Botrytis cinerea及B. elliptica偵測用分子標記之建立

Establishment of molecular markers for detection and diagnosis of Botrytis cinerea and B. elliptica

陳隆鐘(L. C. Chen) ； 陳天枝(T. Z. Chen) ； 陳惠玲(H. L. Chen) ； 葉瑩(Y. Yeh)

《植物病理學會刊》 7卷4期 (1998/12) Pp. 177-188

https://doi.org/10.6649/PPB.199812_7(4).0003

摘要

將Botrytis cinerea菌株WFFr103與B. elliptica菌株EPL803之粒腺體核酸經限制酵素EcoRI處理後，分別與載體pBluescript SK+接合，再轉型至E. coli D H5α菌株，各構築一基因庫，並自轉殖株回收之mtDNA片段製備成的核酸探針與其餘菌株之全DNA進行點雜配反應，測試結果以核酸探針pBE83-8 (0.9 kb)可偵測至只含10^(-2) ng之灰黴病菌全量核酸之高敏感度，核酸探針pBE83-4B (1.2 kb)與所有供試B. cinerea和B. elliptica菌株之核酸同源性最高，皆有反應出現。另以隨機增幅核酸多型性分析，篩選出引子OPB - 17，其增幅灰黴病菌DNA時，皆有專一性300 bp、600 bp及800 bp左右之條帶產生，並進一步將此些片段進行轉殖及製備成探針pBCE30、pBCE60及pBCE80。以南方氏核酸雜合反應分析，其與Botrytis cinerea和B. elliptica菌株皆有反應產生，並分別進行核酸序列解序設計引子對PB30-1F/PB30-1R、PB60-1F/PB60-1R和PBCE80-1F/PB80-1R，其中引子對PB80-1F/PB80-1R能專一性自B. cinerea增幅出890 bp的條帶，和從B. elliptica增幅出381 bp的條帶，但對Sclerotium rolfsii與Mucor sp. 則無任何產物產生。

關鍵字

灰黴病菌 ； 粒線體核酸探針 ； 聚合 ； 連鎖反應

並列摘要

Botrytis cinerea is a widespread pathogen producing infections on more than 200 hosts. Hosts attack can occur before harvesting or later during transport or storage. And B. elliptica causal agent of leaf blight, inflicts serious losses cut flowers of lily in Taiwan. However, B. cinerea and B. elliptica have become one of the most important threats to the production, import and export of cut flowers and some important crops. However, detection of the fungus at the latent stage befor packing for transitting is difficult because time consuming and tedious by classical methods. In this study we describe the development of DNA probe and polymerase chain reaction analysis to detection of B. cinerea and B. elliptica. A genomic library of mitochondrial DNA, digested with EcoRI restriction enzyme from isolates of WFFr103、EPL803 of B.cinerea and B. elliptica was constructed in the vector of pBluescript (SK+), and transformed into E. coli DH5α. Randomly selected recombinant clones designated as pBE83-8 (0.9 kb)、pBE83-4B (1.2 kb)、 pBE83-7 (1.1 kb)、pBC26-9 (1.2 kb)、pBC29-9 (0.7 kb) and pBC29-2 (1.2 kb) were nonradioactive labelled with NEBlot-Phototope^(TM). The sensitivity of DNA probe Be 83-8(0.9 kb) is highest up to 10^(-5)μg among all DNA probes from dot-blot hybridization analyses with total DNA of B. cinere a and B. elliptica . But the DNA probe of Be 83-4B(1.2 kb) hybridized with all isolates of B. cinere a and B. elliptica. According to RAPD band patterens analyses with primer OPB-17 to ampify DNA of B. cinerea and B.elliptica have the 300 bp、600 bp and 800 bp fragments in amplified DNA after gel electrophoresis. These 300 bp、600 bp and 800 bp fragments transformant clones were prepared for DNA probes, asp BCE3 0、pBCE60 and pBCE80. And the high specificity of DNA probes of pBCE30、pBCE60 and pBCE80 were proved with Southern hybridzation with all isolates of B. cinere a and B. elliptica . For sets of primers of PB30-1R/PB30-1F、PB60-1R/PB60-1F and PB80-1F/PB80-1R were developed from the nucleotide sequences analysing of insert DNAs in pBCE30、pBCE60 and pBCE80, respectively, useing total DNAs of B. cinere a、B. elliptica and another check fungus isolates as templates in PCR assay. The PB80-1F/PB80-1R primers set specifically amplified 890 bp DNA fragments from DNA of B. cinerea, and amplified 381 bp DNA fragments from DNA of B. elliptica but not DNA from any another check isolates as Sclerotium rolfsii and Mucor sp. Using of this highly specific and reliable detection could prove valuable for regulatory, epidemiological and ecological studies.

並列關鍵字

Botrytis cinerea ； B. elliptica ； DNA probes ； polymerase chain reaction analysis