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Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

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Abstract

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

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References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  2. Andrews JH, Harris RF (2000) The ecology and biogeography of microorganisms on plant surfaces. Annu Rev Phytopathol 38:145–180

    Article  PubMed  Google Scholar 

  3. Araújo WL, Marcon J, Maccheroni WJ, Van Elsas JD, Van Vuurde JWL, Azevedo JL (2002) Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914

    Article  PubMed  CAS  Google Scholar 

  4. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2006) New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microbiol 72:5734–5741

    Article  PubMed  CAS  Google Scholar 

  5. Becker R, Behrendt U, Hommel B, Kropf S, Ulrich A (2008) Effects of transgenic fructan-producing potatoes on the community structure of rhizosphere and phyllosphere bacteria. Fems Microbiol Ecol 66:411–425

    Article  PubMed  CAS  Google Scholar 

  6. Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlini C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol 70:6147–6156

    Article  PubMed  CAS  Google Scholar 

  7. Carroll BJ, McNeil DL, Gresshoff PM (1985) Isolation and properties of soybean [Glycine max (L.) Merr.] mutants that nodulate in the presence of high nitrate concentrations. Proc Natl Acad Sci U S A 82:4162–4166

    Article  PubMed  CAS  Google Scholar 

  8. Chelius MK, Triplett EW (2001) The diversity of Archaea and Bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263

    PubMed  CAS  Google Scholar 

  9. Cole JR, Chai B, Marsh TL, Farris RJ, Wang Q, Kulam SA, Chandra S, McGarrell DM, Schmidt TM, Garrity GM, Tiedje JM et al (2003) The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31:442–443

    Article  PubMed  CAS  Google Scholar 

  10. Deamer DW, Crofts A (1967) Action of Triton X-100 on chloroplast membranes. Mechanisms of structural and functional disruption. J Cell Biol 33:395–410

    Article  PubMed  CAS  Google Scholar 

  11. Dent KC, Stephen JR, Finch-Savage WE (2004) Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet). J Microbiol Methods 56:17–26

    Article  PubMed  CAS  Google Scholar 

  12. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  13. Felsenstein J (1989) PHYLIP—phylogeny of inference package (version 3.2). Cladistics 5

  14. Gagne S, Richard C, Roussean H, Antoun H (1987) Xylem-residing bacteria in alfalfa roots. Can J Microbiol 33:996–1000

    Article  Google Scholar 

  15. Garbeva P, Overbeek LS, Vuurde JW, Elsas JD (2001) Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. Microb Ecol 41:369–383

    PubMed  CAS  Google Scholar 

  16. Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biometrika 40:237–264

    Google Scholar 

  17. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914

    CAS  Google Scholar 

  18. Hung PQ, Kumar SM, Govindsamy V, Annapurna K (2007) Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties. Biol Fertil Soils 44:155–162

    Article  Google Scholar 

  19. Idris R, Trifonova R, Puschenreiter M, Wenzel WW, Sessitsch A (2004) Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense. Appl Environ Microbiol 70:2667–2677

    Article  PubMed  CAS  Google Scholar 

  20. Ikeda S, Rallos LEE, Okubo T, Eda S, Inaba S, Mitsui H, Minamisawa K (2008) Microbial community analysis of field-grown soybeans with different nodulation phenotypes. Appl Environ Microbiol 74:5704–5709

    Article  PubMed  CAS  Google Scholar 

  21. Ikeda S, Tsurumaru H, Wakai S, Noritake C, Fujishiro K, Akasaka M, Ando K (2008) Evaluation of the effects of different additives in improving the DNA extraction yield and quality from Andosol. Microbes Environ 23:159–166

    Article  Google Scholar 

  22. Ikeda S, Watanabe KN, Minamisawa K, Ytow N (2004) Evaluation of soil DNA from arable land in Japan using a modified direct-extraction method. Microbes Environ 19:301–309

    Article  Google Scholar 

  23. Jiao J-Y, Wang H-X, Zeng Y, Shen Y-M (2006) Enrichment for microbes living in association with plant tissues. J Appl Microbiol 100:830–837

    Article  PubMed  Google Scholar 

  24. Kadivar H, Stapleton AE (2003) Ultraviolet radiation alters maize phyllosphere bacterial diversity. Microb Ecol 45:353–361

    Article  PubMed  CAS  Google Scholar 

  25. Kemp PF, Aller JY (2004) Bacterial diversity in aquatic and other environments: what 16S rDNA libraries can tell us. Fems Microbiol Ecol 47:161–177

    Article  PubMed  CAS  Google Scholar 

  26. Kent AD, Triplett EW (2002) Microbial communities and their interactions in soil and rhizosphere ecosystems. Annu Rev Microbiol 56:211–236

    Article  PubMed  CAS  Google Scholar 

  27. Kerkhof L, Santoro M, Garland J (2000) Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis. FEMS Microbiol Lett 184:95–101

    Article  PubMed  CAS  Google Scholar 

  28. Kuklinsky-Sobral J, Araujo WL, Mendes R, Geraldi IO, Pizzirani-Kleiner AA, Azevedo JL (2004) Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ Microbiol 6:1244–1251

    Article  PubMed  CAS  Google Scholar 

  29. Leveau JHJ (2007) The magic and menace of metagenomics: prospects for the study of plant growth-promoting rhizobacteria. Eur J Plant Pathol 119:279–300

    Article  CAS  Google Scholar 

  30. Lindahl V, Bakken LR (1995) Evaluation of methods for extraction of bacteria from soil. Fems Microbiol Ecol 16:135–142

    Article  CAS  Google Scholar 

  31. Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883

    Article  PubMed  CAS  Google Scholar 

  32. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, Van der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606

    Article  Google Scholar 

  33. Meixner C, Vegvari G, Ludwig-Müller J, Gagnon H, Steinkellner S, Staehelin C, Gresshoff P, Vierheilig H (2007) Two defined alleles of the LRR receptor kinase GmNARK in supernodulating soybean govern differing autoregulation of mycorrhization. Physiol Plant 130:261–270

    Article  CAS  Google Scholar 

  34. Normander B, Prosser JI (2000) Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions. Appl Environ Microbiol 66:4372–4377

    Article  PubMed  CAS  Google Scholar 

  35. Offre P, Pivato B, Siblot S, Gamalero E, Corberand T, Lemanceau P, Mougel C (2007) Identification of bacterial groups preferentially associated with mycorrhizal roots of Medicago truncatula. Appl Environ Microbiol 73:913–921

    Article  PubMed  CAS  Google Scholar 

  36. Oka-Kira E, Kawaguchi M (2006) Long-distance signaling to control root nodule number. Curr Opin Plant Biol 9:496–502

    Article  PubMed  CAS  Google Scholar 

  37. Okubo T, Ikeda S, Kaneko T, Eda S, Mitsui H, Sato S, Tabata S, Minamisawa K (2009) Nodulation-dependent communities of culturable bacterial endophytes from stems of field-grown soybeans. Microbes Environ doi:10.1264/jsme2.ME09125

  38. Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Bioinformatics 12:357–358

    Article  CAS  Google Scholar 

  39. Pimentel IC, Glienke-Blanco C, Gabardo J, Stuart RM, Azevedo JL (2006) Identification and colonization of endophytic fungi from soybean (Glycine max (L.) Merril) under different environmental conditions. Braz Arch Biol Technol 49:705–711

    Article  Google Scholar 

  40. Rasche F, Hödl V, Poll C, Kandeler E, Gerzabek MH, Van Elsas JD, Sessitsch A (2006) Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure. Fems Microbiol Ecol 56:219–235

    Article  PubMed  CAS  Google Scholar 

  41. Rasche F, Trondl R, Naglreiter C, Reichenauer TG, Sessitsch A (2006) Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum annum L.). Can J Microbiol 52:1036–1045

    Article  PubMed  CAS  Google Scholar 

  42. Reiter B, Sessitsch A (2006) Bacterial endophytes of the wildflower Crocus albiflorus analyzed by characterization of isolates and by a cultivation-independent approach. Can J Microbiol 52:140–149

    Article  PubMed  CAS  Google Scholar 

  43. Reiter B, Wermbter N, Gyamfi S, Schwab H, Sessitsch A (2003) Endophytic Pseudomonas spp. populations of pathogen-infected potato plants analysed by 16S rDNA- and 16S rRNA-based denaturating gradient gel electrophoresis. Plant Soil 257:397–405

    Article  CAS  Google Scholar 

  44. Rickwood D, Ford T, Graham J (1982) Nycodenz: a new nonionic iodinated gradient medium. Anal Biochem 123:23–31

    Article  PubMed  CAS  Google Scholar 

  45. Saito A, Ikeda S, Ezura H, Minamisawa K (2007) Microbial community analysis of the phytosphere using culture-independent methodologies. Microbes Environ 22:93–105

    Article  Google Scholar 

  46. Saito A, Kawahara M, Ikeda S, Ishimine M, Akao S, Minamisawa K (2008) Broad distribution and phylogeny of anaerobic endophytes of cluster XIVa clostridia in plant species including crops. Microbes Environ 23:73–80

    Article  Google Scholar 

  47. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  48. Sato S, Kotani H, Nakamura Y, Kaneko T, Asamizu E, Fukami M, Miyajima N, Tabata S (1997) Structural analysis of Arabidopsis thaliana chromosome 5. I. Sequence features of the 1.6 Mb regions covered by twenty physically assigned P1 clones. DNA Res 4:215–219

    Article  PubMed  CAS  Google Scholar 

  49. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506

    Article  PubMed  CAS  Google Scholar 

  50. Schloss PD, Handelsman J (2006) Introducing SONS, a tool for operational taxonomic unit-based comparisons of microbial community memberships and structures. Appl Environ Microbiol 72:6773–6779

    Article  PubMed  CAS  Google Scholar 

  51. Seghers D, Wittebolle L, Top EM, Verstraete W, Siciliano SD (2004) Impact of agricultural practices on the Zea mays L. endophytic community. Appl Environ Microbiol 70:1475–1482

    Article  PubMed  CAS  Google Scholar 

  52. Sessitsch A, Reiter B, Peifer U, Wilhelm E (2002) Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. Fems Microbiol Ecol 39:23–32

    Article  PubMed  CAS  Google Scholar 

  53. Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25:13–19

    Article  Google Scholar 

  54. 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 25:4876–4882

    Article  PubMed  CAS  Google Scholar 

  55. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: 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

    Article  PubMed  CAS  Google Scholar 

  56. Tian X, Cao L, Tan H, Han W, Chen M, Liu Y, Zhou S (2007) Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. Microb Ecol 53:700–707

    Article  PubMed  Google Scholar 

  57. Ulrich K, Ulrich A, Ewald D (2008) Diversity of endophytic bacterial communities in poplar grown under field conditions. Fems Microbiol Ecol 63:169–180

    Article  PubMed  CAS  Google Scholar 

  58. Wang HX, Geng ZL, Zeng Y, Shen YM (2008) Enriching plant microbiota for a metagenomic library construction. Environ Microbiol 10:2684–2691

    Article  PubMed  CAS  Google Scholar 

  59. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267

    Article  PubMed  CAS  Google Scholar 

  60. Yang CH, Crowley DE, Borneman J, Keen NT (2001) Microbial phyllosphere populations are more complex than previously realized. Proc Natl Acad Sci U S A 98:3889–3894

    Article  PubMed  CAS  Google Scholar 

  61. Zhou J, Xia B, Treves DS, Wu LY, Marsh TL, O'Neill RV, Palumbo AV, Tiedje JM (2002) Spatial and resource factors influencing high microbial diversity in soil. Appl Environ Microbiol 68:326–334

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by Special Coordination Funds for Promoting Science and Technology, by Grant-in-Aid for Scientific Research (no. 17658034) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a grant of Genomics for Agricultural Innovation (PMI-0002) from Ministry of Agriculture, Forestry and Fisheries of Japan.

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Authors and Affiliations

  1. Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan

    Seishi Ikeda, Takashi Okubo, Lynn E. E. Rallos, Shima Eda, Hisayuki Mitsui & Kiwamu Minamisawa

  2. Kazusa DNA Research Institute, 2-6-7 Kazusakamatari, Kisarazu, Chiba, 292-0818, Japan

    Takakazu Kaneko, Shusei Sato, Yasukazu Nakamura & Satoshi Tabata

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  1. Seishi Ikeda
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  2. Takakazu Kaneko
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  3. Takashi Okubo
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  4. Lynn E. E. Rallos
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Correspondence to Seishi Ikeda.

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Supplementary Fig. 1

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Supplementary Table 1

Sequence similarities of RISA amplicons to known species (gif 149 kb)

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Ikeda, S., Kaneko, T., Okubo, T. et al. Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems. Microb Ecol 58, 703–714 (2009). https://doi.org/10.1007/s00248-009-9566-0

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