Molecular characterization of diazotrophic bacteria isolated from rhizosphere of wheat cropping system from central plain region of Punjab

Soil is a hot spot for microbial diversity, however, the excessive use of agrochemicals have reduced natural microflora of soil. Soil samples were collected from central plain region of Punjab and geo-referenced. Physicochemical properties of the soil samples ranged from 5.9-8.7 (pH), 0.13-0.51 dSm-1 electrical conductivity (EC), 0.26 - 0.77% organic carbon (OC), 14 -119 ppm (ammonical N) and 28 -119 ppm (nitrate N). Variable diazotrophic population was obtained on eight different nitrogen free media. Diazotrophic count was found to be positively affected by OC; whereas, it was negatively affected by pH, EC, ammonical and nitrate nitrogen. A total of 169 diazotrophs were isolated and characterized using cultural, morphological and biochemical techniques and tentatively identified as diverse genera of Pseudomonas sp., Bacillus sp., Azotobacter sp., Rhizobium sp., Azospirillum sp., Beijerinckia sp. and Derxia sp. Using molecular techniques, sixty seven isolates were found to be positive for amplification of nif H. Based on unweighted pair group method with arithmetic mean (UPGMA) clustering, dendrogram was obtained and the representative cultures were identified as Xanthomonas sp., Beijerinckia indica, Flavobacterium johnsoniae, Pseudoxanthomonas suwonensis, Lysinibacillus sphaericus, Stenotrophomonas maltophilia and Pseudomonas aeruginosa.   Key words: Biochemical characterization, diazotrophic count, nitrogen fixing bacteria, physicochemical properties, nif H, 16S rDNA.