Resistance Monitoring Of Plutella Xylostella In Eastern China And Characterization Of Resistance To Flubendiamide Mediated By The I4790M Mutation Of Ryanodine Receptor

The diamondback moth,Plutella xylostella(L.),which belongs to Lepidoptera Plutellidae,is an important economic pest to cruciferous vegetable in the world.Diamide insecticidestarget insect ryanodine receptors(RyRs).Since 2007,this class of insecticide products has been put on the market for pest control.For less than three years,the field populations of P.xylostella from Guangdong Province evolved more than 2000-fold resistance to chlorantraniliprole.In this work,wemonitoriedthe resistance status of new insecticides including chlorantraniliprole,in order to provide informative knowledge for formulating rational pesticide use strategies.CRISPR/Cas9 gene editing technique was empolyed to construct a homozygous strain of P.xylostella withthe ryanodine receptor I4790M mutation.The contribution of PxRyR I4790M mutation to diamide resistance was determined in the gene edited strain.The genetic mode of resistance to flubendiamide was studied by reciprocal crosses between the mutant strain and the susceptible WH-S strain.Further,the linkage between I4790M mutation and resistance phenotype to flubendiamide was confirmed by genetic analysis.Our studydemonstrated the perspective of reverse genetics approach for in vivo functional verification of both the known resistance genes and the new genes recently discovered.1.Resistance monitoring and detection of resistance-related PxRyR mutations in P.xylostella from Estern ChinaThe resistance status of P.xylostella to commonly used pesticides in Eastern China was determined during 2017-2018.The populations of P.xylostella collected from Hangzhou has higher levels of resistance than thosefrom Shanghai and Nanjing.The results showed that resistance levels of P.xylostella field populationswere 11-385 fold to abamectin,11-1268 fold to chlorantraniliprole,4-215 fold to chlorfenapyr,39-386 fold to indoxacarb,and 1-27 fold to spinosad.The monitoring data indicates that resistance levels of the field populationsto spinosad is relatively low,and it can still be used for controlling the P.xylostella field populations in this region.Resistance monitoring data also showed field populationsdeveloped moderate to high levels of resistance to chlorantraniliprole a few years after its commercialization,and it use should be suspended in the short-term.Resistance levels among different areaswere quietdifferent for a specific insecticide,therefore,the chemical control recipe should be adjusted timelybased on the monitoring data.Prevous studies indicated that PxRyR G4946E and 14790M mutations were associated with diamide resistance.So,resistance associated PxRyR mutations were detected in P.xylostella populations from Estern China.Allele frequencies of G4946E,I4790M anda new mutation I4790K ranged from 18.3%-90.3%,0%-8.3%and 0%-43.3%,respectively.2.Establishment of the gene-edited PxRyR I4790M strain of P.xylostella and its susceptibility to insecticidesByusing the CRISPR/Cas9 technique,the PxRyR I4790M mutation was successfully introduced into the WH-S strain of P.xylostella.Through marker assisted selection,a geneedited strain with homozygous I4790M mutation was established in the G3 generation.This strain was named as I4790M.Compared to the background WH-S strain,the I4790M strain obtained 67-fold resistance to flubendiamide,and medium to low levels of resistance to chlorantraniliprole(11-fold)and cyantraniliprole(3.9-fold).However,variations of susceptibility to other insecticides(indoxacarb,beta-cypermethrin and chlorfenapyr)were within 2-fold.The results provided direct evidence that the I4790M mutation of PxRyR confersdiamide resistance in P.xylostella,and revealed that this point mutation contributes different levels of resistance to phthalic acid(flubendiamide)andanthranilic diamides(chlorantraniliprole and cyantraniliprole).3.Resistance inheritance and genetic linkage of resistance to flubendiamidewith I4790M mutation inthe I4790M strain of P.xylostellaThe resistance level of the I4790M mutant strain to flubendiamide was 67-fold,and F1 progeny from reciprocal crosses between the 14790 and WH-S strains were 1.6-and 2.0-fold resistant to flubendiamide.Dominance levels(D values)were-0.76 and-0.69 respectively,indicating that resistance inheritance mode conferred by the I4790M mutation was autosomal and recessive.The results of backcross analysis and genotyping datademonstrated that the I4790M mutation of PxRyR was tightly linked to flubendiamide resistance in P.xylostella.The above resuts confirmed the causal relationship between PxRyR I4790M mutation and diamide resistance in P.xylostella.Our finding will also enrich our understanding on the action mode of flubendiamide and the resistance mechanisms by target insects.