Dixit et al.

amended with 5 mM/L tryptophan for 24 h (Bric et al ., 1991). After centrifugation

at 10,000 rpm for 15 min, supernatant was collected separately. 100 µl of 10 mM

o -phosphoric acid and 4 ml of Salkowaski’s reagent (1ml of 0.5 mM FeCl ₃ in 35%

of HClO ₄ ) were added to 2ml of supernatant of each strain and incubated at room

temprature for 25 min for developing the pink color. Absorbance was measured at

535 nm using UV-VIS spectrophotometer (Labtronics-model LT 2800, India). Un-

inoculated broth supernatant with Salkowaski’s reagent was taken as control.

HcN production: HCN (hydrogen cyanide) production was determined by

modified method of Bakker and Shippers (1987). Log phase culture was streaked

on to agar plate of respective media amended with 4.4g/l glycine with simultaneous

addition of filter paper soaked in 0.5% picric acid in 1% Na ₂ CO ₃ in the lid of plates.

The control plates were un-inoculated. The plates were sealed with parafilm and

incubated at 28±2°C for 72 h; development of brown color was examined for

HCN production.

Siderophore production: Siderophore production was examined on Chrome-

azurol S (CAS) medium following the method of Schwyn and Neiland (1987). Log

phase cultures of isolated root nodule endophytic bacteria were spot inoculated

onto CAS agar plates and incubated at 28±2°C for 72 h formation of orange to

yellow halo around the colonies showed the production of siderophore.

ammonia production: Ammonia production was examined on peptone broth

following the method of Lannette et al ., 1985. Log phase cultures of isolated

endophytic bacteria were inoculated in peptone broth incubated at 28±2°C for 72

h. Yellow to brown precipitation were observed after adding of Nessler’s reagent.

antagonistic activity: Antagonistic properties of isolates were tested against

Fusarium oxysporum on potato dextrose agar (250g peeled potato, 20g glucose,

15g agar, 1000ml distilled water, pH 6.5) plates using the dual culture technique

(Skidmore and Dickinson, 1976). A loopfull log phase culture (24h old) of each

bacterial strain was separately spot inoculated apart from 5mm agar disc containing

mycelial growth of the fungal pathogen. Plates were incubated at 28±2°C for

3-7 days. The zone of inhibition (%) was recorded by using the formula: 100 ×

( C-T )/ C , where,

radial growth in control;

radial growth in dual

culture.

Results and Discussion

Plant samples collected from different field from various seven provinces had

different soil pH varying from 6.8 to 7.8 (Table 1). Growth of lentil at various soil

pH has also been reported by Jaiswal et al ., 2011.

100

Int. J. Bioinformatics and Biological Sci.: v.2 n.1&2, p. 95-109. March & June 2014