A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.)

Objective: Maize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield. Methodology: Bacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction. Results: Culturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere. Conclusion: The study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere. Keywords: Azospirillum, Diazotrophs, 16S rRNA, nifH, Metagenomics