Journal of Korean Society of Forest Science (한국산림과학회지)

Korean Society of Forest Science (한국산림과학회)
권호 표지

PCR Detection and Sequence Analysis of the rDNA ITS Regions of Rhizina undulata

Rhizina undulata rDNA ITS 영역의 PCR 검정 및 염기배열 분석

  • Lee, Sun Keun (Department of Forest Resources Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Lee, Jong Kyu (Department of Forest Resources Protection, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Kim, Kyung Hee (Division of Forest Insect Pests and Disease, Korea Forest Research Institute) ;
  • Lee, Seung Kyu (Division of Forest Insect Pests and Disease, Korea Forest Research Institute) ;
  • Lee, Sang Yong (Department of Forest Resources Protection, College of Forest and Environmental Sciences, Kangwon National University)
  • 이선근 (강원대학교 산림자원보호학과) ;
  • 이종규 (강원대학교 산림자원보호학과) ;
  • 김경희 (국립산림과학원 산림병해충과) ;
  • 이승규 (국립산림과학원 산림병해충과) ;
  • 이상용 (강원대학교 산림자원보호학과)
  • Received : 2007.06.14
  • Accepted : 2007.07.11
  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

To investigate genetic diversity and PCR detection of Rhizina undulata, PCR detection and sequence analysis of rDNA ITS region of R. undulata in soil were analyzed and developed. The length of partial 18S rDNA from four R. undulata isolates were 1,375 nt. The sequence similarity of R. undulata isolates was 100%. The rDNA ITS regions of R. undulata isolates were 585 nt long. Nucleotide sequencing of the ITS regions showed that PDK-1, PTT-1 and PDJ-9 isolates had 100% sequence identity. But, PDS-5 isolate differed from the three isolates by two nucleotide substitution. R. undulata-specific primers designed by the sequence of ITS region were used in PCR detection of R. undulata. PCR products about 525 bp size, which is specific to R. undulata, were amplified from total DNAs of R. undulata isolates. To assay the sensitivity of PCR detection by R. undulata ITS-specific primer, purely cultured mycelial suspension of R. undulata was serially diluted and mixed with 100g of sterile sandy loam soil, respectively. And then, PCR products of total DNAs extracted from each mycelium-soil mixtures were analysed. The PCR protocol could detected up to 1ng mycelium of R. undulata within 100g of soil.

Rhizina undulata의 PCR 검정 및 유전적 특성 분석을 목적으로, rDNA ITS 영역의 염기배열 해석 및 PCR 방법에 의한 토양으로부터 R. undulata의 진단법을 개발하였다. 18S rDNA 부분의 염기서열 분석 결과, 공시한 4종의 균주 모두 1,375 nt의 크기로 동일하였으며, 염기배열도 100% 일치하였다. 한편, rDNA ITS 영역의 염기배열은 585 nt이었고, PDK-1, PTT-1 및 PDJ-9 균주는 염기배열이 100% 동일하였으나, PDS-5균주에서는 두 곳에서 염기의 치환이 발견되었다. 이와 같은 염기배열을 분석하여 제작한 R. undulata rDNA ITS 영역 특이적 primer를 이용한 PCR 검정 결과, R. undulata 균주들에서만 약 525 bp 크기의 ITS 영역 특이적인 증폭산물이 검출되었다. PCR 방법에 의하여 검출할 수 있는 토양 중의 R. undulata 최소 균사량의 한계를 확인하기 위해서, 순수 배양한 R. undulata 균사현탁액을 순차 희석하여 100g의 사양토에 혼합한 다음, 농도별로 균사 혼합한 각각의 토양 시료로부터 추출한 total DNA의 PCR 증폭산물을 분석한 결과, PCR 방법에 의하여 100g의 토양 중에 1 ng의 R. undulata 균사가 함유되어 있는 경우까지 검출이 가능하였다.

Keywords

Acknowledgement

Supported by : 강원대학교

References

  1. 李相龍, 令完圭 1990 소나무 리지나뿌리썩음病에 關한 研究-Rhizina undulata의 生理的 特性 및 病原性. 韓國林學會誌 79: 322-329
  2. 이상용, 이선근, 이종규, 김경희, 이승규. 2006. 리지나뿌 리썩음병균 분리주들의 배양 특성 및 RAPD에 의한 유 전적 다양성 분석. 한국임학회지 95: 388-392
  3. 李昌根, 呂運鴻, 金敎秀, 金京姬. 1982. 잣나무잎녹病等 樹木病害 3種에 關한 研究. 林業試驗場研究報于書. 29: 253-262
  4. 八幡ー彦, 作山建. 1982.マ、ツっちくらげ病の病原菌の捕 捉とマツ枯損進行との闘係.日本林學會東北支部會誌 34: 111-112
  5. Chao, C.C., Lee, J.T and Tsung, C.C. 2004. Identification of Clinically Relevant Viridans Group Streptococci by Sequence Analysis of the 16s-23s Ribosomal DNA Spacer Region. Journal of Clinical Microbiology 42(6): 2651-2657 https://doi.org/10.1128/JCM.42.6.2651-2657.2004
  6. Cullen, S.W. and Hirsch, P.R. 1998. Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Soil Biology and Biochemistry 30: 983-993 https://doi.org/10.1016/S0038-0717(98)00001-7
  7. Germrnen, J. 1971. Rhizina undulata. A review of research in the Netherlands. European Journal of Forest Pathology1: 1-6 https://doi.org/10.1111/j.1439-0329.1971.tb00283.x
  8. Gibson, I,A.S, 1970. Disease of Pinus patula review. Commonw. Forest Review 49: 267-274
  9. Kageyama, K., Komatsu, T and Suga, H. 2003. Refined PCR protocol for detection of plant pathogens in soil. The Phytopathological Society of Japan. 69: 153- 160
  10. Kikuchi, K., Matsushita, N., Guerin-Laguette, A., Ota, A. and Suzuki K. 2000. Detection of Tricholoma matsutake by specific ITS primers. Mycological Research 104(12): 1427-1430 https://doi.org/10.1017/S0953756200002653
  11. Kreader C.A. 1996. Relief of amplification inhibition in PCR with bovine serum albumin of T4 gene 32 protein. Applied and Environmental Microbiology 62: 1102- 1106
  12. Lee, J.K., Lee, S.Y, Lee, S.J. and Kim, K.H. 2005. Fruiting body development of a root pathogenic fungus, Rhizina undulata, after forest tire in eastern coastal pine forests of Korea. Forest Research 1: 33-37
  13. Lu, L., Li, J. and Cang Y. 2002. PCR-Based Sensitive Detection of Medicinal Fungi Hericium Species from Ribosomal Internal Transcribed Spacer(ITS) Sequences. Pharmaceutical Society of Japan. 25(8): 975-980
  14. Peterson, S.W. and Kurtzman, C.P. 1991. Ribosomal RNA sequence divergence among sibling species of yeasts. Systematic and Applied Microbiology 14: 124-129 https://doi.org/10.1016/S0723-2020(11)80289-4
  15. Porteus, L.A., Armstrong, J.L., Seidler, R.J. and Watrud, L.S. 1994. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification. Journal of Applied Bacteriology 74: 78-85
  16. Ranjard, L., Poly, F., Lata, J.C., Mougel, C., Thioulouse, J. and Nazaret, S. 2001. Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Applied and Environmental Microbiology 67(10): 4479-4487 https://doi.org/10.1128/AEM.67.10.4479-4487.2001
  17. Sugita, T, Nishikawa, A., Ikeda, R. and Shinoda, T. 1999. Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. Journal of Clinical Microbiology 37: 1985-1993
  18. Thompson, J.H. and Tattar, TA. 1973. Rhizina undulata associated with disease of 80-year-old red spruce in Vermont. Plant Disease Reporter 57: 394-396
  19. Weir, J.R. 1915. Observation on Rhizina inflata. Journal of Agricultural Research 4: 93-95
  20. White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. pp. 315-322. In : M.A Innis, D.H. Gelfand, J.J. Snisky and T.J. White, ed. PCR Protocols. Academic Press, London