Diversity of Bradyrhizobium Strains Isolated from Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of 16S rDNA and Low-Molecular-Weight Rna Profiles

Abstract

The Bradyrhizobium species currently admitted are three (B. japonicum, B. elkanii and B. liaoningense), but this number is expected to increase with the analysis of more strains isolated from non-previously studied legumes. In the Canary Islands a large number of endemic shrub legumes of great agronomic interest grow. Tagasaste (Chamaecytisus proliferus) has specially been used for centuries as fodder on the Canaries and other parts of the world. In previous works with some rhizobia isolates from tagasaste and other endemic woody legumes, it was shown that these legumes were nodulated by a group of Bradyrhizobium strains phenotypically and genetically diverse (Leon-Barrios et al., 1991; Santamaria et al., 1997; Vinuesa et al., 1998). In this study, thirty-six strains of slow-growing rhizobia isolated from endemic woody legumes were characterized by analysis of PCR-amplified and restricted 16S rDNA, and by LMW RNA profiles. The isolates, incapable of nodulating soybean, were compared with reference strains of genus Bradyrhizobium. Both techniques showed similar results, clustering the isolates in two main groups. The PCR-amplified 16S rDNA was individually restricted with four endonucleases, and the combined RFLP patterns used for cluster analysis by UPGMA. This analysis showed a larger group (29 strains) containing only Canarian isolates (BTA-1 group), and two strains of Lupinus. One smaller group of nine strains (BGA-1 group) clustered with B. japonicum strains. None of the isolates grouped with the strains of B. elkanii. RNA profiles in the 5S rRNA zone showed a pattern identical to B. japonicum for both groups. However, tRNA profiles of both groups were different from that of B. japonicum, although BGA-1 group profiles are more similar to this species. According to previous results (Velazquez et al., 1998), the different species present different tRNA profiles. Thus, the results obtained from both techniques suggest that some Canarian isolates appear to be new species of genus Bradyrhizobium. This idea is also supported by partial sequencing of 16S rRNA gene (Vinuesa et al., 1998). However, new data will be needed to confirm it, especially total DNA hydridization. The finding of two strains of Bradyrhizobium sp. (Lupinus) clustering at a similar level of 100% with the BTA-1 group by ARDRA analysis was a surprise. It had been found (Vinuesa, 1998) that RFLP analysis of rDNA operon could separate a group of ten Canarian isolates among a vast collection of bradyrhizobia, isolates from different parts of the world, giving strong evidence that they could represent a local population of bradyrhizobia confined to the Canary Islands. However, the fact of two strains of Lupinus collected in geographically unrelated locations (Sevilla and Madrid, Spain-mainland), that can nodulate tagasaste and clustering in the same ARDRA group as the “Canarian group”, means that more investigation should be carried out to clear up this matter. Data reflecting total genome variation will be very interesting.