Rhizobial and non-Rhizobial nodulators of Pueraria phaseoloides

Abstract

Pueraria phaseoloides is a widely grown legume cover crop in Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop. We endeavored to isolate the whole array of diazotrophs colonizing the root nodules of this economically-significant cover crop. Four isolates were isolated by streaking a macerate of an active nodule from Pueraria phaseoloides and the identification of Rhizobial and Non-Rhizobial species was carried out using colony and cell morphology. One isolate from Pueraria phaseoloides (Sub 1), a gram negative bacterium contoured by a coccobacillus cell shape, (suggesting a likely non-Rhizobial identity), was a potent nodulator of Pueraria phaseoloides seedlings. A further three cultures (Sub 2, Sub 3 and Sub 4) too were able to nodulate Pueraria phaseoloides seedlings but were not as effective as Sub 1 in their nodulation potency. All four bacteria secreted to the extra-cellular medium cellulases suggesting their likely involvement in nodule formation and also showed characteristic patterns of motility to the chemoattractant proline. DNA extracted from Sub 1, Sub 3 and Sub 4 gave a PCR amplicon of the anticipated size (360 bp) using universal nifH primers, which indicated that the genetic foundation for the production of a unit of the nitrogenase enzyme, was found in the genome of these isolates. In summary, we have unearthed here, a strong, likely non-Rhizobial nodulator, in the legume cover crop Pueraria phaseoloides, and three other bacilli bacteria, perhaps Rhizobia, capable of efficient nodulation. Further characterization of these isolates using molecular biology tools is ongoing.