臺灣晚疫病菌抗藥性調查及歐西比基礎感受性建立

Abstract

晚疫病菌 Phytophthora infestans 從19世紀發現至今，一直都是馬鈴薯與番茄上的重要病害，其在田間病勢發展迅速，若無法提前預防，很容易讓整個田區遭殃，故農民仍仰賴施藥防治。長期施用藥劑下，病原菌將會產生抗藥性，令防治更加困難。在國外，P. infestans 相繼出現具扶吉胺 (fluazinam)、曼普胺 (mandipropamid)、歐西比 (oxathiapiprolin) 抗性的菌株，其中歐西比為近十年新開發的作用機制，具有許多良好特性。在臺灣，P. infestans 相關研究發表稀少，田間菌株的族群及對各藥劑的感受性許久未更新，因此本實驗利用 2018至2024 年所分離的 37 株 P. infestans 菌株分析現今臺灣主流菌系，並透過含藥培養基 (amended agar medium) 觀察已推行的達滅芬 (dimethomorph)、曼普胺 (mandipropamid)、亞托敏 (azoxystrobin)、安美速 (amisulborn)、滅脫定 (ametoctradin)、滅達樂 (metalaxyl)、扶吉胺等藥劑之感受性，最後建立臺灣 P. infestans 菌株對歐西比的基礎感受性 (baseline sensitivity)。經由粒線體五個基因座的單核苷酸多型性進行族群分析，確認 2018 至 2025 年間的臺灣 P. infestans 為 US-11 菌系。在藥劑感受性研究中，發現 2023、2024 年所分離的 P. infestans 菌株在 CAA (carboxylic acid amides) 類藥劑作用標的 CesA3 (cellulose synthases 3) 上出現變異，CesA3 的第 1109 位胺基酸由纈胺酸 (valine, V) 替換為白胺酸 (leucine, L)，使具有該變異的 P. infestans 菌株對達滅芬的 EC50 值為 0.56 至 7.24 mg/L。此外，2024年所分離的菌株，在 QoI 類 (quinine-outside inhibitor) 藥劑作用位點細胞色素b (cytochrome b) 上也發生變異，細胞色素 b 的第 142 位胺基酸由甘胺酸 (glycine, G) 替換為丙胺酸 (alanine, A)，使具有該變異的 2024 年菌株擁有對亞托敏的抗性 (EC50 值 > 200)。按推薦稀釋倍數 1000 倍稀釋 23% 亞托敏藥劑，將番茄葉片浸泡稀釋藥液後，番茄葉片仍會遭到具 G142A 的 P. infestans 菌株感染，再次證明其對亞托敏的抗性。所有年份的 P. infestans 菌株均保有前人報導之滅達樂抗藥性。最後，本研究中臺灣的 P. infestans 菌株則對其它藥劑曼普胺、安美速、滅脫定、歐西比、扶吉胺高度敏感，其中對歐西比 EC50 值為 0.000010 至 0.000018 mg/L，可作為臺灣的晚疫病菌對歐西比之基礎感受性，供後人參考。綜上所述，本研究證實臺灣 P. infestans 族群並未出現劇烈變化，但既有 US-11 菌系卻變異出對達滅芬、亞托敏的抗藥性。本研究結果為目前農藥使用現況提出警示，顯示有必要持續監測田間抗藥性變化，並建立更為永續的殺菌劑管理策略。

Late blight (Phytophthora infestans) has been an important disease to potatoes and tomatoes industry since the Great Famine. This disease can spread rapidly in the field, and devastate the entire field without early prevention. To manage this disease, farmers still rely on fungicide application for the control. However, continuous use of fungicide may lead to the development of fungicide resistance. In other countries, P. infestans isolates resistant to fluazinam, mandipropamid, and oxathiapiprolin have been reported. Among them, oxathiapiprolin is a recent addition to fungicide options, developed within the past ten years, and has attracted attention due to its novel action and high effectiveness. In Taiwan, studies on P. infestans are limited, and information on the population structure and fungicide sensitivity of field isolates has not been updated for a long time. To address this gap, this study analyzed 37 P. infestans isolates collected between 2018 and 2024 to characterize the current dominant population in Taiwan. In addition, we evaluated their sensitivity to several commercial fungicides, including dimethomorph, mandipropamid, azoxystrobin, amisulbrom, ametoctradin, metalaxyl, and fluazinam by amended agar medium. Furthermore, we established a baseline sensitivity of P. infestans to oxathiapiprolin. Population analysis based on single-nucleotide polymorphisms (SNPs) at five mitochondrial loci confirmed that the P. infestans populations in Taiwan remain to be US-11 lineage. In the fungicide sensitivity tests, isolates collected in 2023 and 2024 were found to carry a mutation at the target site of carboxylic acid amide (CAA) fungicides: CesA3 (cellulose synthase 3). Valine (V) was substituted to leucine (L) at amino acid position 1109, and isolates carrying this mutation showed elevated EC50 values to dimethomorph (0.56 to 7.24 mg/L). In addition, a mutation at the cytochrome b gene, the target site of QoI (quinone outside inhibitor) fungicides, was detected in our 2024 isolates. A substitution from glycine (G) to alanine (A) at position 142 (G142A) resulted in high resistance to azoxystrobin (EC50 > 200 mg/L). Infection assays using tomato leaves soaked in azoxystrobin (23%) at the recommended dilution (1000x) confirmed that isolates carrying G142A were still capable of causing disease, further demonstrating their resistance. All isolates across years remained resistant to metalaxyl, consistent with previous reports. On the other hand, all isolates used in the experiment were showed highly sensitive to mandipropamid, amisulbrom, ametoctradin, oxathiapiprolin, and fluazinam. Notably, the EC50 value for oxathiapiprolin was as low as 0.000010 to 0.000018 mg/L, which could be a reference for baseline sensitivity of P. infestans in Taiwan. In conclusion, this study demonstrates that the P. infestans population in Taiwan has remained stable, with US-11 as the dominant lineage. However, resistance to dimethomorph and azoxystrobin has emerged within this lineage, likely driven by prolonged fungicide use. These findings give warning of the need for continuous monitoring and more sustainable fungicide management strategies.