Isolated from root nodules of (Širj.) Vassilcz. grown in Tibet, China, cells of the bacterial strains CCBAU 85039 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 species. In the phylogeny based on 16S rRNA gene sequences, they were most similar to CFN 42 (98.2 % similarity) and USDA 2370 (97.6 %). Sequence analyses of the housekeeping genes , and 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 . Cross-nodulation tests and sequencing of and genes showed that these two strains were symbiotic bacteria that nodulated , , , , and . Based on the results, the novel species sp. nov. is described to accommodate the two strains. The type strain is CCBAU 85039 (=LMG 24453 =CGMCC 1.7071). The DNA G+C content of this strain is 59.7 mol% ( ).


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  1. de Lajudie, P., Willems, A., Pot, B., Dewettinck, D., Maestrojuan, G., Neyra, M., Collins, M. D., Dreyfus, B., Kersters, K. & Gillis, M.(1994). Polyphasic taxonomy of rhizobia: emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. nov., Sinorhizobium saheli sp. nov., and Sinorhizobium teranga sp. nov. Int J Syst Bacteriol 44, 715–733.[CrossRef] [Google Scholar]
  2. de Lajudie, P., Willems, A., Nick, G., Moreira, F., Molouba, F., Hoste, B., Torck, U., Neyra, M., Collins, M. D. & other authors(1998). Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov. Int J Syst Bacteriol 48, 369–382.[CrossRef] [Google Scholar]
  3. De Ley, J.(1970). Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J Bacteriol 101, 738–754. [Google Scholar]
  4. De Ley, J., Cattoir, H. & Reynaerts, A.(1970). Quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[CrossRef] [Google Scholar]
  5. Eardly, B. D., Young, J. P. & Selander, R. K.(1992). Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes. Appl Environ Microbiol 58, 1809–1815. [Google Scholar]
  6. Frank, B.(1889). Über die Pilzsysymbiose der Leguminosen. Ber Dtsch Bot Ges 7, 332–346 (in German). [Google Scholar]
  7. 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]
  8. Garcia-Fraile, P., Rivas, R., Willems, A., Peix, A., Martens, M., Martinez-Molina, E., Mateos, P. F. & Velazquez, E.(2007).Rhizobium cellulosilyticum sp. nov., isolated from sawdust of Populus alba. Int J Syst Evol Microbiol 57, 844–848.[CrossRef] [Google Scholar]
  9. Gaunt, M. W., Turner, S. L., Rigottier-Gois, L., Lloyd-Macgilp, S. A. & Young, J. P.(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, 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. Gu, C. T., Wang, E. T., Sui, X. H., Chen, W. F. & Chen, W. X.(2007). Diversity and geographical distribution of rhizobia associated with Lespedeza spp. in temperate and subtropical regions of China. Arch Microbiol 188, 355–365.[CrossRef] [Google Scholar]
  12. Haukka, K., Lindström, K. & Young, J. P. W.(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. Hou, B. C., Wang, E. T., Li, Y., Jia, R. Z., Chen, W. F., Man, C. X., Sui, X. H. & Chen, W. X.(2009). Rhizobial resource associated with epidemic legumes in Tibet. Microb Ecol 57, 69–81. [Google Scholar]
  14. Jarvis, B. D. W., Van Berkum, P., Chen, W. X., Nour, S. M., Fernandez, M. P., Cleyet-Marel, J. C. & Gillis, M.(1997). Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int J Syst Bacteriol 47, 895–898.[CrossRef] [Google Scholar]
  15. Jordan, D. C.(1982). Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int J Syst Bacteriol 32, 136–139.[CrossRef] [Google Scholar]
  16. Kalita, M., Stepkowski, T., Łotocka, B. & Małek, W.(2006). Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia. Arch Microbiol 186, 87–97.[CrossRef] [Google Scholar]
  17. Kämpfer, P., Buczolits, S., Albrecht, A., Busse, H. J. & Stackebrandt, E.(2003). Towards a standardized format for the description of a novel species (of an established genus): Ochrobactrum gallinifaecis sp. nov. Int J Syst Evol Microbiol 53, 893–896.[CrossRef] [Google Scholar]
  18. Kumar, S., Tamur, K. & Nei, M.(2004).mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  19. Laranjo, M., Alexandre, A., Rivas, R., Velázquez, E., Young, J. P. & Oliveira, S.(2008). Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species. FEMS Microbiol Ecol 66, 391–400.[CrossRef] [Google Scholar]
  20. Lin, D. X., Man, C. X., Wang, E. T. & Chen, W. X.(2007). Diverse rhizobia that nodulate two species of Kummerowia in China. Arch Microbiol 188, 495–507.[CrossRef] [Google Scholar]
  21. Liu, X. Y., Wang, E. T., Li, Y. & Chen, W. X.(2007). Diverse bacteria isolated from root nodules of Trifolium, Crotalaria and Mimosa grown in the subtropical regions of China. Arch Microbiol 188, 1–14.[CrossRef] [Google Scholar]
  22. Martínez-Romero, E., Segovia, L., Mercante, F. M., Franco, A. A., Graham, P. & Pardo, M. A.(1991).Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees. Int J Syst Bacteriol 41, 417–426.[CrossRef] [Google Scholar]
  23. NZ Rhizobia(2008).The current taxonomy of rhizobia [Updated 16 November 2008]. http://www.rhizobia.co.nz/taxonomy/rhizobia.html.
  24. Quan, Z. X., Bae, H. S., Baek, J. H., Chen, W. F., Im, W. T. & Lee, S. T.(2005).Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor. Int J Syst Evol Microbiol 55, 2543–2549.[CrossRef] [Google Scholar]
  25. Ramírez-Bahena, M. H., García-Fraile, P., Peix, A., Valverde, A., Rivas, R., Igual, J. M., Mateos, P. F., Martínez-Molina, E. & Velázquez, E.(2008). Revision of the taxonomic status of the species Rhizobium leguminosarum (Frank 1879) Frank 1889AL, Rhizobium phaseoli Dangeard 1926AL and Rhizobium trifolii Dangeard 1926AL. R. trifolii is a later synonym of R. leguminosarum. Reclassification of the strain R. leguminosarum DSM 30132 (=NCIMB 11478) as Rhizobium pisi sp. nov. Int J Syst Evol Microbiol 58, 2484–2490.[CrossRef] [Google Scholar]
  26. Segovia, L., Young, J. P. & Martínez-Romero, E.(1993). Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov. Int J Syst Bacteriol 43, 374–377.[CrossRef] [Google Scholar]
  27. Tan, Z. Y., Xu, X. D., Wang, E. T., Gao, J. L., Martinez-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]
  28. Tan, Z. Y., Hurek, T., Vinuesa, P., Müller, P., Ladha, J. K. & Reinhold-Hurek, B.(2001). Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S–23S ribosomal DNA intergenic spacer-targeted PCR. Appl Environ Microbiol 67, 3655–3664.[CrossRef] [Google Scholar]
  29. Terefework, Z., Kaijalainen, S. & Lindström, K.(2001). AFLP fingerprint as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis. J Biotechnol 91, 169–180.[CrossRef] [Google Scholar]
  30. Tighe, S. W., de Lajudie, P., Dipietro, K., Lindström, K., Nick, G. & Jarvis, B. D. W.(2000). Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System. Int J Syst Evol Microbiol 50, 787–801.[CrossRef] [Google Scholar]
  31. Turner, S. L. & Young, J. P. W.(2000). The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. Mol Biol Evol 17, 309–319.[CrossRef] [Google Scholar]
  32. van Berkum, P., Beyene, D. & Eardly, B. D.(1996). Phylogenetic relationships among Rhizobium species nodulating the common bean (Phaseolus vulgaris L.). Int J Syst Bacteriol 46, 240–244.[CrossRef] [Google Scholar]
  33. van Berkum, P., Badri, Y., Elia, P., Aouani, M. E. & Eardly, B. D.(2007). Chromosomal and symbiotic relationships of rhizobia nodulating Medicago truncatula and M. laciniata. Appl Environ Microbiol 73, 7597–7604.[CrossRef] [Google Scholar]
  34. Vincent, J. M.(1970). The cultivation, isolation and maintenance of rhizobia. In A Manual for the Practical Study of the Root-Nodule Bacteria, pp. 1–13. Edited by J. M. Vincent. Oxford: Blackwell Scientific.
  35. Vinuesa, P., Silva, C., Lorite, M. J., Izaguirre-Mayoral, M. L., Bedmar, E. J. & Martínez-Romero, E.(2005). 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]
  36. Yan, X. R., Chen, W. F., Fu, J. F., Lu, Y. L., Xue, C. Y., Sui, X. H., Li, Y., Wang, E. T. & Chen, W. X.(2007).Mesorhizobium spp. are the main microsymbionts of Caragana spp. grown in Liaoning Province of China. FEMS Microbiol Lett 271, 265–273.[CrossRef] [Google Scholar]

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[PDF file of Supplementary Figs S1 and S2](82 KB)


Electrophoretic protein patterns showing the relationships among strains CCBAU 85039 and CCBAU 85027 and reference strains of phylogenetically related species. Only strain CCBAU 85039 was used to construct the dendrogram, because strain CCBAU 85027 had an almost identical protein profile.


[PDF file of Supplementary Tables S1 and S2](73 KB)


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