Microbial Ecology

, Volume 50, Issue 1, pp 82–89 | Cite as

Screening for Putative PGPR to Improve Establishment of the Symbiosis Lactarius deliciosus-Pinus sp.

  • J. Barriuso
  • M. T. Pereyra
  • J. A. Lucas García
  • M. Megías
  • F. J. Gutierrez Mañero
  • B. Ramos


A screening for plant growth promoting rhizobacteria (PGPR) was carried out in the mycorrhizosphere of wild populations of Pinus pinea and P. pinaster, and in the mycosphere of associated Lactarius deliciosus. A total of 720 bacteria were isolated, purified, and grouped by morphological criteria. Fifty percent of the isolates were selected and tested for aminocyclopropanecarboxylic acid (ACC) degradation, auxin and siderophore production, and phosphate solubilization. Thirty eight percent of the isolates showed at least one of the evaluated activities. Nutrient-related traits were associated with P. pinaster, whereas hormone production traits predominated in P. pinea. These activities were found mostly in Gram positive isolates. After PCR-RAPDs (random amplified polymorphic DNA) analysis, 10 groups appeared with 85% similiarity when considering all isolates, indicating the low diversity in the system. One strain of each group was identified by 16S rDNA sequencing. Our results suggest that P. pinaster selects for mycorrhizosphere bacteria that mobilize nutrients, whereas P. pinea selects for bacteria that have the capacity to increase root growth via production of plant growth regulators.


Plant Growth Promote Rhizobacteria Siderophore Production Lactarius Tree Nursery Bioedit Sequence Alignment Editor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was financed by project FEDER 1FD97-1441. We thank Linda Hamalainen for editorial help.


  1. 1.
    Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils 12:39–45CrossRefGoogle Scholar
  2. 2.
    Benizri E, Courtade A, Picard C, Guckert A (1998) Role of maize root exudates in the production of auxins by Pseudomonas fluorescens M.3.1. Soil Biol Biochem 30:1481–1484CrossRefGoogle Scholar
  3. 3.
    Bowen GD, Rovira AD (1999) The rhizosphere and its management to improve plant growth. Adv Agron 66:1–103Google Scholar
  4. 4.
    Burdman S, Jurkevith E, Okon Y (2000) Recent advances in the use of plant growth promoting rhizobacteria (PGPR) in agriculture. Microbial Interations in Agriculture and Forestry 2:229–250Google Scholar
  5. 5.
    Cattelan AJ, Hartel PG, Fuhrmann JJ (1999) Screeening for plant growth-promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63:1670–1680Google Scholar
  6. 6.
    Clark AG, Lanigan CMS (1993) Prospects for estimating nucleotide divergence with RAPDs. Mol Biol Evol 10:1096–1111PubMedGoogle Scholar
  7. 7.
    de Freitas JR, Banerjee MR, Germida JJ (1997) Phosphate-solubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Biol Fertil Soils 24:358–364CrossRefGoogle Scholar
  8. 8.
    di Cello F, Bevivino A, Chiarini L, Fani R, Paffetti D, Tabacchioni S Dalmastri C (1997) Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Appl Environ Microbiol 63:4485–4493PubMedGoogle Scholar
  9. 9.
    Enebak SA, Wei G, Kloepper JW (1997) Effects of plant growth-promoting rhizobacteria on loblolly and slash pine seedlings. Forest Sci. 44:139–144Google Scholar
  10. 10.
    Frey-Klett P, Pierrat JC, Garbaye J (1997) Location and survival of Mycorrhiza helper Pseudomonas fluorescens during establishment of ectomycorrhizal symbiosis between Laccaria bicolour and Douglas fir. Appl Environ Microbiol 63:139–144Google Scholar
  11. 11.
    Garbaye J (1994) Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phyto. 128:197–210Google Scholar
  12. 12.
    Glick BR, Karaturovic DM, Newell PC (1995) A novel procedure for rapid isolation of plant growth promoting pseudomonads. Can J Microbiol 41:533–536Google Scholar
  13. 13.
    Gutierrez Mañero FJ, Acero N, Lucas JA, Probanza A (1996) The influence of native rhizobacteria on European alder [Alnus glutinosa (L.) Gaertn.] growth. II. Characterization of growth promoting and growth inhibiting strains. Plant Soil 182:67–74Google Scholar
  14. 14.
    Gutierrez Mañero FJ, Ramos Solano B, Probanza A, Mehouachi J, Tadeo FR, Talon M (2001) The plant growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins. Physiol Plantarum 111:1–7CrossRefGoogle Scholar
  15. 15.
    Gutierrez Manero FJ, Ramos B, Lucas Garcia JA, Probanza A, Barrientos ML (2002) Systemic induction of terpenic compounds in D. lanata. J Plant Physiol 160:105–113Google Scholar
  16. 16.
    Hall, IR, Yun, W, Amicucci, A (2003) Cultivation of edible ectomycorrhizal mushrooms. Trends Biotechnol 21(10): 433–438CrossRefPubMedGoogle Scholar
  17. 17.
    Hirsch AM, Fang Y, Asad S, Kapulnik Y (1997) The role of phytohormones in plant-microbe symbioses. Plant Soil 194:171–184CrossRefGoogle Scholar
  18. 18.
    Kloepper JW, Scrhoth MN, Miller TD (1980) Effects of rhizosphere colonization by plant growth-promoting rhizobacteria on potato plant development and yield. Phytopathology 70:1078–1082Google Scholar
  19. 19.
    Louws FJ, Rademaker JLW, de Bruijn FJ (1999) The three Ds of PCR-based genomic analysis of Phytobacteria: diversity, detection and disease diagnosis. Annu Rev Phytpathol 37:81–125CrossRefGoogle Scholar
  20. 20.
    Lucas Garcia JA, Probanza A, Ramos B, Gutierrez Manero FJ (2001) Genetic variability of rhizobacteria from wild populations of four Lupinus species based on PCR-RAPDs. J Plant Nutr Soil Sci 164:1–7CrossRefGoogle Scholar
  21. 21.
    Lynch JM, (1990) The rhizosphere. Wiley-Interscience, Chichester, EnglandGoogle Scholar
  22. 22.
    Marilley L, Aragno M (1999) Phylogenetic diversity of bacterial communities differing in degree of proximity of Lolium perenne and Trifolium repens roots. Appl Soil Ecol 13:127–136CrossRefGoogle Scholar
  23. 23.
    Marten P, Brueckner S, Berg G (2001) Biological plant protection using rhizobacteria–an environmentally friendly alternative for biological control of soilborne and seedborne phytopathogenic fungi. Gesunde Prlanzen 53:224–234Google Scholar
  24. 24.
    Nei M, Miller JC (1990) A simple method for estimating average number of nucleotide substitutions within and between populations from restriction data. Genetics 125:873–879PubMedGoogle Scholar
  25. 25.
    Probanza A, Mateos JL, Lucas JA, Ramos B, de Felipe MR, Gutierrez Manero FJ (2001) Effects of inoculation with PGPR Bacillus and Pisolitus tinctorius on Pinus pinea L. growth, bacterial rhizosphere colonization and mycorrhizal infection. Microbial Ecol 41:140–148Google Scholar
  26. 26.
    Rainey PB (1999) Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ Microbiol 1:243–257CrossRefPubMedGoogle Scholar
  27. 27.
    Ramamoorthy V, Viswanathan R, Raguchander T, Prakasam V, Samiyappan R (2001) Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and diseases. Crop Protection 20:1–11CrossRefGoogle Scholar
  28. 28.
    Ramos Solano, B, Pereyra de la Iglesia, MT, Probanza, A, Lucas Garcia, JA, Megiás, M, Gutíerrez Mañero, FJ (2003) Screening for PGPR in the rhizosphere of Cistus ladanifer to improve reforestation of degraded mediterranean ecosystems. Plant Soil (in press)Google Scholar
  29. 29.
    Ramos B, Pozuelo JM, Acero N, Gutierrez Mañero FJ (1998). Seasonal variations of Bacillus isolated from the rhizosphere of Elaeagnus angustifolia L. Orsis 13:7–16Google Scholar
  30. 30.
    Requena N, Perez-Solis E, Azcon-Aguilar C, Jeffries P, Barea JM (2001) Management of indigenous plant-microbe symbioses aids restoration of desertified ecosystems. Appl Environ Microbiol 67:495–498CrossRefPubMedGoogle Scholar
  31. 31.
    Rodriguez H, Rossolini GM, Gonzalez T, Li J, Glick BR (2000) Isolation of a gene from Burholderia cepacia IS16 encoding a protein that facilitates phosphatase activity. Curr Microbiol 40:362–366CrossRefPubMedGoogle Scholar
  32. 32.
    Selvadurai EL, Brown AE, Hamilton JTG (1991) Production of indole-3-acetic acid analogues by strains of Bacillus cereus in relation to their influence on seedling development. Soil Biol Biochem 23:401–403CrossRefGoogle Scholar
  33. 33.
    Shannon CE, Weaver V (1949) The Mathematical Theory of Communication. University of Illinois Press, Urbana, IL, 117 PpGoogle Scholar
  34. 34.
    Smith, SE, Read, DJ (1997) Mycorrhizal Symbiosis. Academic Press, San Diego, USAGoogle Scholar
  35. 35.
    Ulrike E, Rogall T, Blocker H, Emde M, Bottger EG (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16s ribosomal RNA. Nucleic Acids Res 17:7843–7853PubMedGoogle Scholar
  36. 36.
    Van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453–483CrossRefPubMedGoogle Scholar
  37. 37.
    Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255:571–586CrossRefGoogle Scholar
  38. 38.
    Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Kirchevsdy MI, Moore LH, Moore WEC, Murray RGE, Stackerbrandt E, Starr MP, Turper HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Sys Bacteriol 37:463–464Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • J. Barriuso
    • 1
  • M. T. Pereyra
    • 1
  • J. A. Lucas García
    • 1
  • M. Megías
    • 2
  • F. J. Gutierrez Mañero
    • 1
  • B. Ramos
    • 1
  1. 1.Facultad FarmaciaUniversidad San Pablo CEUMadridSpain
  2. 2.Facultad FarmaciaUniversidad de SevillaSevillaSpain

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