The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation of Antifungal Substances by Bacillus amyloliquefaciens IUB158-03 and Antagonistic Activity against Pathogenic Fungi

Bacillus amyloliquefaciens IUB158-03이 생산하는 항진균물질의 분리와 항균활성

  • Published : 2009.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

For the control of pathogenic microorganisms, Bacillus spp. were isolated from diseased pepper fruits in Korea. Among them, Bacillus sp. IUB158-03 showed high inhibitory effect on mycelial growth and spore germination of C. gloeosporioides and Botrytis cinerea. The strain was identified as B. amyloliquefaciens IUB158-03 based on its physiological, biochemical characteristics and Microlog analysis. The highest level of antifungal substances by B. amyloliquefaciens IUB158-03 were obtained when the bacterium was cultured in medium containing 2% soluble starch, 3% yeast extract, 0.5% tryptone, 0.5% $NH_4H_2PO_4$, and 1% NaCl (pH 6.0) at $25^{\circ}C$ for 72 hrs. The antifungal substances were purified by butanol extraction, silica gel column chromatography, preparative thin layer chromatography, and high performance liquid chromatography. The purified antifungal substance was confirmed $R_f$ 0.27 by TLC. This substance exhibited antifungal activity against Fusarium solani, Rhizoctonia solani, Botrytis cineria, Alternata alternaria of plant pathogenic fungi and Trichophyton mentagrophytes, Epidermophyton floccosum, Cryptococcus neoformans of human pathogenic fungi.

병원성 미생물을 방제하기 위하여 국내의 고추 재배지에서 탄저병균에 감염된 고추로부터 Bacillus속 균주를 분리하였다. 이들 중 고추탄저병균인 C. gloeosporioides 균주와 식물에 회색곰팡이병을 일으키는 Botrytis cinerea 균주에 대한 길항작용이 가장 우수한 Bacillus sp. IUB158-03을 선발하였다. 선발된 균주의 형태적, 생리 생화학적 특성과 MicroLog 방법을 이용하여 동정된된 균주는 B. amyloliquefaciens IUB158-03로 명명하였다. 항진균물질 생산을 위한 최적배지 조건은 2% soluble starch, 3% yeast extract, 0.5% tryptone, 0.5% $NH_4H_2PO_4$, 1% NaCl의 pH 6.0에서 $25^{\circ}C$, 72시간 배양한 추출물에서 가장 높은 항진균활성을 나타내었다. B. amyloliquefaciens IUB158-03이 생산한 항진균물질을 butanol로 추출한 후 silica gel column chromatography, preparative thin layer chromatography, and HPLC을 이용하여 항진균물질을 정제하였다. 정제된 항진균물질은 TLC plate 상에서 $R_f$값이 0.27인 것이 확인되었으며, 이 물질은 식물병원균인 Fusarium solani, Rhizoctonia solani, Botrytis cineria, Alternata alternaria와 인체병원성 진균인 Trichophyton mentagrophytes, Epidermophyton floccosum, Cryptococcus neoformans 등에만 선택적으로 높은 항진균 활성을 보였다.

Keywords

References

  1. 문병주, 강진동, 이재필, 손지희, 차외진, 노성환. 1997. Bacillus licheniformis N1과 B. megaterium N4을 이용한 들깨 잎마름병 (Alternaria alternata) 생물학적 방제. 한국균학회 균학회소식. 12(1):73.
  2. Adams, P. B. 1990. The potential of mycoparasites for biological control of plant diseases. Annu. Rev. Phytopathol. 28:59-72. https://doi.org/10.1146/annurev.py.28.090190.000423
  3. Backhouse, D. and A. Stewart. 1989. Ultrastructure of antagonism Sclerotium cepiviorum by Bacillus subtilis. J. Phytopathology. 124:207-214. https://doi.org/10.1111/j.1439-0434.1989.tb04916.x
  4. Baker C. J., J. Stavely, R. Tomas, C. A. Myron, S. Janet and S. Macfall. 1983. Inhibitory effect of Bacillus subtilis on Uromyces phasoli and on development of rust pustules on bean leaves. J. Phytophathology. 73(8):1148-1152. https://doi.org/10.1094/Phyto-73-1148
  5. Becker, J. O. 1993. Control of soil-borne pathogens with living bacteria and fungi: status and outlook. Pestic. Sci. 37:355-363. https://doi.org/10.1002/ps.2780370408
  6. Berdy, J. 1989. The discovery of new bioactive microbial metabolites: Screening and identification, In Bushell. M. E. and U. Grafe(ed), Bioactive metabolites from microorganisms, Elsevier, Amsterdam. 3-25.
  7. Besson, F., F. Peyponx, G. Michel and L. Delcombe. 1978. Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis on Micrococcus leteus. Biochem. Biophys. Res. Commun. 81: 297-304. https://doi.org/10.1016/0006-291X(78)91532-2
  8. Brannen, P. M. and Kenney D. S. 1997. Kodiak-a successful biological- control product for suppression of soil-borne plant pathogens of cotton. J. Indust. Microbiol. Biotechnol. 19:169-171. https://doi.org/10.1038/sj.jim.2900439
  9. Burpee, L. L. and L. G. Goulty. 1984. Evaluations of fungicides for control of pink and gray snow mold on creeping bentgrass. pp.6-7, In; Turfgrass Research Annual Report, R. W. Sheard(ed). Univ. of Guelph, Ontario. 38.
  10. Cook, R. J. 1985. Biological control of plant pathogens: Theory to application. J. Phytopathology. 75:25-28. https://doi.org/10.1094/Phyto-75-25
  11. Fravel, D. R., W. J. Connick and J. A. Lewis. 1998. Formulation of microorganisms to control plant diseases. pp. 187-202. In H. D. Burges(eds). Formulation of microbial biopesticides, beneficial microorganisms and nematodes and seed treatments. Kluwer Academic Publishers, Dordrecht, London.
  12. Gueldner, R. C., C. C. Reilly, P. L. Pusey, C. E. Costello, R. F. Arrendale, R. H. Cox, D. S. Himmelbach, F. G. Crumley and H. G. Cutler. 1988. Isolation and identification of iturins as antifungal peptides in biological control of peach brown rot with Bacillus subtilis. J. Agri. Food. Chem. 36:366-370 https://doi.org/10.1021/jf00080a031
  13. Harold J. Benson. 1990. Microbiological Applications; A Laboratory Manual in General Microbiology. 5th ed. Dubuque, Iowa: Wm. C. Brown Publishers.
  14. 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, Williams Wilkins, U. S. A.
  15. Huang, T. C. and M. C. Chang. 1975. Studies on Xanthobacidin, a new antibiotic from Bacillus subtilis active against Xanthomonas. Bot. Bull. Acad. Sinica. 16:137-148.
  16. Jeong, Y. K, Y. Shin, M. J. Jung, W. H. Joo and J. S. Choi. 2002. Structural analysis of the antifungal antibiotic from Bacillus sp. YJ-63. Kor. J. Microbiol. Biotechnol. 30(1):21-25.
  17. Kharbanda, P. D., J. Yang, P. Beatty, S. Jensen and J. P. Tewari. 1997. Potential of a Bacillus spp. to control blackleg and other diseases of canola (Abstracts). J. Phytopathology. 87:51.
  18. Kloeper, J. W., J. Leong, M. Teintze and M. N. Schroth. 1980. Pseudomonas siderophore: A mechanism explaning disease suppressive soil. Curr. Microbiol. 4:317-320. https://doi.org/10.1007/BF02602840
  19. Lifshitz. R., M. Y. Windham and R. Baker. 1986. Mechanism biological control of pre-emergence damping-off of pea by seed treatment with Trichoderma spp. J. Phytopathology. 76:720-725. https://doi.org/10.1094/Phyto-76-720
  20. Lim, C. S., J. Y. Kim, T. H. Lee, Y. G. Park and Y. H. Han. 2002. Antifungal activity of the isolated Bacillus licheniformis against red-pepper anthracnose-causing Colletotrichum coccides. Kor.J. Mycology. 14(2):73-74.
  21. Lounes, A., A. Lebrihi, C. Benslimane and G. Lefebvre. 1996. Regulation of spiramycin synthesis in Streptomyces ambofaciens: effects of glucose and inorganic phosphate. Appl. Microbiol. Biotechol. 45:204-211. https://doi.org/10.1007/s002530050671
  22. Maget-Dana, R. M. Ptak, F. Peypoux and G. Michel. 1985. Poreforming properties of a lipopeptide antibiotic: Iturin A. Biochiem. Biophys. Acta. 815:405-409. https://doi.org/10.1016/0005-2736(85)90367-0
  23. Mandeel, Q. and R. Baker. 1993. Mechanisms involved in biological control of Fusarium wilt of cucumber with strains of nonpathogenic Fusarium oxysporum. J. Phytopathology. 81:462-469. https://doi.org/10.1094/Phyto-81-462
  24. Mari, M., M. Guizzardi and G. C. Partella. 1996. Biological control of gray mold in pears by antagonistic bacteria. Biological Control. 7:30-37. https://doi.org/10.1006/bcon.1996.0060
  25. Paulitz, T. C. and J. E. Loper. 1991. Lack of a role for fluorescent siderophore prodution in the biological control of Phythium damping-off of cucumber by a strain of Pseudomonas putida. J. Phytopathology. 81:930-935. https://doi.org/10.1094/Phyto-81-930
  26. Schiewe, A. and K. Mendgen. 1992. Identification of antagonists for biological control of the post harvest pathogens Pezicula malicorticis and Nectria galligena on apple. J. Phytopathology. 134:229-237. https://doi.org/10.1111/j.1439-0434.1992.tb01231.x
  27. Shen, S. S., O. H. Choi, S. M. Lee and C. S. Park. 2002. In vitro and in vivo activities of a biocontrol agent. Serratia plymuthica A21-4, against Phytophthora capsici. Plant pathol. J. 18(4): 221-2240. https://doi.org/10.5423/PPJ.2002.18.4.221
  28. Yoshie, Y., I. Katsushige, U. Yoshihisa, O. Atsuko, T. Kazutoh, K. Ikunoshin and N. Hiroshin. 1993. Isolation, structures, and antifungal activities of new Aureobasidins. J. Antibiotics. 46: 1347-1354. https://doi.org/10.7164/antibiotics.46.1347
  29. Victor Lorian, M. and D. Editor. 1991. Antibiotics in laboratory medicine (3nd). Williams & Wilkins. 17-52.
  30. Waksman, S. A. and A. T. Heinrich. 1943. The nomenclature and classification of the Actinomycetes. J. Bacteriol. 46: 337-341.
  31. Wang, S. L., I. L. Shin, Y. W. Liang and C. H. Wang. 2002. Purification and characterization of two antifungal chitinases extracellularly produced by Bacillus amyloliquefaciens. J. Agric. Food. Chem. 50: 2241-2248. https://doi.org/10.1021/jf010885d
  32. Zuber, P., M. M. Nakano, and M. A. Marahicl. 1993. Peptidc antibiotics, Pp 897-916. In A. L. Sonenshein et al (ed). Bacillus subtilis and Other Gram-Positive Bacteria, American Society for Microbiology, Washington, D. C.

Cited by

  1. The Optimal Culture Conditions and Antifungal Activity of Culture Extract from Oudemansiella mucida vol.39, pp.2, 2011, https://doi.org/10.4489/KJM.2010.39.2.091
  2. Difference of Catechins Extracted Level when Fermented Sun-dried Salt and Green Tea vol.12, pp.11, 2012, https://doi.org/10.5392/JKCA.2012.12.11.278
  3. Toxicity and Characteristics of Antifungal Substances Produced by Bacillus amyloliquefaciens IUB158-03 vol.19, pp.11, 2009, https://doi.org/10.5352/JLS.2009.19.11.1672
  4. Antagonistic Mechanisms and Culture Conditions of Isolated Microbes Applied for Controlling Large Patch Disease in Zoysiagrass vol.33, pp.4, 2015, https://doi.org/10.7235/hort.2015.15066