Resistance Monitoring In East China And A309V Mutation Of PxGluClα Subunit Associated With High Abamectin Resistance In Plutellaxylostella

Plutella xylostella (diamondback moth, DBM) is one of the most destructive pests of cruciferous vegetables. Its short life cycle, high fecundity, and extensive selection pressure from insecticides, have resulted in rapid evolution of resistance to various classes of insecticides and it has become one of the pests which are the most difficult to control in the world. The avermectins are a group of fermentation products from a soil actinomycete Streptomyces avermitilis. They are 16-membered macrocyclic lactones with potent nematicidal, acaricidal and insecticidal activities. The abamectin has high efficiency against DBM. However, with intensively use, the field-evolved resistance to abamectin in DBM has become more and more serious. There are evidences to show the resistance is associated with the insensitivity of target genes. Therefore, it is necessary to understand the molecular mechanisms of abamectin resistance in P. xylostella, which will be helpful to set up an effective resistance detection and management strategy. In this paper, resistance of field populations to a wide range of insecticides were monitored in East China and the full-length cDNA sequences of the PxGluCla subunit from Roth-Abm strain and Roth strain were cloned. Sequencing results indicated that there is a point mutation (A309V) in Roth-Abm strain. Later the relationship between A309V mutation of PxGluCla subunit and abamectin resistance in P. xylostella was investigated. And also the frequency of mutation in ten field populations (resistance ratio<300-folds) were screened. All results are as follows:1. Resistance monitoring of field populations in East ChinaResistance to 11 insecticides was measured by leaf-dipping method in the field populations collected from East China (Hefei, Nanjing, Kunshan, Jinan). Different field populations have different resistance pattern. Resistance of DBM to Beta-cypermethrin was moderate to high level with resistance factors ranging from 30.5-fold to 785.9-fold. Resistance to spinosad and indoxacarb was low to moderate level with resistance factors ranging from 2.9-fold to 29.6-fold,5.5-fold to 26.1-fold. Resistance to abamectin was moderate to high level with resistance factors ranging from 24.5-fold to 332.6-fold. DBM was sensitve to chlorantraniliprole, but we found the resistance ratio of Hefei population in 2012 to chlorantraniliprole was 103.6-fold. DBM showed low level resistance to chlorfluazuron, diafenthiuron, tebufenozide, chlorfenapyr, cartap and Bt. Therefore, the use of insecticide to control DBM in the field shoud according the resistance monitoring result in order to avoiding the development of resistance.2. Relationship between A309V mutation of PxGluCla subunit and abamectin in P. xylostellaThe full-length cDNA sequences of the PxGluCla subunit from Roth-Abm strain (resistance ratio is 11500-fold) and Roth strain were cloned. Sequencing analysis indicated that there is a point mutation (A309V) in Roth-Abm strain. A resistance regression strain (Roth-Abm-Reg) was used to investigate the relationship between the A309V mutation of PxGluCla subunit and the resistance to abamectin. When Roth-Abm-Reg strain was treated with 0 mg/L,10 mg/L,20 mg/L,40 mg/L,80 mg/L of abamectin respectively, the PxGluCl-Wal allele frequency in survivors were 7.14%,15%,20%,32.5%,55%. It means the PxGluClV309 allele frequency increases along with the increasion of the concentration of abamectin treated. The V309 allele frequency of PxGluCla was 95.7% in Roth-Abm strain (with 11500-fold resistance), but only 7.14% in Roth-Abm-Reg strain (with 1700-fold resistance). It suggests that the point mutation A309V is related to extremely high level resistance to abamectin in Roth-Abm strain. The A309V mutation frequency of ten field populations (resistance ratio<300-fold) was screened, no A309V mutation was detected in these populations. These results illustrate that low levels of abamectin resistance could be mediated by resistance mechanisms other than the A309V mutation of PxGluCla subunit. Thus the role of A309V mutation of PxGluCla subunit in the high level resistance to amamectin need to be verified by functional experiment.