Skip to main content
SpringerLink
Log in

Pseudomonas spp. isolates with high phosphate-mobilizing potential and root colonization properties from agricultural bulk soils under no-till management

  • Original Paper
  • Published:
Biology and Fertility of Soils Aims and scope Submit manuscript

Abstract

Seven phosphate-mobilizing pseudomonads were isolated, identified, and characterized in terms of their biofertilizer potential and root-colonizing properties. Pseudomonas protegens (ex-fluorescens) CHA0 was used for comparative purposes. Four isolates (LF-MB1, LF-P1, LF-P2, and LF-P3) clustered with members of the “Pseudomonas fluorescens complex,” whereas the other three (LF-MB2, LF-V1, and LF-V2) clustered with members of the “Pseudomonas putida/Pseudomonas aeruginosa complex.” Assays in buffered liquid growth medium supplemented with tricalcium phosphate enabled the separation of the isolates into two groups: group A (LF-P1, LF-P2, LF-P3, and LF-V1) solubilized P from 151 up to 182 μg mL−1, and group B (LF-MB1, LF-MB2, and LF-V2) solubilized less than 150 μg P mL−1. All isolates displayed acid and alkaline phosphatase activities. With the exception of LF-MB2, all isolates were able to degrade phospholipids from lecithin. Additionally, all isolates exhibited extracellular protease activity, and four isolates produced hydrogen cyanide, two traits that are related to biocontrol of phytopathogens. To study root colonization in non-sterile soil, isolates were doubly tagged with gfp and a tetracycline resistance cassette. After 15 days of competition with the indigenous bacterial flora, all tagged isolates colonized soybean roots at counts ranging from 7.6 × 105 to 1.7 × 107 CFU g−1. The results indicate that there are already efficient phosphate-mobilizing pseudomonads adapted to agricultural bulk soils under no-till management in Argentina and thus having excellent potential for use as biofertilizers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Abd-Alla MH (1994) Use of organic phosphorus by Rhizobium leguminosarum biovar. viceae phosphatases. Biol Fert Soils 18:216–218. doi:10.1007/BF00647669

    Article  CAS  Google Scholar 

  • Agaras B, Wall LG, Valverde C (2011) Specific enumeration and analysis of the community structure of culturable pseudomonads in agricultural soils under no-till management in Argentina. Appl Soil Ecol. doi:10.1016/j.apsoil.2011.11.016

  • Ahmad F, Mabood Husain F, Ahmad I (2011) Rhizosphere and root colonization by bacterial inoculants and their monitoring methods: a critical area in PGPR research. In: Ahmad I, Ahmad F, Pichtel J (eds) Microbes and microbial technology: agricultural and environmental applications. Springer, New York, pp 363–391. doi:10.10077978-1-4419-7931-5_14

    Chapter  Google Scholar 

  • Álvarez CR, Torres Duggan M, Chamorro E, D’ambrosio D, Taboada MA (2009) Descompactación de suelos franco limosos en siembra directa: efectos sobre las propiedades edáficas y los cultivos. Cienc Suelo 27:159–169

    Google Scholar 

  • Barrett M, Morissey JP, O’Gara F (2011) Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence. Biol Fertil Soils 47:729–743. doi:10.1007/s00374-011-0605-x

    Article  Google Scholar 

  • Beauchamp CJ, Kloepper JW (2003) Spatial and temporal distribution of a bioluminescent-marked Pseudomonas putida on soybean root. Luminescence 18:346–351. doi:10.1002/bio.747

    Article  PubMed  Google Scholar 

  • Bodilis J, Hedde M, Orange N, Barray S (2006) OprF polymorphism as a marker of ecological niche in Pseudomonas. Environ Microbiol 8:1544–1551. doi:10.1111/j.1462-2920.2006.01045.x

    Article  PubMed  CAS  Google Scholar 

  • Browne P, Rice O, Miller SH, Burke J, Dowling DN, Morrissey JP, O’Gara F (2009) Superior inorganic phosphate solubilization is linked to phylogeny within the Pseudomonas fluorescens complex. App Soil Ecol 43:131–138. doi:10.1016/j.apsoil.2009.06.010

    Article  Google Scholar 

  • Campitelli P, Aoki A, Gudelj O, Rubenacker A, Sereno R (2010) Selección de indicadores de calidad de suelo para determinar los efectos del uso y prácticas agrícolas en un área piloto de la región central de Córdoba. Cienc Suelo 28:223–231

    Google Scholar 

  • Cantú MP, Becker A, Camilo Bedano J, Schiavo HF (2007) Evaluación de la calidad de suelos mediante el uso de indicadores e índices. Cienc Suelo 25:173–178

    Google Scholar 

  • Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho H, Sa T (2005) Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol Biochem 37:1970–1974. doi:10.1016/j.soilbio.2005.02.025

    Article  CAS  Google Scholar 

  • Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:141–145. doi:10.1093/nar/gkn879

    Article  Google Scholar 

  • Collavino MM, Sansberro PA, Mroginski LA, Aguilar OA (2010) Comparison of in vitro solubilization activity of diverse phosphate-solubilizing bacteria native to acid soil and their ability to promote Phaseolus vulgaris growth. Biol Fertil Soils 46:727–738. doi:10.1007/s00374-010-0480-x

    Article  Google Scholar 

  • 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 24:358–364. doi:10.1007/s003740050258

    Article  Google Scholar 

  • De Souza JT, Mazzola M, Raaijmakers JM (2003) Conservation of the response regulator gene gacA in Pseudomonas species. Environ Microbiol 5:1328–1340. doi:10.1046/j.1462-2920.2003.00438.x

    Article  PubMed  Google Scholar 

  • De Werra P, Péchy-Tarr M, Keel C, Maurhofer M (2009) Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0. Appl Environl Microbiol 75:4162–4174. doi:10.1128/AEM.00295-09

    Article  Google Scholar 

  • De-Bashan LE, Bashan Y (2004) Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997–2003). Water Res 38:4222–4246. doi:10.1016/j.watres.2004.07.014

    Article  PubMed  CAS  Google Scholar 

  • Deepa CK, Dastager SG, Pandey A (2010) Isolation and characterization of plant growth promoting bacteria from non-rhizospheric soil and their effect on cowpea (Vigna unguiculata (L.) Walp.) seedling growth. W J Microbiol Biotech 26:1233–1240. doi:10.1007/s11274-009-0293-y

    Article  CAS  Google Scholar 

  • Devliegher W, Syamsul MA, Verstraete W (1995) Survival and plant growth promotion of detergent-adapted Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7NSK2. App Envriron Microbiol 61:3865–3871

    CAS  Google Scholar 

  • Dubuis C, Rolli J, Lutz M, Défago G, Haas D (2006) Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression. Appl Environ Microbiol 72:2606–2613. doi:10.1128/AEM.72.4.2606-2613.2006

    Article  PubMed  CAS  Google Scholar 

  • Egan SV, Yeoh HH, Bradbury JH (1998) Simple picrate paper kit for determination of the cyanogenic potential of cassave flour. J Sci Food Agric 76:39–48

    Article  CAS  Google Scholar 

  • Ferreras L, Magra G, Besson P, Kovalevski E, García F (2007) Indicadores de calidad física en suelos de la región pampeana norte de Argentina bajo siembra directa. Cienc Suelo 25:159–172

    Google Scholar 

  • Gould WD, Hagedorn C, Bardinelli TR, Zablotowicz RM (1985) New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats. Appl Environ Microbiol 49:28–32

    PubMed  CAS  Google Scholar 

  • Gyaneshwar P, Naresh KG, Parekh LJ (1998) Effect of buffering on the phosphate-solubilizing ability of microorganisms. W J Microbiol Biotech 14:669–673

    Article  CAS  Google Scholar 

  • Gyaneshwar P, Naresh KG, Parekh LJ, Poole PS (2002) Role of soil microorganisms in improving P nutrition of plants. Plant Soil 245:83–93. doi:10.1023/A:1020663916259

    Article  CAS  Google Scholar 

  • Haas D, Défago G (2005) Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 3:307–319. doi:10.1038/nrmicro1129

    Article  PubMed  CAS  Google Scholar 

  • Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60:579–598. doi:10.1007/s13213-010-0117-1

    Article  Google Scholar 

  • Herisgtad B, Hamilton M, Heersink J (2001) How to optimize the drop plate method for enumerating bacteria. J Microbiol Meth 44:121–129

    Article  Google Scholar 

  • Jha B, Gandhi Pragash M, Cletus J, Raman G, Sakthivel N (2009) Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonas aeruginosa, P. plecossicida and P. mosselii. W J Microbiol Biotech 25:573–581. doi:10.1007/s11274-008-9925-x

    Article  CAS  Google Scholar 

  • Joseph S, Jisha MS (2009) Buffering reduces phosphate solubilizing ability of selected strains of bacteria. W J Agricul Sci 5:135–137

    CAS  Google Scholar 

  • Kämpfer P (2007) Taxonomy of phosphate solubilizing bacteria. In: Velázquez E, Rodríguez-Barrueco C (eds) Developments in plant and soil sciences: first international meeting on microbial phosphate solubilization. Springer, Dordrecht, pp 101–106

    Chapter  Google Scholar 

  • Lugtenberg B, Kamilova F (2009) Plant-growth promoting rhizobacteria. Annu Rev Microbiol 63:541–556. doi:10.1146/annurev.micro62.081307.162918

    Article  PubMed  CAS  Google Scholar 

  • Malboobi MA, Owla P, Behbahani M, Sarokhani E, Moradi S, Yakhchali B, Deljo A, Heravi KM (2009) Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates. W J Microbiol Biotech 25:1471–1477. doi:10.1007/s11274009-0037-z

    Article  CAS  Google Scholar 

  • Martín L, Velazquez E, Rivas R, Mates PF, Martínez-Molina E, Rodríguez-Barrueco C, Peix A (2007) Effect of inoculation with a strain of Pseudomonas fragi in the growth and phosphorus content of strawberry plants. In: Velázquez E, Rodríguez-Barrueco C (eds) Developments in plant and soil sciences: first international meeting on microbial phosphate solubilization. Springer, Dordrecht, pp 309–315

    Chapter  Google Scholar 

  • Meyer JB, Frapolli M, Keel C, Maurhofer M (2011) A novel molecular marker for studying phylogeny and diversity of phosphate-solubilizing pseudomonads: the pyrroloquinoline quinone biosynthetic gene pqqC. Appl Environ Microbiol 77:7345–7354. doi:10.1128/AEM.05434-11

    Article  PubMed  CAS  Google Scholar 

  • Murphy J, Riley JP (1962) A modified single solution method for determination of phosphate. Anal Chim Acta 27:31–36

    Article  CAS  Google Scholar 

  • Nannipieri P, Giagnoni L, Landi L, Renella G (2011) Role of phosphatase enzymes in soil. In Bunemann EK et al (eds) Phosphorus in Action. Soil Biology 26. Springer Verlag, Berlin Heidelberg, pp 215-241.

  • Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265–270. doi:10.1111/j.1574-6968.1999.tb13383.x

    Article  PubMed  CAS  Google Scholar 

  • Oliveira CA, Alves VMC, Marriel LE, Gomes EA, Scotti MR, Carneiro NP, Guimaraes CT, Schaffert RE, Sá NMH (2009) Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome. Soil Biol Biochem 41:1782–1787. doi:10.1018.j.soilbio.2008.01.012

    Article  CAS  Google Scholar 

  • Picard C, Bosco M (2007) Genotypic and phenotypic diversity in populations of plant-probiotic Pseudomonas spp. colonizing roots. Naturwissenschaften 95:1–16. doi:10.1007/s00114-007-0286-3

    Article  PubMed  Google Scholar 

  • Picard C, Di Cello F, Vestura M, Fani R, Guckert A (2000) Frequency and biodiversity of 2,4-diacetylphloroglucinol producing bacteria isolated from the maize rhizosphere at different stages of plant growth. Appl Environ Microbiol 66:948–955

    Article  PubMed  CAS  Google Scholar 

  • Picone L, Capozzi I, Zamuner E, Echeverría H, Sainz Rozas H (2007) Transformaciones de fósforo en un molisol bajo sistemas de labranza contrastantes. Cienc Suelo 25:99–107

    Google Scholar 

  • Polonenko DR, Scher FM, Kloepper JW, Singleton CA, Laliberte M, Zaleska I (1987) Effects of root colonizing bacteria on nodulation of soybean roots by Bradyrhizobium japonicum. Can J Microbiol 33:498–503. doi:10.1139/m87-083

    Article  Google Scholar 

  • Ramachandran K, Srinivasan V, Hamza S, Anandaraj M (2007) Phosphate solubilizing bacteria isolated from the rhizosphere soil and its growth promotion on black pepper (Piper nigrum L.) cuttings. In: Velázquez E, Rodríguez-Barrueco C (eds) Developments in plant and soil sciences: first international meeting on microbial phosphate solubilization. Springer, Dordrecht, pp 325–331

    Chapter  Google Scholar 

  • Ramette A, Frapolli M, Défago G, Moenne Y (2003) Phylogeny of HCN synthase-encoding hcnBC genes in biocontrol fluorescent Pseudomonas and its relationship with host plant species and HCN synthesis ability. Mol Plant Microbe Interact 16:525–535. doi:10.1094/MPMI.2003.16.6.508

    Article  PubMed  CAS  Google Scholar 

  • Ramette A, Frapolli M, Fischer-LeSaux M, Gruffaz C, Meyer JM, Défago G, Sutra L, Moënne-Loccoz Y (2011) Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin. Syst Appl Microbiol. doi:10.1016/j.syapm.2010.10.005

  • Ravindra NP, Raman G, Narayanan KB, Sakthivel N (2008) Assesment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiol 8:230. doi:10.1186/1471-2180-8-230

    Article  Google Scholar 

  • Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Aust J Plant Physiol 28:897–906. doi:10.1071/PP01093

    Google Scholar 

  • Richardson AE, Simpson RJ (2011) Soil microorganisms mediating phosphorus availability. Plant Physiol 156:989–996. doi:10.1104/pp.111.175448

    Article  PubMed  CAS  Google Scholar 

  • Richardson AE, Barea JM, McNeill AM, Pringet-Combaret C (2009) Acquisition of P and N in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 321:305–339. doi:10.1007/s11104-009-9895-2

    Article  CAS  Google Scholar 

  • Rodríguez H, Fraga R (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotech Adv 17:319–339. doi:10.1016/s0734-9750(99)000142

    Article  Google Scholar 

  • Sacherer P, Défago G, Hass D (1994) Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiol Lett 116:155–160

    Article  PubMed  CAS  Google Scholar 

  • Selvakumar G, Joshi P, Suyal P, Mishra PK, Joshi GK, Bisht JK, Bhatt JC, Gupta HS (2011) Pseudomonas lurida M2RH3 (MTCC9245), a psychrotolerant bacterium from the Uttarakhand Himalayas, solubilizes phosphate and promotes wheat seedling growth. W J Microbiol Biotech 27:1129–1135. doi:10.1007/s11274-010-0559-4

    Article  CAS  Google Scholar 

  • Singh JS, Pandey VC, Singh DP (2011) Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agri Ecosys Environ 140:339–353. doi:10.1016/j.agee2011.01.017

    Article  Google Scholar 

  • Tamura K, Dudley K, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Mol Biol Evol 24:1596–1599. doi:10.1093/molbev/msm092

    Article  PubMed  CAS  Google Scholar 

  • Tao G, Tian S, Cai M, Xie G (2008) Phosphate solubilizing and mineralizing abilities of bacteria isolated from soils. Pedosphere 18:515–523. doi:10.1016/S1002-0160(08)60042-9

    Article  CAS  Google Scholar 

  • Trujillo ME, Veláquez E, Miguélez S, Jimenez MS, Mateos PF, Martínez-Molina E (2007) Characterization of a strain of Pseudomonas fluorescens that solubilizes phosphates in vitro and produces high antibiotic activity against several microorganisms. In: Velázquez E, Rodríguez-Barrueco C (eds) Developments in plant and soil sciences: first international meeting on microbial phosphate solubilization. Springer, Dordrecht, pp 265–268

    Chapter  Google Scholar 

  • Vassilev N, Vassileva M, Nicolaeva I (2006) Simultaneous P solubilizing and biocontrol activity of microorganism: potential and future trends. Appl Microbiol Biotech 71:137–144. doi:10.1007/s00253-006-0380-z

    Article  CAS  Google Scholar 

  • Viruel E, Lucca ME, Siñeriz F (2011) Plant growth promotion traits of phosphobacteria isolated from Puna, Argentina. Arch Microbiol 193:489–496. doi:10.1007/s00203-011-0692-y

    Article  PubMed  CAS  Google Scholar 

  • Wall LG (2011) The BIOSPAS consortium. In: de Bruijn FJ (ed) Handbook of molecular microbial ecology I. Wiley-Blackwell, New Jersey, pp 299–306

    Chapter  Google Scholar 

Download references

Acknowledgements

This work was supported by grants PAE 36976-PID 52 and PME 2006-1730 (Agencia Nacional de Promoción Científica y Tecnológica, Argentina), PIP 112-494 200801-02271 (CONICET, Argentina), and PUNQ 0395/07 (Universidad Nacional de Quilmes, Argentina). We thank Ana María Zamponi (CONICET) for her technical assistance and Carolina Fernández for providing laboratory facilities to use the optical microscope. LF and BA hold a Ph.D. fellowship from CONICET. LGW and CV are members of CONICET.

Author information

Authors and Affiliations

  1. Laboratorio de Microbiología Agrícola, Departamento de Agronomía, Universidad Nacional del Sur, San Andrés s/n Altos del Palihue, Bahía Blanca, 8000, Buenos Aires, Argentina

    Leticia Fernández

  2. Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal, B1876BXD, Buenos Aires, Argentina

    Betina Agaras, Luis G. Wall & Claudio Valverde

  3. Laboratorio de Suelos Salinos y Sódicos, Departamento de Agronomía, Universidad Nacional del Sur, San Andrés s/n Altos del Palihue, Bahía Blanca, 8000, Buenos Aires, Argentina

    Pablo Zalba

Authors
  1. Leticia Fernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Betina Agaras
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pablo Zalba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Luis G. Wall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Claudio Valverde
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leticia Fernández.

Additional information

Leticia Fernández and Betina Agaras equally contributed to this paper. Rizobacter Argentina S.A. has priority access to the bacterial isolates reported here.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fernández, L., Agaras, B., Zalba, P. et al. Pseudomonas spp. isolates with high phosphate-mobilizing potential and root colonization properties from agricultural bulk soils under no-till management. Biol Fertil Soils 48, 763–773 (2012). https://doi.org/10.1007/s00374-012-0665-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00374-012-0665-6

Keywords

Search

Navigation