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Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii

  • Babita Kumari Jha
  • Mohandass Gandhi Pragash
  • Jean Cletus
  • Gurusamy Raman
  • Natarajan SakthivelEmail author
Original Paper

Abstract

Of 80 fluorescent pseudomonad strains screened for phosphate solubilization, three strains (BFPB9, FP12 and FP13) showed the ability to solubilize tri-calcium phosphate (Ca3(PO4)2). During mineral phosphate solubilization, decrease of pH in the culture medium due to the production of organic acids by the strains was observed. These phosphate solubilizing strains produced indole-3-acetic acid (IAA) and protease as well as exhibited a broad-spectrum antifungal activity against phytopathogenic fungi. When tested in PCR using the gene-specific primers, strain BFPB9 showed the presence of hcnBC genes that encode hydrogen cyanide. On the basis of phenotypic traits, 16S rRNA sequence homology and subsequent phylogenetic analysis, strains BFPB9, FP12 and FP13 were designated as Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii, respectively. Present investigation reports the phosphate solubilization potential and biocontrol ability of new strains that belong to P. plecoglossicida and P. mosselii. Because of the innate potential of phosphate solubilization, production of siderophore, IAA, protease, cellulase and HCN strains reported in this study can be used as biofertilizers as well as biocontrol agents.

Keywords

Fluorescent pseudomonads Phosphate solubilization Indole-3-acetic acid Protease Antifungal activity 

Notes

Acknowledgments

We thank the Department of Biotechnology, Ministry of Science and Technology, Government of India for financial assistance.

References

  1. Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils 12:39–45. doi: 10.1007/BF00369386 CrossRefGoogle Scholar
  2. Ayyadurai N, Ravindra Naik P, Sakthivel N (2007) Functional characterization of antagonistic fluorescent pseudomonas associated with rhizospheric soil of rice (Oryza sativa L.). J Microbiol Biotechnol 17:919–927Google Scholar
  3. Baker R, Elad Y, Sneh B (1986) Physical, biological and host factors in iron competition in soils. In: Swinburne TR (ed) Iron siderophores and plant diseases. Plenum Press, New York, pp 77–84Google Scholar
  4. Bano N, Musarrat J (2003) Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr Microbiol 46:324–328. doi: 10.1007/s00284-002-3857-8 CrossRefGoogle Scholar
  5. Bano N, Musarrat J (2004) Characterization of a novel carbofuran degrading Pseudomonas sp. with collateral biocontrol and plant growth promoting potential. FEMS Microbiol Lett 231:13–17. doi: 10.1016/S0378-1097(03)00894-2 CrossRefGoogle Scholar
  6. Bric M, John M, Bostock R, Silverstone SE (1991) Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538Google Scholar
  7. Brown AE, Hamilton JTG (1993) Indole-3-ethanol produced by Zygorrhynchusmoeller, and indole-3-acetic acid analogue with antifungal activity. Mycol Res 96:71–74CrossRefGoogle Scholar
  8. Cattelan AJ, Hartel PG, Furhmann FF (1999) Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63:1670–1680Google Scholar
  9. Chen YP, Rekha PD, Arun AB, Shen FT, Lai WA, Young CC (2006) Phosphate solubilizing bacteria from subtropical soil and their tri-calcium phosphate solubilizing abilities. Appl Soil Ecol 34:33–41. doi: 10.1016/j.apsoil.2005.12.002 CrossRefGoogle Scholar
  10. de Souza JT, Raaijmakers JM (2003) Polymorphisms within the PrnD and PltC genes from pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp. FEMS Microbiol Ecol 43:21–34. doi: 10.1111/j.1574-6941.2003.tb01042.x Google Scholar
  11. Dey R, Pal KK, Bhatt DM, Chauha SM (2004) Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria. 159:371–394Google Scholar
  12. Gaind S, Gaur AC (2002) Impact of fly ash and phosphate solubilising bacteria on soybean productivity. Bioresour Technol 58:313–315. doi: 10.1016/S0960-8524(02)00088-3 CrossRefGoogle Scholar
  13. Glick BR (1995) The enhancement of plant growth by free living bacteria. Can J Microbiol 41:109–117CrossRefGoogle Scholar
  14. Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiol 26:192–195CrossRefGoogle Scholar
  15. Gulati A, Rahli P, Vyas Pratibha (2008) Characterization of phosphate solubilizing fluorescent pseudomonads from the rhizosphere of sea buckthorn growing in the cold deserts of Himalayas. Curr Microbiol 56:73–79. doi: 10.1007/s00284-007-9042-3 CrossRefGoogle Scholar
  16. Gurusiddaiah S, Weller MD, Sarkar A, Cook JR (1986) Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp. Antimicrob Agents Chemother 29:488–495Google Scholar
  17. Hameeda B, Harish Kumar Reddy Y, Rupela OP, Kumar GN, Reddy Gopal (2006) Effect of carbon substrates on rock phosphate solubilization by bacteria from composts and macrofauna. Curr Microbiol 53:298–302. doi: 10.1007/s00284-006-0004-y CrossRefGoogle Scholar
  18. Higgins DG, Bleashy AT, Fuchs R (1992) Clustal V: improved software for multiple sequence alignment. Comput Appl Biosci 8:189–191Google Scholar
  19. Homma Y, Suzui T (1989) Role of antibiotic production in suppression of radish damping-off by seed bacterization with Pseudomonas cepacia. Ann Phytopathol Soc Jpn 55:643–652Google Scholar
  20. Illmer P, Schinner F (1995) Solubilization of inorganic calcium phosphates-solubilization mechanisms. Soil Biol Biochem 3:257–263. doi: 10.1016/0038-0717(94)00190-C CrossRefGoogle Scholar
  21. Kapoor KK, Mishra MM, Kuhkreja K (1989) Phosphate solubilization by soil microoraganisms – a review. Indian J Microbiol 29:119–127Google Scholar
  22. Kim HY, Schlictman D, Shankar S, Xie Z, Chakrabarty MA, Kornberg A (1998) Alginate, inorganic polyphosphate, GTP and ppGpp synthesis co-regulated in Pseudomonas aeruginosa: implications for stationary phase survival and synthesis of RNA/DNA precursors. Mol Microbiol 27:717–725. doi: 10.1046/j.1365-2958.1998.00702.x CrossRefGoogle Scholar
  23. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. doi: 10.1007/BF01731581 CrossRefGoogle Scholar
  24. King EO, Ward MK, Raney DE (1954) Two simple media for demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307Google Scholar
  25. Kraus J, Loper EJ (1995) Characterization of a genomic region required for production of the antibiotic pyoluterin by the biological control agent Pseudomonas fluorescens Pf5. Appl Environ Microbiol 61:849–854Google Scholar
  26. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163. doi: 10.1093/bib/5.2.150 CrossRefGoogle Scholar
  27. Lehinos V (1994) Effects of pH and glucose on auxin production of phosphate-solubilizing rhizobacteria in vitro. Microbiol Res 149:135–138Google Scholar
  28. Lehinos V, Vacek O (1994) Biosynthesis of auxin by phosphate-solubilizing rhizobacteria from wheat (Triticum aestivum) and rye (Secale cereale). Microbiol Res 149:31–35Google Scholar
  29. Martinez Noel GMA, Madrid EA, Botín R, Lamattina L (2001) Indoleacetic acid attenuates disease severity in potato-Phytophthora infestans interaction and inhibits the pathogen growth in vitro. Plant Physiol Biochem 39:815–823. doi: 10.1016/S0981-9428(01)01298-0 CrossRefGoogle Scholar
  30. Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS (2001a) Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183:6454–6465. doi: 10.1128/JB.183.21.6454-6465.2001 CrossRefGoogle Scholar
  31. Mavrodi OV, Gardener BBM, Mavrodi DV, Bonsall RF, Weller DM, Thomashow LS (2001b) Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. Phytopathology 91:35–43. doi: 10.1094/PHYTO.2001.91.1.35 CrossRefGoogle Scholar
  32. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pre-treatment of lignocellulosic biomass. Bioresour Technol 96:673–686. doi: 10.1016/j.biortech.2004.06.025 CrossRefGoogle Scholar
  33. Musarrat J, Bano N, Rao RAK (2000) Isolation and characterization of 2, 4-dichlorophenoxyacetic acid acid-catabolizing bacteria and their biodegradation efficiency in soil. World J Microbiol Biotechnol 16:495–497. doi: 10.1023/A:1008945720327 CrossRefGoogle Scholar
  34. Nielsen TH, Christophersen C, Anthoni U, Sorensen J (1999) Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54. J Appl Microbiol 86:80–90. doi: 10.1046/j.1365-2672.1999.00798.x CrossRefGoogle Scholar
  35. Nielsen TH, Thrane C, Christophersen C, Anthoni U, Sorensen J (2000) Structure, production characteristics and fungal antagonism of tensin-a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578. J Appl Microbiol 89:992–1001. doi: 10.1046/j.1365-2672.2000.01201.x CrossRefGoogle Scholar
  36. O’Sullivan DJ, O’Gara F (1992) Traits of fluorescent Pseudomonas sp. involved in suppression of plant root pathogens. Microbiol Rev 56:662–676Google Scholar
  37. Pandey A, Palni LMS (1998) Isolation of Pseudomonas corrugate from Sikkim Himalaya. World J Microbiol Biotechnol 14:411–413. doi: 10.1023/A:1008825514148 CrossRefGoogle Scholar
  38. Pandey A, Trivedi P, Kumar B, Palni LMS (2006) Characterization of a phosphate solubilizing and antagonistic strain of Pseudomonas putida (BO) isolated from a sub-alpine location in the Indian central Himalaya. Curr Microbiol 53:102–107. doi: 10.1007/s00284-006-4590-5 CrossRefGoogle Scholar
  39. Peix A, Rivas R, Mateos PF, Martinez-Molina E, Rodriguez-Barrueco C, Velazquez E (2003) Pseudomonas rhizosphaerae sp. nov., a novel species that actively solubilizes phosphate in vitro. Int J Syst Evol Microbiol 53:2067–2072. doi: 10.1099/ijs.0.02703-0 CrossRefGoogle Scholar
  40. Peix A, Rivas R, Santa-Regina I, Mateos PF, Martinez-Molina E, Rodriguez-Barrueco C, Velazquez E (2004) Pseudomonas lutea sp. nov., a novel phosphate-solubilizing bacterium isolated from the rhizosphere of grasses. Int J Syst Evol Microbiol 54:847–850. doi: 10.1099/ijs.0.02966-0 CrossRefGoogle Scholar
  41. Pfender WF, Kraus J, Loper EJ (1993) A genomic region from Pseudomonas fluorescens Pf-5 required for pyrrolnitrin production and inhibition of Pyrenophora tritici-repentis in wheat straw. Phytopathology 83:1223–1228. doi: 10.1094/Phyto-83-1223 CrossRefGoogle Scholar
  42. Pierson LS, Thomashow SL (1992) Cloning of heterologous expression of phenazine biosynthesis locus from Pseudomonas aureofaciens. Mol Plant Microbe Interact 53:330–339Google Scholar
  43. Pikovskaya RI (1948) Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologiya 17:363–370Google Scholar
  44. Raaijmakers J, Weller DM, Thomashow LS (1997) Frequency of antibiotic producing Pseudomonas spp. in natural environments. Appl Environ Microbiol 63:881–887Google Scholar
  45. Ramette A, Frapolli M, Defago G, Monenne 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.2003.16.6.525 CrossRefGoogle Scholar
  46. Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Aust J Plant Physiol 28:8797–8906Google Scholar
  47. Rodriguez H, Fraga R (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv 17:319–339. doi: 10.1016/S0734-9750(99)00014-2 CrossRefGoogle Scholar
  48. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  49. Sakthivel N, Gnanamanickam SS (1987) Evaluation of Pseudomonas fluorescens for suppression of sheath rot disease and for enhancement of grain yields in rice (Oryza sativa L.). Appl Environ Microbiol 53:2056–2059Google Scholar
  50. Sakthivel N, Gnanamanickam SS (1989) Incidence of different biovars of Pseudomonas fluorescens in flooded rice rhizospheres in India. Agric Ecosyst Environ 25:287–298. doi: 10.1016/0167-8809(89)90126-6 CrossRefGoogle Scholar
  51. Sakthivel N, Mortensen NC, Mathur BS (2001) Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques. Appl Microbiol Biotechnol 56:435–441. doi: 10.1007/s002530100641 CrossRefGoogle Scholar
  52. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society of Microbiology, Washington, DC, pp 607–654Google Scholar
  53. Sorensen D, Nielsen HT, Christophersen C, Sorensen J, Gajhede M (2001) Cyclic lipoundecapeptide amphisin from Pseudomonas sp. strain DSS73. Acta Crystallogr Sect Crystallogr Struct Commun 57:1123–1124. doi: 10.1107/S0108270101010782 CrossRefGoogle Scholar
  54. Sunish Kumar R, Ayyadurai N, Pandiaraja P, Reddy VA, Venkateswarlu Y, Prakash O, Sakthivel N (2005) Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. J Appl Microbiol 98:145–154. doi: 10.1111/j.1365-2672.2004.02435.x CrossRefGoogle Scholar
  55. Sutra L, Rojas MA, de los Rios GEJ, Saux Fischer-Le M, Jimenez P, Reche P, Bonneau S, Mathieu-Daude F, McClelland M (2001) Erwinia toletana sp. nov., associated with Pseudomonas savastanoi-induced tree knots. Int J Syst Evol Microbiol 54:2217–2222Google Scholar
  56. Vassilev N, Vassileva M, Nikolaeva I (2006) Simultaneous P-solubilizing and biocontrol activity of microorganisms. Appl Microbiol Biotechnol 71:137–144. doi: 10.1007/s00253-006-0380-z CrossRefGoogle Scholar
  57. Weisburg WG, Barns SM, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Babita Kumari Jha
    • 1
  • Mohandass Gandhi Pragash
    • 1
  • Jean Cletus
    • 1
  • Gurusamy Raman
    • 1
  • Natarajan Sakthivel
    • 1
    Email author
  1. 1.Department of BiotechnologyPondicherry UniversityKalapetIndia

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