比較以PCR為基礎之方法檢測臺灣甜瓜帶萎凋病菌種苗之效率

Comparison of the efficiency of PCR-based methods for detecting Fusarium oxysporum f. sp. melonis seeds and seedlings in Taiwan

張再得(Chang, T.-D.) ； 吳榮彬(Wu, Z.-B.) ； 蘇士閔(Su, S-M) ； 林盈宏(Lin, Y.-H.)

《植物醫學》 62卷1期 (2020/03) Pp. 1-11

https://doi.org/10.6716/JPM.202003_62(1).0001

摘要

由Fusarium oxysporum f. sp. melonis（Fom）所引起的甜瓜萎凋病為甜瓜栽培過程中的限制因子。Fom能以種子進行傳播，且臺灣曾有Fom race 2感染甜瓜之記錄，為了減少Fom傳播所造成之經濟損失，有效的種子與種苗健康檢測法可在甜瓜種植前或種子（苗）出口前及早偵測種苗或種子帶菌情形，將有助於避免此病原菌的傳播與危害。聚合酶連鎖反應（polymerase chain reaction, PCR）與即時聚合酶連鎖反應（real-time PCR），因具有快速、高靈敏、高專一性的特點，近年來被大量的用於植物病原的檢測工作上。本研究使用國際上已發表對Fom race 2具專一性引子對Fa15F/Fa15R，開發出臺灣甜瓜萎凋病菌帶菌種子之檢測方法，係利用一般PCR技術（conventional PCR）、SYBR green螢光染劑即時定量聚合酶鏈鎖反應技術（SYBR green-based real-time PCR）及TaqMan probe螢光染劑即時定量聚合酶鏈鎖反應技術（TaqMan probe-based real-time PCR），開發出適用於臺灣甜瓜萎凋病中常見的Fom菌株與帶菌種子之檢測方法。結果顯示，本研究開發之檢測方法皆可有效檢測出10%至50%帶菌率之甜瓜種子；另在甜瓜帶菌幼苗檢測中，檢出率以SYBR green-based real-time PCR之檢測方式，針對幼苗下胚軸檢體達100%，為最高檢出率之系統。未來將進一步利用本研究開發的甜瓜萎凋病菌檢測技術應用在甜瓜種子及種苗診斷偵測實務上，以降低病原菌之危害衝擊。

關鍵字

甜瓜病害 ； 防檢疫 ； 病原真菌檢測 ； 即時核酸檢測技術

並列摘要

Fusarium oxysporum f. sp. melonis (Fom) is a destructive phytopathogen that causes a seed-borne Fusarium wilt disease on melons (FWM). It is vitally important to recognize melon seedlings or seeds infected by Fom as rapidly as possible, preferably before the appearance of visual symptoms, in order to prevent the introduction and dissemination of Fom from diseased to healthy melon plants as much as possible, which could reduce the impact of FWM outbreaks. Development of a specific and efficient tool for Fom detection is important. For the Fom molecular detection purpose, we developed three molecular methods to detect Fom-contaminated seedlings and seeds of melon by using PCR, SYBR green-based real-time PCR, and TaqMan probe-based real-time PCR assays. The data indicated that the race 2-specific primers Fa15F/Fa15R and the novel TaqMan probe TDCpr1 were specific to Fom in Taiwan. We were able to obtain all positive results by using the three detection methods when the DNA template was 10% to 50% (Fom-contaminated/Fom-free) of Fom-infected seeds. The detection rates of PCR, TaqMan probe-based real-time PCR, and SYBR green-based real-time PCR assays were 0%, 52.38%, and 100%, respectively, when the asymptomatic melon seedlings (1 day after plating) were used as test samples. These data indicated that the SYBR green-based real-time PCR assay with the primer set Fa15F/Fa15R has high applicability for the early detection of Fom race 2-infected seeds and seedlings of melons. This Fom race 2 SYBR green-based real-time PCR assay has the potential to serve as a rapid, specific, and sensitive tool for the routine detection of seeds and seedlings of melon contaminated by Fom race 2.

並列關鍵字

melon disease ； quarantine prevention ； pathogenic fungus detection ； real time nucleic acid detection techniques