Isolation, enumeration, and characterization of diazotrophic bacteria from paddy soil sample under long-term fertilizer management experiment

A study was undertaken to determine the free-living culturable diazotrophic bacteria of paddy soils from a long-term fertilizer management experiment. Long-term application of different fertilizers significantly affected the population of free-living diazotrophs. Out of 165 distinct bacterial morphotypes observed during the isolation process, only 32 were positive for both acetylene reduction assay (ARA), and nifH gene screening. The ARA activity of the isolates ranged from 1.8 to 2,844.7 nmol ethylene h -1 mg protein -1. The 16S rRNA analysis identified the isolates to be members of 13 different genera viz. Bacillus, Pseudomonas, Paenibacillus, Serratia, Ochrobactrum, Lysinibacillus, Burkholderia, Brevundimonas, Herbaspirillum, Novosphingobium, Sphingomonas, Xanthomonas, and Azorhizobium. Though partial nifH gene sequencing of diazotrophic isolates showed good consistency with that of 16S rRNA-based identification, some nifH sequences were similar to a variety of uncultured nitrogen-fixing bacteria. The diversity of free-living diazotrophic bacteria and the wide distribution of nifH sequences indicate the potential contribution of these microorganisms to nitrogen input to paddy fields.