Isolation and Characterization of Insoluble Phosphate-Solubilizing Bacteria with Antifungal Activity

항진균능을 가진 불용성 인산 가용화 세균의 분리 및 특성

  • Park, Ki-Hyun (Department of Biotechnology, Miryang National University) ;
  • Son, Hong-Joo (Department of Biotechnology, Miryang National University)
  • Published : 2006.09.30

Abstract

To develop multifunctional microbial inoculant, an insluble phosphate-solubilizing bacterium with antifungal activity was isolated from plant rhizospheric soil. On the basis of its morphological, cultural and physiological characteristics and Biolog analysis, this bacterium was identified as Pseudomonas fluorescens RAF15. P. fluorescens RAF15 showed antifungal activities against phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. fluorescens RAF15 were 1.5% of glucose, 0.005% of urea, 0.3% $MgCl_2{\cdot}6H_2\;0.01%\;of\;MgSO_4{\cdot}7H_2O\;0.01%,\;of\;CaCl_2{\cdot}2H_2O$, and 0.05% of NaCl along with initial pH 7.0 at $30^{\circ}C$. The soluble phosphate production under optimum condition was 863 mg/L after 5 days of cultivation. The solubilization of insoluble phosphates was associated with a drop in the pH of the culture medium. P. fluorescens RAF15 showed resistance against different environmental stresses like $10-35^{\circ}C$ temperature, 1-4% salt concentration and pH 2-11 range. The strain produced soluble phosphate to the culture broth with the concentrations of 971-1121 mg/L against $CaHPO_4$, 791-908 mg/L against $Ca_3(PO_4){_2}$, and 844 mg/L against hydroxyapatite, respectively. However, the strain produced soluble phosphate to the culture broth with the concentrations of 15 mg/L against $FePO_4$, and 5 mg/L against $AlPO_4$, respectively.

다기능성 농업용 미생물 제제를 개발하기 위하여 항진균능을 가진 불용성 인산 가용화 세균을 식물의 근권 토양으로부터 분리하였다. 분리균주의 분류학적 위치를 검토한 결과, Pseudomonas fluorescens RAF15로 동정되었다. P. fluorescens RAF15는 농작물의 잿빛곰팡이병균 Botrytis cinerea와 줄기썩음병균 Rhizoctonia solani의 생육을 억제할 수 있었다. 불용성 인산인 $Ca_3(PO_4){_2}$로부터 가용성 인산을 생성하기 위한 최적 배지 및 배양조건은 glucose 1.5%, urea 0.005%, $MgCl_2{\cdot}6H_2\;0.3%,\;MgSO_4{\cdot}7H_2O\;0.01%,\;CaCl_2{\cdot}2H_2O$ 0.01%, NaCl 0.05%, 배양온도 $30^{\circ}C$ 및 초기 pH 7.0이었으며, 최적조건에서 배양 5일 후 863 mg/L의 가용성 인산이 생성되었다. 불용성 인산 가용화는 유기산 생성에 의한 배양액의 pH 감소와 밀접한 관계가 있었다. P. fluorescens RAF15는 $10-35^{\circ}C$, 1-4% 염 농도 및 pH 2.0-11.0의 범위에서도 가용성 인산을 생성할 수 있었다. 본 균주는 $CaHPO_4,\;Ca_3(PO_4){_2}$ 및 hydroxyapatite에서 각각 971-1121 mg/L, 791-908 mg/L 및 844 mg/L의 가용성 인산을 생성하였다. 그러나 $FePO_2$$AlPO_4$의 경우, 생성된 가용성 인산의 농도는 각각 18 mg/L, 5 mg/L이었다.

Keywords

References

  1. Shoda, M. 2000. Bacterial control of plant diseases. J. Biosci. Bioeng. 89, 515-521 https://doi.org/10.1016/S1389-1723(00)80049-3
  2. Sholberg, P.L., A. Marchi, and J. Bechard. 1995. Biocontrol of postharvest diseases of apple using Bacillus spp. isolated from stored apples. Can. J. Microbiol. 41, 247-252 https://doi.org/10.1139/m95-034
  3. Subhash, C.V., K.L. Jagdish, and K.T. Anil. 2001. Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J. Biotechnol. 91, 127-141 https://doi.org/10.1016/S0168-1656(01)00333-9
  4. Narsian, V. and H.H. Patel. 2000. Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biol. Biochem. 32, 559-565 https://doi.org/10.1016/S0038-0717(99)00184-4
  5. Vassilev, N., M.T. Baca, M. Vassileva, I. Franco, and R. Azcon. 1995. Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium. Appl. Microbiol. Biotechnol. 44, 546-549 https://doi.org/10.1007/BF00169958
  6. Kobayashi, D.Y. and N.E.H. El-Bararad. 1996. Selection of bacterial antagonists using enrichment cultures for the control of summer patch disease in Kentucky Bluegrass. Curr. Microbiol. 32, 106-111 https://doi.org/10.1007/s002849900019
  7. Chaurasia, B., A. Pandey, L.M.S. Palni, P. Trivedi, B. Kumar, andN. Colvin. 2005. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro. Microbiol. Res. 160, 75-81 https://doi.org/10.1016/j.micres.2004.09.013
  8. Patel, V.J., S.R. Tendulkar, and B.B. Chattoo. 2004. Bioprocess development for the production of an antifungal molecules by Bacillus licheniformis BC98. J. Biosci. Bioeng. 98, 231-235 https://doi.org/10.1016/S1389-1723(04)00274-9
  9. Nautiyal, C.S. 1997. Selection of chickpea-rhizosphere-competent Pseudomonas fluorescens NBRI1303 antagonistic to Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Phythium sp. Curr. Microbiol. 35, 52-58 https://doi.org/10.1007/s002849900211
  10. Gupta, R., R. Singal, A. Shankar, R.C. Kuhad, and R.K. Saxena. 1994. A modified plate assay for screening phosphate solubilizing microorganisms. J. Gen. Appl. Microbiol. 40, 255-260 https://doi.org/10.2323/jgam.40.255
  11. Nautiyal, C.S. 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265-270 https://doi.org/10.1111/j.1574-6968.1999.tb13383.x
  12. Son, H.J., G.T. Park, M.S. Cha, and M.S. Heo. 2006. Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere. Biores. Technol. 97, 204-210 https://doi.org/10.1016/j.biortech.2005.02.021
  13. Macfaddin, J.F. 1980. Biochemical Tests for Identification of Medical Bacteria. The Williams and Wilkins Co., Baltimore
  14. Barrow, G.I. and R.K.A. Felthanm. 1993. Cowan and Steel's Manual for the identification of medical bacteria. Cambridge University Press, New York
  15. Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams. 1994. Bergey's Manual of Determinative Bacteriology, 9th ed. The Williams and Wilkins Co., Baltimore
  16. Rodriguez, H., T. Gonzalez, and G. Selman. 2000. Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains. J. Biotechnol. 84, 155-161 https://doi.org/10.1016/S0168-1656(00)00347-3
  17. Clesscerl, L.S., A.E. Greenberg, and A.D. Eaton. 1998. Standard methods for the examination of water and wastewater, 20th ed.APHA-AWWA-WEF. Washington, D.C
  18. Nautiyal, C.S., S. Bhadauria, P. Kumar, H. Lal, R. Mondal, and D. Verma. 2000. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol. Lett. 182, 291-296 https://doi.org/10.1111/j.1574-6968.2000.tb08910.x
  19. Van Elas, J.D., J.T. Trevors, and E.M.H. Wellington. 1997. Modern soil microbiology. Marcel Dekker, Inc., New York
  20. Whitelaw, M.A., T.J. Harden, and K.R. Helyar. 1999. Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biol. Biochem. 31, 655-665 https://doi.org/10.1016/S0038-0717(98)00130-8
  21. Kim, K.Y., D. Jordan, and H.B. Krishnan. 1997. Rahnella aquatilis, a bacterium isolated from soybean rhizosphere, can solubilize hydroxyapatite. FEMS Microbiol. Lett. 153, 273-277 https://doi.org/10.1016/S0378-1097(97)00246-2
  22. Illmer, P., A. Barbato, and F. Schinner. 1995. Solubilization of hardly-soluble AlPO4 with P-solubilizing microorganisms. Soil Biol. Biochem. 27, 265-270 https://doi.org/10.1016/0038-0717(94)00205-F