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Plant and Soil

, Volume 356, Issue 1–2, pp 83–99 | Cite as

Molecular characterisation of the diazotrophic bacterial community in uninoculated and inoculated field-grown sugarcane (Saccharum sp.)

  • Doreen Fischer
  • Barbara Pfitzner
  • Michael Schmid
  • Jean L. Simões-Araújo
  • Veronica M. Reis
  • William Pereira
  • Ernesto Ormeño-Orrillo
  • Brigitte Hai
  • Andreas Hofmann
  • Michael Schloter
  • Esperanza Martinez-Romero
  • Jose Ivo Baldani
  • Anton Hartmann
Regular Article

Abstract

To identify active diazotrophs in sugarcane, 16S rRNA and nifH transcript analyses were applied. This should help to better understand the basis of the biological nitrogen fixation (BNF) activity of a high nitrogen fixing sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropédica, RJ, Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with inoculation. The five-strain mixture developed by EMBRAPA-CNPAB was used as inoculum. Root and leaf sheath samples were harvested in the third year of cultivation to analyse the 16S rRNA and nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a wide diversity of nifH sequences from previously uncharacterised Ideonella/Herbaspirillum related phylotypes in sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots was found. These results were confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus and B. tropica were detected in leaf sheaths and roots. Known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using molecular analyses. Two strains of the inoculum mix were identified at the late summer harvest.

Keywords

Sugarcane (Saccharum sp.) Biological nitrogen fixation Molecular community analysis nifH transcripts 16S ribosomal RNA 

Notes

Acknowledgements

Financial support from Deutsche Forschungsgemeinschaft (Grant Ha 1708/9), EMBRAPA and the Helmholtz Zentrum München is greatly acknowledged. Financial support came also from INCT/CNPq (proc. no 573828/2008-3) and CT-AGRO (proc. no 480178/2005-4) projects for the fellowship.

Supplementary material

11104_2011_812_MOESM1_ESM.doc (32 kb)
Table S1 Number of sequences achieved from the different sugarcane plant tissues grown in field with and without the mixed inoculant and nitrogen fertilisation (DOC 32 kb)
11104_2011_812_MOESM2_ESM.doc (64 kb)
Table S2 Organisms and AccessionNumbers (GenBank) of nifH phylogeny used for calculation (DOC 63 kb)
11104_2011_812_MOESM3_ESM.doc (104 kb)
Table S3 Accession-Numbers (GenBank) of 16S rRNA and nifH sequences obtained in this study and used for calculation of phylogeny (DOC 104 kb)

References

  1. Abu Kwaik Y, Pederson LL (1996) The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol Microbiol 21:543–556PubMedCrossRefGoogle Scholar
  2. Ando S, Goto M, Meunchang S, Thongra-ar P, Fujiwara T, Hayashi H, Yoneyama T (2005) Detection of nifH sequences in Sugarcane (Saccharum officinarum L.) and pineapple (Ananas comosus [L.] Merr.). Soil Sci Plant Nutr 51:303–308CrossRefGoogle Scholar
  3. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2005) At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl Environ Microbiol 71:7724–7736PubMedCrossRefGoogle Scholar
  4. Biggs IM, Stewart GR, Wilson JR, Critchley C (2002) 15N natural abundance studies in Australian commercial sugarcane. Plant Soil 238:21–30CrossRefGoogle Scholar
  5. Boddey RM, Polidoro JC, Resende AS, Alves BJ, Urquiaga S (2001) Use of the 15N natural abundance technique for the quantification of the contribution of N2 fixation to sugarcane and other grasses. Aust J Plant Physiol 28:889–895Google Scholar
  6. Boddey RM, Urquiaga S, Alves BJ, Reis VM (2003) Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil 252:139–149CrossRefGoogle Scholar
  7. Boddey RM, Soares LHB, Alves BJR, Urquiaga S (2008) Bio-ethanol production in Brazil. In: Pimentel D (ed) Biofuels, solar and wind as renewable energy systems. Springer, USA, p 504Google Scholar
  8. Buddrus-Schiemann K, Schmid S, Schreiner K, Welzl G, Hartmann A (2010) Root colonization by Pseudomonas sp. DSMZ 13134 and impact on the indigenous rhizosphere bacterial community of barley. Microb Ecol 60:381–393PubMedCrossRefGoogle Scholar
  9. Burbano CS, Liu Y, Roesner KL, Reis VM, Caballero-Mellado J, Reinhold-Hurek B, Hurek T (2011) Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field-grown sugarcane plants and in Norway spruce. Environ Microbiol Reports 3:383–389CrossRefGoogle Scholar
  10. Cavalcante VA, Doebereiner J (1988) A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108:23–31CrossRefGoogle Scholar
  11. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucl Acids Res 37(suppl 1):D141–D145PubMedCrossRefGoogle Scholar
  12. da Silva MF, de Oliveira PJ, Xavier GR, Rumjanek NG, Reis VM (2009) Inoculants containing polymers and endophytic bacteria for the sugarcane crop. Pesquisa Agropecuaria Brasileira 44:1437–1443CrossRefGoogle Scholar
  13. Doebereiner J (1961) Nitrogen-fixing bacteria of the genus Beijerinckia Derx in the rhizosphere of sugarcane. Plant Soil 15:211–216CrossRefGoogle Scholar
  14. Doebereiner J (1995) Isolation and identification of aerobic nitrogen fixing bacteria. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic, London, pp 134–141Google Scholar
  15. Doebereiner J (1997) Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biol Biochem 29:771–774CrossRefGoogle Scholar
  16. Doebereiner J, Day JM, Dart PJ (1972) Nitrogenase activity in the rhizosphere of sugarcane. J Gen Microbiol 71:103–116Google Scholar
  17. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376PubMedCrossRefGoogle Scholar
  18. Fitch WM (1966) An improved method of testing for evolutionary homology. J Mol Biol 16:9–16PubMedCrossRefGoogle Scholar
  19. Fuentes-Ramirez LE, Caballero-Mellado J, Sepúlveda J, Martínez-Romero E (1999) Colonization of sugarcane by Gluconacetobacter diazotrophicus is inhibited by high N-fertilisation. FEMS Microbiol Ecol 29:117–128CrossRefGoogle Scholar
  20. Hoefsloot G, Termorshuizen AJ, Watt DA, Cramer MD (2005) The contribution of diazotrophic bacteria to the nitrogen budget of a commercially grown South African sugarcane cultivar. Plant Soil 277:85–96CrossRefGoogle Scholar
  21. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  22. Hurek T, Handley L, Reinhold-Hurek B, Piché Y (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 15:233–242PubMedCrossRefGoogle Scholar
  23. James EK, Olivares FL (1998) Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs. CRC Crit Rev Plant Sci 17:77–119CrossRefGoogle Scholar
  24. Kuykendall DL (2005) Genus I. Bradyrhizobium Jordan 1982. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s Manual of Systematic Bacteriology, 2nd edition, Volume 2, the Proteobacteria. Springer, New York, pp 438–443CrossRefGoogle Scholar
  25. Laguerre G, Nour SM, Macheret V, Sanjuan J, Drouin P, Amarger N (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiol 147:981–993Google Scholar
  26. Lima E, Boddey RM, Doebereiner J (1987) Quantification of biological nitrogen fixation associated with sugarcane using 15N aided balance. Soil Ecol Biochem 19:165–170CrossRefGoogle Scholar
  27. Ludwig W, Strunk O, Westram R, Richter L, Meier H et al (2004) ARB: a software environment for sequence data. Nucl Acids Res 32:1363–1371PubMedCrossRefGoogle Scholar
  28. Magnani GS, Didonet CM, Cruz LM, Picheth CF, Pedrosa FO, Souza EM (2010) Diversity of endophytic bacteria in Brazilian sugarcane. Genet Mol Res 9:250–258PubMedCrossRefGoogle Scholar
  29. Miller DN (2001) Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J Microbiol Meth 44:49–58CrossRefGoogle Scholar
  30. Muyzer G, Hottentraeger S, Teske A, Wawer C (1996) Denaturing gradient gel electrophoresis of PCR-ampliced 16S rDNA—a new molecular approach to analyze the genetic diversity of mixed microbial communities. In: Akkermans ADL, Van Elsas JD, De Brujin F (eds) Molecular microbial ecology manual. pp. 3.4.4/1-23. Kluwer Academic, DordrechtGoogle Scholar
  31. Olivares FL, Baldani VLD, Reis VM, Baldani JI, Doebereiner J (1996) Occurrence of endophytic diazotroph Herbaspirillum spp. in roots, stems and leaves predominantly of gramineae. Biol Fertil Soils 21:197–200CrossRefGoogle Scholar
  32. Oliveira ALM, Urquiaga S, Doebereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215CrossRefGoogle Scholar
  33. Oliveira ALM, Canuto EL, Silva EE, Reis VM, Baldani JI (2004) Survival of endophytic diazotrophic bacteria in soil under different moisture levels. Braz J Microbiol 35:295–299CrossRefGoogle Scholar
  34. Oliveira ALM, Canuto EL, Urquiaga S, Reis VM, Baldani JI (2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with endophytic diazotrophic bacteria. Plant Soil 284:23–32CrossRefGoogle Scholar
  35. Oliveira ALM, Stoffels M, Schmid M, Reis VM, Baldani JI, Hartmann A (2009) Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur J Soil Biol 45:106–113CrossRefGoogle Scholar
  36. Ormeño-Orrillo E, Rogel MA, Lloret L, López A, Martínez J, Vinuesa P, Martínez-Romero E (2009) Rhizobial diversity in different land use systems in the rain forest of Los Tuxtlas, Mexico. In: Barois I, Huising EJ, Okoth P, Trejo D, De Los Santos M (eds), Below-ground biodiversity in Sierra Santa Marta, Los Tuxtlas, Veracruz, México. Xalapa, México. p. 65–84Google Scholar
  37. Poly F, Monrozier LJ, Bally R (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol 152:95–103PubMedCrossRefGoogle Scholar
  38. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W et al (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl Acids Res 35:7188–7196PubMedCrossRefGoogle Scholar
  39. Reis VM, Estrada-de los Santos P, Tenorio-Salgado S, Vogel J, Stoffels M, Guyon S, Mavingui P, Baldani VLD, Schmid M, Baldani JI, Balandreau J, Hartmann A, Caballero-Mellado J (2004) Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium. Int J Syst Evol Microbiol 54:2155–2162PubMedCrossRefGoogle Scholar
  40. Reis VM, Urquiaga S, Pereira W, Hipolito G, De Barros JC et al (2008) Reposta de duas variedades de cana-de-acucar a inoculacao com bacterias diazotroficas. Anais do 9o Congresso Nacional da STAB Maceia: Universidade Federal de Alagoas 1: 681–686Google Scholar
  41. Reiter B, Pfeifer U, Schwab H, Sessitsch A (2002) Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica. Appl Environ Microbiol 68:2261–2268PubMedCrossRefGoogle Scholar
  42. Roesch LFW, Olivares FL, Passaglia LMP, Selbach PA et al (2006) Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply. World J Microbiol Biotechnol 22:967–974CrossRefGoogle Scholar
  43. Roesch LFW, Camargo FAO, Bento FM, Triplett EW (2008) Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 302:91–104CrossRefGoogle Scholar
  44. Ruschel AP, Henis Y, Salati E (1975) Nitrogen-15 tracing of N-fixation with soil-grown sugarcane seedlings. Soil Biol Biochem 7:181–182CrossRefGoogle Scholar
  45. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  46. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506PubMedCrossRefGoogle Scholar
  47. Sevilla M, Burris RH, Gunapala N, Kennedy C (2001) Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif—mutants strains. Mol Plant Microbe Interact 14:358–366PubMedCrossRefGoogle Scholar
  48. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101:11030–11035PubMedCrossRefGoogle Scholar
  49. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  50. Terakado-Tonooka J, Ohwaki Y, Yamakawa H, Tanaka F, Yoneyama T, Fujihara S (2008) Expressed nifH genes of endophytic bacteria detected in field-grown sweet potatoes (Ipomoea batatas L.). Microbes Environ 23:89–93PubMedCrossRefGoogle Scholar
  51. Thaweenut N, Hachisuka Y, Ando S, Yanagisawa S, Yoneyama T (2011) Two seasons’ study on nifH gene expression and nitrogen fixation by diazotrophic endophytes in sugarcane (Saccharum spp. hybrids): expression of nifH genes similar to those of rhizobia. Plant Soil 338:435–449CrossRefGoogle Scholar
  52. Toewe S, Wallisch S, Bannert A, Fischer D, Hai B, Haesler F, Kleineidam K, Schloter M (2011) Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils. J Microbiol Meth 84:406–412CrossRefGoogle Scholar
  53. Urquiaga S, Cruz KH, Boddey RM (1992) Contribution of nitrogen fixation to sugarcane: nitrogen-15 and nitrogen-balance estimates. Soil Sci Soc Am J 56:105–114CrossRefGoogle Scholar
  54. Weisburg WG, Barns SM, Pelletier DA, Lane D (1991) 16S ribosomal DNA amplification for phylogeneic study. J Bacteriol 173:697–703PubMedGoogle Scholar
  55. Yoneyama T, Muraoka T, Kim TH, Dacanay EV, Nakanishi Y (1997) The natural 15N abundance of sugarcane and neighbouring plants in Brazil, the Philippines and Miyako (Japan). Plant Soil 189:239–244CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Doreen Fischer
    • 1
  • Barbara Pfitzner
    • 1
  • Michael Schmid
    • 1
  • Jean L. Simões-Araújo
    • 2
  • Veronica M. Reis
    • 2
  • William Pereira
    • 2
  • Ernesto Ormeño-Orrillo
    • 3
  • Brigitte Hai
    • 4
  • Andreas Hofmann
    • 1
  • Michael Schloter
    • 4
  • Esperanza Martinez-Romero
    • 3
  • Jose Ivo Baldani
    • 2
  • Anton Hartmann
    • 1
  1. 1.Helmholtz Zentrum München, German Research Center for Environmental Health, Department Microbe-Plant InteractionsNeuherbergGermany
  2. 2.EMBRAPA-CNPAB SeropédicaSeropédicaBrazil
  3. 3.Centro de Ciencias GenomicasUniversidad Nacional Autonoma de Mexico, UNAMCuernavacaMexico
  4. 4.Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Soil Ecology, Department of Terrestrial EcogeneticsNeuherbergGermany

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