Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Molecular Characteristics of Phytophthora katsurae Using PCR-SSCP Analysis

PCR-SSCP 분석에 의한 Phytophthora katsurae의 분자생물학적 특성

  • Lee, Sun-Keun (Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Jang, Ha-Na (Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Lee, Dong-Hyeon (Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Lee, Sang-Hyun (Division of Forest Insect Pests and Diseases, Korea Forest Research Institute) ;
  • Lee, Sang-Yong (Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Lee, Jong-Kyu (Department of Forest Environment Protection, College of Forest and Environmental Sciences, Kangwon National University)
  • 이선근 (강원대학교 산림환경과학대학 산림환경보호학과) ;
  • 장하나 (강원대학교 산림환경과학대학 산림환경보호학과) ;
  • 이동현 (강원대학교 산림환경과학대학 산림환경보호학과) ;
  • 이상현 (국립산림과학원 산림병해충과) ;
  • 이상용 (강원대학교 산림환경과학대학 산림환경보호학과) ;
  • 이종규 (강원대학교 산림환경과학대학 산림환경보호학과)
  • Received : 2010.11.30
  • Accepted : 2011.05.28
  • Published : 2011.08.31
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Abstract

Phytophthora katsurae is the fungus responsible for chestnut ink disease. The objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis of rDNA-ITS region, elongation factor 1 alpha gene and ${\beta}$-tubulin gene could be used for rapid identification and genetic diversity of P. katsurae, and to assess the potential use of the SSCP technique as a diagnostic tool for P. katsurae. Each regions amplified by PCR using primers designed to overlap the genus Phytophthora were characterized for the Phytophthora species. PCR products were denatured and electrophoresed for SSCP analysis. P. katsurae isolates showed an unique pattern in SSCP analysis and were easily distinguished from other Phytophthora species used as the control. This indicates that SSCP analysis is an useful technique for distinguishing Phytophthora species from genetically close relatives, and show that the SSCP analysis of each region is an efficient detection tool for P. katsurae. But PCR-SSCP analysis of single-gene may have difficulty in distinguishing P. katsurae from other Phytophthora species. Therefore, PCR-SSCP analysis of multi-genes can be useful for rapid and effective identification of P. katsurae.

우리나라에서 분리한 P. katsurae의 유전적 특성을 구명하기 위하여 국내에서 분리한 P. katsurae를 대상으로 nuclear DNA(nDNA)의 ${\beta}$-tubulin (BTU)과 Elongation facter 1 alpha (EF1A) 그리고 rDNA ITS 부위의 PCR-SSCP 분석을 실시하여, P. katsurae와 Phytophthora 속에 속하는 다양한 종들의 각 부위를 대상으로 유전적 유연관계를 비교분석 하고 동정에 이용하고자 하였다. 각각의 Phytophthora 속에서 변이가 가장 많이 발생하는 부위를 포함하여 증폭 시킬 수 있도록 각 부위의 공통 염기배열로부터 제작된 primer는 Phytophthora 종에 특이적인 반응을 나타냄으로서 동정 및 진단에도 유용하게 활용될 수 있을 것으로 판단되었다. SSCP 분석 결과는 국내 P. katsurae 균주와 공시한 다른 Phytophthora 속 균주들과의 구분이 가능하였으며, Phytophthora 종 간의 구분도 가능하였다. 그러나 한 가지 부위만을 이용한 PCR-SSCP 분석은 Phytophthora 종 간의 구분이 어려운 경우도 있었다. 따라서 보다 정확하고 명확한 Phytophthora 종의 유전적 다양성 분석 및 동정을 위하여서는 단일 부위에 의한 PCR-SSCP보다는 복수 부위에 의한 PCR-SSCP를 실시하는 것이 바람직한 것으로 확인되었다.

Keywords

References

  1. 김만조, 황명수, 김선창, 이욱. 2007. 한국의 밤나무 재배품종에 대한 밤나무혹벌 내충성 검정. 한국임학회지 96: 295-299.
  2. 김태욱. 2002. 한국의 수목. 교학사 pp. 64-65.
  3. 이상현, 김경희, 변재경, 이종규, 문병주. 2006. 밤나무 재배지에 서의 생물적, 환경적 요인이 밤나무 줄기마름병 발생에 미치는 영향. 한국임학회지 95: 139-144.
  4. Abreu, C. G., Carvalho, L., Gomes, M. J., Colaco, J. and Cardoso, A. O. 1999. Assessment of resistance to chestnut ink disease. Acta Hort. 494: 363-367.
  5. Blair, J. E., Coffey, M. D., Park, S. Y., Geiser, D. M. and Kang, S. 2007. A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences. Fungal Gen. Biol. 45: 266-277.
  6. Boutard, A. 2001. The western chestnut: More information on chestnuts and ink disease. The Western Chestnut Growers Assn., Inc. 3: 6-11.
  7. Brasier, C. M. and Jung, T. 2006. Recent developments in Phytophthora diseases of trees in natural ecosystems in Europe. Progress in Research on Phytophthora diseases in Forest trees. 5-16, Farnham.
  8. Chang, T. T., Yang, W. W. and Wang, W. Y. 1996. Use of random amplified polymorphic DNA markers for the detection of genetic variation in Phytophthora cinnamomi in Taiwan. Bot. Bul. Aca. Sinica 37: 165-171.
  9. Ersek, T., Schoelz, J. E. and English, J. T. 1994. PCR Amplication of species-specific DNA sequences can distinguish among Phytophthora species. Appl. Environ. Microbiol. 60: 2616-2621.
  10. Hayashi, K. 1991. PCR-SSCP: A simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods and Applications 1: 34-38. https://doi.org/10.1101/gr.1.1.34
  11. Hong, C., Gallegly, M. E., Richardson, P. A., Kong, P. and Moorman, G. W. 2008. Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America. Fed. Eur. Microbiol. Soc. 285: 203-211. https://doi.org/10.1111/j.1574-6968.2008.01226.x
  12. Katsura, K. 1976. Two new species of Phytophthora causing damping-off of cucumber and trunk rot of chestnut. Trans. Mycol. Soc. Japan 17: 238-242.
  13. Jyan, M. H., Huang, L. C., Ann, P. J. and Liou, R. F. 2002. Rapid detection of Phytophthora infestans by PCR. Plant Pathol. Bull. 11: 45-56.
  14. Ko, W. H. and Chang, H. S. 1979. Phytophthora katsurae, a new name for P. castaneae. Mycologia 71: 840-844. https://doi.org/10.2307/3759198
  15. Kong, P., Hong, C. X., Richardson, P. A. and Gallegly, M. E. 2003. Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora. Fungal Gen. Biol. 39: 238-249. https://doi.org/10.1016/S1087-1845(03)00052-5
  16. Kong, P., Hong, C. X., Tooley, P. W., Ivors, K., Garbelotto, M. and Richardson, P. A. 2004. Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1. Soc. Appl. Microbiol. 38: 433-439. https://doi.org/10.1111/j.1472-765X.2004.01510.x
  17. Kroon, L. P. N. M., Bakker, F. T., Van den Bosch, G. B. M., Bonants, P. J. M. and Flier, W. G. 2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences. Fungal Gen. Biol. 41: 766-782. https://doi.org/10.1016/j.fgb.2004.03.007
  18. Kong, P., Richardson, P. A. and Hong, C. 2005. Direct colony PCR-SSCP for detection of multiple pythiaceous oomycetes in environmental samples. J. Microbiol. Meth. 61: 25-32. https://doi.org/10.1016/j.mimet.2004.10.019
  19. Mahuku, G., Peters, R. D., Platt, H. W. and Daayf, F. 2000. Random amplified polymorphic DNA (RAPD) analysis of Phytophthora infestans isolates collected in Canada during 1994 to 1996. Plant Pathol. 49: 252-260. https://doi.org/10.1046/j.1365-3059.2000.00450.x
  20. Martin, F. N. and Tooley, P. W. 2003. Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes. Mycologia 95: 269-284. https://doi.org/10.2307/3762038
  21. Oh, E. S., Lee, J. K., Lee, S. H. and Kim, K. H. 2007. Chestnut ink disease caused by Phytophthora katsurae. J. Forest Sci. 23: 65-71.
  22. Ristaino, J. B., Madritch, M., Trout, C. L. and Parra, G. 1998. PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora. Appl. Environ. Microbiol. 64: 948-954.
  23. Shen, G., Wang, Y. C., Zhang, W. L. and Zheng, X. B. 2005. Development of a PCR assay for the molecular detection of Phytophthora boehmeriae in infected cotton. J. Phytopathol.153: 291-296. https://doi.org/10.1111/j.1439-0434.2005.00971.x
  24. Verma, L. R. and Sharma, R. C. 1999. Diseases of horticultural crops: fruits. Indus Publishing 226-228.
  25. Vettraino, A. M., Morel, O., Perlerou, C., Diamandis, S. and Vannini, A. 2005. Occurrence and distribution of Phytophthora species in European chestnut stands, and their association with ink disease and crown decline. Eur. J. Plant Pathol. 111: 169-180. https://doi.org/10.1007/s10658-004-1882-0
  26. Vettraino, A. M., Natili, G., Anselmi, N. and Vannini, A. 2001. Recovery and pathogenicity of Phytophthora species associated with a resurgence of ink disease in Castanea sativa in Italy. Plant Pathol. 50: 90-96. https://doi.org/10.1046/j.1365-3059.2001.00528.x
  27. Willem, A. M., Wil, J. V., Elena, I., Arthur, W. A. M. de cock., Peter, J. M. B. and Rob, P. 1998. Natural hybrids of Phytophthora nicotianae and Phytophthora cactorum demonstrated by isozyme analysis and random amplified polymorphic DNA. Phytopathology 88: 922-929. https://doi.org/10.1094/PHYTO.1998.88.9.922