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小菜蛾對美文松的抗藥性遺傳研究

By Chia-Li Lin and 林佳麗

Abstract

Diamondback moth (Plutella xylostella L.), which causes enormous damage on cruciferous vegetables, can develop resistance very rapidly to insecticides. To effectively manage insecticides resistance, it is necessary to understand the inheritance of insecticide resistance and evaluate the speed of resistant development in diamondback moth. In this study, we used mevinphos an example to investigate the inheritance of insecticide resistance in P. xylostella. Based on our previous studies that acetylcholinesterase (AChE) amino acid substitution associated with resistance to mevinphos, two pure lines, a wild-type SHggt strain and resistant SHMTCN strain carrying A298S, G324A and F386V amino acid substitutions, were established and performed reciprocal cross experiments, bioassays, and mutation frequency analysis. The results showed that the LC50 of parental, SHggt and SHMTCN, and two reciprocal cross progeny, F1and F1’, were 14.9, 775.6, 334.1 and 363.3 μg / ml. The degree of dominance of F1 and F1'' were 0.57 and 0.62. A plateau was found on the log dose –Probit lines of backcrossing and self-cross progeny. These results indicate that the inheritance of mevinphos-resistance in P. xylostella is an incomplete dominant and autosomal trait governed by polygene. In addition, the frequency of AChE gene (ace1) mutation in P. xylostella was found highly correlated with the LC50, the R2 is 0.97. The relaxation of SHMTCN show the resistant ratio 52.01-fold at first generation and decline to 5.48-fold at eleventh generation prove the unstable mevinphos-resistane in P. xylostella. The results show faster development of resistance than recessive inheritance. Therefore, the future of resistant management strategy is to use high doses mevinphos to kill all heterozygous, and to continue monitoring of resistance trends, to rotate insecticides with different mode of action, in order to avoid or delay the resistance in diamondback moth.小菜蛾 (Plutella xylostella L.) 為十字花科蔬菜的重要害蟲,也是最容易對防治藥劑產生抗藥性的害蟲之一。為有效管理抗藥性問題,有必要進一步了解小菜蛾對殺蟲劑之抗藥性遺傳特性,以評估小菜蛾的抗藥性發展速度。本研究以美文松為例,利用本實驗室先前研究發現與美文松抗藥性相關的乙醯膽鹼酯酶 (Acetylcholinesterase, AChE) A298S、G324A與F386V胺基酸置換建立攜帶AChE感性基因型的 SHggt品系與抗性基因型的SHMTCN品系,並進行互交、生物檢定與突變頻度分析。初步結果發現感性與抗性親代,以及兩個互交子代F1及F1’ 小菜蛾的半致死濃度分別為14.9, 775.6, 334.1及363.3 μg/ ml,F1及F1’ 小菜蛾的顯性度 (Degree of dominance) 分別為0.57及0.62。這些結果顯示小菜蛾對美文松的抗藥性是沒有母體效應的不完全顯性遺傳。經由F1與感性親代回交及F1自交的子代死亡率Probit值與美文松濃度作圖,發現劑量死亡反應曲線為具有平台的椅型曲線,表示由單基因控制,但在卡方檢測不符合單基因模型,進而進行間接卡方檢測,則符合2個基因模型。小菜蛾對美文松抗藥性穩定度,第一至第十一世代其抗性倍率從52.10倍衰退到5.48倍,可見小菜蛾對美文松的抗藥性不穩定。第一步偵測小菜蛾AChE基因 (Pxae1) 變異頻度發現G892T、G971C、T1156G點突變頻度與半致死濃度高度相關,相關度達0.97。綜合上述結果得知小菜蛾對美文松抗藥性主要與ae1點突變相關,屬於不完全顯性遺傳,抗藥性發展速率較隱性遺傳者快。因此,未來在小菜蛾對美文松或其他有機磷殺蟲藥劑的抗藥性管理應使用高劑量藥劑的防治策略來殺死偏顯性的異型合子,並且持續監測抗藥性的發展趨勢,適時輪用不同作用機制的藥劑,以達到避免或延遲抗藥性產生的目的。中文摘要 1 英文摘要 2 目錄 3 表目錄 5 圖目錄 5 壹、 前言 6 貳、 前人研究 8 一、 小菜蛾簡介 8 (一) 小菜蛾形態特徵與生活史 8 (二) 小菜蛾的重要性與防治歷史 8 (三) 台灣現行防治方法 9 二、 殺蟲藥劑─美文松 10 (一) 美文松之分類 10 (二) 美文松作用機制 11 (三) 美文松應用歷史 11 三、 抗藥性遺傳研究 12 (一) 抗藥性產生之機制 12 (二) 抗藥性遺傳 13 參、 材料與方法 17 小菜蛾飼養 17 小菜蛾純系建立 17 遺傳雜交 18 生物檢定 19 數據分析 19 抗藥性穩定度 20 抗藥性基因點突變偵測 21 (一) 樣本收集 21 (二) DNA萃取 21 (三) 聚合酶連鎖反應 21 (四) 聚合酶連鎖反應-限制酵素多型性 22 肆、 結果 24 美文松感性與抗性純系小菜蛾之建立 24 生物檢定與抗藥性遺傳研究 24 抗藥性基因點突變偵測 25 抗藥性基因點與抗藥性相關性 25 抗藥性穩定度 25 伍、 討論 26 美文松感性與抗性純系小菜蛾之建立 26 生物檢定與抗藥性遺傳研究 27 抗藥性穩定度 28 陸、 結論 31 柒、 引用文獻 32 捌、 圖表 3

Topics: Plutella xylostella, mevinphos, inheritance of resistance, acetylcholinesterase
Year: 2015
OAI identifier: oai:ir.lib.nchu.edu.tw:123456789/85977
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