PRODUCTION OF TRICHODERMA CULTURE

PRODUCTION OF TRICHODERMA CULTURE

The soil of our factory command areas are having abundant source of fungal diseases & deficient of several nutrients therefore, need of application of Trichoderma spp in our soil to improve soil health.

Trichoderma is a very effective biological mean for plant disease management especially the soil born. It is a free-living fungus which is common in soil and root ecosystems. It is highly interactive in different mechanisms like competition, antibiosis, mycoparasitism, hyphal interactions, and enzyme secretion.

Working mechanism of Trichoderma:

Disease Control: Trichoderma is a potent biocontrol agent and used extensively for soil born diseases. It has been used successfully against pathogenic fungi belonging to various genera, viz. Fusarium, Phytopthara, Scelerotia etc.

Plant Growth Promoter: Trichoderma strains solubilize phosphates and micronutrients. The application of Trichoderma strains with plants increases the number of deep roots, thereby increasing the plant's ability to resist drought.

Biochemical Elicitors of Disease: Trichoderma strains are known to induce resistance in plants. Three classes of compounds that are produced by Trichoderma and induce resistance in plants are now known. These compounds induce ethylene production, hypersensitive responses and other defense related reactions in plant cultivars.

Transgenic Plants: Introduction of endochitinase gene from Trichoderma into plants such as tobacco and potato plants has increased their resistance to fungal growth. Selected transgenic lines are highly tolerant to foliar pathogens such as Alternaria alternata, A. solani, and Botrytis cirerea as well as to the soil-borne pathogen, Rhizectonia spp.

Bioremediation: Trichoderma strains play an important role in the bioremediation of soil that are contaminated with pesticides and herbicides. They have the ability to degrade a wide range of insecticides: organochlorines, organophosphates and carbonates.

Preparation of PDA media:

Culture media are essential for isolation of fungi and maintaining them in a pure culture either in test tubes or in Petri plates. The fungal cultures may be grown in liquid media (broths) or on solid agar media. (Agar agar is a solidifying material obtained from sea seed weed like Geladium sp., which melts on boiling and solidifies upon cooling).

Potato dextrose agar : (BAM Media M127 and potato dextrose broth are common microbiological growth media made from potato infusion, and dextrose. Potato dextrose agar (abbreviated "PDA") is the most widely used medium for growing fungi and bacteria.

Potato dextrose agar (PDA):

Potato	200 g (sliced washed unpeeled)
Dextrose	20 g
Agar agar	20 g
Streptomycin	0.05 g
Distilled water	~ 1000 ml. (pH : 7)

Method of making PDA: 

1. Wash 200 g potato, peel off the skin, and slice them into small pieces.
2. Cook the sliced potato in 500 ml. water for 30 minutes in an open vessel or pressure cooker for 20 minutes.
3. Collect the potato extract by filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form).
4. Mix with Dextrose, Agar and make the volume to 1 litre with distilled water and boil to dissolve.
5. Autoclave 15 min at 121.6°C
6. After solidifying, the tubes / Petridishs / flasks are arranged in a wire basket and stored in a clean room for further use.
7. It can store in at 5-25 °C for a month.

Procedure for checking pH: Stir the medium with a glass rod and take a small bit of pH indicator paper and dip it in the medium. Take it out and compare the colour development with the colour chart given on the cover sheet of the filter paper book. If pH is above 7, add a few drops of 0.1N Hydrochloric acid (HCl), stir well and again check the pH for neutral. Contrastingly, if the pH of the medium is below 7, add a few drops of 0.1 N Sodium hydroxide (NaOH) to increase.

Sterilization (microbiology):

Steam sterilization Invented by Charles Chamberland in 1880

Steam sterilization exposes each item to direct stream contact at required temperature and pressure for the specified time.

Steam sterilization is primary used for heat-stable materials such as – Glassware, surgical instrument & different type of biological Medias

There are two types of stream sterilizers

•         Gravity displacement Autoclave
•         High speed prevacuum sterilizers

The recommendations for sterilization in an autoclave are 15 minutes at 121-124 °C (200 kPa). The temperature should be used to control and monitor the process; the pressure is mainly used to obtain the required steam temperature. Alternative conditions, with different combinations of time and temperature, are given below.

Temperature (°C)	Approximate corresponding pressure (kPa)	Minimum sterilization time (min)
126-129	250 (~2.5 atm)	10
134-138	300 (~3.0 atm)	5

Dry heat sterilization: Dry heat sterilization is one of the earliest forms of sterilization practice

It used as product that may be degraded when exposed to stream or moisture but which can with stand high temperatures

Examples of items sterilized by dry heat sterilization: Mental surgical instrument, needles, glassware, powders, oils, etc.

Preparations to be sterilized by dry heat are filled in units that are either sealed or temporarily closed for sterilization. The entire content of each container is maintained in the oven for the time and at the temperature given in the table below. Other conditions may be necessary for different preparations to ensure the effective elimination of all undesirable microorganisms.

Temperature (°C)	Minimum sterilization time (min)
160	180
170	60
180	30

Procedure of rearing of trichoderma:

Maintaining nucleus culture:

1. Isolation of trichoderma spp from soil of that area with help of serial-dilution method on selected media.
2. Purification of available strain by sub culturing
3. Purified fungi maintained nucleus culture on regular interval sub culturing.

Mass culturing of trichoderma:

1. It can be mass cultured by liquid media (broth) or cereals grains but we have used waste of Corcyra boxes.
2. Make 200 gm waste of Corcyra boxes in plastic bag and plug with norbsorbent cotton.
3. Steam Sterilized all pkts ar desired pressure & temperature.
4. Sterilized pkt keep in inoculation room for inoculation.
5. Before inoculation laminar air flow surface sterilized by sprit or expose of UV lights for 15-20 min.
6. Sterilized pkts pore with trichoderma desired species @ 1 nucleus culture plate pore 10 pkts.
7. Inoculated Trichoderma pkts keep in culture room for development of fungus at 20-25 °C temperature.

Commercial culturing of trichoderma:

1. We can be make commercial form of Trichoderma in Talcum powder, Charcol, Pressmud, liquid form or any other organic martial
2. Collect pressmud keep in heap (1 fit height, 1 mt wide & desirable length) and broken all lamps and removed all unwanted material.
3. Pressmud moist with water.
4. Add Corcyra maize @ 0.5 bag/qt of pressmud.
5. Mixed mass culture pkt @ 20 pkts in 1 qt of pressmud before mixing make peast with help of mixer grander.
6. Fungal suspension spore sprayed on pressmud mix thoroughly.
7. Leave for 7-10 days for fungal devolvement charning of culture every 2^nd day for increasing aeration.
8. Packing of culture in white bags @ 4 kg / bag.
9. It can be store up to 60 days at room temperature.

Precaution:

1. Purification of species is most important activity.
2. Maintaining of nucleus culture.
3. Avoiding contamination during inoculation.
4. Adding water in pressmud is not run-off.