Monitoring Of Pesticide Resistance And The Mutation Frequencies Of Target Genes In Tetranychus Urticae

The two-spotted spider mite,Tetranychus urticae Koch,is one of the most damaging agricultural pest mites worldwide.It has broad host range including more than 1100 species of vegetables,fruits,maize,cotton,and ornamentals.The T.urticae is mainly active and causes damage on the underside of leaves through the adult and larval mites.It punctures the leaf cells with its cheliceral stylets and feeds on the leaking cell contents,causing the leaf chlorosis and even plant death,resulting in the serious threat to the crop quality and production.Chemical application is one of the most important measures for controlling T.urticae field populations in China.However,due to the unreasonable and unscientific application of the chemicals,T.urticae has developed different resistance to various insecticides and acaricides including organophosphates?OPs?,carbamates,synthetic pyrethroids,and even some recently developed compounds.Among the elucidated resistance mechanisms,target resistance is one of the main important mechanisms.Various point mutations in a series of well-defined acaricide target genes were found to be responsible for T.urticae resistance to major acaricides.In the present study,firstly,we investigated the resistance of seven field populations of T.urticae to 12 pesticides in China,using the optimized leaf dipping method,in order to illustrate the resistance level of T.urticae in different areas.Secondly,in order to examine the abamectin resistance of T.urticae rapidly,two molecular techniques named cleaved amplified polymorphic sequences?CAPS?and Loop-mediated isothermal amplification?LAMP?were established.Lastly,the mutation frequencies of target genes related to pesticide resistance were determined using different molecular biology method,providing the molecular basis of T.urticae against different chemicals.The mains contents and results of this study are as follows.1.We investigated the resistance of seven field populations of T.urticae to 12 pesticides in China,using optimized leaf dipping method.As determined by the standard bioassay,compared to the Lab-SS strain,all seven field populations exhibited high to extremely high resistance to abamectin?the maximum of RF is 1809.51?.For the traditional acaricides,all seven field populations had low RF values for pyridaben,bifenthrin,and profenofos,but the LC₅₀0 values were very high?the maximum is2783.89 mg/L?,this indicated these three chemicals had lower toxicity to the T.urtciae mites.For the newly developed chemicals including B-azolemiteacrylic and spinetoram,the T.urtciae field populations were proved to be susceptible?the RF value below 5?,and most field populations were low or moderate resistance to cyflumetofen,cyenopyrafen and bifenazate?the RF value between 5 to 40?.2.The DNA fragment in glutamate-gated chloride channel gene 3?GluCl3?from T.urticae susceptible and resistant populations were cloned.The differences of GluCl3 between T.urticae susceptible and resistant populations were also compared and analyzed.The results confirmed that the G326E mutation was existed in T.urticae filed populations in China.The cleaved amplified polymorphic sequences?CAPS?and Loop-mediated isothermal amplification?LAMP?techniques were established based on the point mutation in GluCl3.For the CAPS technique,the PCR production was digested with restriction enzyme Hinf I and the individual genotype of single mite could be identified though the number of bands in electrophoresis detection.In the LAMP technique,whether the G326E mutation occurred in the mites could be evaluated though the color of reaction product.3.The mutation frequencies of target genes related to pesticide resistancein T.urticae field populations were determined using molecular biology method.The mutations G314D and G326E for abamectin resistance were not observed in the Lab-SS strain,in the T.urticae field populations,the frequency of G314D and G326E mutations ranged from 28.33 to 63.64%and from 0 to 95%,respectively.The frequency of G119S and A201S mutations for OP resistance was 50%and 0%,respectively,in the Lab-SS strain.For the field populations,the frequencies ranged from 33.33 to 56.67%and from 5.00 to 43.33%,respectively.For L1024V,A1215D,and F1538I mutations in the voltage-gated sodium channel gene,no L1024V mutation was detected in any of the populations.However,the A1215D and F1538I frequency were 25%and 81.67%in the Lab-SS strain;for the field populations,the frequencies of these two mutations were all above 70%.No point mutations or amino acid substitutions were detected at positions 126,136,141,161,or 262 in the cytochromeb?Cytb?gene probably related to bifenazate resistance.