A total of 128 bacterial test strains originated from Astragalus, Caragana, Gueldenstaedtia, Medicago, Melilotus, Oxytropis, Trifolium, and Vicia grown in Tibet were characterized phenotypically and genomically. Based upon the consensus of grouping results, they were identified as 16 putative species. Twenty-five test strains belonging to seven putative species of Agrobacterium, Bradyrhizobium, and Rhizobium might be nonsymbiotic bacteria and the remaining 103 test strains were symbiotic bacteria belonging to Mesorhizobium, Rhizobium, and Sinorhizobium meliloti. Although no novel taxon was detected in the symbiotic bacteria, several characters including the alkaliphilic psychrotolerance revealed that the Tibetan rhizobia could be ecotypes adapted to the local conditions. The results also demonstrated that frequent lateral transfer of symbiotic genes might have happened in the Tibetan rhizobia since nodC genes similar to that of S. meliloti were found in several Rhizobium test strains and all the Mesorhizobium species had very similar nodC genes despite their genomic background. All of these findings demonstrated that the Tibetan rhizobia were an important resource for further studies on rhizobial ecology and application.

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Rhizobium tibeticum sp. nov., a symbiotic bacterium isolated from Trigonella archiducisnicolai (Š irj.) Vassilcz. Isolated from root nodules of Trigonella archiducis-nicolai (Š irj.) Vassilcz. grown in Tibet, China, cells of the bacterial strains CCBAU 85039 T and CCBAU 85027 were Gram-negative, aerobic, motile, non-spore-forming rods that formed colonies that were semi-translucent and opalescent on yeast extract-mannitol agar. In numerical taxonomy, SDS-PAGE analysis of whole-cell proteins and DNA-DNA hybridization, the two strains were very similar and were different from reference strains of defined Rhizobium species. In the phylogeny based on 16S rRNA gene sequences, they were most similar to Rhizobium etli CFN 42 T (98.2 % similarity) and R. leguminosarum USDA 2370 T (97.6 %). Sequence analyses of the housekeeping genes recA, atpD and glnII and the 16S-23S rRNA intergenic spacer, phenotypic characteristics and cellular fatty acid profiles strongly suggested that these two strains represented a novel species within Rhizobium. Crossnodulation tests and sequencing of nifH and nodA genes showed that these two strains were symbiotic bacteria that nodulated Trigonella archiducis-nicolai, Medicago lupulina, Medicago sativa, Melilotus officinalis, Phaseolus vulgaris and Trigonella foenum-graecum. Based on the results, the novel species Rhizobium tibeticum sp. nov. is described to accommodate the two strains. The type strain is CCBAU 85039 T (5LMG 24453 T 5CGMCC 1.7071 T ). The DNA G+C content of this strain is 59.7 mol% (T m ).The genus Rhizobium was first described by Frank (1889) to accommodate all symbiotic nitrogen-fixing bacteria associated with legumes. After a series of revisions, some earlier described species have been moved into Bradyrhizobium (Jordan, 1982), Sinorhizobium (de Lajudie et al., 1994) and Mesorhizobium (Jarvis et al., 1997), while many novel species and genera of rhizobia within the Alphaproteobacteria and Betaproteobacteria have been reported (NZ Rhizobia, 2008). The current genus Rhizobium includes about 30 species of fast-growing, acid-producing rhizobia, which can be differentiated from Sinorhizobium species on the basis of 16S rRNA gene sequence-based phylogeny. With the exception of Rhizobium cellulosilyticus (Garcia-Fraile et al., 2007), most of them can induce root nodules on a certain range of legume species.In a survey of rhizobial resources in Tibet, 12 strains of root-nodule bacteria were isolated with yeast mannitol agar (YMA) medium according to the method of Vincent (1970) from root nodules of Trigonella archiducis-nicolai (Širj.) Vassilcz., an ephemeral legume growing in grasslands at altitudes of 3700-4000 m. Six of them were identified as Sinorhizobium meliloti, and six as Rhizobium strains (Hou et al., 2009). Among the Rhizobium strains, CCBAU 85039 T and CCBAU 85027 were classified as a small group by numerical taxonomy, amplified 16S rRNA gene restriction analysis, amplified 16S-23S intergenic spacer restriction analysis and 16S rRNA gene phylogeny (Hou et al., 2009). To...

Four rhizobial strains, designated CCBAU 85046 T , CCBAU 85051, CCBAU 85048 and CCBAU 85049, isolated from root nodules of Oxytropis glabra grown in Tibet, China, were previously defined, using amplified 16S rRNA gene restriction analysis, as a novel group within the genus Rhizobium. To clarify their taxonomic position, these strains were further analysed and compared with reference strains of related bacteria using a polyphasic approach. The 16S rRNA gene analysis showed that the four isolates formed a distinct phylogenetic lineage in the genus Rhizobium. The isolates showed highest sequence similarity (97.8 %) to Rhizobium indigoferae CCBAU 71042 T . Phenotypic and physiological tests, DNA-DNA hybridization, phylogenetic analyses of housekeeping genes recA, atpD and glnII and fatty acid profiles also indicated that these four strains constitute a novel group distinct from recognized species of the genus Rhizobium. , CCBAU 85048, CCBAU 85049 and CCBAU 85051 as a novel group belonging to the genus Rhizobium based on restriction fragment length polymorphism analysis of amplified 16S rRNA genes. To clarify the taxonomic status of this group, in the present study the four strains were further characterized and compared with reference strains by using a polyphasic approach.The four test strains and reference strains for species of the genus Rhizobium were obtained from the corresponding culture collection centres and maintained on YMA medium (Vincent, 1970) at 4 u C for temporary storage. Genomic DNA was extracted from the strains according to the protocol of Terefework et al. (2001) and was used as a template in the amplification of different genes or specific Abbreviation: IGS, intergenic spacer.The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA, 16S-23S rRNA intergenic spacer, atpD, recA and glnII gene sequences of strain CCBAU 85046 T are EU256434, EU288745, EU288670, EU288696 and DQ985380, respectively. The GenBank/ EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains CCBAU 85051, CCBAU 85048 and CCBAU 85049 are GU938296, GU938297 and GU938298, respectively. DNA fragments. The 16S rRNA gene of each of the four strains was amplified using primers fD1 and rD1 as described previously (Weisburg et al., 1991) and sequenced directly (van Berkum et al., 1996). The 16S rRNA gene sequences of type strains of species of the genus Rhizobium were obtained from the GenBank database. All sequences were aligned using the CLUSTAL W program in MEGA 4.0 software (Tamura et al., 2007). Phylogenetic distances were calculated according to the model of Jukes & Cantor (1969). Phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximumlikelihood (Felsenstein, 1985) methods with bootstrap values based on 1000 replicates. The topology of the maximum-likelihood tree (not shown) was similar to that of the neighbour-joining tree (Fig. 1). In the phylogenetic tree ( Fig. 1 T , which showed sequence similarities ranging between 97.6 and 96.9 %.Since the 16S-23S intergeni...

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