【作者】 王东；

【导师】 王开运；

【作者基本信息】 山东农业大学 ， 农药学， 2010， 博士

【摘要】 棉铃虫Helicoverpa armigera (Hubner)是北方棉区重要害虫,即使Bt棉的广泛种植依然不能使我们忽视其重要的害虫地位。目前,棉铃虫已经对多种药剂产生抗性,也为该虫的防治带来重重困难。多杀菌素是由土壤放线菌。刺糖多孢菌(Saccharopolyspora spinosa)经有氧发酵后产生的胞内次级代谢产物。自1997年注册登记以来,如今已经在37个国家150多种作物上用于鳞翅目等害虫的防治。其独特的作用机理获得了广大用户的好评,也使之成为抗性治理策略中的潜力药剂。为了探讨棉铃虫对多杀菌素的抗性发展规律、抗性风险及抗性机理,我们筛选了抗多杀菌素的棉铃虫品系并进行了相关的研究。研究结果如下：1、抗多杀菌素棉铃虫品系的室内筛选及抗性风险评估以泰安田间种群为起始种群,用多杀菌素进行抗性选育。在筛选前期(1.7代),抗性发展缓慢,仅达到原来的3.4倍。从第9代开始抗性迅速增长,经过15代的筛选后,抗性达到24.1倍,属于中抗水平；经过多杀菌素筛选后,棉铃虫对毒死蜱、灭多威、氰戊菊酯、阿维菌素和虫螨腈的敏感性并没有显著变化(LD99水平),表明多杀菌素与所测定的五种药剂没有产生交互抗性；对多杀菌素抗性和敏感品系进行生物学研究发现,抗性品系种群存活率下降50%左右,种群发育时间延长4-5天,单雌产卵量仅为敏感品系的一半,净增殖率(Ro)和内禀增长率(‰)分别为敏感品系的25.35%和65%。本研究结果表明,棉铃虫对多杀菌素的抗性发展比较缓慢,但仍具有产生中等抗性的风险。交互抗性问题并不能显著影响多杀菌素对棉铃虫的防效,多杀菌素仍是治理抗性棉铃虫的合理有效的轮用药剂。多杀菌素在棉铃虫种群上会产生抗性代价,在多杀菌素缺失的情况下,这有利于抗性种群的敏感性恢复。因此,田间实行药剂轮用,间断性暂停多杀菌素的使用,对延缓多杀菌素抗性有着重要意义。2、棉铃虫对多杀菌素的抗性机理研究以多杀菌素抗性和敏感品系为研究对象,采用增效试验、酶学测定及基因分析方法,发现PBO和TPP都能够明显降低棉铃虫对多杀菌素的抗性,抗性比分别降低到原来的31.8%和68.0%,而增效剂DEM对抗多杀菌素试虫的抗药性没有显著影响；抗性试虫细胞色素P450氧脱甲基活性是敏感试虫的8.26倍,羧酸酯酶和谷胱甘肽-S-转移酶分别为1.02和1.04倍；CYP6AEl4基因在抗性品系中过量表达,是相对敏感品系的7.73倍,其次是CYP9A12、CYP9A18、CYP6B2和CYP6B7基因在抗性品系中的表达量是敏感品系的2.45-3.53倍,CYP6AE12在两个品系中的表达量几乎一致,而CYP9A14和CYP6B6在抗性品系中的表达量降低,仅为敏感品系的0.87和0.70倍。这些结果表明细胞色素P450氧脱甲基酶的活性增强是棉铃虫对多杀菌素产生抗性的重要机制。进一步而言,CYP6AE14基因是多杀菌素抗性形成的关键基因,CYP9A12、CYP9A18、CYP6B2和CYP6B7也很可能是作为辅助因子推动抗性的发展。因此,在田间适当选用PBO作为增效剂可以提高多杀菌素对棉铃虫的毒力,延缓其抗性的发展。3、多杀菌素对棉铃虫生长发育及其解毒酶活性的影响通过测定棉铃虫在多杀菌素作用下的生物学特征、解毒酶活性及解毒酶基因转录方面的变化,从而更好地理解抗性代价的形成、抗性发生的原因和发展特性。研究发现,试验浓度下的多杀菌素对棉铃虫不仅仅具有致死效应(增加幼虫死亡率；降低化蛹率、羽化率及孵化率)还具有亚致死效应(降低虫重、蛹重；延长幼虫期、预蛹期、蛹期；缩短成虫期；降低单雌产卵量),通过这些影响来改变棉铃虫种群密度及发展动态。在试验浓度下,多杀菌素能够明显诱导棉铃虫P450氧脱甲基酶活性增强,这种诱导效应与浓度呈正相关。在处理48 h后,多杀菌素最高可以诱导P450氧脱甲基酶比活力达到原来的5.8倍。然而,在相同的浓度下,多杀菌素对羧酸酯酶和谷胱甘肽-S-转移酶的比活力都没有明显的影响。通过接触试验发现,多杀菌素可以显著诱导CYP6AE14的表达,处理组是对照组的4.15倍。其次是CYP6B2、CYP6B7、CYP9A12和CYP9A18基因,也分别被诱导3.32、2.79、2.6和2.07倍。CYP9A14和CYP6B6基因没有被诱导增加,反而被抑制。以上结果都表明,单次诱导和多次筛选后对种群生物学特性、酶学特性及基因表达的影响趋势是一致的,存在着一定的关系,这说明诱导在抗性形成的过程中起到一定的作用,是抗性形成的一种方式。从量上来看,多次筛选后的改变并不是单次诱导简单的相加,在失去药剂的情况下,这种诱导效应很可能会恢复到正常水平,这也证实了棉铃虫对多杀菌素的抗性发展缓慢的原因。4、次生物质与多杀菌素联合作用在试验浓度下,与次生物质(棉酚、单宁、槲皮素)或Bt没有显著改变多杀菌素对棉铃虫生物学特性的影响(如降低化蛹率、蛹重；延长幼虫期、蛹期；降低繁殖力)。这些结果表明,寄主植物中次生物质和Bt对多杀菌素亚致死效应的影响不大。从生物学的角度来看,次生物质对多杀菌素的影响可以忽略,但这并不代表田间使用时我们可以忽略次生物质。许多研究表明,次生物质可以通过改变昆虫体内的某些机理进而影响杀虫剂的防治效果。更多还原

【Abstract】 The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae), is one of the most important lepidopteran pests on a wide range of crops in the north of China. By now, serious resistance of H. armigera to conventional insecticides has resulted in many problems in controlling of this pest. Spinosad is a naturally derived fermentation product of soil actinomycete Saccharopolyspora spinosa. Since it was registered as an insecticide in 1997, spinosad has been used on over 150 various crops in 37 countries for controlling the pest especially lepidoptera larvae. Because of its unique action mechanism, popular spinosad become the potential insecticide for management of resistance. In order to clarify the dynamic development of spinosad resistance, the state of cross-resistance and the resistant mechanism, we selected a spinosad-resistant strain of H. armigera with topical application in the laboratory and documented a series of related studies. The results are as follows:1. The selection of spinosad-resistant strain of H. armigera and the research of cross-resistanceThe parental strain was collected from Tai’an and selected with topical application in the laboratory. The development of resistance was slow during the early selection process (G1-G7), which was just about 2.73-fold increase in resistance compared with the parental strain. Exponential increase of resistance was found from the ninth generation. After 15 generations of selection, the resistance level to spinosad in the selected strain was up to 24.1-fold compared with the parental strain. After selected with spinosad, the susceptibilities of this moderate resistance strain against tested insectcides (chlorpyrifos, methomyl, fenvalerate, avermectin and chlorfenapyr) did not changed significantly, indicating that there was no cross-resistance existed between spinosad and tested five pesticides. Comparing the resistant with the susceptible strain, we found the survival of the resistant strain was half compared with susceptible strain, the development of resistant H. armigera larvae was prolonged about 4-5 days, the number of eggs laid per resistant female was 359, which was half of the susceptible female’s, Net replacement rate (R0) and the intrinsic rate of population increase (rm) from the resistant strain were 25.35% and 65% compared with susceptible strain.Our results showed, even though the development of resistance is slow, H. armigera still has the potential to development moderate resistance. Cross-resistance of spinosad with other pesticides is not a significant factor that could prevent the effective use of spinosad against H. armigera. Spinosad should be recommended for rotational use to control the resistant H. armigera population. Spinsoad conferred the fitness cost in H. armigera, suggesting relaxation of selection pressure was likely to favor reversion to susceptibility for the H. armigera population. Therefore, the development of resistance to spinosad in H. armigera should be delayed by rational resistance management measures such as pesticide rotations.2. The study of resistant mechanism of H. armigera to spinosadFrom the studies of synergism, enzyme and gene, we found PBO and TPP could decrease significantly the toxicity of spinosad against H. armigera, and the resistant ratio reduced to 31.8% and 68.0% respectively. However, DEM exhibited no significant effects on toxicity of spinosad; Compared with CarE or GST activity, increasing activity of ODM was found in the resistant strain with difference at significant level, which was 8.26 times of that in the parental strain; CYP6AE14 from the resistant strain was overexpressed about 7.73-fold than that of the susceptible strain. CYP9A12, CYP9A18, CYP6B2 and CYP6B7 were ranged from 2.45-3.53-fold, but CYP9A14 and CYP6B6 were about 0.87- and 0.70- fold compared with the susceptible strain respectively. Our results indicated that resistance to spinosad in the cotton bollworm might be associated with an increase in cytochrome P450 monooxygenase. In addition, CYP6AE14 was the key gene during the development of spinsoad resistance. CYP9A12, CYP9A18, CYP6B2 and CYP6B7 could be enhancing factors to promote the resistant development. Based on our results, rational applications of PBO might increase the efficacy of spinosad in control of this pest and prolong the useful life of spinosad.3. Effects of spinosad on biological characteristics, detoxifying enzymes and detoxifying genes of H. armigera after treatment with spinosadBy clarification of changes about biological characteristics, detoxifying enzymes and detoxifying genes after treatment with spinosad, we could understand betterly the development of resistant cost, the reason of resistance and the development of resistance.Our found that spinosad not only has lethal effects (decrease in survival of eggs, larvae and pupae) but also has sublethal effects (decrease in weights of larvae and pupae; prolong periods of larvae, prepupae and pupae; shorten the adult longevity; decrease in the number of eggs laid per female). By the lethal and sublethal effects, spinosad changed the density and development of population.Spinosad at the tested concentration could induce ODM activity significantly. The induction was in a time-, dose- and population-specific manner. Greatest induction was found to be 5.8-fold in the Taian population after exposure to spinosad for 48 h. However, exposures to spinosad did not affect significantly CarE and GST activities. After treatment, the greatest induction was found in the expression of CYP6AE14 among all tested gene, which was 4.15-fold compared to the control. CYP6B2、CYP6B7、CYP9A12 and. CYP9A18 were induced to 3.32-,2.79-,2.60-,2.07- fold respectively, CYP9A14 and CYP6B6 were suppressed.Our results showed, there are the same tendency to biological characteristics, detoxifying enzymes and detoxifying genes between single induction and multiple selection, indicating insecticide induction may play an important role during the development of spinosad resistance. Based on our data, development of resistance is not due to simple addition of every single inductions. In the absence of spinosad, the induction may favor reversion to original level, which indicates the development of resistance to spinosad in H.armigera is slow.4. The effects of xenobiotics on spinosad-resistant costIn the tested concentrations, xenobiotics (gossypol, tannin, quercetin) and Bt did not change significantly effects of spinosad on biological characteristics of H.armigera (e.g. decrease in pupation ratio and pupal weight; prolong periods of larvae and pupae; decrease in fertility). These results indicated H.armigera has strong adaptability to the host. From the sight of biology, xenobiotics of hosts have little effect on spinsoad. However, we could not neglect the exist of xenobiotics because some researches about effects of xenobiotics on insecticide toxicity have been reported.更多还原