri wastes as substrate for mass production of trichoderma harzanium

Pranab Dutta / BC Das *

Biocontrol offers safe, cheap and environmental friendly management of plant diseases and avoids or minimizes the usage of harmful fungicides. In biocontrol of plant diseases fungal antagonist like Trichoderma spp. Are extensively uses for control of several soil borne diseases and have also additional advantage of growth promoting effect. Many commercial formulations in solid form based on these antagonists have been developed worldwide.

Trichoderma is a genus of asexually reproducing fungi present in nearly all temperate and tropical soils. The genus is a strong saprophytic, fast grower, prolific producers of spore and powerful antibiotic producers. These properties make these fungi ecologically varied diversity and can be used to produce a wide range of product of commercial and ecological interest. Trichoderma has become a model biocontrol agent in the management of plant Disease as apart of IDM because of following characters:

o Produces high yield and high quality conidial biomass.
o Possess rhizosphere competence.
o Ability to thrive over a wide range of stress condition.
o Having mycoparasitic ability against large number of soil borne pathogens.
o Stimulate plant resistance and defence mechanism.
o Compatible with large numbers of pest.
o Easy to mass multiply.
o The final product has shelf life more than 6 months.

MODE OF ACTION

o Mycoparasitism through production of enzymes and coiling.
o Antibiosis through production of antibiotics.
o Competition for food ad space.

SELECTION OF TRICHODERMA STRAIN

During last 19 years we have search for a suitable biocontrol agent to be used as seed treating and soil treating agent we successes in identifying a strain of T. harzianum (DPP-105). With this a formulation "Biogaurd" has been formulated to fulfil the demand of farmers.

The different process involved in the production of "Biogaurd" are
o Inoculam production
o Selection of culture medium
o Growth and mass multiplication
o Harvesting and drying
o Scaling up and
o Formulations

Three methods viz. solid fermentation, liquid fermentation and deep tank fermentation technology are followed for multiplication of Trichoderma. Out of these solid fermentation is easy, popular and widely used by the farmers.

SOLID FERMENTATION PROCESS

Improved selective medias have been developed for cultivation of Trichoderma spp. Solid media are prepared comprising easily available organics such as wheat bran, saw dust, grain bran, wheat straw, rice straw, sorghum grain, banana leaves, banana peeled skin, farmyard manure (FYM). Presently, FYM and wheat bran-saw dust is extensively used to produce biocontrol agent of T. harzainum and T. viride.

Well decomposed powdered FYM mixed with 1% peptone and half parts water are autoclaved at 30 lbs pressure for 1 hour intermittently for 2 times and heaped in a clean shady place. The stock culture of Trichoderma spp. Multiplied on potato dextrose agar (PDA) medium for 15 days inoculated under aseptic conditions, 500 ml of homogenous spore suspension prepared in a mixer is used for 10 kg of FYM. Heaps are then covered with polythene sheets (to prevent loss of moisture) for 30 days. Heaps are mixed thoroughly at an interval of 7 days. After 30 days of incubation this preparation is ready for application.

LIQUID FERMENTATION

This process involves production of large number of conidia mainly intended for seed treatment, soil application and foliar spray. The Trichoderma based liquid bioformulation developed at AAU i.e., Org-Trichojal with having shelf life of 16 months has found to be highly effective in farmers field for management of plant pathogens and enhance plant growth parameter and plant defence metabolite. The product has been commercialized through private entrepreneur.

DEEP TANK LIQUID FERMENTATION TECHNOLOGY

This involves mass production of fungus. In this technique Trichoderma spp. Are mass cultured in commercially available inexpensive ingredients (molasses and brewer's yeast). The rapid mass production of antagonists for seed treatment formulation, the deep tank liquid fermentation mostly used.

GROWTH AND MASS MULTIPLICATION:

The fermenter medium used to grow and mass multiply Trichoderma spp. Consists of Molasses (30 kg) + Brewers yeast (5 g) + 1 litre water (fermenter medium of above concentrations is used. Procedure for mass production of Trichoderma spp. Is given below:

o Autoclave both starter and fermented medium for 1 hour
o Inoculate starter medium with efficient strain of Trichoderma and shake on rotary shaker
o Inoculate fermented media with starter inoculam and supply regularly at for 10-15 days by shaking/bubbling
o Filter through cotton muslin using funnel
o Air dry or freeze dry the mat for 3 days find grind into powder this contain mycelium and chlamydospores
o Powder is diluted with different diluents and used as dusts, granules, pellets, wettable powders, emulsifiable concentration
o Produce is viable for 6 months, if stored at 50C and 3 months if stored at 200C
o Maximum biomass is obtained generally after 15 days of inoculation.

FORMULATIONS

Different formulations are available and prepared for the usage as given below.
1. Powder formulation: It is done by mixing fermenter biomass with talcum powder as carrier.
2. Encapsulation in organic polymer like sodium alginate.
3. Pelleting biomass and bran with sodium alginate.
4. FYM, banana leaves, banana peeled skin, wheat bran for soil application.
5. Molasses enriched charcoal powder granules.
6. Liquid coating formulations bio-protectant as powder on which suspension of aqueous binder is sprayed on seeds to form 0.1 mm thick layer.
7. As spray form emulsifiable concentrations with 10 spores in all of the above formulations. It is necessary to maintain level of 1-10 cfu ml-1 or g.

MATERIALS REQUIRED FOR PREPARATION OF FORMULATION

1. Freshly isolated native Trichoderma harzianum pure culture
2. PDA media
3. Substrates
4. Distilled water
5. Poly propylene bags/saline bottle
6. Racks
7. Rubber bands
8. Laminar flow cabinet
9. Inoculating needle
10. Spirit lamp
11. Cotton (both absorbent and non absorbent)
12. Spirit lamp
13. Methylated spirit
14. Ethyl Alcohol
15. Petri plates
16. Cork borer
17. Measuring cylinder
18. Muslin cloth
19. Plastic Bucket and mugs
20. BOD incubator

METHODS OF APPLICATION

The methods presently used for effective management of diseases includes the application of Trichoderma by any of these methods viz., broadcast application, furrow application, root zone application, seed coating or seed treatment, wound application.

a. Broadcasting
Broadcasting is used for soil application. The quantity required is high and varies from 125-250 kg ha -1. It is mixed with FYM and broadcasted on the soil and then incorporated into the soil through harrowing. It is used to control Sclerotinia rot of vegetables, damping off of vegetables, stem rot of soybean. Trichoderma is applied by this method at least 2 weeks before sowing of crops.

b. Furrow application
It is comparatively economical to broadcasting and is followed in nurseries. The quantity required 130-160 kg ha-1. It should be applied in the open furrows covered with the soil. Trichoderma is applied by this method 15 days before sowing of the seeds.

c. Root zone application
This is used in wide spaces crop such as plantation and fruit crops. It is done in peach and plum against silver leaf diseases.

d. Seed treatment
In this method of application, the seeds are first mixed with water 5-7 ml kg-1, so that the seed surface becomes moist. Take known quantity of seed formulation (Powder form) 4 g kg-1 seed, mix thoroughly by agitating the container so that the powder adheres to the seed surface. Then treated seeds should be dried I shade for two hours and sown immediately after treatment. This formulation can used to apply on and around the root zone.

This helps good seed germination, seedling emergence and finally good plant stands. It also gives protection in the rhizosphere against root rots, stem rot, seedling blight, damping off, collar rot, white mold, rhizome rot of various crops such as chick pea, cabbage, cauliflower, French bean, brinjal, chilli, cucumber, potato, ginger, rice, sunflower caused by Rhizoctoniasolani, Pyhiumapphanidermataum, Sclerotiniasclerotiorum, Fusarium oxysporum, F. moniliforme, Sclerotium rolfsii etc.

ECONOMICS

Cost of production (Excluding laboratory equipment) : Rs. 30 kg-1
Selling price : Rs. 60 kg-1

ADVANTAGES

o Parasital activity takes place on different stages of insect ontogenesis.
o The fungus possesses the high speed of growth, huge reproductive ability and high specificity.
o It is able to remain for a long time in nature without the decline of infectivity.
o The effectiveness of this product depends very much on climatic condition (temp 25-280C and relative air humidity of 80-90%), the methods of application and doses.
o Easy to deliver.
o Improve plant growth, flowering and increases yield.
o Safe to the environment, human beings, animals.
o The no risk of development of resistance in pathogen.
o Compatible with biofertilizer like rhizobium, azospirrillium etc.

PRECAUTIONS

o Formulation must free of contamination.
o Store in cool and dark place, avoid direct sunlight.
o It uses for seed treatment, the treated seeds must be dried in shade at least for 6 hrs prior to sowing.
o Application must be done in cooler hrs of a day.
o Culture must be periodically re-isolate from the crop rhizosphere of phyllo sphere.
o Avoid direct contact on skin.


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CAU, Imphal.
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