Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

한국의 고추 탄저병을 일으키는 Colletotrichum 5종의 신속한 검출을 위한 포자 PCR 및 qPCR 방법

  • Haejun Jeong (Department of Plant Medicine, Sunchon National University) ;
  • Jonghan Yoon (Department of Plant Medicine, Sunchon National University) ;
  • Hoyoung Park (Department of Plant Medicine, Sunchon National University) ;
  • Min Son (Department of Agricultural Biotechnology, Seoul National University) ;
  • Sook-Young Park (Department of Plant Medicine, Sunchon National University) ;
  • Kwang-Hyung Kim (Department of Agricultural Biotechnology, Seoul National University)
  • 정해준 (순천대학교 식물의학과) ;
  • 윤종한 (순천대학교 식물의학과) ;
  • 박호영 (순천대학교 식물의학과) ;
  • 손민 (서울대학교 농생명공학부) ;
  • 박숙영 (순천대학교 식물의학과) ;
  • 김광형 (서울대학교 농생명공학부)
  • Received : 2024.07.20
  • Accepted : 2024.08.14
  • Published : 2024.09.30
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Abstract

Pepper anthracnose, caused by Colletotrichum spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of Colletotrichum spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

Colletotrichum spp.에 의해 감염되는 고추 탄저병은 고추 생산량에 경제적인 감소를 초래한다. 분자 진단은 병원균의 신속한 동정과 살균제 저항성을 결정하는 데 중요한 역할을 하는데, 이를 위한 병원균의 분리, 게놈 DNA 추출 및 유전자 염기서열 분석은 시간이 소요된다. 이 연구에서는 게놈 DNA 추출이나 특별한 처리 과정 없이 Colletotrichum 종의 포자를 사용하는 방법을 적용하였다. 방법의 명확성을 위해 ITS 기반의 primer 쌍을 이용하여 PCR 및 qPCR 검정으로 민감도를 분석하였다. PCR과 qPCR 실험 모두 탄저병균 1개의 포자에서도 검출되었다. 이 결과 1,000개 포자가 1 pg의 게놈 DNA와 동일하였다. QoI 계열 살균제 감수성 검정을 위한 cytb 유전자 증폭은 단일포자에서도 검출이 가능하지만, cycle 수를 35 cycle로 늘릴 경우 염기서열 정보를 안정적으로 확보할 수 있을 만큼의 PCR 산물이 확보되었다. 또한, V8-Juice 한천 배지에 10% 간 고추를 첨가할경우 일반적으로 사용되는 감자 한천 배지에 비해 3.2-6.0배 더 많은 포자가 형성되어 포자 기반 연구를 위해 적용 가능할 것이다. 본 연구를 종합하면, 이 연구는 PCR 및 qPCR 분석을 통해 고추 탄저병의 분자 동정을 위한 최소 포자량 및 바코드 유전자를 포함한 표적 유전자 증폭에 포자를 활용할 수 있는 유용한 기술을 제공한다.

Keywords

Acknowledgement

This work was supported by the Rural Development Administration of Korea (RS-2022-RD010350).

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