| Two-spotted spide mite Tetranychus urticae, which is a worldwide agricultural pest mites, was controlled by chemical pesticides as the main measurements in field, but the mite has serious resistance to many insecti cides in recent years because of using insecticides incorrectly, which lead to the control times increase each year and seriously affect the development of pollution- free agriculture. with the development of study in pesticide structure, activity and resistance mechanism, It was proposed that the program of using different pesticides not only one at same field. In this study, three kinds of insecticides, spirodiclofen, avermectin, fenpropathrin, were selected to indicate multiple resistance mechanism and resistance monitoring to Tetranychus urticae. Through breeding by the three mixed insecticides to obtain multiple resistant strains of T. urticae, then the major detoxification enzymes MFOs, Car Es and GSTs and gene expression changes were analyzed between mixed resistance strains and susceptible strains in order to clarify the molecular mechanism of multiple resistance, to establish rapid multi-resistant molecular monitoring technology, to provide theoretical basis for multiple resistance management of field.1. To obtain multiple resistant strains(Mix-R) of T. urticae in laboratory condition. T. urticae were treated by mixture of spirodiclofen, avermectin and fenpropathrin for continuous 59 generations, the LC50 of mixture increased from 2.725 mg/L to 304.327mg/L, resistance factor reached 110.584 times, that of avermectin increased from 1.83 mg/L to 1207.76 mg/L, resistance factor reached 658.44 times; that of fenpropathrin increased fro m 35.75 mg/L to 6108.63 mg/L, resistance factor reached 170.88 times; that of spirodiclofen increased from 45.31 mg/L to 2805.62 mg/L, resistance factor reached 61.93 times.2. Using biochemical analysis, the activity difference of Car Es, MFOs, GSTs between multiple resistant(Mix-R) and Susceptible strain(SS) were tested.The results showed that GSTs, MFOs and Car Es total activity and specific activity of Mix-R strains increased significantly by compared with SS strain, the relative ratio exceed one, which indicate The mixture of avermectin, spirodiclofen and fenpropathrin had inductive effect on detoxifying enzyme of T. urticae. GSTs vitality of the adult mites was obviously higher than other stages, the activity of MFOs and Car Es specific in egg stage was significantly higher than other stages. The Km Constant value of GSTs, MFOs and Car Es of the resistant strains lower than that of the SS strain, the maximum velocity(Vmax)of the resistant strains higher than the susceptible strain, which show that the Mix-R strain detoxifying enzyme-substrate affinity greater than the susceptible strain.3. Using RT-q PCR technology, ELFn as reference gene, the expression amount of total 10 gene of T. urticae, including Tu GSTd01, Tu GSTd05, Tu GSTd06 and Tu GSTd09 of GSTs, CYP392D8, CYP392E10, CYP392A6, CYP392A16 of P450 enzymes, Tu CCE35, Tu CCE36 of Car Es Enzymes were analyzed. The result sshowed the expression amount of Mix- R strains higher than Fe-R, Av-R, Sp-R and susceptible strains, which indicate that these genes were very related to mixture multi-resistant of avermectin, fenpropathrin and Spirodiclofen of T. urticae.4. By combined with allele genotyping technology, multiplex PCR and semi-quantitative RT-PCR method, the mutation sites of F1538 I of anti- fenpropathrin and G326 E of anti-avermectin of T. urticae. By gene expression analysis, F1538 I mutations expression is 1.02 times of the SS, 2.33 times of wild homozygotes; G326 E mutations expression is 1.48 times the SS, 1.09 times the wild homozygotes. Mix-R strains gene frequencies and expression levels higher than the susceptible strain. So the method can be used to detect sodium ion channel mutation, glutamate-gated chloride channels associated with resistance genes fastly and correctly, and also can be used to monitor multi-resistant of T. urticae in the field. |
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