Resistance Monitoring And Preliminary Study On Resistance Mechanism Of Abamectin In Chilo Suppressalis(lepidoptera: Pyralidae)

The rice stem borer,Chilo suppressalis(Walker)(Lepidoptera: Pyraidae)is an important pest of rice.It damages crops such as rice and water oat by boring and feeding,resulting in the decline of crop yield and quality.At present,chemical control is still the main meansure to control C.suppressalis.Due to the long-term and large-scale use of insecticides,rice stem borer has developed resistance to most of registerd insecticides.In 2008,chlorantraniliprole became the main insecticide for controling the rice stem borer.With the extensive applying,C.suppressalis has developed high resistance to chlorantraniliprole in Zhejaing,Jiangxi and Hunan province.Abamectin recovered to be the first choice to control C.suppressalis.Therefore,it is vital to clarify the resistance status of the field populations to abamectin.In this study,the resistance level of rice stem borer to abamectin was monitored in 38 fields populations collected from 8 provinces from 2019 to 2020,and the target and metabolic resistance mechanism of abamectin were also studied.The results could provide guidance for the resistance management of C.suppressalis.1.Resistance Monitoring of C.suppressalis against abamectin and emamectin benzoateThe resistance of 38 field populations to abamectin were monitored by topical application method during 2019-2020.The results showed that 73.7% of the monitored populations were susceptible to low resistance,and 21.1% of the monitored populations showed moderate level resistance,but Cangnan population collected from Zhejiang province reached high level resistance(RR=142.3).Among the 19 field populations monitored in 2020,47.4% of the monitored populations were susceptible to low level resistance,and 26.3% of the monitored populations showed moderate level resistance.There are 6 populations of Youxian,Hunan,Shanggao,Nanchang,Fengcheng and Cangnan have reached high level resistance(RR=121.6-143.3),more high resistance populations thanthat of 2019.The resistance of 31 field populations to emamectin benzoate were monitored during2019 to 2020.The results showed that the susceptible to low resistance populations accounted for 87.5% of the monitored population,the moderate level resistance populations accounted for 12.5%,they are Youxian population in Hunan province(RR=22.3)and Cangnan population in Zhejiang province(RR=33.2),respectively.Among the 15 field populations monitored in 2020,53.3% of the monitored populations were susceptible to low level resistance,and 46.7%(RR=13.8-46.2)were moderate level resistance,there are more moderate resistance populations than that of 2019.In conclusion,the resistance to abamectin and emamectin benzoate exhibited an upward trend during 2019-2020.There were significant regional differences,appearing on the field populations with moderate and high levels resistance to abamectin and emamectin benzoate mainly concentrated in Zhejiang,Jiangxi and Hunan provinces,while the field populations from other provinces showed relatively sensitive.2.Cloning and sequence analysis of chlorine glutamate channel gene of C.suppressalisBased on the mutation of Glu Cl,the mechanism of target resistance to abamectin was studied.The full-length c DNA sequences of CSGlu Cl were cloned to detect target mutations in four field resistant populations(CN19,DC19,YX19,RA19)collected in 2019 and susceptible strains(SS).The results showed that D339 G point mutation and 133-bp deletion mutation were found in the resistant population.Among them,D339 G is located in the conserved domain after the transmembrane domain TM3,while the 133-bp deletion mutation leads to the premature termination of the translation process,resulting in partial deletion of the transmembrane domain TM3 and complete deletion of TM4.The statistical results showed that the frequency of these two mutations was positively correlated with the resistance of abamectin in 4 populations.However,no mutations were found in 2020 field populations.In addition,there are three kinds of alternative spliceosomes in exon 3 and exon 9 of CsGluCl.Statistical results showed that the splices were mainly 3A and 3B at exon 3,and the frequency of 3A was negatively correlated with the resistance to abamectin,while the frequency of 3B showed positively correlation.The splices were mainly 9A and 9C at exon9,and the frequency of 9C negatively correlated with the resistance to abamectin,while the frequency of 9A showed positively correlation.The expression levels of different spliceosomes were detected by Quantitative Reverse Transcription-PCR(RT-q PCR)technique,and then converted into the proportion of spliceosomes in the exon expression.The results showed that the frequencies of 3A and 9C negatively correlated with the resistance to abamectin.However,3B and 9A were positively correlated with the resistance to abamectin,which was similar to the statistical results.By comparing the statistical results of spliceosome frequency in 2020 with that in 2019,there is no significant difference in susceptible strain,but significant difference in field populations.At exon 3,the frequency of3 A decreased,while the frequency 3C increased and no significant differences in 3B.At exon9,the frequency of 9A in high resistant populations(YX20 and CN20)decreased,while the frequency of 9C increased,but it was still significantly higher than that of the susceptible strain.The correlation between the frequencies of 3A and 9C,3B and 9A and abamectin resistance kept similar of that in 2019.3.Metabolic resistance mechanism of C.suppressalis to abamectin31 differentially expressed genes were screened out between susceptible strains(SS)and field resistant populations(CN19,YX19 and YY19)by transcriptome sequencing.The up-regulated genes were mainly concentrated on the metabolic process,and it was noted that the two P450 genes(CYP6B5-like and CYP6AB45)were related to the metabolic resistance of abamectin.The synergistic effects of PBO,TPP and DEM on abamectin were also determined with field resistant population(CN20)and susceptible strain(SS-YZ18).The results showed that PBO and TPP had a 3.2-fold and 3.4-fold synergistic effect on abamectin in resistant population,respectively.In conclusion,cytochrome P450 participate in the metabolic resistance of abamectin,and carboxylesterase may be involved in the metabolic resistance mechanism of abamectin.