Plant and Soil

, Volume 287, Issue 1–2, pp 23–33 | Cite as

Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils

  • R. Rivas
  • A. Peix
  • P. F. MateosEmail author
  • M. E. Trujillo
  • E. Martínez-Molina
  • E. Velázquez
Original Paper


Within rhizobia, two species nodulating chickpea, Mesorhizobium ciceri and Mesorhizobium mediterraneum, are known as good phosphate solubilizers. For this reason, we have analysed the ability to solubilize phosphate of a wide number of strains isolated from Cicer arietinum growing in several soils in Spain. The aim of this work was to analyse microbial populations nodulating chickpea, that are able to solubilize phosphates, using molecular techniques. In the present work we analyzed 19 strains isolated from effective nodules of C. arietinum growing in three soils from the North of Spain. Nineteen strains showed ability to solubilize phosphate in YED-P medium. These strains were separated into 4 groups according to the results obtained by 879F-RAPD fingerprinting. The 16S rDNA sequencing of a representative strain from each group allowed the identification of strains as belonging to the genus Mesorhizobium. Strains from groups I and II showed a 99.4% and 99.2% similarity with M. mediterraneum UPM-CA142T, respectively. The strains from group III were related to M. tianshanense USDA 3592T at a 99.4% similarity level. Finally, the strain from group IV was related to M. ciceri USDA 3383T with a 99.3% similarity. The LMW RNA profiles confirmed these results. Strains from groups I and II showed an identical LMW RNA profile to that of M. mediterraneum UPM-CA142T; the profile of strains from group III was identical to that of M.␣tianshanense USDA 3592T and the profile of strains from group IV was identical to that of M. ciceri USDA 3383T. Different 879F-RAPD patterns were obtained for strains of the group I, group II and the M.␣mediterraneum type strain (UPM-CA142T). The 879-RAPD patterns obtained for group III also differed from the pattern shown by M. tianshanense USDA 3592T. Finally, the patterns between group IV and M. ciceri USDA 3383T were also different. These results suggest that groups I and II may be subspecies of M. mediterraneum, group III a subspecies of M. tianshanense and group IV a subspecies of M. ciceri. Nevertheless, more studies are needed to establish the taxonomic status of strains isolated in this study.


Phosphate solubilizing bacteria rhizobia Mesorhizobium chickpea 


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This work was supported by the Junta de Castilla y León and the DGICYT (Dirección General de Investigación Científica y Técnica).


  1. Bergersen FJ (1961) The growth of Rhizobium in synthetic media. Aust J Biol Sci 14:349–360Google Scholar
  2. Beringer JE (1974) R factors transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198PubMedGoogle Scholar
  3. Bidle KD, Fletcher M (1995) Comparison of free-living and particle-associated bacterial communities in the Chesapake Bay by stable low-molecular-weight RNA analysis. Appl Environ Microbiol 61:944–952PubMedGoogle Scholar
  4. Chen WX, Li GS, Qi YL, Wang ET, Yuan HL, Li JL (1991) Rhizobium huakuii sp. nov. isolated from the root nodules of Astragalus sinicus. Int J Syst Bacteriol 41:275–280Google Scholar
  5. Chen W, Wang E, Wang S, Li Y, Chen X, 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 45:153–159PubMedGoogle Scholar
  6. Chen WM, Laevens S, Lee TM, Coenye T, de Vos P, Mergeay M, Vandamme P (2001) Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51:1729–1735PubMedGoogle Scholar
  7. Cruz-Sánchez JM, Velázquez E, Mateos P, Martínez-Molina E (1997) Enhancement of resolution of low molecular weight RNA profiles by staircase electrophoresis. Electrophoresis 18:1909–1911PubMedCrossRefGoogle Scholar
  8. de Lajudie P, Willems A, Nick G, Moreira F, Molouba F, Hoste B, Torck U, Neyra M, Collins MD, Lindström K, Dreyfus B, Gillis M (1998) Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov. Int J Syst Bacteriol 48:369–382PubMedGoogle Scholar
  9. de la Puente-Redondo VA, García del Blanco N, Gutiérrez Martín CB, García-Peña FJ, Rodríguez Ferri EF (2000) Comparison of different PCR approaches for typing of Francisella tularensis strains. J Clin Microbiol 38:1016–1022Google Scholar
  10. Fischer-Le Saux M, Viallard V, Brunel B, Normand P, Boemare NE (1999) Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov. Int J Syst Bacteriol 49:1645–1656PubMedGoogle Scholar
  11. Haas H, Budowle B, Weiler G (1994) Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments. Electrophoresis 15:153–158PubMedCrossRefGoogle Scholar
  12. Halder AK, Mishra AK, Chakrabartty PK (1990) Solubilization of phosphatic compounds by Rhizobium. Indian J Microbiol 30:311–314Google Scholar
  13. Herrera-Cervera JA, Caballero-Mellado J, Laguerre G, Tichy HV, Requena N, Amarger N, Martínez-Romero E, Olivares J, Sanjuán J (1999) At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil. FEMS Microbiol Ecol 30:87–97CrossRefGoogle Scholar
  14. Höfle MG (1988) Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach. J Microbiol Methods 8:235–248CrossRefGoogle Scholar
  15. Igual JM, Valverde A, Rivas R, Mateos PF, Rodríguez-Barrueco C, Martínez-Molina E, Cervantes E, Velázquez E 2003 Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli. Plant Soil 254, 115–123Google Scholar
  16. Jarabo-Lorenzo A, Velázquez E, Pérez-Galdona R, Vega-Hernández MC, Martínez-Molina E, Mateos PF, Vinuesa P, Martínez-Romero E, León-Barrios M (2000) Restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA and low molecular weight RNA profiling in the characterisation of rhizobial isolates from shrubby legumes endemic to the Canary Islands. Syst Appl Microbiol 23:4–18Google Scholar
  17. Jarvis BDW, Pankhurst CE, Patel JJ (1982) Rhizobium loti, a new species of legume root nodule bacteria. Int J Syst Bacteriol 32:378–380Google Scholar
  18. Jarvis BDW, van Berkum P, Chen WX, Nour SM, Fernandez MP, Cleyet-Marel JC, Gillis M (1998) Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum and Rhizobium tianshanense to Mesorhizobium gen. nov. Int J Syst Bacteriol 47:895–898Google Scholar
  19. Jonhson FJ (1990) Detection method of nitrogen (total) in fertilizers. In: Elrich K (ed) Methods of analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, USA pp 17–19Google Scholar
  20. Kimura M 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedCrossRefGoogle Scholar
  21. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) Molecular evolutionary genetics analysis software. Arizona State University. Tempe, Arizona. USAGoogle Scholar
  22. Moulin L, Munive A, Dreyfus B, Boivin-Masson C (2001) Nodulation of legumes by members of β–subclass of Proteobacteria. Nature 411:948–950PubMedCrossRefGoogle Scholar
  23. Nakamura LK, Roberts MS, Cohan FM (1999) Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49:1211–1215PubMedGoogle Scholar
  24. Nour SH, Fernández MP, Normand P, Cleyet-Marel JC (1994) Rhizobium ciceri, sp. nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.). Int J Syst Bacteriol 44:511–522PubMedGoogle Scholar
  25. Nour SH, Cleyet-Marel JC, Normand P, Fernandez MP (1995) Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Int J Syst Bacteriol 45:640–648PubMedGoogle Scholar
  26. Nuswantara S, Fujie M, Yamada T, Malek W, Inaba M, Kaneko Y, Murooka Y (1999) Phylogenetic position of Mesorhizobium huakuii subsp. rengei, a symbiont of Astragalus sinicus cv. Jpn J Biosci Bioeng 87:49–55CrossRefGoogle Scholar
  27. Palomo JL, Velázquez E, Mateos PF, García-Benavides P, Martínez-Molina E (2000) Rapid identification of Clavibacter michiganensis subspecies sepedonicus based on the stable low molecular weight RNA (LMW RNA) profiles. Eur J Plant Pathol 106:789–793CrossRefGoogle Scholar
  28. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448PubMedCrossRefGoogle Scholar
  29. Peix A, Rivas-Boyero AA, Mateos PF, Rodríguez-Barrueco C, Martínez-Molina E, Velázquez E. (2001). Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol Biochem 33:103–110CrossRefGoogle Scholar
  30. Perret X, Staehelin C, Broughton WJ (2000) Molecular basis of symbiotic promiscuity. Microbiol Mol Biol Rev 64:180–201PubMedCrossRefGoogle Scholar
  31. Rivas R, Velázquez E, Valverde A, Mateos PF, Martínez-Molina E (2001) A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis 22:1086–1089PubMedCrossRefGoogle Scholar
  32. Rivas R, Velázquez E, Palomo JL, Mateos P, García-Benavides P, Martínez-Molina E. (2002a). Rapid identification of Clavibacter michiganensis subspecies sepedonicus using two primers random amplified polymorphic DNA (TP-RAPD) fingerprints. Eur J Plant Pathol 108:179–184CrossRefGoogle Scholar
  33. Rivas R, Velázquez E, Willems A, Vizcaíno N, Subba-Rao NS, Mateos PF, Gillis M, Dazzo FB, Martínez-Molina E (2002b) A new species of Devosia that forms a nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L. f.) Druce. Appl Environ Microbiol 68:5217–5222CrossRefGoogle Scholar
  34. Saitou N, Nei M (1987) A neighbour-joining method: a new method for reconstructing phylogenetics trees. Mol Biol Evol 44:406–425Google Scholar
  35. Sprinzl M, Moll J, Meissner F, Hatmann T (1985) Compilation of tRNA sequences. Nucleic Acid Res 13:1–49Google Scholar
  36. Sy A, Giraud E, Jourand P, García N, Willems A, de Lajudie P, Prin Y, Neyra M, Gillis M, Boivin-Masson C, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. J Bacteriol 183: 214–220PubMedCrossRefGoogle Scholar
  37. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 24:4876–4882CrossRefGoogle Scholar
  38. Velázquez E, Cervantes E, Igual JM, Peix A, Mateos PF, Benamar S, Moiroud A, Wheeler CT, Dawson J, Labeda D, Rodríguez-Barrueco C, Martínez-Molina E (1998a) Analysis of LMW RNA profiles of Frankia strains by staircase electrophoresis. Syst Appl Microbiol 21:539–545Google Scholar
  39. Velázquez E, Cruz-Sánchez JM, Mateos PF, Martínez-Molina E (1998b) Analysis of stable low molecular weight RNA profiles of members of the family Rhizobiaceae. Appl Environ Microbiol 64:1555–1559Google Scholar
  40. Velázquez E, Calvo O, Cervantes E, Mateos PF, Tamame M, Martínez-Molina E (2000) Staircase electrophoresis profiles of stable low molecular weight RNA as yeast fingerprinting. Int J Syst Evol Microbiol 50:917–923PubMedGoogle Scholar
  41. Velázquez E, Igual JM, Willems A, Fernández MP, Muñoz E, Mateos PF, Abril A, Toro N, Normand P, Cervantes E, Gillis M, Martínez-Molina E (2001a) Description of Mesorhizobium chacoense sp. nov. that nodulates Prosopis alba in the Chaco Arido region (Argentina). Int J Syst Evol Microbiol 51:1011–1021Google Scholar
  42. Velázquez E, Martínez-Romero E, Rodríguez-Navarro DN, Trujillo ME, Daza A, Mateos PF, Martinez-Molina E, Van Berkum P (2001b) Characterization of rhizobial isolates of Phaseolus vulgaris by staircase electrophoresis of low molecular weight RNA. Appl Environ Microbiol 67:1008–1010CrossRefGoogle Scholar
  43. Velázquez E, Trujillo ME, Peix A, Palomo JL, García-Benavides P, Mateos P, Ventosa A, Martínez-Molina E (2001c) Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms. Syst Appl Microbiol 24:490–499CrossRefGoogle Scholar
  44. Vincent JM (1970) The cultivation, isolation and maintenance of rhizobia. In: Vincent JM (ed) A manual for the practical study of root-nodule. Blackwell Scientific Publications, Oxford pp 1–13Google Scholar
  45. Wang ET, van Berkum P, Sui XH, Beyene D, Chen WX, Martínez-Romero E (1999) Diversity of rhizobia associated with Amorpha fruticosa isolated from chinese soils and description of Mesorhizobium amorphae sp. nov. Int J Syst Bacteriol 49:51–65PubMedCrossRefGoogle Scholar
  46. Wieser M, Busse HJ (2000) Rapid identification of Staphylococcus epidermidis. Int J Syst Evol Microbiol 50:1087–1093PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • R. Rivas
    • 1
  • A. Peix
    • 2
  • P. F. Mateos
    • 3
    Email author
  • M. E. Trujillo
    • 1
  • E. Martínez-Molina
    • 3
  • E. Velázquez
    • 1
  1. 1.Departamento de Microbiología y Genética, Lab. 209, Edificio Departamental de BiologíaUniversidad de SalamancaSalamancaSpain
  2. 2.Instituto de Recursos Naturales y AgrobiologíaCSICSalamancaSpain
  3. 3.Departamento de Microbiología y Genética, Instituto Hispano Luso de Investigaciones AgrariasUniversidad de SalamancaSalamancaSpain

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