開發臺灣尖鐮孢菌之分子檢測技術

Development of the molecular methods for rapid detection of Fusarium oxysporum in Taiwan

尖鐮孢菌（Fusarium oxysporum）寄主範圍相當廣泛，可感染超過100種作物，並使作物發生萎凋病。本病原為土壤傳播性病原真菌，可藉由根部傷口或是自然開口侵入植物，受感染之作物會出現葉片黃化、植株萎凋等病徵。目前對尖鐮孢菌感染所造成的病害，目前尚無較為有效且安全之防治法可避免萎凋病發生時所造成的危害。因此監測田間尖鐮孢菌族群量，或為避免此病原菌於田間傳播進而減少此病害大發生的可行策略之一。為了達到快速檢測田間尖鐮孢菌之目的，本研究針對尖鐮孢菌建立一套快速檢測平台。自尖鐮孢菌基因組中設計專一性之引子對LNHFnF-1/LNHFnR-1與探針pLNH，搭配聚合酶連鎖反應（PCR）與即時聚合酶連鎖反應（real-time PCR）技術，進行引子專一性及靈敏度分析，並將檢測反應條件最佳化後，導入香蕉黃葉病田間檢體的田間分子檢測工作中。結果顯示，本研究開發之檢測方法對臺灣來源之尖鐮孢菌具專一性，且兩項real-time PCR技術（包含SYBR green-based real-time PCR與TaqMan probe-based real-time PCR）對具病徵的香蕉假莖中香蕉黃葉病菌（F. oxysporum f. sp. cubense）之帶菌檢出率為100%。在PCR、SYBR green-based real-time PCR及TaqMan probe-based real-time PCR三項技術中，對無病徵香蕉假莖中的香蕉黃葉病菌之帶菌檢出率，以該兩項real-time PCR檢測方法對病原的檢出率為最高，皆達93.8%。未來希望能將此檢測平台應用於各種尖鐮孢菌帶菌檢體之檢測工作，於田間及早篩檢出帶菌檢體，藉此擬定適當的防治策略，進而有效減少此類病害發生時造成的經濟損失。

"Fusarium oxysporum, soil-borne pathogenic fungus, can host more than 100 kinds of crops and lead to withering crop disease. The counterpart can be rooted through the wound or natural opening invasion, resulting in leaf yellowing, withering symptoms. There are several specialized forms (known as forma specialis, f. sp.), which can infect different hosts. F. oxysporum, the top 5 fungal pathogens in the world, can infect more than 100 different hosts, provoking severe losses in crops; furthermore, there are no chemicals that can control Fusarium wilt safely, economically and effectively. Therefore, this study aims to develop molecular methods of rapid detection of F. oxysporum based on PCR, SYBR green-based realtime PCR, and TaqMan probe-based real-time PCR assays with a novel primer set LNHFnF-1/LNHFnR-1. The results illustrate that the primer set was specific to F. oxysporum in Taiwan. By using the two real-time PCR detection methods, all the results can be positive. At the same time, meet F. oxysporum f. sp. cubense (Foc)infected symptomatic banana (including mild, moderate, or severe symptoms of pseudostems) by the two real-time PCR assays. When detecting the asymptomatic banana pseudostems, its results, detection rates, PCR, TaqMan probe-based real-time PCR, and SYBR green-based real-time PCR assays were 43.8% (7/16), 93.8% (15/16), and 93.8% (15/16), respectively. This can indicate that the two realtime PCR assays with the primer set LNHFnF-1/LNHFnR-1 has high applicability for the early detection of Foc-infected pseudostems of banana. Two real-time PCR assays, therefore, have the potential to serve as the rapid, specific, and sensitive tools for the routine detection of F. oxysporum in the field. "