Chickpea Plant Feasibility Study

2. Materials and methods

2.1. Rhizosphere soil sampling and isolation of PGPR

Rhizosphere soil of chickpea plants growing in the agricultural field near the university (26o42’ 30.88” N and 88o20’ 52.97” E) was collected during the month of December. 1g soil was suspended in 10 ml of standard 0.85 % saline and vortexed for 10 min to get a uniform suspension. Serial dilutions were made by sequentially adding 1 ml from 10-1 till 10-7. The serially diluted suspensions (10-1 to10-7) were spread plated on nutrient agar and incubated at 37 ºC for 24 h. The isolated colonies with distinct colony morphology were selected, further streaked to get pure cultures and stored in glycerol at -20oC. All the discrete isolated strains were tested for their plant growth promoting (PGP) attributes as mentioned below.

2.2. Plant growth promoting traits of bacterial isolates

2.2.1. Nitrogen fixation

Bacterial isolate was grown in Asbhy’s N-free liquid medium containing (g l-1) mannitol, 15; MgSO4.7H2O, 0.2; K2HPO4, 0.2; CaCl2, 0.2; FeCl3,0.05 and 1 drop of Na2MoO4 (10 % w/v ) at 37 ºC for 24h. The culture was then streaked on Asbhy’s N-free agar plates and incubated at 37 ºC for 24h. Production of ammonia by the isolate was quantified by the method of Goswami et al. (2014). Bacterial culture grown in Asbhy’s N-free liquid medium was

Bacterial culture was grown in LB broth (pH 5.0) supplemented with tryptophan (1 mg ml-1) at 37 °C under 120 rpm shaking (120 rpm) for 2 days followed by centrifugation at 6000 rpm for 10 min. 1 ml of supernatant was combined with 2 ml of Salkowski’s reagent (150 ml of 95-98 % H2SO4, 7.5 ml of 0.5M FeCl3.6H2O and 250 ml distilled water) and incubated for 30 min at room temperature (25 oC). Development of pink colour indicates IAA production and its optical density was recorded at 530 nm. Concentration of IAA was estimated using standard curve prepared with 1-20 μg ml-1