Isolation and molecular characterisation of diazotrophic growth-promoting bacteria from wheat rhizospheric soils of Punjab

Abstract

Diazotrophs are nitrogen-fixing bacteria which possess the nifH gene that codes for the nitrogenase enzyme involved in reduction of atmospheric dinitrogen to ammonia. Seventy-two diazotrophic bacteria were isolated using eight nitrogen-free media from wheat rhizospheric soil. The diazotrophic population was found to be negatively related to soil nitrogen, whereas a positive correlation was observed with organic carbon and electrical conductivity of soil. The isolates were initially identified on the basis of cultural, morphological, and biochemical characterisation. Various diazotrophic isolates were screened for functional activities. Thirty-seven isolates were acetylene reduction assay positive, among which 28 isolates exhibited nitrogenase activity ranging from 22.3 to 72.0 nmol C2H4/h. The majority of isolates were able to produce indole acetic acid ranging from 11.2 to 23.0 µg/mL and only a few diazotrophs could solubilise phosphate. These isolates showed amplification with two nifH primers (nifH1 and nifH2), thereby confirming their diazotrophic potential. The positive nifH isolates were further characterised using restriction fragment length polymorphism of 16S rDNA to reveal diversity among them. Based on UPGMA clustering and partial sequencing of 16S rDNA, the isolates were identified as Azotobacter sp., Azospirillum sp., Stenotrophomonas maltophilia, Stenotrophomonas sp., Sphingomonas paucimobilis, Rhizobium larrymoorei, Pseudomonas aeruginosa, and Xanthomonas oryzae.