Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-f ixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplif ied 165 rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 165 rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16s rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 165 rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 165 rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow-to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve a t the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 165 rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16s rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SOZT, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 165 rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huaut/ense is significant because in the reconstruction of the phylogeny of R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum .

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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 ...

Besides nodulating the original trap host, the isolates formed nitrogen-fixing symbioses with Phaseolus vulgaris. Only half of the isolates nodulated alfalfa (Medicago sativa), but these did not form nitrogen-fixing symbioses. Rhizobium tropici also formed nitrogen-fixing symbioses with Medicago ruthenica. A total of 56 distinctive multilocus electrophoretic types (ETs) were identified among 94 of the 106 isolates which were analysed for variation in electrophoretic mobility of 12 enzyme loci. One isolate (USDA 1920) possessed a unique El, while the ETs of the other isolates formed two weakly divergent subgroups approximately equal in size. It was concluded from small subunit rRNA gene sequences of eight isolates of Medicago ruthenica that they belonged to the genus Rhizobium and not to the genus Sinorhizobium which is more commonly associated with Medicago. Genomic similarity, determined from DNA hybridization analysis, between USDA 1920 and the strain representing the remaining isolates (USDA 1844) was lower than 20%. Based upon these observations it was concluded that at least three genomic species of rhizobia form nitrogen-f ixing symbioses with Medicago ruthenica. One of these genomic species is R. tropici, another is represented by the single isolate USDA 1920 and the name Rhizobium mongolense is proposed for the third genomic species represented by USDA 1844.

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Our study showed widespread occurrence of cystic echinococcosis in cattle, which may have a role in the lifecycle of this serious zoonosis.

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