Mycorrhiza is the product of an association between a fungus and plant root. Vesicular-Arbus Mycorrhiza (vam) is formed by the symbiotic association between certain phycomycetous fungi and angiosperm roots.

Staining and observation of VAM fungi

Materials required

· Lactic acid

· Phenol

· Trypan blue

· Glycerol

· KOH

· Con. HCL

· Forceps

· Paster pipette

· Filter paper

· Glass slide

· Cover slip

· Conical flasks (250ml)

· Funnel

· Beaker(500ml,250ml)

· Measuring cylinder (1000ml)

· Boiling tubes

· Surgical needle

· Blade

· Tissue paper

· Wash bottle

Preparation of lactophenol: Mix 50ml lactic acid, 50ml phenol and 100ml glycerol in 100 ml water

Preparation of trypan blue: 0.5gm in 100ml water

Method

· Collect fine terminal feeder roots, wash thoroughly in tap water and cut it into small pieces (1-2cm)

· Add 10% KOH to a boiling tube along with root bits and boil it for 1-2 hr in water bath.

· Pour off KOH

· Add 2% hcl TO NEUTRALIZE koh (5MIN.)

· Stain roots with trypan blue in lactophenol (3min)

· Pour off trypan blue and add keep it in lactophenol

Quantification of root Colonization

The number of root segments are counted and expressed as a percentage of total root segments in the sample.

% colonization = Number of colonized segments X 100

Total number of segments examined

Isolation of VAM fungi ( Wet sieving and decanting method)

Materials

· Sieves (850,250,105 and 45 µm)

· Vermiculate

· Perlite

· Cowdung

· Soil

Method

· Mix a volume of soil (200g) in water (1000ml) in a measuring cylinder/ beaker, mix well and allow heavier particles to settle down.

· Pour liquid through a coarse sieve ( generally 850 µm) to remove large pieces of organic matter. Collect the liguid which passes through this sieve.

· Pass this suspension through a sieve fine enough to retain the desired spores, generally (250,105,45µm).

· Collect the liquid in separate conical flask with through washing.

· Filter the liquid through Whatman No.I filter paper.

· Observe it under a microscope.

Preparing inoculums for large-scale production

Materials

Vermiculate 65 kg

Perlite 20 kg

Cowdung 5 kg

Soil 10 kg

Ginigrass strip/ Sorghum seeds

· Sterilize the mixture in an autoclave for 1 hour at normal conditions

· Dip the Gini grass strip in 50% alcohol for 5minutes and wash thoroughly with tap water

· Fill the earthenware pots with sterile substrate (3/4).

· Spread mother inoculums over the substrate, sow surface sterilized seeds and plant gini grass strip.

· Observe the colonization frequency of AMF after 1-2 months ( for 50% colonization)

· Both segments of colonized roots containing hypha and/ or vesicles and subsrate containing fungal spores may be used as starting fungal propagules for mass production.

MASS multiplication of VAM

Vermiculate 65 kg

Perlite 20 kg

Cowdung 5 kg

Soil 10 kg

Ginigrass strip / Sorghum seeds

Plastic sheet

Digger

Sterilization of substrate

· Spread the substrate ( usually 100kg/ tank ) in a clean cement tank and make 4-5 small holes and pour 50% formaldehyde (500ml in one tank).

· Cover the tank with a plastic sheet and keep it for one week. Remove the cover after one week and mix it again.

· Make two to three channels in the substrate and fill it with mother inoculums ( remove the shoot portion and add soil along with chopped roots from the pre developed inoculums ( ˜from 2 to 3 earthenware pots).

· Sow surface sterilized seeds and/ or plant gini grass strip, cover evenly and watering it at regular intervals.

· Quality test on AM colonization in root samples is carried out on 30^th and 45^th day.

· Stock plants are grown for 60 days (8 weeks). The inoculums is obtained by cutting all the roots of stock plants. The inoculums