Isolation and Characterization of Putative Nitrogen-Fixing Endophytes from Three Distinct Wild Rice Species in Sri Lanka

Abstract

There are 22 recorded wild rice species on earth, of which 5 are found in Sri Lanka. Knowledge on the endophytic diazotrophs of wild rice species, which can be adopted for paddy cultivation, is still at a fledgling stage of discovery. The identification of stably maintained endophytic diazotrophic bacteria in the genus Oryza requires further attention, especially due to skyrocketing global prices of chemical fertilizers. The main objective of our study was the isolation and characterization of endophytic and rootsurface colonizing nitrogen fixers from three distinct wild rice species found in Sri Lanka, namely Oryza eichingeri, Oryza granulata and Oryza nivara. Endophytic nitrogen fixers were isolated from surface– sterilized stems, leaves, and roots, while root surface colonizing bacteria were isolated from washed roots.

There were eleven endophytic bacteria and four root surface colonizing bacteria isolated by using nitrogenfree yeast mannitol/malate agar plates; six isolates from O. granulata, five isolates from O. eichingeri and four isolates from O. nivara. Out of the 15 isolates, 13 were Gram negative and 2 stained Gram positive. Six of the eleven isolates secreted three enzymes tested, primarily pectinases, proteases, and cellulases, suggesting their prospective involvement in endophytic lifestyles. Most isolates showed either bulls eyes, dendritic or featureless patterns of swarming chemotaxis for the chemoattractant proline, a common promoter of bacterial motility. We also tested the genomes of the isolates for the presence of the nifH gene. DNA from seven isolates gave a PCR amplicon of the expected size (360 bp) using universal nifH primers, which proves that the genetic foundation for the production of nitrogenase reductase subunit was found in these seven isolates. We also tested each of the bacterial isolates against a devastating fungal pathogen of rice (Rhizoctonia solani). We found 4 potential candidates that show partial inhibitory activity against the growth of this rice sheath blight causative agent.