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【目的】二斑叶螨Tetranychus urticae Koch是世界性的重要农业害螨,其抗药性发展严重阻碍了对该螨的科学防控。为了实现田间二斑叶螨对菊酯类杀虫剂抗药性的早期快速检测,本研究拟建立二斑叶螨对联苯菊酯的特异性等位基因PCR(PCR amplification of specific alleles,PASA)检测技术。【方法】玻片浸渍法测定了联苯菊酯对二斑叶螨不同种群的毒力,克隆了二斑叶螨钠离子通道结构域IIIS6的DNA片段,基于该片段中包含的抗性和敏感种群中的点突变,建立了PASA检测技术,并应用于二斑叶螨不同种群中抗性基因F1538I点突变频率的检测。【结果】与室内相对敏感种群相比,二斑叶螨北京通县和海南三亚种群均对联苯菊酯产生了抗药性,LC50分别为1 982.6 mg/L和2 767.4 mg/L,抗性倍数分别为6.0倍和8.4倍;PASA检测结果表明二斑叶螨室内相对敏感种群中存在杂合子个体,F1538I点突变频率为10.0%,而北京通县和海南三亚种群中该突变频率则分别达50.0%和53.3%。【结论】建立的PASA技术可以快速检测二斑叶螨田间种群是否存在与击倒抗性相关的基因点突变,从而判断其对菊酯类杀虫剂的抗药性发展。
【Objective】 Tetranychus urticae Koch is a worldwide agricultural pest mite and the development of its resistance has seriously hindered the scientific control of the mite. In order to detect the resistance of two-spotted spider mite to pyrethroid insecticides early in the field, we aimed to establish PCR amplification of specific alleles (PASA) technology. 【Method】 The toxicity of bifenthrin to different populations of Tetranychus urticae was determined by glass slide dipping method. The DNA fragment of IIIS6 of two-spotted spider mite sodium ion channel was cloned. Based on the resistance and sensitivity contained in this fragment, PASA detection technique was established and applied to detect the point mutation frequency of resistance gene F1538I in different populations of Tetranychus urticae. [Result] Compared with the indoor relative sensitive population, the populations of Beijing two-spotted spider mites, Beijing Tongxian and Hainan Sanya, developed resistance to bifenthrin with LC50 of 1 982.6 mg / L and 2 767.4 mg / L respectively, 6.0 and 8.4 times respectively. The results of PASA showed that there were heterozygous individuals in the relatively sensitive indoor population of two-spotted spider mites, the mutation frequency of F1538I point was 10.0%, while the frequency of mutation in Beijing Tongxian and Hainan Sanya was 50.0 % And 53.3%. 【Conclusion】 The established PASA technique can rapidly detect the point mutation of gene associated with knock-down resistance in the field population of two-spotted spider mite and determine its resistance development to pyrethroid insecticides.