小菜蛾对氯虫苯甲酰胺的抗性机制及溴氰虫酰胺对小菜蛾生物学特性的影响

发布时间：2018-08-23 11:26

【摘要】：小菜蛾(Plutella xylostella L.)属鳞翅目(Lepidoptera)菜蛾科(Plutella),是一种世界性害虫,对十字花科蔬菜危害严重。目前,国内外防治小菜蛾仍然以使用化学农药为主。但是,由于小菜蛾自身具有较强的繁殖力和环境适应力,以及杀虫剂不合理大量使用等因素,使得小菜蛾已对多种杀虫剂产生了较严重的抗性。邻甲酰氨基苯甲酰胺类杀虫剂是一类作用于鱼尼丁受体的新型杀虫剂,通过干扰钙离子的释放,从而扰乱昆虫正常的生理活动,导致昆虫取食停止,最终引起死亡。因其独特的作用机理和显著的杀虫效果,以及对有益节肢动物毒害作用小和环境安全等特点,是目前防治小菜蛾等鳞翅目害虫最受欢迎的药剂之一,但其抗性问题也很快凸显出来。因此,我们系统研究了小菜蛾对氯虫苯甲酰胺的抗性机理和抗性特征,以及溴氰虫酰胺对小菜蛾的亚致死效应,以期为该类药剂的科学使用提供指导。主要结果如下:1.通过连续以浸叶法用氯虫苯甲酰胺筛选小菜蛾52代,获得抗性达48.2倍的抗氯虫苯甲酰胺品系。通过抗性发展规律发现,F1~F8抗性发展比较缓慢,随后抗性发展速度逐渐加快,但是并没有出现抗性突增的现象。抗性稳定性试验显示,小菜蛾对氯虫苯甲酰胺的抗性不稳定,将抗性品系在不接触药剂的情况下饲养6代后,抗性倍数下降至14.9倍。表明该抗性属易恢复性抗性。2.交互抗性结果显示,抗氯虫苯甲酰胺小菜蛾品系对氟苯虫酰胺和溴氰虫酰胺分别产生了7.29和3.31倍的交互抗性;而对毒死蜱、高效氯氰菊酯、甲氨基阿维菌素苯甲酸盐、溴虫腈、多杀菌素、氟铃脲和虫酰肼则未产生交互抗性。因此可以使用这些药剂与氯虫苯甲酰胺交替轮换使用,以延缓抗性。3.抗性遗传实验表明,将抗性品系和敏感品系进行正反交,测定氯虫苯甲酰胺对其后代的毒力指数,结果表明小菜蛾对氯虫苯甲酰胺的抗性是常染色体、不完全显性遗传。回交后代的LD-P线在死亡率为50%处没有明显的斜坡,说明小菜蛾对氯虫苯甲酰胺的抗性由多个基因控制;同时,适合性检测也进一步证明了以上结果。4.采用组建生命表法分析小菜蛾抗性品系的相对适合度,结果表明抗氯虫苯甲酰胺品系的生物适合度仅为0.77,适合度相对较低。与敏感品系相比,抗性品系小菜蛾幼虫历期延长了约1.5d,蛹期也明显延长;卵孵化率、幼虫羽化率及单雌产卵量均显著降低;另外其内禀增长率和净增值率也均有所降低。这些结果均表明小菜蛾抗氯虫苯甲酰胺品系的生长发育和繁殖较对照有退化现象。5.通过比较敏抗品系小菜蛾酶活性发现,抗性品系小菜蛾细胞色素P450的活性明显高于敏感品系,约为敏感品系的4.26倍;而酯酶和谷胱甘肽-S-转移酶两个代谢酶,以及过氧化物酶和过氧化氢酶两个保护酶的活性在两个品系之间并没有明显差异。酶抑制剂增效试验结果表明,加入PBO和氯虫苯甲酰胺混用,对小菜蛾的毒力明显增加,说明细胞色素P450在小菜蛾对氯虫苯甲酰胺抗性形成中起了重要作用。采用荧光PCR的方法测定P450基因在转录水平上的差异,发现CYP6BF1V1在抗性品系中的表达量明显高于敏感品系,约为敏感品系的8.9倍,另外CYP6BG1和CYP6CV2表达量也有所升高,分别为敏感品系的3.5和3.2倍。因此我们推测这三个基因应该是导致小菜蛾对氯虫苯甲酰胺产生抗性的根本原因之一。6.为研究小菜蛾对氯虫苯甲酰胺的靶标抗性机制,本试验克隆了小菜蛾鱼尼丁受体基因(PxRyR)的全长,为15,643bp,开放阅读框为15,372bp,能够编码5,123个氨基酸。将PxRyR的氨基酸序列与其他20个物种的鱼尼丁受体进行比对分析,构建进化树。对氨基酸二级结构分析发现C末端存在5个跨膜区,能形成功能性的Ca2+通道。用MOTIF Search进一步分析其结构域,发现小菜蛾鱼尼丁受体包括RYR结构域、RIH结构域、三磷酸肌醇和鱼尼丁受体共同控制钙离子释放结构域、MIR结构域等。用DNAMAN对敏抗种群鱼尼丁受体氨基酸序列进行比对,没有发现氨基酸的突变。但是荧光定量结果显示,抗氯虫苯甲酰胺小菜蛾品系中鱼尼丁受体的转录水平是敏感品系的6.0倍,靶标受体的量变也是其产生抗性的重要原因。7.用LC20和LC50浓度的溴氰虫酰胺处理小菜蛾48h后分别观察对亲代和F1代的影响。与对照相比,溴氰虫酰胺处理后小菜蛾亲代4龄幼虫的历期、蛹期明显延长,化蛹率和羽化率显著降低,并且与浓度呈正比;溴氰虫酰胺处理后的雌雄蛹重均小于对照组,但是两个浓度处理之间没有差异;另外,LC50处理能够明显降低小菜蛾的单雌产卵量,而LC20处理和对照之间并没有显著性差异;表明溴氰虫酰胺对小菜蛾的生长发育和繁殖表现出显著的不利性。但是对其F1代继续观察发现,除LC50溴氰虫酰胺处理后能够明显延长F1代的卵的历期外,其他一系列生物学指标均没有明显变化。
[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.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433.4

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