Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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A Duplex PCR for Detection of Phytophthora katsurae Causing Chestnut Ink Disease

밤나무 잉크병균, Phytophthora katsurae의 검출을 위한 Duplex PCR

  • Lee, Dong-Hyeon (Department of Forest Environment Protection, Kangwon National University) ;
  • Lee, Sun-Keun (Department of Forest Environment Protection, Kangwon National University) ;
  • Kim, Hye-Jeong (Department of Forest Environment Protection, Kangwon National University) ;
  • Lee, Sang-Hyun (Division of Forest Insects and Diseases, Korea Forest Research Institute) ;
  • Lee, Sang-Yong (Department of Forest Environment Protection, Kangwon National University) ;
  • Lee, Jong-Kyu (Department of Forest Environment Protection, Kangwon National University)
  • 이동현 (강원대학교 산림환경보호학과) ;
  • 이선근 (강원대학교 산림환경보호학과) ;
  • 김혜정 (강원대학교 산림환경보호학과) ;
  • 이상현 (국립산림과학원 산림병해충과) ;
  • 이상용 (강원대학교 산림환경보호학과) ;
  • 이종규 (강원대학교 산림환경보호학과)
  • Received : 2012.05.31
  • Accepted : 2012.06.08
  • Published : 2012.06.30
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Abstract

Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 ${\mu}g/ml$ and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from $1{\times}10^6$ to $1{\times}10^2$ cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were $1{\times}10^5$ cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.

Phytophthora katsurae는 밤나무 잉크병의 원인이 되는 병원성 균류이다. P. katsurae를 검출하기 위하여 2개의 duplex PCR primer set을 제작하였다(SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R). SOPC 1F/1R과 SOPC 1-1F/1-1R primer pair는 sequence characteristic amplification regions(SCAR)를 이용하여 제작하였고, KatI 3F/5R primer pair는 internal transcribed spacer(ITS) 부위로부터 제작하였다. 추출한 genomic DNA를 1 mg/ml에서 1 ng/ml까지 10배씩 순차적으로 희석하여 균사량에 따른 Duplex PCR의 민감도를 확인한 결과, 2개의 primer set은 각각 1 ${\mu}g/ml$와 100 ng/ml까지 검출이 가능하였다. 유주포자를 $1{\times}10^6$부터 $1{\times}10^2$ cells/ml까지 10배씩 순차적으로 희석하고 genomic DNA를 추출하여 P. katsurae의 유주포자량에 의한 검출 한계를 확인한 결과, 2개의 primer set 모두 $1{\times}10^5$ cells/ml까지 검출할 수 있었다. 각각의 primer set은 PCR 검출을 실시하였을 때 P. katsurae 균주에서만 PCR 산물이 증폭된 반면에, Phytophthora 16종에서는 P. katsurae에 특이적인 PCR 증폭산물이 생성되지 않았다. 따라서, 2개의 primer set을 이용한 Duplex PCR은 P. katsurae의 신속하고 효과적인 검출에 유용하게 활용될 수 있을 것이다.

Keywords

References

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