Novel nitrogen-fixing bacteria isolated from the root nodules of the Pea plant

Rhizobia is a large group of bacteria developing symbiotic relation with their host plants by forming root nodules and fixing atmospheric nitrogen. The bacteria belonging to this group are important from agriculture and environmental perspective. Three strains of rhizobia were collected from Indian Trans-Himalaya region, isolated from root nodules of Pea plant. The characterization was done using 16S rRNA, atpD and recA genes. Based on 16S rRNA gene sequencing, isolated strains showed ≥99.9% sequence similarity to the members within the Rhizobium leguminosarum group. Phylogenetic analysis based on sequences of atpD, recA and 92 core genes indicated that two of the three strains were closely related to the group Rlaguerreae while one strain was closely related to the Rhidalgonense group. Two strains closely related to the group R. laguerreae have 60.8 mol% of DNA G+C content. Based on characterization studies, these two strains represent a novel species, Rhizobium indicum sp.nov. The study provided a detailed taxonomic species description of these novel species.

The nodC and nifH genes play an important role in nodulation and nitrogen fixation respectively. These genes were amplified for all three strains which confirmed their genetic potential for symbiosis.  All three strains have nodC gene sequence similar to the strains of Rhizobium leguminasarum that belong to the symbiovar viciae and isolated from different countries.

Genome studies revealed that the complete genome, chromosome and plasmids of these strains are comparable in size to the other strains of Rhizobium leguminasarum. Researchers also examined the genome sequences of the two strains to identify the compliment of nod genes in these strains. Both strains contained a cluster of 12 nod genes. Phylogenetic analysis revealed that both these strains are closely related to Rhizobium laguerreae followed by Rruizarguesonis and R. leguminosarum.

Morphological analysis revealed that the strains are rod-shaped, gram-negative and motile. Colonies were translucent, cream-white and convex. A total of 20 different cellular fatty acids in different quantities were detected in these three strains. Overall, the fatty acid compositions of the newly isolated strains were identical to the type strains of Rhizobium leguminosarumRhizobium laguerreae and Rhizobium ecuadorense, with differences in their proportions. The strain were positive for oxidase and catalase.

To conclude, both the novel strains are closely related to each other and can be differentiated from other species of the genus Rhizobium by their housekeeping gene sequences, ANI values, genome nucleotide analysis, and phenotypic and chemotaxonomic features.