Molecular Characterization of Rhizobia from Revertants of Non-nodulating Cultivar and Normal Cultivar of Chickpea

The normal nodulating chickpea cultivar (HC5) and revertant of non-nodulating cultivar ICC 4993 NN designated as ICC 4993 (R) were used to study and characterize the rhizobia infecting both cultivars. On the basis of growth characteristic and nodulation, 43 rhizobial isolates from revertant of non-nodulating cultivar and 8 rhizobial isolates from normal nodulating cultivar were selected. Heterogeneity of the rhizobia infecting both of the cultivars was estimated by ERIC (Enterobacterial Repetitive Intergenic Consensus) as well as RFLP (Restriction Fragment Length Polymorphism) analysis of 16S rDNA sequence. Based on the presence of different ERIC profiles, rhizobial isolates from cv. ICC4993 (R) formed eight different clusters and those from cv. HC5 formed three clusters at 80% similarity. A combined dendrogram of all the mesorhizobial isolates from the two cultivars showed two clusters at 70% similarity and eight subclusters at 80% similarity level. Similarly, RFLP patterns showed that rhizobial isolates from cv. ICC4993 (R) formed eleven clusters while those from cv. HC5 formed two clusters at 80% similarity. A combined dendrogram of mesorhizobial isolates from the two cultivars formed twelve clusters at 80% level of similarity. Using both methodologies, heterogeneity (if any) of mesorhizobia nodulating cvs. ICC4993 (R) and HC5 could not be ascertained. Further sequencing of partially amplified 16S rDNA of three rhizobial isolate from cv. ICC4993 (R) and one from cv. HC5 showed more than 98% similarity with Mesorhizobium muleiense and Mesorhizobium mediterraneum. The phylogenetic analysis of 16S rRNA partial sequence revealed 11 monophyletic clades. The isolates NN78 and HC 1065 were clustered along with Mesorhizobium mediterraneum strain PECA20 while NNs13 and NN90 formed a separate cluster.