臺灣水稻徒長病菌族群之遺傳組成、致病力及藥劑感受性分析

Abstract

水稻徒長病 (Bakanae disease) 是臺灣水稻古老病害之一，已被記載超過 100年的歷史。近年來水稻徒長病在臺灣各地似有逐漸流行的趨勢，本病崛起的原因，除了農友的稻種消毒操作不確實之外，很可能與田間病原菌族群變異，產生高致病力或具藥劑耐受性的菌株有關，這也成為臺灣水稻生產的潛在威脅。本研究與農業試驗所及各區農業改良場合作，分析由全臺各地稻種、稻苗、田間植株上分離所得 900 株可能之水稻徒長病菌菌株。接著以針對 TEF (translation elongation factor-1 alpha gene) 新設計之專一性引子對 FfTef-F/FfTef-R 輔助形態鑑定，確認菌株為Fusarium fujikuroi 者共 663 株，再根據 2013 年韓國 Jeong 氏等人發表之 F. fujikuroi B14 全解序基因體之序列，設計並建立水稻徒長病菌之高通量簡單重複性序列 (Simple sequence repeats, SSRs) 分子標誌平臺。本研究共開發 48 對引子對，選用其中 16 對進行全臺菌株之分子分群，結果顯示臺灣水稻徒長病菌族群主要分為四群 (A1、A2、B1、B2)。為瞭解其病原性，本研究根據分子分群結果選出 24 株代表性菌株，對八個水稻品種 (Budda、IR78581-12-3-2-2、豐錦、越光、臺南 11 號、高雄 139 號、臺稉 16 號及臺農 71 號) 進行接種，結果顯示相較於 A1、A2、B1 族群，B2 族群屬於弱病原性菌株，且在試驗之菌株與水稻品種中並未發現臺灣水稻徒長病菌有生理小種的存在，而水稻品種 Budda 則對所測試之代表性菌株有明顯之抗性，臺南 11 號則在商業品種中具有較佳抗性。另外針對臺中地區，選取所有早期 (1998及2000年) 菌株 8 株及近期 (2012年) 之代表性菌株4株，對不同時代之水稻領先品種臺南 11 號、臺農 67 號以及臺農 71 號進行接種試驗，發現過去與現在之菌株其病原性並無顯著變化。利用交配型引子對 GFmat1a/1b 與 GFmat2c/2d 對選用收集之菌株進行分析，發現臺灣水稻徒長病菌菌株存在兩種交配型 MAT-1 與 MAT-2 (比例為 171:307)，顯示在適合之環境條件下，F. fujikuroi 可能行有性生殖，增加其族群內遺傳變異性。在藥劑耐受性方面，本研究利用一般常用於稻種消毒之撲克拉 (prochloraz) 與得克利 (tebuconazole) 對 1998、2000 及 2012 年共 60 株代表性菌株進行試驗，發現 2012 年整體菌株對撲克拉藥劑之耐受性有提升之現象，顯示臺灣徒長病菌已演化出抗藥性，其中 B2 族群對撲克拉有較低耐受性，而對得克利則有較高耐受性，另外亦檢測出 1 株耐撲克拉之菌株及 3 株耐得克利之菌株。本研究之成果，有助於瞭解臺灣田間水稻徒長病菌族群之演變，相關資訊亦可供未來抗病育種及防治措施擬定之參考。

Bakanae disease is an old disease in Taiwan. It was first reported around 100 years ago. Since 2009, the Bakanae disease of rice has become more prevalent in many rice growing areas all around Taiwan, which has been a potential risk to rice production. The epidemic can be caused by ineffective seed sterilization or the evolution of highly virulent or fungicide-resistant Fusarium fujikuroi strain(s). Collaborating with Taiwan Agricultural Research Institute and seven Agricultural Research and Extension Stations, we totally collected 899 isolates originated from rice seeds, diseased seedlings from breeding farms, and diseased rice plants in selected patty fields. We designed a specific primer pair FfTef-F/FfTef-R based on the TEF gene (translation elongation factor-1 alpha gene), and identify a total of 663 F. fujikuroi isolates. A high-throughput SSR (simple sequence repeat) marker system was also established to reveal the population structure of F. fujikuroi. A total of 48 SSR markers were developed based on the whole-genome sequences of a F. fujikuroi isolate B14, from which 16 SSR markers were chosen to analyze the collected isolates. The result showed that there are mainly four subpopulations (A1, A2, B1, and B2) in Taiwan. In the virulence analysis, we chose 24 F. fujikuroi isolates representative for different genetic clusters and different geographical regions, to inoculate a set of eight selected cultivars. The result showed that compared to the isolates in A1, A2, and B1 clusters, the isolates in B2 cluster displayed lower virulence. Different physiological races were not observed among the 24 isolates. Among the rice cultivars under test, Budda exhibited high resistance to all the 24 representative isolates, and TN11 showed relatively higher resistance than other commercial cultivars. To evaluate the evolution of F. fusarium pathogenicity in Taichung, we used all the 8 available early isolates (1998 and 2000) and 4 representative recent isolates (2012) to inoculate three mainstream cultivars in different eras. No significant difference was found between early and recent isolates on TN11, TNG67, and TNG71. Determined by two specific primer pairs GFmat1a/GFmat1b and Gfmat2c/GFmat2d targeting MAT1 and MAT2 genes, respectively, the mating type composition in F. fujikuroi in Taiwan was found to be MAT1:MAT2 = 171:307. The coexistence of both mating types indicated the possibility of increased genetic diversity due to meiotic recombination of F. fujikuroi under suitable environmental condition. In the fungicide tolerance analysis, we used prochloraz and tebuconazole to test 60 representative isolates from 1998, 2002, and 2012. The 2012 isolates were less sensitive to prochloraz than the isolates from 1998 and 2000, indicating that fungicide-resistant strains are present in Taiwan. The B2 subpopulation showed less tolerance to prochloraz but higher tolerance to tebuconazole. We also detected one prochloraz-tolerant isolate and three tebuconazole-tolerant isolates. This study can facilitate our understanding of the evolution of the F. fujikuroi population in Taiwan, and provide useful information for resistance breeding and the development of control measures in the future.