Microbial Ecology

, Volume 57, Issue 4, pp 611–623 | Cite as

Soil Amendment with Pseudomonas fluorescens CHA0: Lasting Effects on Soil Biological Properties in Soils Low in Microbial Biomass and Activity

  • Andreas Fließbach
  • Manuel Winkler
  • Matthias P. Lutz
  • Hans-Rudolf Oberholzer
  • Paul Mäder
Original Article


Pseudomonas fluorescens strains are used in agriculture as plant growth-promoting rhizobacteria (PGPR). Nontarget effects of released organisms should be analyzed prior to their large-scale use, and methods should be available to sensitively detect possible changes in the environments the organism is released to. According to ecological theory, microbial communities with a greater diversity should be less susceptible to disturbance by invading organisms. Based on this principle, we laid out a pot experiment with field-derived soils different in their microbial biomass and activity due to long-term management on similar parent geological material (loess). We investigated the survival of P. fluorescens CHA0 that carried a resistance toward rifampicine and the duration of potential changes of the soil microflora caused by the inoculation with the bacterium at the sowing date of spring wheat. Soil microbial biomass (Cmic, Nmic) basal soil respiration (BR), qCO2, dehydrogenase activity (DHA), bacterial plate counts, mycorrhiza root colonization, and community level substrate utilization were analyzed after 18 and 60 days. At the initial stage, soils were clearly different with respect to most of the parameters measured, and a time-dependent effect between the first and the second set point were attributable to wheat growth and the influence of roots. The effect of the inoculum was small and merely transient, though significant long-term changes were found in soils with a relatively low level of microbial biomass. Community level substrate utilization as an indicator of changes in microbial community structure was mainly changed by the growth of wheat, while other experimental factors were negligible. The sensitivity of the applied methods to distinguish the experimental soils was in decreasing order Nmic, DHA, Cmic, and qCO2. Besides the selective enumeration of P. fluorescens CHA0 rif+, which was only found in amended soils, methods to distinguish the inoculum effect were DHA, Cmic, and the ratio of Cmic to Nmic. The sampling time was most sensitively indicated by Nmic, DHA, Cmic, and qCO2. Our data support the hypothesis—based on ecosystem theory—that a rich microflora is buffering changes due to invading species. In other words, a soil-derived bacterium was more effective in a relatively poor soil than in soils that are rich in microorganisms.


  1. 1.
    Aguirre de Carcer D, Martin M, Mackova M, Macek T, Karlson U, Rivilla R (2007) The introduction of genetically modified microorganisms designed for rhizoremediation induces changes on native bacteria in the rhizosphere but not in the surrounding soil. ISME J 1:215–223CrossRefGoogle Scholar
  2. 2.
    Alexander M (1982) Most probable number method for microbial populations. In: Segoe S (ed) Methods of soil analysis part 2, chemical and microbiological properties. ASA-SSSA, Madison, US, pp 815–820Google Scholar
  3. 3.
    Anderson TH, Domsch KH (1993) The metabolic quotient for CO2 (qCO2) as a specific activity parameter to assess the effects of environmental conditions, such as pH, on the microbial biomass of forest soils. Soil Biol Biochem 25:393–395CrossRefGoogle Scholar
  4. 4.
    Compeau G, Al-Achi BJ, Platsouka E, Levy SB (1988) Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Appl Environ Microbiol 54:2432–2438PubMedGoogle Scholar
  5. 5.
    Fal R, Faw R, Rac R (1996) Referenzmethoden der Eidg. landwirtschaftlichen Forschungsanstalten - 1. Bodenuntersuchung zur Düngeberatung, Zürich-ReckenholzGoogle Scholar
  6. 6.
    Fließbach A, Oberholzer H-R, Gunst L, Mäder P (2007) Soil organic matter and biological soil quality indicators after 21 years of organic and conventional farming. Agric Ecosys Environ 118:273–284CrossRefGoogle Scholar
  7. 7.
    Fuchs J-G, Moënne-Loccoz Y, Défago G (2000) The laboratory medium used to grow biocontrol Pseudomonas sp. Pf153 influences its subsequent ability to protect cucumber from black root rot. Soil Biol Biochem 32:421–224CrossRefGoogle Scholar
  8. 8.
    Garland JL (1996) Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biol Biochem 28:213–221CrossRefGoogle Scholar
  9. 9.
    Garland JL, Mills AL (1991) Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl Environ Microbiol 57:2351–2359PubMedGoogle Scholar
  10. 10.
    Garthright WE, Blodgett RJ (2003) FDA’s preferred MPN methods for standard, large or unusual tests, with a spreadsheet. Food Microbiol 20:439–445CrossRefGoogle Scholar
  11. 11.
    Haas D, Defago G (2005) Biological control of soil-borne pathogens by fluorescent pseudomonads. Nature Rev Microbiol 3:307–319CrossRefGoogle Scholar
  12. 12.
    Joergensen RG, Mueller T (1996a) The fumigation extraction method to estimate soil microbial biomass: calibration of the kEC-factor. Soil Biol Biochem 28:25–31CrossRefGoogle Scholar
  13. 13.
    Joergensen RG, Mueller T (1996b) The fumigation extraction method to estimate soil microbial biomass: calibration of the kEN-factor. Soil Biol Biochem 28:33–37CrossRefGoogle Scholar
  14. 14.
    Keel C, Schnider U, Maurhofer M, Voisard C, Laville J, Burger U, Wirthner P, Haas D, Defago G (1992) Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol Plant-Microb Interact 5:4–13Google Scholar
  15. 15.
    King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  16. 16.
    Kloepper JW, Leong J, Teintze M, Schroth MN (1980) Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 286:885–886CrossRefGoogle Scholar
  17. 17.
    Kloepper JW, Lifshitz R, Zablotowicz RM (1989) Free-living bacterial inocula for enhancing crop productivity. Trends Biotechnol 7:39–44CrossRefGoogle Scholar
  18. 18.
    Kozdroj J (1995) Effect of genetically modified Pseudomonas fluorescens introduced into soil contaminated with copper(II) on microbial community diversity in the soil and rhizosphere. World J Microbiol Biotechnol 11:546–548CrossRefGoogle Scholar
  19. 19.
    Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883PubMedCrossRefGoogle Scholar
  20. 20.
    Mäder P, Fließbach A, Dubois D, Gunst L, Fried P, Niggli U (2002) Soil fertility and biodiversity in organic farming. Science 296:1694–1697PubMedCrossRefGoogle Scholar
  21. 21.
    Mäder P, Fließbach A, Dubois D, Gunst L, Jossi W, Widmer F, Oberson A, Frossard E, Oehl F, Wiemken A, Gattinger A, Niggli U (2006) The DOK experiment (Switzerland). In: Raupp J, Pekrun C, Oltmanns M, Köpke U (eds) Long-term field experiments in organic farming. Koester, Bonn, pp 41–58Google Scholar
  22. 22.
    Marilley L, Vogt G, Blanc M, Aragno M (1998) Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis. Plant and Soil 198:219–224CrossRefGoogle Scholar
  23. 23.
    Marx J (2004) The roots of plant–microbe collaborations. Science 304:234–236PubMedCrossRefGoogle Scholar
  24. 24.
    Mascher F, Hase C, Moenne-Loccoz Y, Defago G (2000) The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil. Appl Environ Microbiol 66:1662–1667PubMedCrossRefGoogle Scholar
  25. 25.
    Mascher F, Moenne-Loccoz Y, Schnider-Keel U, Keel C, Haas D, Defago G (2002) Inactivation of the regulatory gene algU or gacA can affect the ability of biocontrol Pseudomonas fluorescens CHA0 to persist as culturable cells in nonsterile soil. Appl Environ Microbiol 68:2085–2088PubMedCrossRefGoogle Scholar
  26. 26.
    Matos A, Kerkhof L, Garland JL (2005) Effects of microbial community diversity on the survival of Pseudomonas aeruginosa in the wheat rhizosphere. Microb Ecol 49:257–264PubMedCrossRefGoogle Scholar
  27. 27.
    Natsch A, Keel C, Hebecker N, Laasik E, Defago G (1997) Influence of biocontrol strain Pseudomonas fluorescens CHA0 and its antibiotic overproducing derivative on the diversity of resident root colonizing pseudomonads. FEMS Microbiol Ecol 23:341–352CrossRefGoogle Scholar
  28. 28.
    Natsch A, Keel C, Pfirter HA, Haas D, Defago G (1994) Contribution of the global regulator gene gacA to persistence and dissemination of Pseudomonas fluorescens biocontrol strain CHA0 introduced into soil microcosms. Appl Environ Microbiol 60:2553–2560PubMedGoogle Scholar
  29. 29.
    Natsch A, Keel C, Troxler J, Zala M, Von Albertini N, Defago G (1996) Importance of preferential flow and soil management in vertical transport of a biocontrol strain of Pseudomonas fluorescens in structured field soil. Appl Environ Microbiol 62:33–40PubMedGoogle Scholar
  30. 30.
    Nichols D (2007) Cultivation gives context to the microbial ecologist. FEMS Microbiol Ecol 60:351–357PubMedCrossRefGoogle Scholar
  31. 31.
    Oehl F, Sieverding E, Ineichen K, Mäder P, Boller T, Wiemken A (2003) Impact of land use intensity on the species diversity of arbuscular mycorrhizal fungi in agro-ecosystems of central Europe. Appl Environ Microbiol 69:2816–2824PubMedCrossRefGoogle Scholar
  32. 32.
    Paul EA, Clark FE (1996) Soil microbiology and biochemistry. Academic, San Diego, p 340, CalGoogle Scholar
  33. 33.
    Phillips JM, Hayman DS (1970) Improved procedures for clearing roots and staining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–160CrossRefGoogle Scholar
  34. 34.
    Raaijmakers JM, Weller DM, Thomashow LS (1997) Frequency of antibiotic-producing Pseudomonas ssp. in natural environments. Appl Environ Microbiol 63:881–887PubMedGoogle Scholar
  35. 35.
    Ritz K (2007) The plate debate: cultivable communities have no utility in contemporary environmental microbial ecology. FEMS Microbiol Ecol 60:358–362PubMedCrossRefGoogle Scholar
  36. 36.
    Schwieger F, Tebbe CC (2000) Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album) linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria. Appl Environ Microbiol 66:3556–3565PubMedCrossRefGoogle Scholar
  37. 37.
    Schwieger F, Willke B, Munch JC, Tebbe CC (1997) Ecological pre-release risk assessment of two genetically engineered, bioluminescent Rhizobium meliloti strains in soil column model systems. Biol Fertil Soils 25:340–348CrossRefGoogle Scholar
  38. 38.
    Speiser B, Tamm L, Maurer V, Berner A, Walkenhorst M, Böhler K, Früh B, Chevillat V (2007) Hilfsstoffliste 2007. Zugelassene und empfohlene Hilfsstoffe für den biologischen Landbau. FiBL, Frick, p 80Google Scholar
  39. 39.
    Steddom K, Menge JA, Crowley D, Borneman J (2002) Effect of repetitive applications of the biocontrol bacterium Pseudomonas putida 06909-rif/nal on citrus soil microbial communities. Phytopathology 92:857–862PubMedCrossRefGoogle Scholar
  40. 40.
    Stutz E, Defago G, Kern H (1986) Naturally occurring fluorescent pseudomonads involved in suppression of black root rot of tobacco. Phytopathology 76:181–185CrossRefGoogle Scholar
  41. 41.
    Tabatabai MA (1982) Soil Enzymes. In: Page AL, Miller RH, Keeney DR (eds) Methods of Soil Analysis, Part 2 Chemical and Microbiological Properties. American Society of Agronomy & Soil Science Society of America, Madison, Wisconsin, pp 903–947Google Scholar
  42. 42.
    Tebbe CC (2003) Dissemination of genetically engineered microorganisms in terrestrial ecosystems: case studies for identifying risk potentials. In: Ecological impact of GMO dissemination in agro-ecosystems, Grossrussbach, Austria, September 27–28, 2002, 2003. OECD (ed.), pp 31–44Google Scholar
  43. 43.
    Vahjen W, Munch J-C, Tebbe CC (1997) Fate of three genetically engineered, biotechnologically important microorganism species in soil: impact of soil properties and intraspecies competition with non-engineered strains. Can J Microbiol 43:827–834CrossRefGoogle Scholar
  44. 44.
    Vahjen W, Munch JC, Tebbe CC (1995) Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level. FEMS Microbiol Ecol 18:317–328CrossRefGoogle Scholar
  45. 45.
    Vance ED, Brookes PC, Jenkinson DS (1987) An extraction method for measuring soil microbial biomass C. Soil Biol Biochem 19:703–707CrossRefGoogle Scholar
  46. 46.
    Vogel (1922) Impfung von Hülsenfrüchten und Nichtleguminosen. Z Pflanzenern Düng 1:531–535Google Scholar
  47. 47.
    Weller DM, Raaijmakers JM, McSpadden Gardener BB, Thomashow LS (2002) Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu Rev Phytopathol 40:309–348PubMedCrossRefGoogle Scholar
  48. 48.
    Widmer F, Rasche F, Hartmann M, Fließbach A (2006) Community structures and substrate utilization of bacteria in soils from organic and conventional farming systems of the DOK long-term field experiment. Appl Soil Ecol 33:294–307CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Andreas Fließbach
    • 1
  • Manuel Winkler
    • 1
    • 4
  • Matthias P. Lutz
    • 2
  • Hans-Rudolf Oberholzer
    • 3
  • Paul Mäder
    • 1
  1. 1.Research Institute of Organic Agriculture (FiBL)AckerstrasseSwitzerland
  2. 2.Plant Pathology Group, Institute of Plant SciencesSwiss Federal Institute of TechnologyZurichSwitzerland
  3. 3.Agroscope Reckenholz-Tänikon Research Station (ART)ZürichSwitzerland
  4. 4.Institute HISCIAArlesheimSwitzerland

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