Deciphering Soil Diazotrophic Diversity in the Wheat-Maize Cropping System of Punjab Using Morphological, Biochemical and Molecular Techniques

Soil is the critical resource as well as a basic medium for the growth of natural micro flora present in it. The soil fertility depends on its physicochemical properties and microbial population diversity. Diazotrophs are the nitrogen fixing bacteria which possess nifH gene that is responsible for coding the nitrogenase enzyme involved in the reduction of atmospheric nitrogen to ammonia. These nitrogen fixing bacteria play an imperative role, function and significance in the soil. Soil samples were collected from the different wheat-maize cropping system of Punjab and analyzed for physicochemical properties as pH, electrical conductivity, organic carbon, soil texture, ammoniacal as well as nitrate nitrogen. Eighty diazotrophic bacteria were isolated on eight different nitrogen free media and characterized culturally, morphologically, biochemically, functionally and using molecular techniques. The diazotrophic nature of the isolates was confirmed by the amplification of nifH gene using two nifH primers viz. nifH1 and nifH2. The nifH positive isolates were further used for 16S rDNA restriction analysis using different enzymes such as Taq1, Rsa I and Hae III. Based on UPGMA clustering, the representative strains were sequenced and identified as Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus subtilis, Paenibacillus sp., Azotobacter vinelendii, Stenotrophomonas maltophilia, Rhizobium larrymorrei, Flavobacterium anhuiense, Sphingomonas paucimobilis, Paenibacillus panacisoli, Azospirillum sp., Pseudomonas putida, Paenibacillus amyloliticus, Bacillus circulans, Paenibacillus polymyxa and Xanthomonas oryzae.