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Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

During a study of endophytic nitrogen-fixing bacteria present in the wild rice species Oryza alta, eight novel isolates were obtained from surface-sterilized roots and classified in the genus Rhizobium on the basis of almost-complete 16S rRNA gene sequence analysis. These strains can nodulate Phaseolus vulgaris and Glycine max. The highly similar protein patterns, DNA fingerprint patterns of insertion sequence-based PCR (IS-PCR) and DNA-DNA hybridizations showed that these novel isolates were members of the same species. The closest phylogenetic relatives of the representative strain Alt 505(T) of the novel group were Rhizobium etli CFN 42(T) and Rhizobium indigoferae CCBAU 71714(T), with 96.2 and 96.0% 16S rRNA gene sequence similarity, respectively. Low DNA-DNA relatedness with the type strains of R. etli, R. indigoferae, Rhizobium hainanense, Rhizobium mongolense and Rhizobium galegae and differences in IS-PCR fingerprinting patterns, SDS-PAGE of proteins, antibiotic resistance, phenotypic tests and comparison of cellular fatty acids with Rhizobium species indicated that the novel group of isolates were distinct from previously described species. Based on these results, we propose to place them in a novel species, as Rhizobium oryzae sp. nov. The type strain is Alt 505(T) (=LMG 24253(T) =CGMCC 1.7048(T)).

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three ‘core’ chromosomal genes ( rrs , glnII and atpD ) and two ‘auxiliary’ symbiotic genes ( nifH and nodB ) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R . gallicum bv. orientale . The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.

Comparative analysis of 70 16S rDNA sequences representing 20 Rhizobium species (including pathogenic Agrobacterium spp.) was conducted using Maximum Likelihood to establish relationships of species using multiple sequences. There is no significant internal division of the Rhizobium clade to suggest that it represents more than one genus. Plant pathogenic (Agrobacterium) species are distributed within the genus. The analysis supported the synonymy of some species (Rhizobium gallicum and Rhizobium mongolense) and the need for comparative investigations of the tumorigenic and nodulating properties of Rhizobium tropici and Rhizobium rhizogenes. Misidentification of some sequences may conceal one or more putative novel species. Some sequences appear to be misidentified because of faulty sequencing or incomplete or inadequate analysis.

Three microbial strains isolated from common beans, 23C2T (Tunisia), Gr42 (Spain) and IE4868 (Mexico), which have been identified previously as representing a genomic group closely related to Rhizobium gallicum , are further studied here. Their 16S rRNA genes showed 98.5–99 % similarity with Rhizobium loessense CCBAU 7190BT, R. gallicum R602spT, Rhizobium mongolense USDA 1844T and Rhizobium yanglingense CCBAU 71623T. Phylogenetic analysis based on recA, atpD, dnaK and thrC sequences showed that the novel strains were closely related and could be distinguished from the four type strains of the closely related species. Strains 23C2T, Gr42 and IE4868 could be also differentiated from their closest phylogenetic neighbours by their phenotypic and physiological properties and their fatty acid contents. All three strains harboured symbiotic genes specific to biovar gallicum. Levels of DNA–DNA relatedness between strain 23C2T and the type strains of R. loessense , R. mongolense , R. gallicum and R. yanglingense ranged from 58.1 to 61.5 %. The DNA G+C content of the genomic DNA of strain 23C2T was 59.52 %. On the basis of these data, strains 23C2T, Gr42 and IE4868 were considered to represent a novel species of the genus Rhizobium for which the name Rhizobium azibense is proposed. Strain 23C2T ( = CCBAU 101087T = HAMBI3541T) was designated as the type strain.

Medicago ruthenica [(L.) Ledebour] is native to inner Mongolia where rhizosphere samples were collected for the isolation of 106 rhizobial cultures. 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 ET, 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-fixing 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.

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfanodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation., Instituto de Biotecnologia y Biologia Molecular

To screen high strength phenol degrading bacteria, we selected 108 rhizobial strains isolated from nodules of eight wild legumes species in the mining tailing region of Shaanxi province, northwest of China, and cultivated them in a basal salt (BS) medium supplemented with different phenol concentrations as a sole carbon source. The results showed that some of the strains could use phenol as sole carbon source. In order to study the characteristics of phenol degradation, the strain CCNWTB701 isolated from Astragalus chrysopteru was used as well, due to the fact that it was very efficient in phenol degradation. The phenol degradation was around 99.5 and 78.3%, with an initial concentration of 900 and 1000 mg/l phenol in 62 and 66 h, respectively. Kinetic studies indicated that the strain had a high KS (743.1 microM) and an extremely high KSI (10,469 microM) in Haldane's model. The phylogenetic analysis based on 16S rRNA gene sequences showed that CCNWTB701 belonged to the Rhizobium genus, and it was closely related to Rhizobium mongolense and Rhizobium gallicum.

Although phosphate solubilization is a character known to be present in species of Mesorhizobium, this property has not been described before in species of Sinorhizobium. The type strains of the three species that nodulate Medicago species, Sinorhizobium meliloti, S. medicae and Rhizobium mongolense, do not solubilize phosphate from bicalcium phosphate in plate culture. We observed phosphate solubilization among isolates we obtained from nodules of Medicago sativa, Melilotus and Trigonella growing in a Spanish soil. Phenotypic and genetic analyses of these isolates led to the conclusion that they were placed within the genus Sinorhizobium with characteristics in common with S. meliloti and S. medicae. The group of strains solubilizing phosphate is distinguishable to strains from S. meliloti and S. medicae basing on LMW RNA profiles, TP-RAPD patterns and SDS-PAGE profiles.