Thirty-one rhizobial strains isolated from nodules of legumes native of Xinjiang, China, were characterized. These strains were classified as belonging to the genus based on amplified 16S rDNA restriction analysis (ARDRA). The strains were distinguished from recognized species using analysis of 16S–23S rDNA intergenic spacers (IGS-RFLP), SDS-PAGE analysis of whole proteins and BOX-PCR; the test strains always formed a distinct cluster with patterns that were quite different from those of the reference rhizobial strains used. According to the phylogenetic analysis based on the 16S rRNA gene, the test strains belonged to the genus , with , and as the closest related species, with 99.6, 99.2 and 99.4 % sequence similarities, respectively, between the type strains of the three species and strain CCBAU 83401. Phylogenetic analyses of the representative strains using IGS and , and genes all confirmed the phylogenetic arrangements obtained using the 16S rRNA gene. The DNA–DNA relatedness values between strain CCBAU 83401 and strains CCBAU 83364, CCBAU 83345 and CCBAU 83523 ranged from 80.8 to 100 %, showing that they belong to the same species. The DNA–DNA relatedness between strain CCBAU 83401 and IIB CIAT 899, IIA CFN 299, LMG 150 and P1-7 were 26.9, 27.7, 38.2 and 22.6 %, respectively, clearly indicating that strain CCBAU 83401 represents a novel species. Phenotypic characterization of four representative strains, CCBAU 83401, CCBAU 83364, CCBAU 83345 and CCBAU 83523, showed several distinctive features that differentiated them from closely related species. The 31 strains had identical and genes, which were very similar to those of the bean-nodulating , and IIB. Based upon these results, the strains from this study are considered to represent a novel species, for which the name sp. nov. is proposed. The DNA G+C content ranged from 65.3 to 66.0 mol% ( ). The type strain is CCBAU 83401 (=LMG 23946=HAMBI 2975), which nodulates but not , or .


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  1. Allen, O. N. & Allen, E. K.(1981).The Leguminosae. A Source Book of Characteristics, Uses and Nodulation. Madison, USA: University of Wisconsin Press.
  2. Chen, W. X., Yan, G. H. & Li, J. L.(1988). Numerical taxonomy study of fast-growing soybean rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int J Syst Bacteriol 38, 392–397.[CrossRef] [Google Scholar]
  3. Chen, W., Wang, E., Wang, S., Li, Y., Chen, X. Q. & Li, Y.(1995). Characteristics of Rhizobium tianshanense sp. nov., a moderately and slowly growing root nodule bacterium isolated from an arid saline environment in Xinjiang, People's Republic of China. Int J Syst Bacteriol 45, 153–159.[CrossRef] [Google Scholar]
  4. Chen, W. X., Tan, Z. Y., Gao, J. L., Li, Y. & Wang, E. T.(1997).Rhizobium hainanense sp. nov., isolated from tropical legumes. Int J Syst Bacteriol 47, 870–873.[CrossRef] [Google Scholar]
  5. Cho, J.-C. & Tiedje, J. M.(2000). Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl Environ Microbiol 66, 5448–5456.[CrossRef] [Google Scholar]
  6. De Ley, J., Cattoir, H. & Reynaerts, A.(1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[CrossRef] [Google Scholar]
  7. Diouf, A., de Lajudie, P., Neyra, M., Kersters, K., Gillis, M., Martínez-Romero, E. & Gueye, M.(2000). Polyphasic characterization of rhizobia that nodulate Phaseolus vulgaris in West Africa (Senegal and Gambia). Int J Syst Evol Microbiol 50, 159–170.[CrossRef] [Google Scholar]
  8. Gao, J. L., Sun, J. G., Li, Y., Wang, E. T. & Chen, W. X.(1994). Numerical taxonomy and DNA relatedness of tropical rhizobia isolated from Hainan province of China. Int J Syst Bacteriol 44, 151–158.[CrossRef] [Google Scholar]
  9. Gaunt, M. W., Turner, S. L., Rigottier-Gois, L., Lloyd-Macgilp, S. A. & Young, J. P. W.(2001). Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol 51, 2037–2048.[CrossRef] [Google Scholar]
  10. Graham, P. H., Sadowsky, M. J., Keyser, H. H., Barnet, Y. M., Bradley, R. S., Cooper, J. E., De Ley, D. J., Jarvis, B. D. W., Roslycky, E. B. & other authors(1991). Proposed minimal standards for the description of new genera and species of root- and stem-nodulating bacteria. Int J Syst Bacteriol 41, 582–587.[CrossRef] [Google Scholar]
  11. Han, T. X., Wang, E. T., Han, L. L., Chen, W. F., Sui, X. H. & Chen, W. X.(2008). Molecular diversity and phylogeny of rhizobia associated with wild legumes native to Xinjiang, China. Syst Appl Microbiol (in press). [Google Scholar]
  12. Haukka, K., Lindström, K. & Young, J. P.(1998). Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America. Appl Environ Microbiol 64, 419–426. [Google Scholar]
  13. Healy, M., Huong, J., Bittner, T., Lising, M., Frye, S., Raza, S., Schrock, R., Manry, J., Renwick, A. & other authors(2005). Microbial DNA typing by automated repetitive-sequence-based PCR. J Clin Microbiol 43, 199–207.[CrossRef] [Google Scholar]
  14. Hurek, T., Wagner, B. & Reinhold-Hurek, B.(1997). Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints. Appl Environ Microbiol 63, 4331–4339. [Google Scholar]
  15. Jensen, M. A., Webster, J. A. & Straus, N.(1993). Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 59, 945–952. [Google Scholar]
  16. Jordan, D. C.(1984). Family III. Rhizobiaceae Conn 1938, 321AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 234–242. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
  17. Jukes, T. H. & Cantor, C. R.(1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
  18. Kimura, M.(1983).The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
  19. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  20. Kwon, S. W., Park, J. Y., Kim, J. S., Kang, J. W., Cho, Y. H., Lim, C. K., Parker, M. A. & Lee, G. B.(2005). Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences. Int J Syst Evol Microbiol 55, 263–270.[CrossRef] [Google Scholar]
  21. Laguerre, G., Mavingui, P., Allard, M. R., Charnay, M. P., Louvrier, P., Mazurier, S. I., Rigottier-Gois, L. & Amarger, N.(1996). Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Appl Environ Microbiol 62, 2029–2036. [Google Scholar]
  22. Maggi Solca, N., Bernasconi, M. V., Valsangiacomo, C., Van Doorn, L. J. & Piffaretti, J. C.(2001). Population genetics of Helicobacter pylori in the southern part of Switzerland analysed by sequencing of four housekeeping genes (atpD, glnA, scoB and recA), and by vacA, cagA, iceA and IS605 genotyping. Microbiology 147, 1693–1707. [Google Scholar]
  23. Marmur, J.(1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.[CrossRef] [Google Scholar]
  24. Marmur, J. & Doty, P.(1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[CrossRef] [Google Scholar]
  25. Navarro, E., Simonet, P., Normand, P. & Bardin, R.(1992). Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer. Arch Microbiol 157, 107–115. [Google Scholar]
  26. Nick, G. & Lindström, K.(1994). Use of repetitive sequences and the polymerase chain reaction to fingerprint the genomic DNA of R. galegae strains and to identify the DNA obtained by sonicating liquid cultures and root nodules. Syst Appl Microbiol 17, 265–273.[CrossRef] [Google Scholar]
  27. Peng, G. X., Tan, Z. Y., Wang, E. T., Reinhold-Hurek, B., Chen, W. F. & Chen, W. X.(2002). Identification of isolates from soybean nodules in Xinjiang region as Sinorhizobium xinjiangense and genetic differentiation of S. xinjiangense from Sinorhizobium fredii. Int J Syst Evol Microbiol 52, 457–462. [Google Scholar]
  28. Ponsonnet, C. & Nesme, X.(1994). Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch Microbiol 161, 300–309. [Google Scholar]
  29. Rademaker, J. L., Hoste, B., Louws, F. J., Kersters, K., Swings, J., Vauterin, L., Vauterin, P. & de Bruijn, F. J.(2000). Comparison of AFLP and rep-PCR genomic fingerprinting with DNA–DNA homology studies: Xanthomonas as a model system. Int J Syst Evol Microbiol 50, 665–677.[CrossRef] [Google Scholar]
  30. Rivas, R., Velázquez, E., Willems, A., Vizcaíno, N., Subba-Rao, N. S., Mateos, P. F., Gillis, M., Dazzo, F. B. & Martínez-Molina, E.(2002). A new species of Devosia that forms a unique nitrogen-fixing root nodule symbiosis with the aquatic legume Neptunia natans (L. f.) Druce. Appl Environ Microbiol 68, 5217–5222.[CrossRef] [Google Scholar]
  31. Rzhetsky, A. & Nei, M.(1993). Theoretical foundation of the minimum-evolution method of phylogenetic inference. Mol Biol Evol 10, 1073–1095. [Google Scholar]
  32. Saitou, N. & Nei, M.(1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. [Google Scholar]
  33. Swofford, D. L.(1993).paup: Phylogenetic Analysis Using Parsimony, version 3.1.1. Champaign, IL: Illinois Natural History Survey.
  34. Tajima, F. & Nei, M.(1984). Estimation of evolutionary distance between nucleotide sequences. Mol Biol Evol 1, 269–285. [Google Scholar]
  35. Tamura, K. & Nei, M.(1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10, 512–526. [Google Scholar]
  36. Tan, Z. Y., Xu, X. D., Wang, E. T., Gao, J. L., Martínez-Romero, E. & Chen, W. X.(1997). Phylogenetic and genetic relationships of Mesorhizobium tianshanense and related rhizobia. Int J Syst Bacteriol 47, 874–879.[CrossRef] [Google Scholar]
  37. Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994).clustalw: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef] [Google Scholar]
  38. Turner, S. L. & Young, J. P.(2000). The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. Mol Biol Evol 17, 309–319.[CrossRef] [Google Scholar]
  39. Valverde, A., Igual, J. M., Peix, A., Cervantes, E. & Velazquez, E.(2006).Rhizobium lusitanum sp. nov., a bacterium that nodulates Phaseolus vulgaris. Int J Syst Evol Microbiol 56, 2631–2637.[CrossRef] [Google Scholar]
  40. Versalovic, J., Schneider, M., de Bruijn, F. J. & Lupski, J. R.(1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5, 25–40. [Google Scholar]
  41. Vinuesa, P., Rademaker, J. L., de Bruijn, F. J. & Werner, D.(1998). Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA and 16S–23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic fingerprinting, and partial 16S rDNA sequencing. Appl Environ Microbiol 64, 2096–2104. [Google Scholar]
  42. Vinuesa, P., Silva, C., Lorite, M. J., Izaguirre-Mayoral, M. L., Bedmar, E. J. & Martínez-Romero, E.(2005a). Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands. Syst Appl Microbiol 28, 702–716.[CrossRef] [Google Scholar]
  43. Vinuesa, P., Silva, C., Werner, D. & Martínez-Romero, E.(2005b). Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol Phylogenet Evol 34, 29–54.[CrossRef] [Google Scholar]
  44. Wang, F. Q., Wang, E. T., Zhang, Y. F. & Chen, W. X.(2006). Characterization of rhizobia isolated from Albizia spp. in comparison with microsymbionts of Acacia spp. and Leucaena leucocephala grown in China. Syst Appl Microbiol 29, 502–517.[CrossRef] [Google Scholar]
  45. Wang, F. Q., Wang, E. T., Liu, J., Chen, Q., Sui, X. H., Chen, W. F. & Chen, W. X.(2007).Mesorhizobium albiziae sp. nov., a novel bacterium nodulating Albizia kalkora in a subtropical region of China. Int J Syst Evol Microbiol 57, 1192–1199.[CrossRef] [Google Scholar]
  46. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef] [Google Scholar]
  47. Wei, W., Jiang, J., Li, X., Wang, L. & Yang, S. S.(2004). Isolation of salt-sensitive mutants from Sinorhizobium meliloti and characterization of genes involved in salt tolerance. Lett Appl Microbiol 39, 278–283.[CrossRef] [Google Scholar]
  48. Yan, A. M., Wang, E. T., Kan, F. L., Tan, Z. Y., Sui, X. H., Reinhold-Hurek, B. & Chen, W. X.(2000).Sinorhizobium meliloti associated with Medicago sativa and Melilotus spp. in arid saline soils in Xinjiang, China. Int J Syst Evol Microbiol 50, 1887–1891. [Google Scholar]

Data & Media loading...


Dendrogram based on IGS-RFLP fingerprints. [PDF](17 KB)


Dendrogram based on SDS-PAGE of proteins. [PDF](17 KB)


Dendrogram based on BOX-PCR fingerprints. [PDF](16 KB)


Extended phylogenetic tree based on 16S rRNA gene sequences. [PDF](23 KB)


Phylogenetic trees based on IGS sequences and , and gene sequences. [PDF](23 KB)


Phylogenetic trees based on and gene sequences. [PDF](20 KB)


Fatty acid profile of sp. nov. and closely related taxa. [PDF](75 KB)

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