Collection of Samples
Root nodulating bacterial strains were isolated from the root nodule of Cicer arietinum (chick pea) according to Vincent (1970), collected from different sites of Dehradun (U.K).
Isolation and Characterization of Root Nodulating Bacteria
The collected nodules were first surface-sterilized with 95% ethanol, then with 0.1% mercuric chloride and finally washed thoroughly with distilled water. strains were obtained by streaking the crushed root nodules on YEMA plates and incubated at 28±2°C .After 2 days of incubation, Rhizobium colonies were obtained. Further streaking, spreading and visual characterization of colony morphology helped in isolation of pure cultures..Biochemical characterization of recovered isolates were done according to Bergey’s Manual of Determinative Bacteriology (Holt et. al., 1994) All the test were carried out with 03 replicates.
Salt and pH tolerance
The ability of the isolated Rhizobial strain to grow in different concentration of salt was tested by streaking them on YEMA medium containing 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0% and 8.0% (wt/vol) NaCl. Differences in pH tolerance were tested in YEM agar. The pH was adjusted to 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5 and 9.0 with HCl or NaOH. All the plates were incubated at 28°C during 72 hours and plates containing basal YEMA medium were used as controls.
Strains were considered salt tolerant and resistant to acidity when growth was similar to the growth in the control plates.
Antibiotic Resistance Pattern of Isolates
All the isolates were tested for antibiotic sensitivity by Kirby-bauer disc diffusion method (Bauer et al; 1966) on YEMA. Cultures were inoculated by swabbing with standard inoculums (corresponding to 0.5 Mc Farland tube over the entire agar surface. The agar surface was allowed to dry for 3-5 minutes before applying the antibiotic disc using sterile forcep. The plates were incubated at 300C for 48 hours, sensitive to an antibiotic was detected by zone of inhibition around the disc. The following antibiotic discs were used, Cefalexin(CN30), Meropenem(MRP10), Polymixin-B(PB50), Netillin(NET30), Amikacin(AK30), Ceftriaxone(CTR30), Clindamycine(CD2), Streptomycin(S25), Amoxycilline(AMX25), Ampicilline(AMP25).
Determination of Carbohydrate Utilization
To determine the capability of isolates to use carbohydrates (Mannitol, Dextrose, Maltose, Galactose, Mannose, Sucrose, Raffinose, Arabinose, Sorbitol, Lactose.) fresh culture was spread on YEMA plates. The agar surface was allowed to dry for 3-5 minutes before applying the carbohydrate disc using sterile forcep. The plates were incubated at 300C for 48 hours. Carbohydrate resistance was detected by zone of inhibition around the disc.
Determination of Fungicide Tolerance
The effect of fungicides on the growth of strains on YEMA medium were tested as described by Bollich et al, (1985) using measurement of diameters of the growth inhibition zones. The fungicides used were:- Thiram (75%), Mancozeb (75%), Zineb (75%), Carbendazim (50%). The concentrations used were 25, 50, 75 and 100 g/l. The cultures were spread over the entire agar surface four disc of filter paper from different concentrations were applied using sterile forcep. The plates were incubated at 300C.The diameters of the inhibited growth zones formed were measured after 24 hours. A plate with filter paper disc without any fungicide was used as control.