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The genetic diversity within 195 rhizobial strains isolated from root nodules of 18 agroforestry species (15 woody and three herbaceous legumes) growing in diverse ecoclimatic zones in southern Ethiopia was investigated by using PCR-RFLP of the ribosomal operon [16S rRNA gene, 23S rRNA gene and the internal transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes] and 16S rRNA gene partial sequence (800 and 1350 bp) analyses. All of the isolates and the 28 reference strains could be differentiated by using these methods. The size of the ITS varied among test strains (500-1300 bp), and 58 strains contained double copies. UPGMA dendrograms generated from cluster analyses of the 16S and 23S rRNA gene PCR-RFLP data were in good agreement, and the combined distance matrices delineated 87 genotypes, indicating considerable genetic diversity among the isolates. Furthermore, partial sequence analysis of 67 representative strains revealed 46 16S rRNA gene sequence types, among which 12 were 100% similar to those of previously described species and 34 were novel sequences with 94-99% similarity to those of recognized species. The phylogenetic analyses suggested that strains indigenous to Ethiopia belonged to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium and Sinorhizobium. Many of the rhizobia isolated from previously uninvestigated indigenous woody legumes had novel 16S rRNA gene sequences and were phylogenetically diverse. This study clearly shows that the characterization of symbionts of unexplored legumes growing in previously unexplored biogeographical areas will reveal additional diversity.

Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia