Root nodule bacteria from Clitoria ternatea L. are putative invasive nonrhizobial endophytes

In this study, bacteria (8 species and 5 genera) belonging to the classes Betaproteobacteria, Gammaproteobacteria, and Sphingobacteria were isolated from root nodules of the multipurpose legume Clitoria ternatea L. and identified on the basis of partial 16S rRNA sequencing. The root nodule bacteria were subjected to phenotypic clustering and diversity studies using biochemical kits, including Hi-Media Carbokit™, Enterobacteriaceae™ identification kit, ERIC-PCR, and 16S ARDRA. All the strains showed growth on Ashby's N-free media over 7 generations, indicative of presumptive nitrogen fixation and further confirmed by amplification of the nifH gene. None of the strains showed the capability to renodulate the host plant, neither alone nor in combination with standard rhizobial strains, which was further confirmed by the absence of nodC bands in PCR assay. The results clearly indicate the common existence of nonrhizobial microflora inside the root nodules of legumes, which were thought to be colonized only by rhizobia and were responsible for N2 fixation in leguminous crops. However, with the recent discovery of nodule endophytes from a variety of legumes, as also observed here, it can be assumed that symbiotic rhizobia are not all alone and that these invasive endophytes belonging to various bacterial genera are more than just opportunistic colonizers of specialized nodule niche.