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Detection of Anthracnose Fungus Colletotrichum circinans by Conventional PCR and Real-time PCR

일반 PCR과 Real-time PCR을 이용한 탄저병균 Colletotrichum circinans 검출

  • 김준영 (단국대학교 자연과학대학 미생물학과)
  • Received : 2018.11.08
  • Accepted : 2018.11.19
  • Published : 2018.12.01

Abstract

Colletotrichum circinans, an anthracnose pathogen, causes serious damage to onions worldwide. In this study, specific molecular markers were developed to detect C. circinans accurately and quickly with both conventional and real-time PCR methods. The cirTef-F/cirTef-R and cirTu-F/cirTu-R primer sets, which are specific for C. circinans, were constructed by analyzing $tef-1{\alpha}$ and ${\beta}-tubulin$ genes in the fungus. Using the conventional PCR method, 100 pg and 1 ng of fungal DNA could be detected using the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Using the real-time PCR method, 10 pg and 100 pg of fungal DNA could be detected more sensitively with the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Detection of C. circinans from the artificially infected onion seeds was possible by using both conventional and real-time PCR methods and the developed cirTef-F/cirTef-R primer set. The PCR markers specific for C. circinans developed in this study may enhance the efficiency of fungal pathogen detection in imported vegetables and seeds.

탄저병균인 Colletotrichum circinans는 세계적으로 양파에 심각한 피해를 주는 병원균이다. 본 연구에서는 일반 PCR방법과 real-time PCR방법으로 C. circinan를 정확하면서도 쉽고 빠르게 검출이 가능한 특이 마커를 개발하였다. $tef-1{\alpha}$ 유전자와 ${\beta}-tubulin$ 유전자를 분석하여 C. circinan를 특이적으로 검출할 수 있는 cirTef-F/cirTef-R set와 cirTu-F/cirTu-R set를 제작하였다. 일반 PCR 방법으로 cirTef-F/cirTef-Rset는 100pg, cirTu-F/cirTu-Rset는 1ng까지 검출이 가능하였고 real-time PCR 방법으로는 각각 10 pg, 100 pg까지 검출이 가능하였다. C. circinans에 인공적으로 감염된 양파 종자에서도 cirTef-F/cirTef-Rset를 사용하여 일반 PCR방법과 real-time PCR 방법 모두 C. circinans검출이 가능하였다. 본 연구에서 개발한 C. circinans 특이 검출마커는 수출입 되는 채소 및 종자에서 빠르고 정확하게 탄저병균인 C. circinans를 검출하는데 사용될 수 있을 것이다.

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Fig. 1. Sequence alignment of translation elongation 1-α genes of Colletotrichum spp. used in this study. Green and sky-blue boxed sequences indicate respectively the position of the designed primer cirTef-F and cirTef-R. GJS08_43: C. theobromicola, E183: C. ignotum, 125331: C. circinans, 112.81: C. circinans, 41028: C. caudatum, 40807: C. higginsianum, 42433: C. lindemuthianum, 40893: C. boninense, 40009: C. coccodes, 46159: C. capsici, 40805: C. acutatum, 40003: C. gloeosporioides, 40808: C. orbiculare.

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Fig. 2. Sequence alignment of β-tubulin genes of Colletotrichum spp. used in this study. Green and sky-blue boxed sequences indicate respectively the position of the designed primer cirTu-F and cirTu-R. 112.81: C. circinans, 125331: C. circinans, 40981: C. liliacearum, 40009: C. coccodes, 40010: C. coccodes, 40805: C. acutatum, 346.37: C. fructi, 40808: C. orbiculare, 40903: C. orbiculare, 40893: C. boninense, 40013: C. dematium.

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Fig. 3. Specificity test of the primer cirTef-F/cirTef-R set against diverse fungal DNAs. M, 1 kb DNA ladder; 1, Colletotrichum circinans CBS 125331; 2, C. circinans CBS 221.81; 3-6, C. acutatum KACC 40805, 43124, 44886, 44887; 7, C. boninense KACC 40893; 8, C. capsica KACC 46159; 9, C. caudatum KACC 41028; 10, 11, C. coccodes KACC 40227, 40808; 12, C. dematium KACC 40013; 13-16, C. gloeosporioides KACC 40003, 40448, 40690, 40892; 17, C. higginsianum KACC 40807; 18, C. liliacearum KACC 40947; 19, C. lindemuthianum KACC 42433; 20, C. musae KACC 40947; 21, C. orbiculare KACC 40808; 22, C. orbiculare KACC 40903; 23, DNA mixture of C. circinans CBS 221.81 and common six molds (Alternaria sp., Aspergillus sp., Cladosporium sp., Fusarium sp., Penicillium sp., Trichoderma sp.); 24, DNA mixture of common six molds.

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Fig. 4. Specificity test of the primer cirTu-F/cirTu-R set against DNAs of common six molds. M1, 1 kb DNA ladder; 1, Colletotrichum circinans CBS 125331; 2, C. circinans CBS 221.81; 3-6, C. acutatum KACC 40805, 43124, 44886, 44887; 7, C. boninense KACC 40893; 8, C. capsica KACC 46159; 9, C. caudatum KACC 41028; 10, 11, C. coccodes KACC 40227, 40808; 12, C. dematium KACC 40013; 13-16, C. gloeosporioides KACC 40003, 40448, 40690, 40892; 17, C. higginsianum KACC 40807; 18, C. liliacearum KACC 40947; 19, C. lindemuthianum KACC 42433; 20, C. musae KACC 40947; 21, C. orbiculare KACC 40808; 22, C. orbiculare KACC 40903; 23, DNA mixture of C. circinans CBS 221.81 and common six molds (Alternaria sp., Aspergillus sp., Cladosporium sp., Fusarium sp., Penicillium sp., Trichoderma sp.); 24, DNA mixture of common six molds; M2, 100 bp DNA ladder.

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Fig. 5. Conventional PCR amplification of different amounts of Colletotrichum cirninans DNA with the primers cirTef-F/ cirTef-R (A) and cirTu-F/cirTu-R (B). M, 1 kb ladder marker; 1, 10 ng; 2, 1 ng; 3, 100 pg; 4, 10 pg; 5, 1 pg; 6, 100 fg; 7, 10 fg; 8, 1 fg.

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Fig. 6. Real-time PCR amplification of Colletotrichum circinans DNA with the primers cirTef-F/cirTef-R (A) and cirTu-F/cirTu-R (B). a, Amplification plot; b, Melting peak analysis; c, Standard curve .Sample 1, 10 ng; Sample 2, 1 ng; Sample 3, 100 pg; Sample 4,0 1 pg.

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Fig. 7. Detection of Colletotrichum circinans by conventional PCR using the primer set cirTef-F/cirTef-R in DNA from infected seeds. M, 1 kb ladder marker; 1, Infected onion seed; 2, Control onion seed.

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Fig. 8. Detection of Colletotrichum circinans by real-time PCR using the primer set cirTef-F/cirTef-R in DNA from infected onion seeds. A, Amplification plot; B, Melting peak analysis; C, Gel electrophoersis; M, 1 kb ladder marke.r

Table 1. Colletotrichum strains used in this study.

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Table 2. Primers designed in this study

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Acknowledgement

Supported by : Animal and Plant Quarantine Agency

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