Resistance Risk Assessment And Bi-PASA Molecular Monitoring Of Flubendiamide In Diamoandback Moth(Plutella Xylostella)

Diamondback moth(Plutella xylostella L.) is one of the most important pests on cruciferous vegetables, and it also was one of the most serious emergence and development of drug-resistant pests. Because of the overlapping generations, the high fecundity, and the applying pestcides disorderly in field, the resistance development of P. xylostella is so fast. The resistance has resulted in many problems in control of this pest. As the ryanodine receptor activator, flubendiamide was efficient to lepidopteran pest and low toxicity to mammals, the channel of insect RyR remains open after acted by flubendiamide, which caused the concentration of calcium ions increased in cell. Sustained release of ions will result in the pest continue to shrink, vomiting, defecation, and then food refusal, eventually to die. With the using of flubendiamide in pest, the problem of pest resistance has become increasingly prominent.In order to investigate the risk of resistance to flubendiamide in diamondback moth, the laboratory resistance screen, biological fitness of resistant strain and risk assesment were carried out in this paper. In addition, the molecular mechanisms and gene mutation frequency for the resistant strain were also studied. This study aims at provides some suggestions for the scientific use of flubendiamide. The obtained results as follows:1. The toxicity of flubendiamide against three strains of the3rd larvae of diamondback moth was tested by topical application, the results showed the72h LD50of flubendiamide to susceptive strain was0.921μg/g, the72h LD50to filed population, BY and ZC, were22.237μg/g and1638.680μg/g, respectively.2. The resistance level to flubendiamide in P. xylostella reached40.72-fold after selection of17generations with the pressure of killing40-80%individuls of the population. The resistance to flubendiamide in P. xylostella developed slowly during the F0to F8, however, the resistance development was faster from the F9to F17.3. The toxicity of flubendiamide-resistant strain larvae to different insecticides were tested by topical application. The results showed that the cross-resistance to lambda-cyhalothrin, chlorantraniliprole and emamectin benzoate was2.75,4.84and1.90-fold, respectively. The resistant strain caused a cross-resistance to chlorantraniliprole and lambda-cyhalothrin, but no cross-resistance to emamectin benzoate was determined. The effects of flubendiamide resistance on P. xylotella fitness were evaluated in terms of developmental and reproductive characteristics by constructing life table of resistance strain and susceptible strain. The results indicated that the fitness value is of0.63in the resistance strain. The resistance strain possessed some developmental disadvantages in reproduce ability. 4. Estimation of realized heritability (h2) of resistance in different screen stages were studied based on the Tabashnik’s method. The results suggested that realized heritability for the17generations was0.135. Assuming that the slope was2.0(δp=0.5), and h2was0.135, it requires8～19generations of P. xylostella to obtain10-fold increase of flubendiamide in LD50under selection pressure at50%-90%mortality for each generation of selection. When h2was0.093, it requires12-27generations.5. According to the mutation of the diamondback moth ryanodine receptor gene reported, we designed Bi-PASA primers to detect the mutation of resistant and sensitive diamondback moth ryanodine receptor gene mutation. This method could detect the gene mutation frequency for the field strain, selected resistance strain and susceptible strain, respectively. The results indicated that the homozygous mutation of the field strain diamondback moth (ZC) was80.77%, the heterozygotes were19.23%, and no wild-type were detected. The homozygous mutation of the field strain diamondback moth (BY) was19.23%, the heterozygotes were73.08%, and the wild-type was7.69%. The heterozygotes of sensitive diamondback moth were16.67%, the wild-type was83.33%, and no homozygous mutations were detected. And for the mutation of flubendiamide resistant diamondback moth, the homozygous mutation was19.23%, the heterozygotes were65.38%, and the wild-type was15.38%.6. The expression abundance of RyR mRNA from P. xylostella larvae in the difference strains was determined using real-time quantitative PCR. The results showed that the expression level of RyR gene from flubendiamide resistant strain was2.938-fold compared with that of the susceptible strain. This indicated that the formation of resistance may be related with the over-expression of receptor.