本土小菜蛾對新穎殺蟲劑之抗藥性研究

Resistance of novel insecticides in diamondback moth (Plutella xylostella) in Taiwan

張嘉哲(Chia-Che Chang)

指導教授 ： 許如君

國立臺灣大學/生物資源暨農學院/植物醫學碩士學位學程/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01475

摘要

小菜蛾 (Plutella xylostella (L.)) 屬鱗翅目昆蟲，被認為是最具破壞性的十字花科蔬菜害蟲，可造成全球損失及防治費用總和高達每年 40~50 億美元。國內小菜蛾防治方法主要以藥劑為主，其登記用藥數量多達 44 種，目前有許多報導指出小菜蛾對許多作用機制不同的殺蟲劑產生抗藥性，造成小菜蛾防治不易，故本研究針對臺灣地區小菜蛾，探討其對 11 種殺蟲劑的感受性，及小菜蛾對較新登記殺蟲劑產生抗藥性之原因。2013 年底至 2014 年初，自九個不同地區採集，以 10 種殺蟲劑的田間施用濃度測試，賜諾特 (spinetoram) 對小菜蛾具最高致死率 (80~100%)。各地區小菜蛾對藥劑反應存在差異，以台南新市、雲林西螺、嘉義太保、宜蘭大同及彰化溪湖等地區的小菜蛾抗藥性較為嚴重。於彰化溪湖連續三年調查 10 種殺蟲劑之登記濃度對田間小菜蛾的致死率均於兩年內顯著降低。進一步以焦磷酸定序技術分析已知與五類殺蟲劑相關的作用標的抗性基因點突變頻度，臺灣地區小菜蛾對合成除蟲菊酯類、有機磷類、芬普尼及二醯胺類殺蟲劑皆具抗藥性。本研究最後使用次世代定序進行龐大的定序工作，於感性品系的 14G 轉錄體中，de novo 組裝出 67,003 條 contigs，每條 contig 平均長度為 822 bp，分類出 172 條細胞色素 P450 (cytochrome P450s, CYP)、38 條麩胱胺硫基轉移酶 (glutathione S-transferases, GST) 及 153 條羧酸酯酶 (carboxylesterases, COE)，此外，共有 478 條 contigs 被歸納在 14 類殺蟲劑的作用標的基因。於三大代謝酵素抗性機制中，具表現量差異的 contig 數目，因滅汀抗性蟲有 10 條 CYP、4 條 GST 及 1 條 COE；脫芬瑞抗性蟲有 1 條 CYP、4 條 GST 及 6 條 COE；剋安勃抗性蟲有 5 條 CYP、3 條 GST 及 1 條 COE；氟大滅抗性蟲有 6 條 CYP、4 條 GST 及 5 條 COE，這些具表現量差異的代謝酵素皆可能與抗藥性有關。針對標的基因點突變，於因滅汀抗性中，PxGABARα1 上有 2 個點突變；於脫芬瑞抗性中共發現 18 個點突變，分別位於 8 條不同的 NADH dehydrogenase 上；於剋安勃及氟大滅抗性中同樣發現在 RyR1 上有 3 個點突變。本研究結果可供田間防治小菜蛾推薦用藥，抗性轉錄體資訊也可提供未來抗性研究、抗性管理及開發新藥之方向。

關鍵字

小菜蛾 ； 剋安勃 ； 氟大滅 ； 脫芬瑞 ； 因滅汀 ； 抗藥性 ； 次世代定序

並列摘要

The diamondback moth (Plutella xylostella) belongs to the Lepidoptera order and is considered to be one of the most destructive pests of cruciferous vegetables, causing significant decreases in crop yields and increases in costs of pest control and management. It is estimated that management costs can reach up to 40 to 50 billion a year. In Taiwan, the control of diamondback moths mostly relies on the 44 insecticides registered for use. However, many current studies have reported differing diamondback moth resistance mechanisms to various pesticides, posing a challenge to diamondback moth control. This study explores the susceptibility of diamondback moths in Taiwan to 10 applied insecticides and also investigates the causes of resistance to new insecticides. Starting from late 2013 until early 2014, we tested the diagnostic doses of 11 applied insecticides in nine distinct regions. Spinetoram showed the highest mortality rates (80~100%). Regional differences were found in diamondback moth insecticide responses; the diamondback moths in Tainan Xinshi, Yunlin Xiluo, Chiayi Taibao, Yilan Datong, and Changhua Xihu showed higher levels of insecticide resistance. Field surveys in Changhua Xihu showed significantly decreasing mortality rates to the 10 types of applied insecticides over the past two years. Further pyrosequencing analysis located five resistance target genes and identified their mutation frequencies to related insecticides. In Taiwan, diamondback moths have developed resistance to pyrethroids, organophosphates, fipronil, and diamides. This study used high-throughput next generation sequencing to obtain 14G transcriptomes of the susceptible strain, resulting in 67,003 contigs by de novo assembly with average lengths of 822 bp; 172 CYPs, 38 GSTs, and 153 COEs of contigs were identified. In addition, 478 contigs were identified and grouped into 14 categories of insecticide target genes. In the emamectin benzoate-resistant strain, 10 CYPs, 4 GSTs, and 1 COE were identified, with ≧ 2 fold difference as compared to susceptible and field strains. In the tolfenpyrad-resistant strain, 4 GSTs, and 6 COE were identified, with ≧ 2 fold difference as compared to susceptible and field strains. In the chlorantraniliprole-resistant strain, 5 CYPs, 3 GSTs, and 1 COE were identified, with ≧ 2 fold difference ascompared to susceptible and field strains. In the flubendiamide-resistant strain, 6 CYPs, 4 GSTs, and 5 COE were identified, with ≧ 2 fold difference as compared to susceptible and field strains. All of these metabolic enzyme genes could be involved in insecticide resistance. In the emamectin benzoate-resistant strain, 2 amino acid substitutions were found on PxGABARα1; in the tolfenpyrad-resistant strain, a total of 18 amino acid substitutions were found on NADH dehydrogenases; in the chlorantraniliprole- and flubendiamide-resistant strains, 3 amino acid substitutions were found on RyR1. The results of this study could be used for future diamondback moth control, and the contig information could also prove useful for future development of insecticides, resistance management, and academic research.

並列關鍵字

Plutella xylostella ； chlorantraniliprole ； flubendiamide ； tolfenpyrad ； emamectin benzoate ； resistance ； next generation sequencing

參考文獻

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