Microbial Ecology

, Volume 53, Issue 3, pp 399–413 | Cite as

Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil

  • Karin Hjort
  • Antje Lembke
  • Arjen Speksnijder
  • Kornelia Smalla
  • Janet K. Jansson


The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.



This work was funded by the EU Project, METACONTROL (QLK 3-2002-2068). Plasmodiophora brassicae spores were kindly provided by Hanna Friberg, Department of Ecology and Crop Production Science, Swedish University of Agricultural Sciences, Uppsala, Sweden.


  1. 1.
    Artursson, V, Finlay, RD, Jansson, JK (2005) Combined bromodeoxyuridine immunocapture and terminal restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation of plant species. Environ Microbiol 7: 1952–1966PubMedCrossRefGoogle Scholar
  2. 2.
    Artursson, V, Jansson, JK (2003) Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae. Appl Environ Microbiol 69: 6208–6215PubMedCrossRefGoogle Scholar
  3. 3.
    Bach, HJ, Tomanova, J, Schloter, M, Munch, JC (2002) Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification. J Microbiol Methods 49: 235–245PubMedCrossRefGoogle Scholar
  4. 4.
    Berg, G, Opelt, K, Zachow, C, Lottmann, J, Götz, M, Costa, R, Smalla, K (2006) The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site. FEMS Microbiol Ecol Online publication date: 2 Nov 2005 doi:10.1111/j.1574-6941.2005.00025.xGoogle Scholar
  5. 5.
    Berg, G, Roskot, N, Steidle, A, Eberl, L, Zock, A, Smalla, K (2002) Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants. Appl Environ Microbiol 68: 3328–3338PubMedCrossRefGoogle Scholar
  6. 6.
    Borneman, J (1999) Culture-independent identification of microorganisms that respond to specified stimuli. Appl Environ Microbiol 65: 3398–3400PubMedGoogle Scholar
  7. 7.
    Cole, JR, Chai, B, Farris, RJ, Wang, Q, Kulam, SA, McGarrell, DM, Garrity, GM, Tiedje, JM (2005) The high-throughput rRNA analysis. Nucleic Acids Res 1(Database Issue): D294–D296Google Scholar
  8. 8.
    Chernin, LS, De la Fuente, L, Sobolev, V, Haran, S, Vorgias, CE, Oppenheim, AB, Chet, I (1997) Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Appl Environ Microbiol 63: 834–839PubMedGoogle Scholar
  9. 9.
    Downing, KJ, Thomson, JA (2000) Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi. Can J Microbiol 46: 363–369PubMedCrossRefGoogle Scholar
  10. 10.
    Girvan, MS, Bullimore, J, Pretty, JN, Osborn, AM, Ball, AS (2003) Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils. Appl Environ Microbiol 69: 1800–1809PubMedCrossRefGoogle Scholar
  11. 11.
    Gomes, NC, Costa, MR, Smalla, K (2004) Simultaneous extraction of DNA and RNA from bulk and rhizosphere soil. In: Akkermans, ADL, van Elsas, JD, de Brujin, FJ (Eds.) Molecular Microbial Ecology Manual, 2nd ed. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 159–169Google Scholar
  12. 12.
    Gooday, GW (1990) The ecology of chitin degradation. Adv Microb Ecol 11: 387–430Google Scholar
  13. 13.
    Hamasaki, K, Long, RA, Azam, F (2004) Individual cell growth rates of marine bacteria, measured by bromodeoxyuridine incorporation. Aquat Microb Ecol 35: 217–227Google Scholar
  14. 14.
    Heuer, H, Krsek, M, Baker, P, Smalla, K, Wellington, EMH (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63: 3233–3241PubMedGoogle Scholar
  15. 15.
    Heuer, H, Smalla, K (1997) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) for studying soil microbial communities. In: Van Elsas, JD, Trevors, JT, Wellington, EMH (Eds.) Modern Soil Microbiology. Marcel Dekker, New York, pp 353–373Google Scholar
  16. 16.
    Heuer, H, Wieland, G, Schönfeld, J, Schönwälder, A, Gomes, NCM, Smalla, K (2001) Bacterial community profiling using DGGE or TGGE analysis. In: Rouchelle, P (Ed.) Environmental Molecular Microbiology: Protocols and Applications. Horizon Scientific Press, Wymondham, UK, pp 177–190Google Scholar
  17. 17.
    Inglis, GD, Kawchuk, LM (2002) Comparative degradation of oomycete, ascomycete, and basidiomycete cell walls by mycoparasitic and biocontrol fungi. Can J Microbiol 48: 60–70PubMedCrossRefGoogle Scholar
  18. 18.
    Kitamura, E, Kamei, Y (2003) Molecular cloning, sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp. PE2, and its domain structure. Appl Microbiol Biotechnol 61: 140–149PubMedGoogle Scholar
  19. 19.
    Kobayashi, DY, Reedy, RM, Bick, J, Oudemans, PV (2002) Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control. Appl Environ Microbiol 68: 1047–1054PubMedCrossRefGoogle Scholar
  20. 20.
    Krechel, A, Faupel, A, Hallmann, J, Ulrich, A, Berg, G (2002) Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood. Can J Microbiol 48: 772–786PubMedCrossRefGoogle Scholar
  21. 21.
    Krsek, M, Wellington, EM (2001) Assessment of chitin decomposer diversity within an upland grassland. Antonie Van Leeuwenhoek 79: 261–267PubMedCrossRefGoogle Scholar
  22. 22.
    Liu, WT, Marsh, TL, Cheng, H, Forney, LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63: 4516–4522PubMedGoogle Scholar
  23. 23.
    Mann, JW, Heintz, CE, Macmillan, JD (1972) Yeast spheroplasts formed by cell wall-degrading enzymes from Oerskovia sp. J Bacteriol 111: 821–824PubMedGoogle Scholar
  24. 24.
    Mazzola, M (2002) Mechanisms of natural soil suppressiveness to soilborne diseases. Antonie Van Leeuwenhoek 81: 557–564PubMedCrossRefGoogle Scholar
  25. 25.
    Metcalfe, AC, Krsek, M, Gooday, GW, Prosser, JI, Wellington, EM (2002) Molecular analysis of a bacterial chitinolytic community in an upland pasture. Appl Environ Microbiol 68: 5042–5050PubMedCrossRefGoogle Scholar
  26. 26.
    Milling, A, Smalla, K, Maidl, FX, Schloter, M, Munch, JC (2004) Effects of transgenic potatoes with an altered starch composition on the diversity of soil and rhizosphere bacteria and fungi. Plant Soil 266: 23–39CrossRefGoogle Scholar
  27. 27.
    Moxham, SE, Buczacki, ST (1983) Chemical composition of the resting spore wall of Plasmodiophora brassicae. Trans Br Mycol Soc 80: 291–304Google Scholar
  28. 28.
    Murakami, H, Tsushima, S, Shishido, Y (2000) Soil suppressiveness to clubroot disease of Chinese cabbage caused by Plasmodiophora brassicae. Soil Biol Biochem 32: 1637–1642CrossRefGoogle Scholar
  29. 29.
    Muyzer, G, Smalla, K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73: 127–141PubMedCrossRefGoogle Scholar
  30. 30.
    Muyzer, G, Teske, A, Wirsen, CO, Jannasch, HW (1995) Phylogenetic relationship of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164: 165–172PubMedGoogle Scholar
  31. 31.
    Narisawa, K, Hashiba, T (1998) Development of resting spores on plates inoculated with a dikaryotic resting spore Plasmodiophora brassicae. Mycol Res 102: 949–952CrossRefGoogle Scholar
  32. 32.
    Pernthaler, A, Pernthaler, J, Schattenhofer, M, Amann, R (2002) Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton. Appl Environ Microbiol 68: 5728–5736PubMedCrossRefGoogle Scholar
  33. 33.
    Rademaker, JLW, Louws, FJ, Rossbach, U, Vinuesa, P, de Bruijn, FJ (1998) Computer-assisted pattern analysis of molecular fingerprints and database construction. In: Akkermans, ADL, van Elsas, JD, de Bruijn, FJ (Eds.) Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands, 7.1.3Google Scholar
  34. 34.
    Radajewski, S, Ineson, P, Parekh, NR, Murrell, JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403: 646–649PubMedCrossRefGoogle Scholar
  35. 35.
    Reguera, G, Leschine, SB (2003) Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda. Arch Microbiol 180: 434–443PubMedCrossRefGoogle Scholar
  36. 36.
    Sessitsch, A, Gyamfi, S, Stralis-Pavese, N, Weilharter, A, Pfeifer, U (2002) RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols. J Microbiol Methods 51: 171–179PubMedCrossRefGoogle Scholar
  37. 37.
    Sindhu, SS, Dadarwal, KR (2001) Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea. Microbiol Res 156: 353–358PubMedCrossRefGoogle Scholar
  38. 38.
    Smalla, K (2004) Culture-independent microbiology. In: Bull, AT (Ed.) Microbial Diversity and Bioprospecting. ASM Press, Washington DC, pp 88–99Google Scholar
  39. 39.
    Smalla, K, Wieland, A, Buchner, A, Zock, A, Parzy, S, Kaiser, S, Roskot, N, Heuer, H, Berg, G (2001) Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol 67: 4742–4751PubMedCrossRefGoogle Scholar
  40. 40.
    Tukey, JW (1953) Some selected quick and easy methods of statistical analysis. Trans NY Acad Sci 16: 88–97Google Scholar
  41. 41.
    Urbach, E, Vergin, KL, Giovannoni, SJ (1999) Immunochemical detection and isolation of DNA from metabolically active bacteria. Appl Environ Microbiol 65: 1207–1213PubMedGoogle Scholar
  42. 42.
    Wang, SL, Chang, WT (1997) Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol 63: 380–386PubMedGoogle Scholar
  43. 43.
    Weisburg, WG, Barns, SM, Pelletier, DA, Lane, DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173: 697–703PubMedGoogle Scholar
  44. 44.
    Weller, DM, Raaijmakers, JM, Gardener, BB, Thomashow, LS (2002) Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu Rev Phytopathol 40: 309–348PubMedCrossRefGoogle Scholar
  45. 45.
    Williamson, N, Brian, P, Wellington, EM (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie Van Leeuwenhoek 78: 315–321PubMedCrossRefGoogle Scholar
  46. 46.
    Worku, Y, Gerhardson, B (1996) Suppressiveness to clubroot, pea root and Fusarium wilt in Swedish soils. J Phytopathol 144: 143–146Google Scholar
  47. 47.
    Yin, B, Crowley, D, Sparovek, G, De Melo, WJ, Borneman, J (2000) Bacterial functional redundancy along a soil reclamation gradient. Appl Environ Microbiol 66: 4361–4365PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Karin Hjort
    • 1
  • Antje Lembke
    • 2
  • Arjen Speksnijder
    • 3
  • Kornelia Smalla
    • 2
  • Janet K. Jansson
    • 1
  1. 1.Department of MicrobiologySwedish University of Agricultural SciencesUppsalaSweden
  2. 2.Federal Biological Research Centre for Agriculture and Forestry (BBA)BraunschweigGermany
  3. 3.Plant Research International B.V.WageningenThe Netherlands

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