利用基因體與轉錄體解析立枯絲核菌 AG-7 對殺菌劑賓克隆的反應機制

Abstract

立枯絲核菌 (Rhizoctonia solani) 為一種重要的植物病原菌，能造成多種作物病害以及嚴重的農業經濟損失，而化學防治是當前防治立枯絲核菌病害的主要方法。在諸多化學藥劑中，賓克隆 (pencycuron) 因其對R. solani的專一性防治效果而被廣泛使用；然而，R. solani對賓克隆的抗性機制尚不明確。本研究針對來自臺灣的164株 R. solani AG-7 分離株進行賓克隆敏感性測試，結果顯示菌株對藥劑的反應存在顯著差異。所有分離株均表現出中至高度的抗性，唯獨分離株214對藥劑高度敏感。其中抗性最強的分離株213之EC₅₀值超過2,000 μg/mL，而最敏感的分離株214則顯示EC₅₀值僅為0.47 μg/mL。為理解這二菌株之間的抗藥反應差異，本研究利用比較基因體學和比較轉錄體學，結果顯示這二菌株在基因體上具有顯著差異。菌株213具有34.46 Mb的二倍體 (AB)結構，而菌株214則為94.97 Mb的五倍體 (AAABB)結構且伴隨高雜合度。根據賓克隆處理下的轉錄體分析結果，抗藥性菌株213顯著上調與解毒作用相關的基因，包括細胞色素P450 (cytochrome P450) 和ABC運輸蛋白 (ABC transporter)；相比之下，感藥性菌株214則表現出多元分歧的基因表現反應，其中與DNA複製、脂質生合成及細胞維持相關的途徑顯著下調。後續針對抗藥性菌株213中的細胞色素P450 (RsP450) 和ABC運輸蛋白 (RsABC1) 進行功能性驗證，然因R. solani無法建構穩定基因轉植菌株，故於感藥性的Monilinia fructicola中進行異源表現以驗證R. solani的基因功能。結果顯示，轉殖RsP450及 RsABC1的M. fructicola菌株皆顯著提升對賓克隆的抗藥性，顯示其部分提升了賓克隆的抗藥性。綜合以上結果，本研究針對R. solani AG-7對於殺菌劑賓克隆抗藥性，提供了比較基因體學、比較轉錄體學、及基因功能驗證的嶄新知識。這些結果不僅加深對賓克隆抗藥性機制的理解，亦為立枯絲核菌之病害管理策略及殺菌劑使用提供了重要認識。

Rhizoctonia solani is a destructive plant pathogen, and it causes severe diseases in various crops, leading to substantial economic losses worldwide. Currently, chemical control is the primary strategy for managing Rhizoctonia diseases. Among a diverse range of chemicals, pencycuron is a fungicide widely used due to its specific efficacy against R. solani. Nonetheless, the resistance mechanisms to pencycuron remain poorly understood. In this study, the sensitivity of 164 R. solani AG-7 isolates from Taiwan to pencycuron was investigated, revealing a wide range of sensitivity. All isolates exhibited moderate to high resistance, except for the isolate 214, which was highly sensitive. The most resistant isolate, 213, exhibited an EC₅₀ value exceeding 2,000 μg/mL, whereas the most sensitive isolate, 214, displayed an EC50 value at 0.47 μg/mL. To elucidate the molecular basis of the different pencycuron resistance between these two isolates, comparative genomics and transcriptomics were employed and the results identified that the isolate 213 has a diploid (AB) genome structure of 34.46 Mb, while the isolate 214 exhibits a pentaploid (AAABB) genome structure of 94.97 Mb with high heterozygosity. The resistant isolate 213 significantly upregulated the detoxification-related genes, including cytochrome P450s and ATP-binding cassette (ABC) transporters; in contrast, the sensitive isolate 214 displayed widespread transcriptional responses, such as downregulation of pathways associated with DNA replication, lipid biosynthesis, and cellular maintenance. Subsequently, functional validation of a cytochrome P450 (RsP450) and an ABC transporter (RsABC1) from the isolate 213, was performed using the pencycuron-sensitive Monilinia fructicola as a heterologous host due to the instability of R. solani transformants. The M. fructicola transformants expressing RsP450 or RsABC1 exhibited significantly elevated resistance to pencycuron, indicating their contribution to pencycuron resistance. Collectively, this study provides novel insights into the comparative genomics, transcriptomics, and gene functions of pencycuron resistance in R. solani AG-7. The findings enhance the understanding of pencycuron resistance and offer new prospects to the development of Rhizoctonia disease management and fungicide usage.