【摘要】：小菜蛾(Plutella xylostella L.)屬鱗翅目(Lepidoptera)菜蛾科(Plutella),是一種世界性害蟲,對十字花科蔬菜危害嚴重。目前,國內(nèi)外防治小菜蛾仍然以使用化學農(nóng)藥為主。但是,由于小菜蛾自身具有較強的繁殖力和環(huán)境適應力,以及殺蟲劑不合理大量使用等因素,使得小菜蛾已對多種殺蟲劑產(chǎn)生了較嚴重的抗性。鄰甲酰氨基苯甲酰胺類殺蟲劑是一類作用于魚尼丁受體的新型殺蟲劑,通過干擾鈣離子的釋放,從而擾亂昆蟲正常的生理活動,導致昆蟲取食停止,最終引起死亡。因其獨特的作用機理和顯著的殺蟲效果,以及對有益節(jié)肢動物毒害作用小和環(huán)境安全等特點,是目前防治小菜蛾等鱗翅目害蟲最受歡迎的藥劑之一,但其抗性問題也很快凸顯出來。因此,我們系統(tǒng)研究了小菜蛾對氯蟲苯甲酰胺的抗性機理和抗性特征,以及溴氰蟲酰胺對小菜蛾的亞致死效應,以期為該類藥劑的科學使用提供指導。主要結(jié)果如下:1.通過連續(xù)以浸葉法用氯蟲苯甲酰胺篩選小菜蛾52代,獲得抗性達48.2倍的抗氯蟲苯甲酰胺品系。通過抗性發(fā)展規(guī)律發(fā)現(xiàn),F1~F8抗性發(fā)展比較緩慢,隨后抗性發(fā)展速度逐漸加快,但是并沒有出現(xiàn)抗性突增的現(xiàn)象。抗性穩(wěn)定性試驗顯示,小菜蛾對氯蟲苯甲酰胺的抗性不穩(wěn)定,將抗性品系在不接觸藥劑的情況下飼養(yǎng)6代后,抗性倍數(shù)下降至14.9倍。表明該抗性屬易恢復性抗性。2.交互抗性結(jié)果顯示,抗氯蟲苯甲酰胺小菜蛾品系對氟苯蟲酰胺和溴氰蟲酰胺分別產(chǎn)生了7.29和3.31倍的交互抗性;而對毒死蜱、高效氯氰菊酯、甲氨基阿維菌素苯甲酸鹽、溴蟲腈、多殺菌素、氟鈴脲和蟲酰肼則未產(chǎn)生交互抗性。因此可以使用這些藥劑與氯蟲苯甲酰胺交替輪換使用,以延緩抗性。3.抗性遺傳實驗表明,將抗性品系和敏感品系進行正反交,測定氯蟲苯甲酰胺對其后代的毒力指數(shù),結(jié)果表明小菜蛾對氯蟲苯甲酰胺的抗性是常染色體、不完全顯性遺傳�；亟缓蟠腖D-P線在死亡率為50%處沒有明顯的斜坡,說明小菜蛾對氯蟲苯甲酰胺的抗性由多個基因控制;同時,適合性檢測也進一步證明了以上結(jié)果。4.采用組建生命表法分析小菜蛾抗性品系的相對適合度,結(jié)果表明抗氯蟲苯甲酰胺品系的生物適合度僅為0.77,適合度相對較低。與敏感品系相比,抗性品系小菜蛾幼蟲歷期延長了約1.5d,蛹期也明顯延長;卵孵化率、幼蟲羽化率及單雌產(chǎn)卵量均顯著降低;另外其內(nèi)稟增長率和凈增值率也均有所降低。這些結(jié)果均表明小菜蛾抗氯蟲苯甲酰胺品系的生長發(fā)育和繁殖較對照有退化現(xiàn)象。5.通過比較敏抗品系小菜蛾酶活性發(fā)現(xiàn),抗性品系小菜蛾細胞色素P450的活性明顯高于敏感品系,約為敏感品系的4.26倍;而酯酶和谷胱甘肽-S-轉(zhuǎn)移酶兩個代謝酶,以及過氧化物酶和過氧化氫酶兩個保護酶的活性在兩個品系之間并沒有明顯差異。酶抑制劑增效試驗結(jié)果表明,加入PBO和氯蟲苯甲酰胺混用,對小菜蛾的毒力明顯增加,說明細胞色素P450在小菜蛾對氯蟲苯甲酰胺抗性形成中起了重要作用。采用熒光PCR的方法測定P450基因在轉(zhuǎn)錄水平上的差異,發(fā)現(xiàn)CYP6BF1V1在抗性品系中的表達量明顯高于敏感品系,約為敏感品系的8.9倍,另外CYP6BG1和CYP6CV2表達量也有所升高,分別為敏感品系的3.5和3.2倍。因此我們推測這三個基因應該是導致小菜蛾對氯蟲苯甲酰胺產(chǎn)生抗性的根本原因之一。6.為研究小菜蛾對氯蟲苯甲酰胺的靶標抗性機制,本試驗克隆了小菜蛾魚尼丁受體基因(PxRyR)的全長,為15,643bp,開放閱讀框為15,372bp,能夠編碼5,123個氨基酸。將PxRyR的氨基酸序列與其他20個物種的魚尼丁受體進行比對分析,構(gòu)建進化樹。對氨基酸二級結(jié)構(gòu)分析發(fā)現(xiàn)C末端存在5個跨膜區(qū),能形成功能性的Ca2+通道。用MOTIF Search進一步分析其結(jié)構(gòu)域,發(fā)現(xiàn)小菜蛾魚尼丁受體包括RYR結(jié)構(gòu)域、RIH結(jié)構(gòu)域、三磷酸肌醇和魚尼丁受體共同控制鈣離子釋放結(jié)構(gòu)域、MIR結(jié)構(gòu)域等。用DNAMAN對敏抗種群魚尼丁受體氨基酸序列進行比對,沒有發(fā)現(xiàn)氨基酸的突變。但是熒光定量結(jié)果顯示,抗氯蟲苯甲酰胺小菜蛾品系中魚尼丁受體的轉(zhuǎn)錄水平是敏感品系的6.0倍,靶標受體的量變也是其產(chǎn)生抗性的重要原因。7.用LC20和LC50濃度的溴氰蟲酰胺處理小菜蛾48h后分別觀察對親代和F1代的影響。與對照相比,溴氰蟲酰胺處理后小菜蛾親代4齡幼蟲的歷期、蛹期明顯延長,化蛹率和羽化率顯著降低,并且與濃度呈正比;溴氰蟲酰胺處理后的雌雄蛹重均小于對照組,但是兩個濃度處理之間沒有差異;另外,LC50處理能夠明顯降低小菜蛾的單雌產(chǎn)卵量,而LC20處理和對照之間并沒有顯著性差異;表明溴氰蟲酰胺對小菜蛾的生長發(fā)育和繁殖表現(xiàn)出顯著的不利性。但是對其F1代繼續(xù)觀察發(fā)現(xiàn),除LC50溴氰蟲酰胺處理后能夠明顯延長F1代的卵的歷期外,其他一系列生物學指標均沒有明顯變化。
[Abstract]:Plutella xylostella L. (Plutella xylostella L.) belongs to Lepidoptera (Plutella), which is a worldwide pest and is harmful to cruciferous vegetables. At present, chemical pesticides are still mainly used to control Plutella xylostella at home and abroad. However, Plutella xylostella has strong fecundity and environmental adaptability, and insecticides are unreasonable. Plutella xylostella has developed serious resistance to various insecticides due to its extensive use. O-formylaminobenzamide insecticides are a new class of insecticides that act on fish nidine receptors. By interfering with the release of calcium ions, they disrupt the normal physiological activities of insects, resulting in the cessation of insect feeding and eventually cause death. The unique mechanism of action, remarkable insecticidal effect, little toxicity to beneficial arthropods and environmental safety are the most popular insecticides against Lepidoptera pests such as Plutella xylostella, but their resistance problems are also highlighted quickly. The main results are as follows: 1. The resistance of 52 generations of Plutella xylostella to chloroform benzamide reached 48.2 times. The resistance of F1~F8 to chloroform benzamide was found by successive leaf soaking. The resistance stability test showed that the resistance of Plutella xylostella to chloroform benzamide was unstable. After rearing the resistant strains for six generations without contact with pesticides, the resistance multiple decreased to 14.9 times. This indicated that the resistance was easy to restore. The results of cross-resistance showed that the chloroform-resistant strains of Plutella xylostella exhibited 7.29 and 3.31 times cross-resistance to fluorobenzamide and deltamethamide respectively, but no cross-resistance to chlorpyrifos, beta-cypermethrin, methamidoavermectin benzoate, bromoconitin, polyfungicides, fluorobollworm urea and tebufenozide. These insecticides were used alternately with chloramphenicol benzamide to delay resistance. 3. Resistance genetic experiments showed that the resistance of diamondback moth to chloramphenicol benzamide was autosomal and incompletely dominant. There was no significant slope at the mortality rate of 50%, indicating that the resistance of Plutella xylostella to chloroform benzamide was controlled by several genes. At the same time, the suitability test further proved the above results. 4. The relative suitability of resistance strains of Plutella xylostella to chloroform benzamide was analyzed by constructed life table method. Compared with the susceptible strain, the larval duration of resistant strain Plutella xylostella was prolonged by about 1.5 days, and the pupae duration was also prolonged. The egg hatching rate, the emergence rate of larvae and the single female spawning rate were significantly decreased. The intrinsic growth rate and the net increment rate were also decreased. The activity of cytochrome P450 in the resistant strain was significantly higher than that in the sensitive strain, which was 4.26 times higher than that in the sensitive strain, while esterase and glutathione S-transferase, peroxidase and hydrogen peroxide were the metabolic enzymes. There was no significant difference between the two protective enzymes. The results of synergism test showed that the toxicity of PBO and chlorobenzamide to Plutella xylostella increased significantly, indicating that cytochrome P450 played an important role in the formation of resistance to chlorobenzamide in Plutella xylostella. The expression of CYP6BF1V1 in resistant strains was 8.9 times higher than that in susceptible strains, and the expression of CYP6BG1 and CYP6CV2 were 3.5 and 3.2 times higher than that in susceptible strains, respectively. In order to study the target resistance mechanism of Plutella xylostella to chloroform benzamide, the PxRyR gene was cloned with a length of 15,643 BP and an open reading frame of 15,372 bp, which could encode 5,123 amino acids. The amino acid secondary structure analysis showed that there were five transmembrane regions at the C-terminal, which could form functional C a2+ channels. Further analysis of the structure domain by MOTIF Search showed that Plutella xylostella nicotine receptor included RYR domain, RIH domain, inositol triphosphate and fish nicotine receptor, which jointly controlled calcium release structure. No amino acid mutation was found by comparing the amino acid sequences of the fish nidine receptor of the sensitive resistant population with DNA MAN. However, the fluorescence quantitative analysis showed that the level of the fish nidine receptor transcription of the chloroform-resistant strain was 6.0 times higher than that of the sensitive strain, and the amount of the target receptor was also an important factor for resistance. Compared with the control, the duration, pupation period, pupation rate and eclosion rate of the 4th instar larvae of Plutella xylostella were significantly prolonged, and the pupation rate and eclosion rate were significantly decreased, which were in direct proportion to the concentration of bromocyanamide. In addition, LC50 treatment could significantly reduce the single female oviposition of Plutella xylostella, but there was no significant difference between LC20 treatment and control. The results showed that bromocyanamide had significant adverse effects on the growth and reproduction of Plutella xylostella. However, further observation of its F1 generation showed that except for LC20 treatment, bromocyanamide had significant adverse effects on the growth and reproduction of Plutella xylostella. LC50 bromocyanamide treatment significantly prolonged the duration of F1 eggs, other biological indicators were not significantly changed.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S433.4

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