二斑叶螨对联苯菊酯抗药性的PASA快速检测技术建立与应用
Establishment and application of the PASA technique of resistance detection to bifenthrin resistance in Tetranychus urticae Koch(Acari: Tetranychidae)
王 玲** 张友军 吴青君 谢 文 王少丽***
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DOI：10.7679/j.issn.2095-1353.2015.059
作者单位：中国农业科学院蔬菜花卉研究所 北京，100081
中文关键词：二斑叶螨，联苯菊酯，抗药性，特异性等位基因PCR
英文关键词： Tetranychus urticae, bifenthrin, insecticide resistance, PCR amplification of specific alleles
中文摘要： 【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。
英文摘要：[Objectives] The two spotted spider mite, Tetranychus urticae Koch, is an important agricultural pest. Pesticide resistance seriously impairs the ability to control this pest. We developed a PCR amplification of specific alleles (PASA) technique in order to detect resistance of T. urticae to pyrethroids. [Methods] The toxicities of bifenthrin to different T. urticae populations were determined using the slide-dip method. The DNA fragment in IIIS6 of the para-type sodium channel protein of T. urticae was cloned. A PASA technique was developed based on the point mutation of F1538I and was used to measure the point mutation frequencies of different T. urticae populations. [Results] Compared to a relatively susceptible strain, field populations collected from Tongxian, Beijing and Sanya, Hainan exhibited resistance to bifenthrin with the LC50 values of 1 982.6 mg/L and 2 767.4 mg/L, which are 6.0- and 8.4-fold respectively, higher than that of the susceptible strain. PASA genotyping results indicate that the F1538I mutation frequency was 10.0% in heterozygotes in the susceptible strain, whereas mutation frequencies as high as 50.0% and 53.3% were found in the Beijing and Hainan populations, respectively. [Conclusion] The PASA technique can be used to detect gene mutations in wild populations of T. urticae that are indicative of resistance to bifenthrin.