Characterisation of plant growth

The fields have variation in soil properties as well as different cultivar variety of

Lens culinaris. Plants were gently uprooted along with rhizospheric soil, kept in

sterile sampling bags and brought immediately to the laboratory for further study.

Isolation of endophytic bacteria from root nodules of Lens culinaris

Endophytic bacteria were isolated from pink colored healthy root nodules of Lens

culinaris . The roots were washed with tap water and healthy nodules were picked

from the roots. Then the nodules were washed thoroughly with sterile distilled

water, surface sterilized in 95% alcohol for 20–30 seconds and subsequently

immerged in 3% sodium hypochlorite solution for 5 minutes. Subsequently

nodules were washed 7–8 times with sterile distilled water to remove the traces

of sodium hypochlorite. Standard microbiological technique was followed for

isolation of root nodulating endophytic bacteria (Dubey and Maheshwari, 2012).

The surface-sterilized nodules were transferred to a sterile culture tubes containing

100μl sterile distilled water. Nodules were crushed with the help of sterile glass

rod. A milky bacterial suspension was obtained that was streaked on the YEMA,

CrYEMA, Pseudomonas Agar medium, and Bacillus Agar medium (Himedia™)

(Vincent, 1970). The plates were incubated at 28±2°C for 72 h and observed for

the specific features of Rhizobium spp., Pseudomonas spp. and Bacillus spp.

The cultures were identified by conducting the tests as described in Bergey’s

Mannual of Determinative Bacteriology (Holt et al ., 1994) and compared with

the standard culture of Rhizobium leguminosarum MTCC99, Sinorhizobium

meliloti MTCC100, Mesorhizobium loti MTCC2378, Pseudomonas fluorescens

MTCC103, Pseudomonas sp. MTCC129, Pseudomonas aeruginosa MTCC1934,

Bacillus sp. MTCC297, Bacillus subtilis MTCC441, Paenibacillus polymixa

MTCC122, procured from Microbial Type Culture Collection Center (MTCC),

Institute of Microbiological Techniques (IMTECH), Chandigarh, India.

characterization of bacterial isolates

Phenotypic Characterization

Differential staining: Gram’s staining of the log phase culture was done to provide

information on presumptive test of isolates following Dubey and Maheshwari

(2012).

colony morphology: Colony colour, form, elevation and margin of bacterial

isolates streaked on YEMA, crYEMA, Pseudomonas Agar medium, and Bacillus

Agar medium (Himedia™) were recorded after their growth (Dubey and

Maheshwari, 2012).

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

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