瓜類細菌性果斑病菌分子偵測技術之研發與應用

Abstract

Acidovorax avenae subsp.citrulli引起的西瓜細菌性果斑病為台灣新發生的嚴重病害, 也可為害洋香瓜及苦瓜.本病菌為種子傳播, 檢測種子以確認種子健康是防治本病害最重要的措施.目前國外應用長出測驗法( grow-out test )來檢測種子, 此法費時費力, 又需大空間, 因此快速之檢測技術有待發展.由於分子檢測技術具有快速、專一及靈敏之特性, 本計畫即擬研發此種技術以應用於果斑病菌之檢疫及防疫上.研發之策略為利用商品化的多組隨機引子對果斑病菌與其他相近細菌進行RADP分析, 以篩選得到果斑病菌特有的DNA片段, 將此片段選殖出並加以定序後, 依據此序列設計數組引子對, 選出能專一性增幅果斑病菌DNA的引子對, 並測試其靈敏度.上年度之研究已獲得可用於PCR的專一性引子對, 本年將以此引子對, 建立快速鑑定果斑病菌的PCR技術, 再探討此PCR技術應用於種子、葉片及果實樣品之偵測時, 樣品製備之方法, 最後建立完整的檢測流程.本研究預期可發展出PCR技術, 以應用於快速且專一性的鑑定分離的細菌是否為果斑病菌及偵測瓜類組織及種子中是否有果斑病菌之存在, 利於未來防疫及檢疫之執行.

Bacterial fruit blotch of watermelon and muskmelon caused by Acidovorax avenae subsp.citrulli ( Aac )is a destructive disease and was observed recently in Taiwan.The pathogen is seed-borne.Seed testing to ensure absence of the pathogen is the most important control measure.Currently, the only reliable method for watermelon seed testing is the grow-out test, which is time-consuming, and requires a large space for testing.The molecular techniques for detection of pathogens are known to be rapid, specific and sensitive.This project, therefore, aims to develop a PCR technique for identification and detection of Aac.The strategy of the development employs amplification of genomic DNAs from Aac and closely related bacteria using a range of RAPD primers, follows by cloning and sequencing of the amplified fragments that are found only in Aac, and a pair of primers specifically amplified the DNA from Aac is designed according to the sequence data.The sensitivity of the Aac-specific primers is also determined.The Aac-specific primer pair has been obtained from last year's research.This year, a PCR technique using this primer pair for rapid and specific identification of Aac is to be developed.In addition, procedures for preparing seed, leaf, and fruit samples for use in PCR amplification will be studied.Finally, the PCR protocols for rapidly identifying and detecting Aac in seeds and other plant tissues will be developed for plant quarantine inspection.