稻熱病菌抗丙烯酸酯類殺菌劑菌株之分子檢測與集群分析

Molecular detection and cluster analysis of Pyricularia oryzae isolates resistant to strobilurin fungicides

Pyricularia oryzae引起的稻熱病是臺灣水稻重要病害之一，為防治稻熱病須頻繁施用化學藥劑，易引發抗藥性風險。為探究稻熱病菌對丙烯酸酯類(strobilurins)殺菌劑抗藥性現況及其分子檢測的可行性，乃以2019年迄2023年採自臺灣各縣市水稻田的192株稻熱病菌單孢菌株，運用定序技術偵測其細胞色素b基因(cyt b)第129、137及143密碼子之變異。結果為第129密碼子有6株菌(3.13%)變異(F129L)，137密碼子皆無變異，143密碼子有171株菌(89.06%)變異(G143A)。測試兩種變異密碼子代表菌株之菌絲半抑制有效濃度(EC50)得知，G143A突變株對百克敏呈低抗藥性或無抗藥性，對亞托敏及三氟敏均呈高抗藥性，但菌株RB225僅呈中等抗藥性；F129L突變株對百克敏及三氟敏皆無抗藥性，但對亞托敏呈低抗藥性。cyt b基因無變異之野生型菌株對上述三種藥劑都無抗藥性。應用t-隨機鄰近嵌入(t-SNE)演算法對供試菌株作集群分析顯示，G143A突變株遍布稻熱病菌各種遺傳背景之集群，而F129L突變株僅見於花蓮及臺東兩縣且屬同一集群。

Rice blast, caused by Pyricularia oryzae, is an important rice disease in Taiwan. Control has relied mainly on fungicides, including the strobilurins (quinone-outside inhibitors [QoIs]). In this study, we investigated the QoIs resistance in 192 P. oryzae single-spore isolates sampled from different paddy fields islandwide. Sequence analysis of the cytochrome b gene (cyt b) revealed that the frequency of the G143A mutation (glycine→alanine) in P. oryzae isolates was 89.06%, and the F129L mutation (phenylalanine→leucine) was 3.13%. However, the G137R (glycine→arginine) mutation was not found. Mycelial EC50 (effect concentration for 50% inhibition) of representative isolates of the mutants indicated that the cyt b gene carrying the G143A mutation was associated with high resistance to azoxystrobin and trifloxystrobin but not to pyraclostrobin. The F129L mutation was associated with low resistance to azoxystrobin but not to pyraclostrobin and trifloxystrobin. The wild-type isolates exhibited no fungicide resistance. Cluster analysis with t-distributed stochastic neighbor embedding (t-SNE) algorithm demonstrated that the G143A mutation appeared in all three genetic backgrounds (clusters), suggesting that field resistance to the strobilurin fungicides in P. oryzae is widespread in Taiwan, while the F129L mutation was restricted to Hualien and Taitung Counties and appeared in one cluster only.