臺灣香蕉黃葉病菌熱帶第四型生理小種分子檢測平台之建立及應用

PCR-based methods for detecting tropical race 4 of Fusarium oxysporum f. sp. cubense and the geographic distribution of F. oxysporum f. sp. cubense collected in Taiwan

林依佳(Lin, Y.-J.) ； 楊峻毓(Yang, J.-J.) ； 商昊德(Shang, H.-T.) ； 張再得(Chang, T.-D.) ； 蔡尚翰(Tsai, S.-H.) ； 張碧芳(Chang, P.-F.) ； 林盈宏(Lin, Y.-H.)

《植物醫學》 62卷3期 (2020/09) Pp. 1-10

https://doi.org/10.6716/JPM.202009_62(3).0001

摘要

根據最新的聯合國糧農組織年報指出，香蕉（Musa spp.）為世界第二大宗的重要果樹。監控病害發生一向是有害生物綜合管理的重要基石，但缺乏快速、準確又可靠的方法來偵測植物病原菌，乃是無法提出有效綜合病害管理策略的主要限制因素之一。本研究希望整合目前獲得最多國際學者引用的兩個聚合酶連鎖反應（polymerase chain reaction, PCR）技術方法，Foc race 4 PCR（針對對象為亞熱帶第四型生理小種）與Foc TR4 PCR（針對對象為熱帶第四型生理小種），開發出適用於檢測臺灣香蕉黃葉病菌（Fusarium oxysporum f .sp. cubense, Foc）之檢測平台，並以大量臺灣來源的檢體，驗證此平台是否能用以輔助快速鑑定出熱帶或亞熱帶第四型（tropical race4, TR4; or subtropical race4, ST4）之臺灣香蕉黃葉病菌，並了解對臺灣香蕉產業最具破壞性的香蕉黃葉病菌熱帶第四型生理小種於臺灣香蕉黃葉病菌中所佔之比例。結果顯示，本研究之檢測方法可有效檢測出熱帶第四型生理小種之香蕉黃葉病菌；對香蕉黃葉病菌來源之基因組DNA（genomic DNA, gDNA）、標準DNA（standard DNA, sDNA）、分生孢子（conidia）、菌絲體（mycelium）的檢測極限分別為100 pg gDNA、10^6 copies sDNA、10^2 conidia、1μg mycelium。本研究並將此套技術導入田間帶菌香蕉檢體之香蕉黃葉病菌篩檢與鑑定工作中，結果顯示，本研究之檢測方法可用於篩檢出香蕉假莖中的香蕉黃葉病菌，且臺灣各地華蕉中之香蕉黃葉病菌目前皆判斷為race4；而熱帶第四型生理小種佔98.6%、亞熱帶第四型生理小種佔1.4%，顯示熱帶第四型生理小種已成為臺灣香蕉黃葉病菌中的優勢菌株。未來將進一步利用此套香蕉黃葉病菌熱帶第四型生理小種快速檢測平台應用在香蕉種苗或田間蕉株之診斷偵測實務上，以降低此病原菌之危害衝擊。

關鍵字

香蕉 ； 香蕉黃葉病 ； 巴拿馬病 ； 生理小種 ； 分子鑑定

並列摘要

Banana (Musa sp.) is cultivated worldwide and is one of the most popular fruits. Based on the statistics of FAO (Food and Agriculture Organization of the United Nations, Rome, Italy), the banana was the second most important fruits in the world. Accurate methods for the identification and characterization of pathogens provide the basis of integrated pest management strategies to counteract diseases. Fusarium oxysporum f. sp. cubense (Foc), a soil-borne fungal pathogen, can result in Fusarium wilt of banana (FWB), which limits harvest amounts of banana. To prevent the outbreak of FWB, and also, dwindle the lost of economic loss of banana yield, it is pivotal to develop a rapid method with specific and fast for disease management. This study used the molecular detection methods (Foc race 4 PCR and Foc TR4 PCR) based on the polymerase chain reaction (PCR) assays for rapid detection of Foc race 4 (including tropical race 4 (TR4) and subtropical race 4 (ST4), which can currently affect bananas in the most banana-producing regions of the world. The detection limit of the Foc race 4 PCR assay (or Foc TR4 PCR assay) was estimated to be 10^5 (or 10^6) copy of the target standard DNA; 10 (or 100) pg of the Foc genomic DNA; 1μg mycelium and 100 spores of Foc. The Foc PCR assays were suitable for the molecular detection of Foc-infected banana pseudostems collected in the field. In addition, a total of 212 banana pseudostem samples infected with Foc from different geographic locations in Taiwan were performed for plate-out assay and the molecular detection assays. The detection rates of Foc race 4 and Foc TR4 were 212/212 (100%) and 209/212 (98.6%), respectively. The results indicated that the Foc TR4 was dominant in the Foc population in Taiwan. The Foc PCR assays have the potential to serve as a rapid and specific tool for the routine Foc detection.

並列關鍵字

Banana ； Fusarium wilt of banana ； Panama disease ； Race ； Molecular identification