Characterization and in planta nitrogen fixation of plant growth promoting endophytic diazotrophic Lysinibacillus sphaericus isolated from rice (Oryza sativa)

This study was conducted to isolate and characterize endophytic diazotrophic isolates with multiple plant growth promoting attributes including biocontrol potential from surface sterilized leaf, stem and root samples of various rice cultivars of Kerala. A total of 18 endophytic diazotrophs were isolated using Dobereiner's semisolid N free bromothymol blue media. All the isolates were found to be positive for in vitro acetylene reduction assay (ARA) while only leaf isolate L1 was able to fix nitrogen in planta. Among the 18 nitrogen fixing endophytes isolated, 3 showed in vitro antifungal activities against Rhizoctonia solani, the rice sheath blight pathogen. The in vivo detached leaf assay confirmed the results. All the selected three isolates were proved to produce important plant hormones Indole 3 Acetic acid (IAA), Gibberllic acid (GA) and Cytokinins (Kinetin). The selected isolates augmented the seed germination and seedling vigor. Morphological, physiological, biochemical characteristics and 16SrDNA sequence analysis identified the isolates as Lysinibacillus sphaericus (L1), Klebsiella pneumoniae (S2) and Bacillus cereus (R2). The nif H gene was also amplified to confirm diazotrphy. The isolate L1, the superior endophytic diazotrophic isolate and S2 were also positive for the production of ACC deaminase. This is the first report of Lysinibacillus sphaericus as a nitrogen fixing and plant growth promoting endophyte with biocontrol activity.