Ectomycorrhizal Inoculum Production | Biotechnology

In this article we will discuss about the requirements and procedure for ectomycorrhizal inoculum production.

Ectomycorrhizae are found associated with forest trees like Pinus, Fagus, Populus, etc. Un­like VAM fungi, these mostly belong to higher fungi like Tuber (Ascomycotina), Amanita, Bo­letus, Pisolithus (Basidiomycotina), etc.

Requirements:

a. Malt agar medium.

b. Fruit bodies/fungal sheath/sclerotia.

c. Inoculating needle.

d. Bunsen flame.

e. Mild detergent/HgCl₂ 100 ppm/30% H₂O₂.

f. Sterile distilled water.

g. Scalpel.

h. Petri dishes.

i. 1-2 litre flasks with wheat/Jowar grains mixed with 3.0% CaC0₃ and 2% gyp­sum.

j. Peat moss enriched with ammonium tartarate asparagine, soyabean meal, malt extract and glucose.

k. Plastic bags (sterile).

l. Bunsen flame.

m. Inoculating needle.

Procedure:

1. Wash fruit bodies in tap water (sheath with root) or sclerotia also to be washed to remove adhering debris followed by keeping in mild detergent for 20-30 minutes.

2. Wash the detergent several times with sterile distilled water and place the fruit bodies in 100 ppm HgCl₂ for 5 minutes or in 30% H₂O₂ for 5-20 seconds.

3. Wash with sterile distilled water 4-5 times.

4. Using a sterile scalpel cut small pieces of the fruit body and inoculate them on malt agar in Petri plates.

5. When growth occurs (2-3 weeks) trans­fer them to slants and maintain pure cultures.

6. Inoculate this to 1 or 2 litre flasks con­taining sterilised wheat/jowar mixed with 3% CaCO₃ and 2% gypsum (CaSO₄) and incubate at room temperature (28°- 30°C) until the grains are completely coveted with mycelium (spawn).

7. Multiply this by inoculating it in grains added to peat moss enriched with am­monium tartarate, asparagine, soyabean meal, malt extract and glucose.

8. Pack in plastic bags, incubate at room temperature (28°-30°C).

9. Within 3-6 weeks inoculum is ready. This is added to seedlings in pots in nurs­ery and then transplanted in fields.