Advertisement

Applied Microbiology and Biotechnology

, Volume 83, Issue 5, pp 897–908 | Cite as

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

  • Daisy Rickli Binde
  • Pâmela Menna
  • Eliane Villamil Bangel
  • Fernando Gomes Barcellos
  • Mariangela HungriaEmail author
Applied Genetics and Molecular Biotechnology

Abstract

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.

Keywords

Bacterial fingerprinting Bacterial taxonomy Biological nitrogen fixation Culture collections Inoculants 16S rRNA 

Notes

Acknowledgments

The work was partially supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), projects 552393/2005-3 and 577933/2008-6, PQ (300698/2007), and PNPD (558455/2008-5). The authors thank also Ligia Maria Oliveira Chueire (Embrapa Soja) for help in several steps of this work and Dr. Allan R.J. Eaglesham for helpful discussion. P. Menna acknowledges fellowships from CNPq (projects 505946/2004-1 and 558455/2008-5), and F.G. Barcellos acknowledges a pro-doc fellowship from CAPES.

References

  1. Alberton O, Kaschuk G, Hungria M (2006) Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean. Soil Biol Biochem 38:1298–1307CrossRefGoogle Scholar
  2. Allen ON, Allen E (1981) The Leguminosae: A source book of characteristics, uses and nodulation. The University of Wisconsin Press, MadisonGoogle Scholar
  3. Barcellos FG, Menna P, Batista JSS, Hungria M (2007) Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian savannah soil. Appl Environ Microb 73:2635–2643CrossRefGoogle Scholar
  4. Batista JSS, Hungria M, Barcellos FG, Ferreira MC, Mendes IC (2007) Variability in Bradyrhizobium japonicum and B. elkanii seven years after introduction of both the exotic microsymbiont and the soybean host in a cerrados soil. Microb Ecol 53:270–284CrossRefGoogle Scholar
  5. de Bruijn FJ (1992) Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacterial. Appl Environ Microb 58:2180–2187Google Scholar
  6. Doignon-Bourcier F, Sy A, Willems A, Torck U, Dreyfus B, Gillis M, de Lajudie P (1999) Diversity of bradyrhizobia from 27 tropical Leguminosae species native of Senegal. Syst Appl Microbiol 22:647–661Google Scholar
  7. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  8. FEPAGRO (Fundação Estadual de Pesquisa Agropecuária) (1999) Culture Collection Catalogue, 8th edn. FEPAGRO, Porto AlegreGoogle Scholar
  9. Germano MG, Menna P, Mostasso FL, Hungria M (2006) RFLP analysis of the rRNA operon of a Brasilian collection of bradyhizobial strains from 33 legumes species. Int J Syst Evol Micr 56:217–229CrossRefGoogle Scholar
  10. Grange L, Hungria M (2004) Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems. Soil Biol Biochem 36:1389–1398CrossRefGoogle Scholar
  11. Hedges SB (1992) The number of replications needed for accurate estimation of the bootstrap p-value in phylogenetic studies. Mol Biol Evol 9:366–369Google Scholar
  12. Hungria M, Campo RJ (2007) Inoculantes microbianos: situação no Brasil. In: Izaguirre-Mayoral ML, Labandera C, Sanjuan J (eds) Biofertilizantes en Iberoamérica: Visión Técnica, Científica y Empresarial. Cyted/Biofag, Montevideo, pp 22–31Google Scholar
  13. Hungria M, Franchini JC, Campo RJ, Graham PH (2005) The importance of nitrogen fixation to soybean cropping in South America. In: Werner W, Newton WE (eds) Nitrogen fixation in agriculture, forestry, ecology and the environment. Springer, Dordrecht Amsterdam, pp 25–42CrossRefGoogle Scholar
  14. Hungria M, Chueire LMO, Megías M, Lamrabet Y, Probanza A, Guttierrez-Mañero FJ, Campo RJ (2006a) Genetic diversity of indigenous tropical fast-growing rhizobia isolated from soybean nodules. Plant Soil 288:343–356CrossRefGoogle Scholar
  15. Hungria M, Campo RJ, Mendes IC, Graham PH (2006b) Contribution of biological nitrogen fixation to the N nutrition of grain crops in the tropics: the success of soybean (Glycine max L. Merr.) in South America. In: Singh RP, Shankar N, Jaiwal PK (eds) Nitrogen nutrition and sustainable plant productivity. Studium Press LLC, Houston, pp 43–93Google Scholar
  16. ILDIS (International Legume Database & Information Service) (2005) Retrieved April 28th. http://www.ildis.org
  17. Jaccard P (1912) The distribution of flora in the alpine zone. New Phytol 11:37–50CrossRefGoogle Scholar
  18. Kaschuk G, Hungria M, Andrade DS, Campo RJ (2006) Genetic diversity of rhizobia associated with common bean (Phaseolus vulgaris L.) grown under no-tillage and conventional systems in Southern Brazil. Appl Soil Ecol 32:210–220CrossRefGoogle Scholar
  19. Kim W, Song M, Song W, Kim K, Chung S, Choi C, Park Y (2003) Comparison of 16 S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis. Ant van Leeuwenhoek 83:125–133CrossRefGoogle Scholar
  20. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  21. Kumar S, Tamura K, Nei M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefGoogle Scholar
  22. Laguerre G, van Berkum P, Amarger N, Prevost D (1997) Genetic diversity of rhizobial symbionts isolated from legume species within the genera Astragalus, Oxytropis, and Onobrychis. Appl Environ Microb 63:4748–4758Google Scholar
  23. MAPA (Ministério da Agricultura, Pecuária e Abastecimento) (2006) Instrução normativa N 10, Retrieved March 21th. http://extranet.agricultura.gov.br/sislegis-consulta/consultarLegislacao.do?operacao=visualizar&id=16735
  24. Menna P, Hungria M, Barcellos FG, Bangel EV, Hess PN, Martinez-Romero E (2006) Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants. Syst Appl Microbiol 29:315–332CrossRefGoogle Scholar
  25. Menna P, Pereira AA, Bangel EV, Hungria M (2009) rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool. Symbiosis 48 (1-3) in press Google Scholar
  26. Mostasso L, Mostasso FL, Dias BG, Vargas MAT, Hungria M (2002) Selection of bean (Phaseolus vulgaris L.) rhizobial strains for the Brazilian Cerrados. Field Crop Res 73:121–132CrossRefGoogle Scholar
  27. Oyaizu H, Naruhashi N, Gamou T (1992) Molecular methods of analysing bacterial diversity: The case of rhizobia. Biodivers Conserv 1:237–249CrossRefGoogle Scholar
  28. Pinto FGS, Hungria M, Mercante FM (2007) Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.). Soil Biol Biochem 39:1851–1864CrossRefGoogle Scholar
  29. Polhill RM, Raven PH (1981) Advances in legume systematics. Royal Botanic Gardens, KewGoogle Scholar
  30. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  31. Sneath PBA, Sokal RR (1973) Numerical taxonomy. WH Freeman & Co, San FranciscoGoogle Scholar
  32. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–7882CrossRefGoogle Scholar
  33. Urtz BE, Elkan GH (1996) Genetic diversity among Bradyrhizobium isolates that effectively nodulate peanut (Arachis hypogaea). Can J Microbiol 42:1121–1130CrossRefGoogle Scholar
  34. Versalovic J, Schneider M, de Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence based PCR (rep-PCR). Meth Mol Cell Biol 5:25–40Google Scholar
  35. Vincent JM (1970) Manual for the practical study of root nodule bacteria. Blackwell, OxfordGoogle Scholar
  36. Vinuesa P, Rademaker JLW, de Bruijn FJ, 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 (16S rDNA) and 16 S–23S rRNA intergenic spacers, repetitive extragenic palindomic PCR genomic fingerprinting, and partial 16S rRNA sequencing. Appl Environ Microbiol 64:2096–2104Google Scholar
  37. Vinuesa P, Leon-Barrios M, Silva C, Willems A, Jarabo-Lorenzo A, Perez-Galdona R, Werner D, Martinez-Romero E (2005) Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int J Syst Evol Microbiol 55:569–575CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Daisy Rickli Binde
    • 1
    • 2
  • Pâmela Menna
    • 1
    • 3
  • Eliane Villamil Bangel
    • 4
  • Fernando Gomes Barcellos
    • 1
    • 2
    • 5
  • Mariangela Hungria
    • 1
    • 2
    • 3
    Email author
  1. 1.Embrapa SojaLondrinaBrazil
  2. 2.Universidade Estadual de LondrinaLondrinaBrazil
  3. 3.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-MCT)BrasíliaBrazil
  4. 4.FEPAGROPorto AlegreBrazil
  5. 5.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)BrasíliaBrazil

Personalised recommendations