(66(4):276-285)甘藷基腐病菌專一性PCR 檢測技術之研發

Foot rot caused by Phomopsis destruens (Harter) Boerema is a major concern for sweet potato production in Taiwan. The conventional methods for the identification of P. destruens are based on culture isolation and morphological analyses, but they are time-consuming and often inconclusive. To assist in a rapid and specific detection of the infection, a polymerase chain reaction (PCR) assay was developed to detect the pathogen from DNA extracted from fungi and plant tissues, respectively. Internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) from P. destruens isolates were used for the specific primers design. The amplification of expected 298 bp PCR products was obtained in all P. destruens isolates, but not for other fungal isolates from sweet potato storage roots including Lasiodiplodia theobrome (B2225), Phomopsis sp. (Ph735), Rhizopus sp., Athelia rolfsii (Sc-3) and Fusarium sp. (Fu245). The PCR assay with the primers could detect 1 ng of DNA template. To enhance the sensitivity of detection, a nested PCR was performed and the detection limit was raised up to 10 pg. In addition, the primers proved efficient in detection of the pathogen in stems and storage roots of infected sweet potato. Positive detection was observed in 93% of the naturally infected stems of sweet potato, a rate which constituted a significant improvement in the identification of P. destruens in sweet potato as compared to conventional methods. These results indicated that the new PCR assay provided a rapid, specific and reliable diagnosis tool for the detection of sweet potato foot rot pathogen. 甘藷基腐病 (foot rot) 是由Phomopsis destruens 所引起，為近年來台灣甘藷生產的主要限制因子之一。目前P. destruens 之鑑定仍依賴傳統方法，以病原菌之組織分離及形態特徵為主，不但耗時費力且成功分離出病原菌之比率不高。本研究依據P. destruens 之去氧核醣核酸的內轉錄區 (internal transcribed spacer; ITS) 序列設計專一性引子對，並利用聚合酶連鎖反應 (polymerase chain reaction; PCR) 建立甘藷基腐病菌之檢測技術。結果顯示，本研究開發之檢測方法可專一性檢測P. destruens，且對經常可於甘藷塊根上分離之Lasiodiplodia theobrome (B2225)、Phomopsis sp. (Ph735)、Rhizopus sp.、Athelia rolfsii (Sc-3) 及Fusarium sp. (Fu245) 等其他真菌並無誤檢反應。此法之靈敏度可達1 ng 之菌絲DNA，若利用巢式PCR (nested PCR) 則靈敏度可提高至10 pg 之菌絲DNA。此外，本檢測方法亦可運用於分析受感染之甘藷莖部與塊根組織。而此技術於感染植物組織之檢測成效良好，93% 之受感染植物樣本可檢測出病原菌，相較於傳統鑑定法之38%，可大幅提升其檢測成功率。此專一性PCR 檢測法具有快速、專一性及靈敏度高之優點，未來可將此技術應用於檢測甘藷是否感染基腐病