Sampling site:
Selected sampling sites were Charfashion Upazila of Bhola district under AEZ 18 (Young Meghna Estuarine Floodplain) and Dumki Upazila of Patuakhali district under AEZ 13 (Ganges Tidal Floodplain) of Bangladesh.
Collection of soil samples: For isolation of rhizobia, soil samples were collected from selected areas. Ten surface soil samples were collected from each location. For the convenience of discussion, the 6 soil samples are referred to as soil -1, soil-2, soil-3, soil-4, soil-5 and soil-6 and these soil samples were collected from Aslampur, Ginnagor, Osmangonj of Charfashion Upazila in Bhola district and Srirampur, PSTU Farm, Jamla of Dumki Upazila in Patuakhali, respectively.
Preparation of the soil sample: Some portions of the collected soil sample were kept in the refrigerator at 40C for isolation of bacteria. The rest portions of soil samples were then air dried ground to pass through a 2 mm sieve and then mixed to from a composite sample. Then these composite samples were kept in clean and sterilized bottles for physical and chemical analysis.
Experimental site:
The laboratory experiment was conducted at the Department of Soil Science and Central Laboratory, Patuakhali Science and Technology University, Dumki, Patuakhali during July 2014 to June 2015.
Soil analysis:
The initial soil samples were analyzed for physical and chemical characteristics. The physical characteristics include textural class and the chemical properties include soil pH, electrical conductivity, organic matter, total N, Exchangeable K, available P and S content.
Culture media:
Yeast mannitol agar media were used for culture of Rhizobium.
Method of isolation:
Enrichment culture technique (in liquid medium) was used for the isolation of bacteria.
Composition of yeast extract mannitol agar:
Mannitol - 10.0 g, K2HPO4- 0.5 g, MgSO4, 7H2O - 0.2 g, NaCl - 0.1 g, Yeast extract - 0.5 g, Distilled water - 1 liter and Agar- 15 g. The medium was prepared and was autoclaved at 1210C and 15 psi for 20 minutes. In the meantime, all accessories like Petridis and pipette (1 ml) was also sterilized by autoclave.
Colony isolation: The growth of Rhizobium was streaking on medium and incubated until pure growth was obtained. Finally, pure Rhizobium was cultured on slant medium as mother culture and stored in the refrigerator. Then different biochemical test is to be done and new mother culture was done after 3-4 months. The colonies showing clear zones around them developed within 48 hours were transferred to agar slants of Yeast mannitol agar medium and allowed to grow at 300±20C for three days. The cultures were then repeatedly plated in the same agar medium till pure strains were obtained and finally 20 bacterial cultures were maintained in the Yeast mannitol agar medium.
Estimation of bacterial population:
The viable cells were calculated by the following formula stated by Somasegaran and Hobben (Somasegaran and Hobben, 1985).
Number of cells/ml (CFU/ml) = [((Number of colonies) × (Dilution factor)} ÷ (Volume per drop)].
Purification of isolates: Six isolates of each Rhizobium were taken from respective cultured media and streaked on respective prepared plate’s media. The streaked plates were incubated at 280C for 2-4 days. Repeated streaking was done until purification.
Identification of Rhizobium isolates: The isolates of Rhizobium obtained from soils were described according to their growth characteristics on solid and liquid Yeast Mannitol Agar media. Some morphological characters such as the shape, size, color, texture of colonies and ability to alter pH and some biochemical characters such as carbohydrate utilization and fermentation, gelatin and starch hydrolysis, Congo red dye absorption.
Preparation and preservation of mother culture: Purified isolates of Rhizobium were transferred into Yeast Mannitol Agar media and preserved for further study.