新疆棉区棉蚜对新烟碱类杀虫药剂抗性监测、风险评估及抗性机制的研究
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摘要
1.采用单头反选育的方法,得到了棉蚜吡虫啉(LC50为0.176mg/L)、啶虫脒(LC50为0.180mg/L)敏感种群；相对于田间原始种群(LCso分别为0.346mg/L和0.312mg/L),棉蚜吡虫啉、啶虫脒敏感种群对吡虫啉、啶虫脒的LCs0分别减少了2倍、1.73倍。采取群体汰选的方式,浸药40次选育的棉蚜吡虫啉、啶虫脒种群,相对于敏感种群,抗性倍数分别为83.27倍(LC50为14.657mg/L)、82.33倍(LCso为14.819mg/L)。采用阈性状分析的方法,获得棉蚜对吡虫啉、啶虫脒的抗性现实遗传力(h2)分别为0.1478和0.1342。进一步预测其抗性发展速度,预计抗性增长100倍时,吡虫啉、啶虫脒分别可使用30.2-38.1代、30.3-38.2代。
2.交互抗性谱的研究结果表明,(1)抗吡虫啉棉蚜种群的交互抗性谱的研究表明,抗性倍数小于5的药剂为吡蚜酮和甲氨基阿维菌素；交互抗性倍数在5-10倍为噻虫嗪、硫丹、阿维菌素、联苯菊酯、毒死蜱、马拉硫磷、丙溴磷和辛硫磷；交互抗性倍数在10-15倍为啶虫脒、高效氯氰菊酯和三唑磷；交互抗性倍数大于15的为氧化乐果。(2)抗啶虫脒棉蚜种群的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为吡蚜酮和甲基阿维菌素；交互抗性倍数在5-10倍为噻虫嗪、联苯菊酯、毒死蜱、马拉硫磷、丙溴磷和辛硫磷；交互抗性倍数在10-15倍为硫丹、阿维菌素、高效氯氰菊酯、三唑磷和氧化乐果；交互抗性倍数大于15的为毗虫啉。
3.抗性监测结果表明,新疆各主要棉区棉蚜对吡虫啉的相对抗性倍数为3.17-4.87倍,对啶虫脒的相对抗性倍数为1.7-22.0倍。2012年和2011年相比,新疆主要棉区棉蚜对吡虫啉和啶虫脒的抗性呈上升趋势。
4.增效剂实验表明,增效剂磷酸三苯酯(TPP)、增效醚(PBO)、顺丁烯二酸二乙酯(DEM),在吡虫啉敏感品系中的增效比分别为1.12、1.09、0.97,在吡虫啉抗性品系中的增效比分别为2.02、1.75、1.05；在啶虫脒敏感棉蚜品系中的增效比为1.02、1.03、1.02,在啶虫脒抗性棉蚜品系中的增效比为1.77、1.61、1.04。TPP和PBO对吡虫啉和啶虫脒有很好的增效作用。
5.解毒酶活性测定表明,棉蚜吡虫啉和啶虫脒抗性品系羧酸酯酶、谷胱甘肽S-转移酶、细胞色素P450s O-脱乙基比活力都高于敏感品系,其中羧酸酯酶、细胞色素P450s O-脱乙基比活力差异都达到了显著水平(P<0.05)。毗虫啉抗性品系三种解毒酶活性分别为敏感品系的3.26倍、1.08倍、1.60倍；啶虫脒抗性品系三种解毒酶活性分别为敏感品系的2.91倍、1.04倍、1.69倍。
6.酶标仪法测定单头棉蚜羧酸酯酶(CarE)活性,吡虫啉、啶虫脒抗性品系单头棉蚜羧酸酯酶(CarE)活性明显高于敏感品系。酶标仪法测定单头棉蚜乙酰胆碱酯酶(AChE)活性,吡虫啉、啶虫脒抗性品系单头棉蚜乙酰胆碱酯酶(AChE)活性明显高于敏感品系。
7.应用实时荧光定量PCR技术分析表明,棉蚜羧酸酯酶基因在吡虫啉抗性品系的相对表达量是敏感品系的1.85倍；棉蚜P450单加氧酶CYPCY3-1基因在吡虫啉抗性品系的相对表达量是敏感品系的1.60倍；棉蚜P450单加氧酶CYPCY3-2基因在吡虫啉抗性品系的相对表达量是敏感品系的2.0倍。棉蚜羧酸酯酶基因啶虫脒抗性品系的相对表达量是敏感品系的1.91倍；棉蚜P450单加氧酶CYPCY3-1基因在啶虫脒抗性品系的相对表达量是敏感品系的1.44倍；棉蚜P450单加氧酶CYPCY3-2基因在啶虫脒抗性品系的相对表达量是敏感品系的1.30倍。由此说明,棉蚜对吡虫啉、啶虫脒的抗性的产生与羧酸酯酶基因、P450单加氧酶CYPCY3-1基因、P450单加氧酶CYPCY3-2基因的过量表达有着密切的关系。
8.应用实时荧光定量PCR技术分析表明,烟碱型乙酰胆碱受体β1亚基基因在吡虫啉抗性品系的相对表达量是敏感品系的0.85倍；在啶虫脒抗性品系的相对表达量是敏感品系的0.36倍,由此说明,棉蚜对吡虫啉、啶虫脒的抗性的产生与烟碱型乙酰胆碱受体β1亚基基因的低水平表达有着密切的关系。
9.根据NCBI上登录的棉蚜乙酰胆碱受体β亚基基因序列(GenBank:JQ627836.1),设计特异性引物检测,结果表明,棉蚜吡虫啉、啶虫脒抗性品系、敏感品系以及新疆农一师、农二师、农三师、农四师、农五师、农六师、农七师、农八师、新疆沙湾、湖北省枣阳市、河南省西华县、安徽省萧县、山西省运城市等地方种群均未检测到突变。
1.The imidacloprid and acetamiprid susceptible strains (LC5o were0.176mg/L and0.180mg/L, respectively) were developed by individual reverse selection. The LC50value of the imidacloprid and acetamiprid susceptible strain had a2and1.73fold decrease compared with the field strain (LC50were0.346mg/L and0.312mg/L, respectively). The resistant strains of A. gossypii were obtained after forty selection with imidacloprid and acetamiprid by group selection. The resistance index of imidacloprid and acetamiprid resistant strain were83.27-fold (LC50was14.657mg/L) and82.33-fold (LC50was14.819mg/L), respectively. The realized heritability (h2) of resistance were0.1478and0.1342by Tabashnik's methods. In order to estimate the speed of resistance, it would take cotton aphid30.2-38.1and30.3-38.2ages of to obtain100-fold resistance to imidacloprid and acetamiprid, which was under breeding pressure of imidacloprid and acetamiprid. The results showed that A. gossypii had the resistance risk to imidacloprid and acetamiprid.
2. The results of cross-resistance to imidacloprid and acetamiprid-resistant strains were analyzed.(1) The results of cross-resistance to imidacloprid-resistant strains showed that the cross-resistaace of imidacloprid less than5folds were pymetrozine and Methyl abamectin, in5-10folds were thiamethoxam, endosulfan, abamectin, bifenthrin, chlorpyrifos, malathion, profenofos and Phoxim, in10-15folds were acetamiprid, beta-cypermethrin and triazophos, more than15folds was Omethoate.
(2) The results of cross-resistance to acetamiprid-resistant strains showed that the cross-resistance of acetamiprid-resistant strains less than5folds were pymetrozine and Methyl abamectin, in5-10folds were thiamethoxam, bifenthrin, chlorpyrifos, malathion, profenofos and Phoxim, in10-15folds were endosulfan, abamectin, beta-cypermethrin, triazophos and Omethoate, more than15folds was imidacloprid.
3. The survey for susceptible levels of cotton aphid to imidacloprid and acetamiprid of each cotton region in xinjiang showed that the relative resistance multiples to imidacloprid and acetamiprid were3.17-4.87folds and1.7-22.0folds. The resistance development speed to acetamiprid was faster than imidacloprid compared with2011in2012.
4. Synergism tests showed that triphenyl phosphate (TPP), piperonyl buoxide (PBO) and diethyl maleate (DEM) resulted in1.12,1.09and0.97-fold synergist ratios, respectively in the imidacloprid-susceptible strain. It was2.02,1.75and1.05-fold SR, respectively in the imidacloprid-resistant strain. It was1.02,1.03and1.02-fold SR, respectively in the acetamiprid-susceptible strain. It was2.02,1.75and1.05-fold SR, respectively in the imidacloprid-resistant strain. TPP and PBO had a good synergistic effect on imidacloprid and acetamiprid.
5. The detoxification enzymes tests showed that the activity of CarE, GST and7-ethoxycoumarin O-deethylase (ECOD) in the resisitant strain were higher than in susceptible strain. Among them, the activity difference of the CarE and ECOD reached significant level (P<0.05). The activity of three kinds detoxification enzymes in imidacloprid resisitant strain were3.26-,1.08-and1.60-folds that of the susceptible strain.The activity of three kinds detoxification enzymes in acetamiprid resisitant strain was2.91-,1.04-,1.69-times that of the susceptible strain.
6. Determination of individual aphid carboxylesterase (CarE) by microplate assay showed the activity of CarE in imidacloprid, acetamiprid resistant Aphis gossypii strains was higher than in susceptible strains. Determination of individual aphid acetylcholinesterase (AChE) by microplate assay showed the activity of AChE in imidacloprid, acetamiprid resistant Aphis gossypii strains was higher than in susceptible strains.
7. The mRNA relative expression level of CarE and P450monooxygenase gene were determined by real-time quantitative PCR. The mRNA relative expression level of CarE gene in imidacloprid resistant strains was1.85-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-1in imidacloprid resistant strains was1.60-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-2in imidacloprid resistant strains was2.00-fold of that of the sensitive strain; The mRNA relative expression level of CarE gene in acetamiprid resistant strains was1.91-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-1in acetamiprid resistant strains was1.44-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-2in acetamiprid resistant strains was1.30-fold of that of the sensitive strain. Therefore, the overexpression of CarE, P450CYPCY3-1and P450CYPCY3-2in Aphis gossypii were closely related with the resisitance to imidacloprid and acetamiprid.
8. The mRNA relative expression level of nAChR beta1subunit were determined by real-time quantitative PCR. The mRNA relative expression level of the imidacloprid resistant strains was0.85-fold of that of the sensitive strain; The mRNA relative expression level of nAChR beta1subunit in acetamiprid resistant strains was0.36-fold of that of the sensitive strain. Therefore, the low level expression of nAChR beta1subunit gene in Aphis gossypii was closely related with the resisitance to imidacloprid and acetamiprid.
9. According to the gene sequence logged on NCBI about Aphis gossypii nAChR betal subunit (GenBank:JQ627836.1), the specific primers were designed to detect the susceptible strain R81T position of arginine(codon:AGA) might be replaced by threonine (codon:ACA) in resistant strains. The results showed that the Aphis gossypii population of imidacloprid, acetamiprid-resistant strain, sensitive strain, main cotton region in Xinjiang, Zaoyang city in Hubei, Xihua County in Henan, Xiao County in Anhui, Yuncheng City in Shanxi all were not detected the mutation.
2.交互抗性谱的研究结果表明,(1)抗吡虫啉棉蚜种群的交互抗性谱的研究表明,抗性倍数小于5的药剂为吡蚜酮和甲氨基阿维菌素；交互抗性倍数在5-10倍为噻虫嗪、硫丹、阿维菌素、联苯菊酯、毒死蜱、马拉硫磷、丙溴磷和辛硫磷；交互抗性倍数在10-15倍为啶虫脒、高效氯氰菊酯和三唑磷；交互抗性倍数大于15的为氧化乐果。(2)抗啶虫脒棉蚜种群的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为吡蚜酮和甲基阿维菌素；交互抗性倍数在5-10倍为噻虫嗪、联苯菊酯、毒死蜱、马拉硫磷、丙溴磷和辛硫磷；交互抗性倍数在10-15倍为硫丹、阿维菌素、高效氯氰菊酯、三唑磷和氧化乐果；交互抗性倍数大于15的为毗虫啉。
3.抗性监测结果表明,新疆各主要棉区棉蚜对吡虫啉的相对抗性倍数为3.17-4.87倍,对啶虫脒的相对抗性倍数为1.7-22.0倍。2012年和2011年相比,新疆主要棉区棉蚜对吡虫啉和啶虫脒的抗性呈上升趋势。
4.增效剂实验表明,增效剂磷酸三苯酯(TPP)、增效醚(PBO)、顺丁烯二酸二乙酯(DEM),在吡虫啉敏感品系中的增效比分别为1.12、1.09、0.97,在吡虫啉抗性品系中的增效比分别为2.02、1.75、1.05；在啶虫脒敏感棉蚜品系中的增效比为1.02、1.03、1.02,在啶虫脒抗性棉蚜品系中的增效比为1.77、1.61、1.04。TPP和PBO对吡虫啉和啶虫脒有很好的增效作用。
5.解毒酶活性测定表明,棉蚜吡虫啉和啶虫脒抗性品系羧酸酯酶、谷胱甘肽S-转移酶、细胞色素P450s O-脱乙基比活力都高于敏感品系,其中羧酸酯酶、细胞色素P450s O-脱乙基比活力差异都达到了显著水平(P<0.05)。毗虫啉抗性品系三种解毒酶活性分别为敏感品系的3.26倍、1.08倍、1.60倍；啶虫脒抗性品系三种解毒酶活性分别为敏感品系的2.91倍、1.04倍、1.69倍。
6.酶标仪法测定单头棉蚜羧酸酯酶(CarE)活性,吡虫啉、啶虫脒抗性品系单头棉蚜羧酸酯酶(CarE)活性明显高于敏感品系。酶标仪法测定单头棉蚜乙酰胆碱酯酶(AChE)活性,吡虫啉、啶虫脒抗性品系单头棉蚜乙酰胆碱酯酶(AChE)活性明显高于敏感品系。
7.应用实时荧光定量PCR技术分析表明,棉蚜羧酸酯酶基因在吡虫啉抗性品系的相对表达量是敏感品系的1.85倍；棉蚜P450单加氧酶CYPCY3-1基因在吡虫啉抗性品系的相对表达量是敏感品系的1.60倍；棉蚜P450单加氧酶CYPCY3-2基因在吡虫啉抗性品系的相对表达量是敏感品系的2.0倍。棉蚜羧酸酯酶基因啶虫脒抗性品系的相对表达量是敏感品系的1.91倍；棉蚜P450单加氧酶CYPCY3-1基因在啶虫脒抗性品系的相对表达量是敏感品系的1.44倍；棉蚜P450单加氧酶CYPCY3-2基因在啶虫脒抗性品系的相对表达量是敏感品系的1.30倍。由此说明,棉蚜对吡虫啉、啶虫脒的抗性的产生与羧酸酯酶基因、P450单加氧酶CYPCY3-1基因、P450单加氧酶CYPCY3-2基因的过量表达有着密切的关系。
8.应用实时荧光定量PCR技术分析表明,烟碱型乙酰胆碱受体β1亚基基因在吡虫啉抗性品系的相对表达量是敏感品系的0.85倍；在啶虫脒抗性品系的相对表达量是敏感品系的0.36倍,由此说明,棉蚜对吡虫啉、啶虫脒的抗性的产生与烟碱型乙酰胆碱受体β1亚基基因的低水平表达有着密切的关系。
9.根据NCBI上登录的棉蚜乙酰胆碱受体β亚基基因序列(GenBank:JQ627836.1),设计特异性引物检测,结果表明,棉蚜吡虫啉、啶虫脒抗性品系、敏感品系以及新疆农一师、农二师、农三师、农四师、农五师、农六师、农七师、农八师、新疆沙湾、湖北省枣阳市、河南省西华县、安徽省萧县、山西省运城市等地方种群均未检测到突变。
1.The imidacloprid and acetamiprid susceptible strains (LC5o were0.176mg/L and0.180mg/L, respectively) were developed by individual reverse selection. The LC50value of the imidacloprid and acetamiprid susceptible strain had a2and1.73fold decrease compared with the field strain (LC50were0.346mg/L and0.312mg/L, respectively). The resistant strains of A. gossypii were obtained after forty selection with imidacloprid and acetamiprid by group selection. The resistance index of imidacloprid and acetamiprid resistant strain were83.27-fold (LC50was14.657mg/L) and82.33-fold (LC50was14.819mg/L), respectively. The realized heritability (h2) of resistance were0.1478and0.1342by Tabashnik's methods. In order to estimate the speed of resistance, it would take cotton aphid30.2-38.1and30.3-38.2ages of to obtain100-fold resistance to imidacloprid and acetamiprid, which was under breeding pressure of imidacloprid and acetamiprid. The results showed that A. gossypii had the resistance risk to imidacloprid and acetamiprid.
2. The results of cross-resistance to imidacloprid and acetamiprid-resistant strains were analyzed.(1) The results of cross-resistance to imidacloprid-resistant strains showed that the cross-resistaace of imidacloprid less than5folds were pymetrozine and Methyl abamectin, in5-10folds were thiamethoxam, endosulfan, abamectin, bifenthrin, chlorpyrifos, malathion, profenofos and Phoxim, in10-15folds were acetamiprid, beta-cypermethrin and triazophos, more than15folds was Omethoate.
(2) The results of cross-resistance to acetamiprid-resistant strains showed that the cross-resistance of acetamiprid-resistant strains less than5folds were pymetrozine and Methyl abamectin, in5-10folds were thiamethoxam, bifenthrin, chlorpyrifos, malathion, profenofos and Phoxim, in10-15folds were endosulfan, abamectin, beta-cypermethrin, triazophos and Omethoate, more than15folds was imidacloprid.
3. The survey for susceptible levels of cotton aphid to imidacloprid and acetamiprid of each cotton region in xinjiang showed that the relative resistance multiples to imidacloprid and acetamiprid were3.17-4.87folds and1.7-22.0folds. The resistance development speed to acetamiprid was faster than imidacloprid compared with2011in2012.
4. Synergism tests showed that triphenyl phosphate (TPP), piperonyl buoxide (PBO) and diethyl maleate (DEM) resulted in1.12,1.09and0.97-fold synergist ratios, respectively in the imidacloprid-susceptible strain. It was2.02,1.75and1.05-fold SR, respectively in the imidacloprid-resistant strain. It was1.02,1.03and1.02-fold SR, respectively in the acetamiprid-susceptible strain. It was2.02,1.75and1.05-fold SR, respectively in the imidacloprid-resistant strain. TPP and PBO had a good synergistic effect on imidacloprid and acetamiprid.
5. The detoxification enzymes tests showed that the activity of CarE, GST and7-ethoxycoumarin O-deethylase (ECOD) in the resisitant strain were higher than in susceptible strain. Among them, the activity difference of the CarE and ECOD reached significant level (P<0.05). The activity of three kinds detoxification enzymes in imidacloprid resisitant strain were3.26-,1.08-and1.60-folds that of the susceptible strain.The activity of three kinds detoxification enzymes in acetamiprid resisitant strain was2.91-,1.04-,1.69-times that of the susceptible strain.
6. Determination of individual aphid carboxylesterase (CarE) by microplate assay showed the activity of CarE in imidacloprid, acetamiprid resistant Aphis gossypii strains was higher than in susceptible strains. Determination of individual aphid acetylcholinesterase (AChE) by microplate assay showed the activity of AChE in imidacloprid, acetamiprid resistant Aphis gossypii strains was higher than in susceptible strains.
7. The mRNA relative expression level of CarE and P450monooxygenase gene were determined by real-time quantitative PCR. The mRNA relative expression level of CarE gene in imidacloprid resistant strains was1.85-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-1in imidacloprid resistant strains was1.60-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-2in imidacloprid resistant strains was2.00-fold of that of the sensitive strain; The mRNA relative expression level of CarE gene in acetamiprid resistant strains was1.91-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-1in acetamiprid resistant strains was1.44-fold of that of the sensitive strain; The mRNA relative expression level of P450CYPCY3-2in acetamiprid resistant strains was1.30-fold of that of the sensitive strain. Therefore, the overexpression of CarE, P450CYPCY3-1and P450CYPCY3-2in Aphis gossypii were closely related with the resisitance to imidacloprid and acetamiprid.
8. The mRNA relative expression level of nAChR beta1subunit were determined by real-time quantitative PCR. The mRNA relative expression level of the imidacloprid resistant strains was0.85-fold of that of the sensitive strain; The mRNA relative expression level of nAChR beta1subunit in acetamiprid resistant strains was0.36-fold of that of the sensitive strain. Therefore, the low level expression of nAChR beta1subunit gene in Aphis gossypii was closely related with the resisitance to imidacloprid and acetamiprid.
9. According to the gene sequence logged on NCBI about Aphis gossypii nAChR betal subunit (GenBank:JQ627836.1), the specific primers were designed to detect the susceptible strain R81T position of arginine(codon:AGA) might be replaced by threonine (codon:ACA) in resistant strains. The results showed that the Aphis gossypii population of imidacloprid, acetamiprid-resistant strain, sensitive strain, main cotton region in Xinjiang, Zaoyang city in Hubei, Xihua County in Henan, Xiao County in Anhui, Yuncheng City in Shanxi all were not detected the mutation.
引文
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