Investigation Of Resistance And Sensitive Gene Resources Of Panonychus Citri

Panonychus citri,also known as the citrus red spider,is an important mite that is found in all citrus production areas around the world.For a long time,chemical control has been the main prevention and control method for P.citri.Due to the small range of activities of P.citri and the opportunity to receive more drugs,coupled with the unreasonable use of acaricides,the problem of mite resistance has become more and more serious.Studies have shown that the mite has developed significant resistance to many types of acaricides.One of the important mechanisms for the development of resistance is the reduction in agent binding caused by mutations in the target.Pyrethroids,as a common class of acaricides,have the advantages of high efficacy,low toxicity and residue and have been used in large quantities for a long time in various production areas in China.However,the background of field application is complex,and different acaricides have diverse targets of action.Therefore,via systematically grasping the current status and dynamic development trend of P.citri resistance in the main citrus production areas in China,and investigating the type,frequency and spatial and temporal distribution of the main resistant and sensitive genes in field populations,we can help to formulate the management strategies of the pest mite and improve the timeliness of chemical prevention and control.Accordingly,this dissertation systematically monitored the resistance levels of 13 geographical populations of P.citri to six acaricides,including fenpermethrin,pyridaben and biflubenzuron,in five citrus production areas in China between 2021 and 2022 by using leaf disc impregnation,in order to grasp the development of resistance in a timely manner and make scientific recommendations on the use of acaricides to the pest mite.Additionally,the contribution of voltage-gated sodium channel(VGSC)target mutations to the resistance of pyrethroids was further analysed by using the transgenic Drosophila technology.The main studies are as follows:1 Monitoring the resistance of six acaricides in field populations of P.citriThe susceptibility of 13 geographical populations of P.citri to six acaricides,including fenpermethrin,pyridaben and biflubenzuron,was systematically determined by leaf impregnation between 2021 and 2022 in Sichuan,Chongqing,Yunnan,Jiangxi and Guangxi.It was found that the level of resistance varied significantly among the geographical populations,except the Wanzhou population with an low susceptibility to the all acaricides,and most of the populations developed moderate and high resistance levels,reaching several hundreds or even tens of thousands of times.Among them,Yuxi,Ganzhou and Nanning populations produced extremely high levels of resistance to avermectin and pyridaben,with relative resistance more than 1000-fold.Bifenazide was effective in killing mates in the field,and the citrus red mite was generally sensitive to this acaricide in all monitored areas(< 10-fold resistance).In addition,the geographical populations of Ruili and Wenshan in Yunnan were less sensitive to the six acaricides,and a certain proportion of resistant individuals were present,as determined by the diagnostic dosimetry.2 The mutation frequency of target resistance allele in field population of P.citriBased on two molecular assays,first-(Sanger sequencing)and second-generation sequencing(SNP amplicon sequencing),the detection of the main target genes of 18 geographical populations of P.citri revealed mutations in the sequences of GluCl,AChE,Cytb,PSST and VGSC genes.Among them,GluCls had mutated loci G314E(GluCl1),A310 E and G325R(GluCl3).Combined with the bioassay results,it was hypothesized that the mutation of G-314 to E might be related to the early development of resistance,while A301E and G325R might play an important role in the development of high resistance levels to this mite.The mutation frequency of H107R(PSST)associated with resistance to pyridaben and azoxystrobin was above 70% in all populations.mutation frequencies at the A352V(AChE),G480A(AChE),G126S(Cytb)and I258T(Cytb)loci varied considerably between geographic populations,ranging from 0 ～ 80%.In addition,three mutant sites in the VGSC gene,including L1031V,F1747L and F1751I,were found in various geographical populations of P.citri,of which F1747L was identified for the first time in pest mites,and the locus was only detected in some geographical populations.The combination of F1747L with the L1031V and F1751I mutations is hypothesized to cause the development of high resistance levels in P.citri.3 Functional validation of the mutant sites of VGSC gene in P.citriBy using transgenic Drosophila technology,L1031V,F1747L and F1751I on the VGSC gene of P.citri were introduced into Drosophila,and single and multiple mutant lines of Drosophila were constructed.The susceptibility of the mutant strains to pyrethroids(type Ⅰ and type Ⅱ)was tested and found to be 2 ～ 5 times more resistant to methothrin.The Drosophila strain with the L1031V + F1747L + F1751I mutation was the least susceptible.The mutant strains showed lower mortality than the wild strain when treated with both single concentrations of permethrin and high performance cypermethrin,and the single mutant locus strains showed higher mortality than the multilocus strains.The F1747L mutation had the least effect on susceptibility,while the combination of L1031V + F1747L + F1751I mutation caused the most significant reduction in susceptibility.Collectively,among the three mutant sites in the VGSC gene,F1747L contributed least to pyrethroid resistance;the combination of three mutations,L1031V,F1747L and F1751I,contributed most to pyrethroid resistance compared to the single and double mutant site(s).In summary,this dissertation systematically monitored and clarified the susceptibility and resistance development of P.citri to common acaricides in various production areas,and also grasped the mutation frequency and spatial and temporal distribution of the main target mutant sites in P.citri.In addition,the F1747L mutant site of VGSC gene was identified for the first time in P.citri,and the contribution of each VGSC mutant site to pyrethroid resistance in P.citri was clarified using transgenic Drosophila technology.The results of this study provide basic data for the field application of acaricides and resistance management to P.citri.