Phylogenetic Relationships among Rhizobium Species Nodulating the Common Bean (Phaseolus vulgaris L.)

Abstract

The phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16s ribosomal DNA genes of these organisms. The bean strains formed four separate clusters. One cluster was composed of Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and R. leguminosarum bv. phaseoli. Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain. Data for species identification were obtained from DNA-DNA reassociation experiments. The levels of DNA relatedness among strains belonging to the three biovars of R. leguminosarum ranged from 58 to 67%. The levels of DNA relatedness between R. leguminosarum bv. phaseoli and R. etli and R. tropici ranged from 43 to 45% and 13 to 16%, respectively. The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.Phaseolus vulgaris L. (common bean) is an agriculturally important legume crop which benefits from a symbiosis with bacteria belonging to the genus Rhizobium. The rhizobia that infect host legumes, such as peas, clovers, and common beans, have been placed in a single species, Rhizobium leguminosarum. This species has been subdivided into three biovars largely on the basis of specificity for host plant infection and nodulation (12). Jordan (12) noted that of the three biovars of R. leguminosarum, R. leguminosarum bv. phaseoli was more distinct than the other two. The reasons for this became apparent when the heterogeneity of strains classified as members of R. leguminosarum bv. phaseoli was described (3). Bean isolates obtained from English fields are only weakly polymorphic (26). In contrast, wide phenotypic and genotypic variations have been reported among strains originating in the Americas (16, 17, 19, 20).Bean strains that originated in Mexico and South America were identified as members of a heterogeneous complex of strongly differentiated phylogenetic lineages, and the data indicated that several species should be recognized (19). One of the deep lineages was subsequently classified as Rhizobium tropici (17). On the basis of 16s ribosomal DNA (rDNA) gene sequences, Willems and Collins (25) showed that the phylogenetic position of R. tropici was distinct from that of R. leguminosarum. However, their analysis was with reference to a clover strain, strain ATCC 14480, because the 16s rDNA sequences of R. leguminosarum bv. phaseoli had not been determined and were not available.Until recently, all bean strains other than R. tropici were classified as R. leguminosai-um. However, there are a number of differences between the nucleotide sequences of a partial analysis of the 16s rDNA genes of bean strains of American origin and the sequence of R. leguminosarum bv. viciae type strain ATCC 10004. This finding led to the suggestion that the bean strains of American origin should be referred to as Rhizobium ...

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