MOD I : RHIZOBIAL INOCULANT

Rhizobium Inoculants

-Submited By

RAJAT MISHRA

6th Sem

B.Tech Biotech GIET,Gunupur

Nobbe and Hiltner introduced a laboratory culture of rhizobia with the name “Nitrogin”.Rhizobia contributes towards better usage of Rhizobia –Legume symbiosis for sustainable crop yield.

Rhizobia Innoculant preparation

1. Isolation Of Rhizobium_:

Rhizobium can be isolated either from soil or root nodules. Root nodules are prefferd for getting contamination free rhizobia.\

` Steps

(1) Healthy root nodules are taken from mature plant and washed with sterile distilled water .

(2)Nodules are externally sterilized by mercuric chloride or sodium hypochlorite.

(3)Suspension is streaked on petriplates containing YEMA. These plates are incubated for 3-4 days at 28-30 °C..colonies appear on surface of medium.

2. Identification

Both rhibozium and agrobacterium grow on YEMA medium and can be distinguished by CRYEMA test.CRYEMA medium is prepared by mixing 2.5ml congo red dye with 1L of YEMA medium .bacteril colonies growing on YEMA are streaked outplates are incubated CRYEMA medium.Plates are incubated at 28-30 °C for 1 week. Rhizobium utilizes congo red dye very slowly and form white,circular and raised colonies whereas agrobacterium form red colonies so these bacterial species can easily be distinguished.

The white coloured colonies are isolated and rhizobial innoculant are produced.

3. Starter Culture

Rhizobium colony is transferred into YEM broth.Innoculated YEM brith is incubated on a rotary starter at 28-30°C. After 4 days suffiecient number of cells are present in YEM broth. It is called mother culture or starter culture.

3. Preparation of Carrier based Innoculum and Curing

A carrier is an inert material with broth so that innoculant can easily be handeled,packed,stored/transported and ues. A variety of carriers are used for eg. Peat, lignite , Farmyard manure, charcoal etc.

The carrier is powdered and dried in sun to get 5% moisture level.It is screened through 100-200 mesh sieve and neutralized by mixing with Calcium Carbonate powder.The carrier is sterilized at 15PSI for 3 hours and dried. The harvested broth (innoculum) obtained is mixed with hand or mechanically . The moisture content is maintained to about 35-40%on dry weight basis.It is left for 2-10days by covering the trays with polythene at 22-24°C .This above process is called as Curing.

3. Packaging And Storage

The cured carrier are packed in polythene bags and kept at a constant room temperature for about a week to facilate the rhizobial cell. Care shpuld be taken to store packet at about 4-15°C in store room.

3. Quality Control of Rhizobium Innoculum

Innoculum quality means the number of effective rhizobial cells present in carrier.

Quality control includes

1. Strain testing, selection skill and control of innoculum density in broth before mixing with carrier.

2. Control of carrier based culture after manufacture .\

3. Control of culture during storage period.

3. Methods of seed innocuation with rhizobial culture

Seed inoculation with aqueous suspension of carrier culture during sowing has releaved the nodulation of good yield of crops. This is indicated by following figure