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Culture-independent detection of changes in root-associated bacterial populations of common bean (phaseolus vulgaris L.) following nitrogen depletion

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Abstract

The structure of root-associated bacterial populations in the legume common bean (Phaseolus vulgaris L.), was studied in plants grown under nitrogen sufficiency and under conditions inducing nitrogen deficiency. Similar cell numbers were obtained in the rhizosphere of nitrogen-amended plants as compared to nitrogen-deficient plants and between various root parts—tip, elongation and branching zones—using DAPI staining. In contrast, a higher proportion of DAPI-stained cells from the nitrogen-amended plants hybridized with a fluorescence-labeled EUB338 probe for theBacteria domain than cells originating from nitrogen-deficient plants. Shifts in the percentages of EUB338-reactive cells—as well as in absolute cell number—hybridizing to fluorescent rRNA-directed probes specific for the α and γProteobacteria and for high GC content gram-positive bacteria in separated root segments were detected between the treatments. No such differences were found using β and δProteobacteria or rRNA group I pseudomonad targeted probes. Denaturating gradient gel electrophoresis profiles of PCR products obtained from the same samples and amplified withBacteria-domain targeted primers supported the results obtained with the whole cell hybridizations. The advantages and drawbacks of the techniques applied are discussed.

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References

  1. Allen ON, Allen EK (1981) The Leguminosae. The University of Wisconsin Press, Madison, WI

    Google Scholar 

  2. Amann R, Binder BJ, Olson RJ, Chishlolm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925

    PubMed  CAS  Google Scholar 

  3. Barcina I, Arana I, Santorum P, Iriberi J, Egea L (1995) Direct viable counts of Gram-positive and Gram-negative bacteria using ciprophloxacin as inhibitor of cellular division. J Microbiol Met 22:139–150

    Article  Google Scholar 

  4. Burdman S, Jurkevitch E, Okon Y (2000) Recent advances in the use of plant growth promoting rhizobacteria (PGPR) in agriculture. In: Subba Rao NS, Dommergures YR (eds) Microbial Interactions in Agriculture and Forestry, Vol 2. Science Publishers, Inc, Enfield, NH, in press

    Google Scholar 

  5. Burdman S, Sarig S, Kigel J, Okon Y (1996) Field inoculation of common bean (Phaseolus vulgaris L.), and chick pea (Cicer arietinum L.) withAzospirillum brasilense strain Cd. Symbiosis 21:41–48

    Google Scholar 

  6. Chanway CP, Turkington R, Holl FB (1991) Ecological implications of specificity between plants and rhizosphere micro-organisms. Adv Ecol Res 21:121–169

    Google Scholar 

  7. Christensen H, Hansen M, Søresen J (1999) Counting and size classification of active soil bacteria by fluorescencein-situ hybridization with an rRNA oligonucleotide probe. Appl Environ Microbiol 65:1753–1761

    PubMed  CAS  Google Scholar 

  8. Curl EA, Truelove B (1986) The Rhizosphere. Springer-Verlag, Berlin

    Google Scholar 

  9. de Freitas JR, Guspta VVSR, Germida JJ (1993) Influence ofPseudomonas syringae R25 andP. putida R105 on the growth and nitrogen fixation (acetylene reduction activity) of pea (Pisum sativum L.) and field bean (Phaseolus vulgaris L.). Biol Fert Soils 16:215–220

    Article  Google Scholar 

  10. Duineveld BM, Rosado AS, van Elsas JD, van Veen JA (1998) Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturating gradient gel electrophoresis and substrate utilization pattern. Appl Environ Microbiol 64:4950–4957

    PubMed  CAS  Google Scholar 

  11. Frederick BA, Klein DA (1994) Nitrogen effects on rhizosphere processes of range grasses from different successional series. Plant Soil 161:241–250

    Article  Google Scholar 

  12. Fuchs BM, Wallner G, Beisker W, Schwippl I, Ludwig W, Amann R (1998) Flow cytometric analysis of thein situ accessibility ofEscherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 64:4973–4982

    PubMed  CAS  Google Scholar 

  13. Fuhrmann J, Wollum II AG (1989) In vitro growth response ofBradyrhizobium japonicum to soybean rhizosphere bacteria. Soil Biol Biochem 21:131–135

    Article  Google Scholar 

  14. Hahn D, Smalla K, Zeyer J (1997). Whole cell hybridization as a tool to studyFrankia populations in root nodules. Physiol Plant 99:696–706

    Article  CAS  Google Scholar 

  15. Hines ME, Evans RS, Sharak Genthner BR, Willis SG, Friedman S, Rooney-Varga JN, Devereux R (1999) Molecular phylogenetic and biochemical studies of sulfate-reducing bacteria in the rhizosphere ofSpartina alterniflora. Appl Environ Microbiol 65:2209–2216

    PubMed  CAS  Google Scholar 

  16. Hiorns WD, Methé BA, Nierzwicki-Bauer SA, Zehr JP (1997) Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences. Appl Environ Microbiol 63:2957–2960

    PubMed  CAS  Google Scholar 

  17. Hirch AM (1992) Development l biology of legume nodulation. New Phytol 122:211–327

    Article  Google Scholar 

  18. Ibijbijen J, Urquiaga S, Ismaili M, Alves BJR, Boddey RM (1996) Effects of arbuscular mycorrhizal fungi on growth, mineral nutrition and nitrogen fixation of three varieties of common bean (Phaseolus vulgaris). New Phytol 134:353–360

    Article  CAS  Google Scholar 

  19. Jurkevitch E, Minz D, Ramati B, Barel G (2000) Prey range characterization, ribotyping and diversity of soil and rhizosphereBdellovibrio spp. isolated on phytopathogenic bacteria. Appl Environ Microbiol 66:2365–2371

    Article  PubMed  CAS  Google Scholar 

  20. Kepner Jr RL, Pratt JR (1994) Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol Rev 58:603–615

    PubMed  CAS  Google Scholar 

  21. Kim J-S, Sakai M, Hosoda A, Matsuguchi T (1999) Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil. Soil Sci Plant Nutr 45:493–497

    CAS  Google Scholar 

  22. Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots by bacteria. Can J Microbiol 38:1219–1232

    Google Scholar 

  23. Knowlton S, Berry A, Torrey JG (1980) Evidence that associated soil bacteria may influence root hair infection of actinorhizal plants byFrankia. Can J Microbiol 26:971–977

    Article  PubMed  CAS  Google Scholar 

  24. Kolb W, Martin P (1988) Influence of nitrogen on the number of N2-fixing and total bacteria in the rhizosphere. Soil Biol Biochem 20:221–225

    Article  CAS  Google Scholar 

  25. Kuske CR, Barns SM, Busch JD (1997) Diverse uncultivated bacterial groups from soils of the arid soutwestern United States that are present in many geographic regions. Appl Environ Microbiol 63:3614–1621

    PubMed  CAS  Google Scholar 

  26. LaRue TA, Patterson TG (1981) How much nitrogen do legumex fix? Adv Agron 34:15–38

    CAS  Google Scholar 

  27. Liljeroth E, Bååth E, Mathiasson I, Lundborg T (1990) Root exudation and rhizoplane bacterial abundance of barley (Hordeum vulgare L.) in relation to nitrogen fertilization and root growth. Plant Soil 127:81–89

    Article  CAS  Google Scholar 

  28. Ludwig W, Amann R, Martinez-Romero E, Schönhuber W, Bauer S, Neef A, Schleifer K-H (1998) rRNA based identification and detection systems for rhizobia and other bacteria. Plant Soil 204:1–19

    Article  CAS  Google Scholar 

  29. Maloney PE, van Bruggen AHC, Hu S (1997) Bacterial community structure in relation to the carbon environment in lettuce and tomato rhizospheres and in bulk soil. Microb Ecol 34:109–117 DOI: 10.1007/s002489900040

    Article  PubMed  CAS  Google Scholar 

  30. Manz W, Wagner M, Amann R, Ludwig W, Schleifer K-H (1994)In situ characterization of the microbial consortia active in two wastewater treatment plants. Wat Res 8:1715–1723

    Article  Google Scholar 

  31. Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H (1992) Phylogenetic oligonucleotide probes for the major subclasses ofProteobacteria: Problems and solutions. System. Appl Microbiol 15:593–600

    Google Scholar 

  32. Muyzer G, Teske A, Wirsen CO, Jannash HW (1995) Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturating gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164:165–172 DOI: 10.1007/s002030050250

    PubMed  CAS  Google Scholar 

  33. Peoples MB, Herridge DF (1990) Nitrogen fixation by legumes in tropical and subtropical agriculture. Adv Agron 44:155–223

    Article  CAS  Google Scholar 

  34. Poulsen LK, Ballard G, Stahl DA (1993) Use of rRNA fluorescencein situ hybridization for measuring the activity of single cells in young and established biofilms. Appl Environ Microbiol 59:1354–1360

    PubMed  CAS  Google Scholar 

  35. Roller C, Wagner M, Amann R, Ludwig W, Schleifer K-H (1994)In situ probing of Gram-positive bacteria with high G+C content using 23S rRNA-targeted oligonucleotides. Microbiol 140:2849–2858

    Article  CAS  Google Scholar 

  36. Ruimy R, Breittmayer B, Boivin V, Christen R (1994) Assessment of the state of activity of individual cells by hybridization with a ribosomal RNA targeted fluorescently labeled oligonucleotidic probe. FEMS Microbiol Ecol 15:207–214

    Article  CAS  Google Scholar 

  37. Schleifer K-H, Amann R, Ludwig W, Rothermund C, Springer N, Dorn S (1992) Nucleic acid probes for the identification andin situ detection of pseudomonads. In: Galli E, Silver S, Witholt B (eds)Pseudomonas Molecular Biology and Biotechnology. ASM Press, Washington, DC, pp 1–134

    Google Scholar 

  38. Schloter M, Hartmann A (1998) Colonization of wheat roots (Triticum eastivum) and endophytic potential of differentAzospirillum brasilense strains studied with strain-specific monoclonal antibodies. Symbiosis 25:159–179

    Google Scholar 

  39. Schultze M, Kondorosi E, Ratet P, Buiré M, Kondorosi A (1994) Cell and molecular biology ofRhizobium-plant interactions. Int Rev Cytol 156:1–75

    Article  CAS  Google Scholar 

  40. Snaidr, Amann R, Huber I, Ludwig W, Schleiffer K-H (1997) Phylogenetic analysis andin-situ identification of bacteria in activated sludge. Appl Environ Microbiol 63:2884–2896

    PubMed  CAS  Google Scholar 

  41. Streeter JG, Stewart Smith R (1998) Introduction of rhizobia into soils—Problems, achievements, and prospects for the future. In: Subba Rao NS, Dommergues YR (eds) Microbial Interactions in Agriculture and Forestry, vol 1, Science Publishers, Enfield, NH

    Google Scholar 

  42. Thies YE, Singleton PW, Bohlool BB (1991) Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia in field grown legumes. Appl Environ Microbiol 57:19–28

    PubMed  CAS  Google Scholar 

  43. Torsvik V, Gosksoyyr J, Daae FL (1990) High diversity of DNA of soil bacteria. Appl Environ Microbiol 56:882–787

    Google Scholar 

  44. Tsai YL, Olson BH (1991) Rapid method for separation of DNA from soil and sediments. Appl Environ Microbiol 57:1070–1074

    PubMed  CAS  Google Scholar 

  45. Turner M, Newman E (1984) Growth of bacteria on roots of grasses: influence of mineral nutrient supply and interactions between species. J Gen Microbiol 130:505–512

    Google Scholar 

  46. Vlassak K, Vanderleyden J, Franco A (1996) Competition and persistence ofRhizobium tropici andRhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures. Biol Fert Soils 21:61–68

    Article  Google Scholar 

  47. Werner D, Berggold R, Jaeger D, Krotzky A, Papen H, Schenk S, Thierfelder H (1988) Plant, microbial and soil factors determing nitrogen fixation in the rhizosphere. Z Pflanzernernähr Bodenk 152:231–236

    Google Scholar 

  48. Yang C-H, Crowley DE (2000) Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl Environ Microbiol 66:345–351

    Article  PubMed  CAS  Google Scholar 

  49. Zhu Y, Pierson III LS, Hawes MC (1997) Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells. Plant Physiol 115:1691–1698

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The-Hebrew University of Jerusalem, P.O.B. 12, 76100, Rehovot, Israel

    E. Schallmach, D. Minz & E. Jurkevitch

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  1. E. Schallmach
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  2. D. Minz
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  3. E. Jurkevitch
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Correspondence to E. Jurkevitch.

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Schallmach, E., Minz, D. & Jurkevitch, E. Culture-independent detection of changes in root-associated bacterial populations of common bean (phaseolus vulgaris L.) following nitrogen depletion. Microb Ecol 40, 309–316 (2000). https://doi.org/10.1007/s002480000072

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  • DOI: https://doi.org/10.1007/s002480000072

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