Isolation and Taxonomical Characterization of Streptomyces sp. JR-24 with Antibacterial Activity of Bacterial Leaf Spot of Pepper (Xanthomonas axonopodis pv. vesicatoria)

고추 세균성 점무늬병원균(Xanthomonas axonopodis pv. vesicatoria)의 항균활성 Streptomyces sp. JR-24 균주의 분리 및 분류학적 특성

  • Han, Song-Ih (Department of Microbial and Nanomaterials, Mokwon University) ;
  • Lee, Hyo-Jin (Department of Microbial and Nanomaterials, Mokwon University) ;
  • Whang, Kyung-Sook (Department of Microbial and Nanomaterials, Mokwon University)
  • 한송이 (목원대학교 미생물나노소재학과) ;
  • 이효진 (목원대학교 미생물나노소재학과) ;
  • 황경숙 (목원대학교 미생물나노소재학과)
  • Received : 2010.12.02
  • Accepted : 2010.12.21
  • Published : 2010.12.31

Abstract

Fifty Actinobacteria strains were isolated from rhizosphere soil of Sasa borealis. In the course of screening for antibacterial activity against bacterial leaf spot of pepper (Xanthomonas axonopodis pv. vesicatoria) of isolates, 12 isolates showed strong antibiotic activity. Basis on the 16S rRNA gene sequence, they were belonging to Streptomyces cluster II. Strain JR-24 exhibited strong antibiotic activity against X. axonopodis pv. vesicatoria, had a minimum inhibitory concentration of 10 ${\mu}l$/disc. The strain JR-24 was most closely related to Streptomyces galbus $DSM40089^T$ (98.1%), Streptomyces longwoodensis $LMG20096^T$ (98%) and Streptomyces capoamus $JCM4734^T$ (97.8%). When assayed with the API 20NE and 50 CHE kit, it is positive for utilization of L-arabinose, D-fructose, D-glucose, D-galactose and hydrolysis of gelatin, protein, starch. The strains contained iso-$C_{14:0}$ (25.93%), iso-$C_{15:0}$ (10.13%), anteiso-$C_{15:0}$ (19.29%) and iso-$C_{16:0}$ (20.35%) as major fatty acids and MK-9 (H4), MK-9 (H6), and MK-9 (H8) as the isoprenoid quinone. Strain JR-24 was suggested new species of genus Streptomyces by nearest neighbors of genotypic relationships and phenotypic characterization. This study was important to microbial resources investigation for environment-friendly agriculture.

조릿대 근권토양으로부터 분리한 방선균 50균주를 대상으로 고추 세균성 점무늬병원균(Xanthomonas axonopodis pv. vesicatoria)의 항균활성 12균주를 선발하였다. 이들 항균활성 12균주의 계통학적 위치를 검토한 결과, 모두 Streptomyces 속의 Cluster II에 속하는 특징을 나타내었다. JR-24 균주는 최소저해 농도(MIC) 10 ${\mu}l$/disc를 나타내었으며, 배양액 5 ${\mu}l$/ml를 처리 하여 12시간 배양한 결과 Xanthomonas axonopodis pv. vesicatoria에 강한 생육저해효과를 나타내어 최우수 균주로 선발되었다. 항균활성 균주 JR-24의 16S rRNA 유전자 염기서열을 검토한 결과, Streptomyces galbus $DSM40089^T$ (X79852)와 98.1%, Streptomyces longwoodensis $LMG20096^T$ (AJ781356)와 98% 그리고 Streptomyces capoamus $JCM4734^T$ (AB045877)와 97.8%의 상동성을 나타내었다. API 20NE와 API 50CHE를 이용하여 JR-24 균주의 생리 생화학적 특성을 확인한 결과, L-arabinose, D-fructose, D-glucose, D-galactose을 이용하며 gelatin, protein, starch에 대하여 분해능이 있는 것으로 확인되었다. 주요지방산으로는 iso-$C_{14:0}$ (25.93%), iso-$C_{15:0}$ (10.13%), anteiso-$C_{15:0}$ (19.29%) 그리고 iso-$C_{16:0}$ (20.35%) 등을 함유하였으며, 퀴논종은 MK-9 ($H_4$) 4.37%, MK-9 ($H_6$) 51.22% 그리고 MK-9 ($H_8$) 49.47%로 동정되었다. Streptomyces sp. JR-24 균주의 계통학적 특성을 근연종인 Streptomyces galbus $DSM40089^T$와 비교한 결과, 다수의 표현형적 및 계통학적 차이를 나타내었다. 본 연구에서 분리된 Streptomyces sp. JR-24는 친환경 미생물제제 개발을 위한 유전자원 확보에 있어서 매우 큰의의가 있을 것으로 사료 된다.

Keywords

References

  1. Bae, M. 1978. Present status and future of antibiotics of agriculture. Kor. J. Microbiol. Biotechnol. 20, 141-148.
  2. Banos, S.B., M.H. Lopez, E.B. Molina, and C.L. Wilson. 2003. Effects of chitosan and plant extracts on growth of Colletotrichum gloeosporioides, anthracnose levels and quality of papaya fruit. Crop Protection 22, 1087-1092. https://doi.org/10.1016/S0261-2194(03)00117-0
  3. Berdy, J. 1989. The discovery of new bioactive microbial metabolites: Screening and identification, pp. 3-27. In M.E. Bushell (ed.), Bioactive metabolites from microorganisms, Elsevier, Amsterdam, Netherlands
  4. Chang, J.Y., H.H. Lee, I.C. Kim, and H.C. Chang. 2001. Characterization of a bacteriocin produced by Bacillus licheniformis cy2. J. Korean Soc. Food Sci. Nutr. 30, 227-233.
  5. Cho, Y.S., C.S. Park, S.K. Lee, Y.K. Lee, C.K. Lim, J.S. Cha, Y.C. Choi, J.Y. Choi, S.K. Her, and I.K. Hwang. 1999. Plant pathology, Seoul University Press.
  6. Euzeby, J.P. 2009. List of Prokaryotic Names with Standing in Nomenclature. http://www.bacterio.cict.fr/
  7. Ghim, S.Y., J.H. Jung, D.Y. Jung, Q.T. Phi, and C.M. Ryu. 2008. Bacillus megaterium KNUC251 for controlling plant disease and accelerating plant growth, and plant disease controlling agent and plant growth accelerant using the same. Korea Patent, 1009637740000
  8. Ghim, S.Y., M.S. Ham, S.J. Park, S.Y. Chen, C.M. Ryu, Y.M. Park, and H.R. Sung. 2009. Ochrobactrum sp. KUDC1013, and plant disease controlling agent and plant growth accelerant using the same. Korea Patent, 1020090033377.
  9. Go, Y.H. 1991. Screening of the new bioactive materials from Actinomyces (I). Ministry of education science and technology. BSG 70110-295-303.
  10. Hadden, J.F. and L.L. Black. 1986. The Status of ripe rot in Louisiana peppers. National Pepper Conferences. pp. 14.
  11. Ikemoto, S., M. Kuraishi, K. Komagata, R. Azuma, R. Suto, and H. Murooka. 1978. Cellular fatty acid composition in Pseudomonas species. J. Gen. Appl. Microbiol. 24, 199-213. https://doi.org/10.2323/jgam.24.199
  12. Kang Y.S., Y. Lee, S.K. Cho, K.H. Lee, B.J. Kim, M. Kim, Y. Lim, and M. Cho. 2009. Antibacterial activity of a disaccharide isolated from Streptomyces sp. strain JJ45 against Xanthomonas sp. FEMS Microbiol. Lett. 294, 119-125. https://doi.org/10.1111/j.1574-6968.2009.01561.x
  13. Kim, B.S., S.S. Moon, and B.K. Hwang. 1999. Isolation, antifungal activity, and structure elucidation of the glutarimide antibiotic, streptimidone, produced by Micromonospora coerulea. J. Agric. Food Chem. 47, 3372-3380. https://doi.org/10.1021/jf981259s
  14. Kim, S.H., Y.D. Lee, W.S. Ha, and H.M. Ro. 1998. A study on the transport phenomena of hydrophobic pesticides influenced by the interfaces of groundwater in unsaturated inorganic porous media. J. KSEE. 20, 1545-1553.
  15. Kloepper, J.W., C.M. Ryu, and S. Zhang. 2004. Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94, 1259-1266. https://doi.org/10.1094/PHYTO.2004.94.11.1259
  16. Lanoot. B., M. Vancanneyt, B. Hoste, K. Vandameulebroecke, M.C. Cnockaert, P. Dawyndt, Z. Liu, Y. Huang, and J. Swings. 2005. Grouping streptomycetes using 16S-ITS RFLP fingerprinting. Res. Microbiol. 156, 755-762. https://doi.org/10.1016/j.resmic.2005.01.017
  17. Lee, H.J. and K.Y. Whang. 2010. Biodiversity and phylogenetic analysis of Streptomyces collected from bamboo forest soil. Kor. J. Microbiol. 46, 262-269.
  18. Lee, J.Y. and B.K. Hwang. 2002. Diversity of antifungal actinomycetes in various vegetative soils of Korea. Can. J. Microbiol. 48, 407-417. https://doi.org/10.1139/w02-025
  19. Mcfaddin, J.F. 1980. Biochemical tests for identification of medical bacteria, pp. 308-320. 2nd ed. Williams and Wilkins, Baltimore, USA.
  20. National Statistical Office (NSO). 2010. The rice plant and red pepper area under cultivation investigation result.
  21. Oh, Y.K. and J.H. Kim. 1997. Effects of residual organochlorine pesticides in the coastal environment on the Cheju Island. J. KSWQ. 13, 317-324.
  22. Paik, S.B. and D.W. Kim. 1995. Screening for phyllospheral antagonistic microorganism for control of red-pepper anthracnose Colletotrichum gloeosporioides. Kor. J. Mycol. 23, 190-195.
  23. Park, J.S., K.S. Whang, and J.S. Cheon. 2005. Procedure of microbial classification and identification. Worldscience. Korea
  24. Park, K.S. and C.H. Kim. 1992. Identification, distribut characteristics of anthracnose fungi of red pepper in Korea. Kor. J. Plant Pathol. 8, 61-69.
  25. Park, S.B. and D.W. Kim. 1995. Screening for phyllosheral anta microorganism for control of red-peper anthracnose Colletotrichum gloeosporioides. Kor. J. Myco. 23, 190-195.
  26. Park, S.M., S.H. Han, and T.S. Yu. 2005. Culture conditions and antifungal activity of Bacillus licheniformis KMU-3 against crop pathogenic fungi. Kor. J. Microbiol. Biotechnol. 33, 112-116.
  27. Prosser, B.L.T. and N.J. Palleroni. 1976. Streptomyces longwoodensis sp. nov. Int. J. Syst. Bacteriol. 26, 319-322. https://doi.org/10.1099/00207713-26-3-319
  28. Shirling, E.B. and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16, 313-340. https://doi.org/10.1099/00207713-16-3-313
  29. Takashi, S. 1987. Cosmopolitan actinomycetes. J. Microorganism 3, 482-492.
  30. Thomson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W; improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  31. Yang, C.S. and J.S. Kim. 2002. Experiment method of soil microorganism. Worldscience. Korea.