| 한국의 고추 탄저병을 일으키는 Colletotrichum 5종의 신속한 검출을 위한 포자 PCR 및 qPCR 방법 |
정해준1,2, 윤종한1,2, 박호영1,2, 손민3, 박숙영1,2 
, 김광형3 |
1순천대학교식물의학과
2순천대학교 IT-Bio 융합시스템(BK21 plus) 협동과정
3서울대학교 농생명공학부 |
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| Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea |
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Haejun Jeong1,2, Jonghan Yoon1,2, Hoyoung Park1,2, Min Son3, Sook-Young Park1,2
, Kwang-Hyung Kim3 |
1Department of Plant Medicine, Sunchon National University, Suncheon 57922, Korea
2Interdisciplinary Program in IT-Bio Convergence System (BK21 plus), Sunchon National University, Suncheon 57922, Korea
3Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea |
Correspondence:
Sook-Young Park, Tel: +82-61-750-5187, Fax: +82-61-750-5187,
Email: spark@scnu.ac.kr
Kwang-Hyung Kim, Tel: +82-2-880-4672, Fax: +82-2-880-2317,
Email: sospicy77@snu.ac.kr
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Received: July 20, 2024 Revised: August 10, 2024 Accepted: August 14, 2024
*Haejun Jeong and Jonghan Yoon contributed equally to this study as co-first authors. |
| Abstract |
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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. |
| Key Words:
Capsicum annuum, Colletotrichum spp., Genomic DNA, Spore PCR, Quantitative real-time PCR |
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