- 期刊
開發檢疫菌Phytophthora ramorum之PCR快速檢測技術
Development of PCR Methods for Rapid Detection of Phytophthora Ramorum, a Quarantine Pathogen of Taiwan
摘要
Phytophthora ramorum Werres, de Cock & Man in't Veld, sp. Nov.是引發櫟樹猝死病（sudden oak death; SOD）之病原菌，但除了櫟樹外，尚可感染杜鵑及珊瑚樹（莢迷）等多種林木，是國際間備受矚目的重要病原菌。P. ramorum肆虐之區域主要分佈於美國及歐洲多個國家如荷蘭、比利時、英國及德國等，目前並未在台灣出現。由於該菌致病性強，且寄主多為重要林木及灌木，若不幸入侵，將嚴重影響生態、景觀與種苗產業，因此P. ramorum已被我國列為重要檢疫病原菌。為開發此病原菌之快速檢測技術，本研究根據YPT基因及核醣體RNA基因之序列分別設計P. ramorum專一性及疫病菌廣效性引子對；PCR分析結果顯示，其具備良好專一性，可偵測之最低量P. ramorum DNA分別為1 ng及10 pg。為檢測病原菌在輸入樣品的存在情形，我們進一步建立以杜鵑葉片誘釣疫病菌，並進行multiplex PCR的實驗流程，可提供檢疫單位使用，以快速檢測P. ramorum的存在情形。
並列摘要
Phytophthora ramorum Werres, De Cock & Man in't Veld, sp. nov., the pathogen of sudden oak death (SOD) and ramorum blight, is known to infect a variety of plants including oaks, Rhododendrom, and Viburnum species. Currently, infection by P. ramorum occurs mainly in Europe and North America. None has been reported in Taiwan. Owing to its threat to woody ornamental plants and the forest ecosystems, P ramorum has become an important quarantine pest of Taiwan. In order to develop a technique for rapid detection of this pathogen, we designed three sets of primers, each specific for P. ramorum (ramyptF1 + ramyptR1), Phytophthora spp. (Phy2S + Phy7A), and Rhododendron pulchrum Scoeet (rdhITSF1 + rdhITSR1), respectively. Specificity of the first two sets of primers was verified by PCR. Further analysis indicated that PCR primed with ramyptF1 and ramyptR1 may amplify P. ramorum DNA with a low limit of 1 ng, while that for Phy2S and Phy7A was 10 pg. To facilitate detection of Phytophthora pathogens which might exist in the imported materials, we established a diagnostic protocol which is comprised of the baiting with the use of leaves of Rhododendron pulchrum G. Don, followed by a multiplex PCR, which contains all three sets of the aforementioned primers. This method provides a useful tool for rapid detection of P. ramorum, and in addition, may help to uncover the possible existence of other Phytophthora spp. as well.
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