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

The normal nodulating chickpea cultivar (HC۵) and revertant of non-nodulating cultivar ICC ۴۹۹۳ NN designated as ICC ۴۹۹۳ (R) were used to study and characterize the rhizobia infecting both cultivars. On the basis of growth characteristic and nodulation, ۴۳ rhizobial isolates from revertant of non-nodulating cultivar and ۸ 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 ۱۶S rDNA sequence. Based on the presence of different ERIC profiles, rhizobial isolates from cv. ICC۴۹۹۳ (R) formed eight different clusters and those from cv. HC۵ formed three clusters at ۸۰% similarity. A combined dendrogram of all the mesorhizobial isolates from the two cultivars showed two clusters at ۷۰% similarity and eight subclusters at ۸۰% similarity level. Similarly, RFLP patterns showed that rhizobial isolates from cv. ICC۴۹۹۳ (R) formed eleven clusters while those from cv. HC۵ formed two clusters at ۸۰% similarity. A combined dendrogram of mesorhizobial isolates from the two cultivars formed twelve clusters at ۸۰% level of similarity. Using both methodologies, heterogeneity (if any) of mesorhizobia nodulating cvs. ICC۴۹۹۳ (R) and HC۵ could not be ascertained. Further sequencing of partially amplified ۱۶S rDNA of three rhizobial isolate from cv. ICC۴۹۹۳ (R) and one from cv. HC۵ showed more than ۹۸% similarity with Mesorhizobium muleiense and Mesorhizobium mediterraneum. The phylogenetic analysis of ۱۶S rRNA partial sequence revealed ۱۱ monophyletic clades. The isolates NN۷۸ and HC ۱۰۶۵ were clustered along with Mesorhizobium mediterraneum strain PECA۲۰ while NNs۱۳ and NN۹۰ formed a separate cluster.

Keywords:

Diversity , ERIC , Nodulation , Mesorhizobium , Phylogenetic analysis , RFLP