| The small brown planthopper,Laodelphax striatellus(Fallén),is a hemipteran insect,which is one of the important pests on cereal crops.Most importantly,L.striatellus can also transfer multiple plant viruses causing seriously production losses of rice,corn and wheat.During the 2000 and around,there were serious occurrences in the eastern areas of China,including Jiangsu,Shandong,Zhejiang,and Anhui province.Meanwhile,rice stripe virus and maize rough dwarf were widely spreading,which caused a serious impact on the production of rice and maize.Typically,chemical control is the major strategy for controlling L.striatellus.Resistance to chemical pesticides has been frequently detected in L.striatellus,causing certain difficulties for chemical control.Precise insecticides resistance detection technology is urgently needed in order to implement early monitoring of insecticide resistance and preventive resistance management.At present,bioassay is the main method to monitor the resistant level of insecticides in L.striatellus.Few studies in practice to monitor resistance with application of the mutations reported,although gene detection has advantages like high sensitivity and no requirement for standard tested insects.Moreover,due to the multi-factor effect of insecticide resistance,the role of these mutations in the development of resistance in the L.striatellus has even not been confirmed.In this study,we firstly determined the relative resistance levels of different populations with bioassays,and then detected the frequency of resistance related genes of different populations.Meanwhile,we used insecticides exposure for screening and determining the frequency changes of resistance related genes before and after the treatment.Thorough the analysis of comparison,we thus determined the relationship between the mutation frequency of insecticide resistance genes and the resistance in fields,and established the foundation for the reliable insecticide resistance gene detection technology.The research results are as follows:The resistance level of deltamethrin,ethiprole,imidacloprid,thiamethoxam and chlorpyrifos was evaluated by micro topical treatment.The LD50 values were as follows:deltamethrin(30.342 ng/insect),chlorpyrifos(10.356 ng/insect),thiamethoxam(1.898 ng/insect),imidacloprid(1.716 ng/insect),ethiprole(0.314 ng/insect),compared with the sensitive strain data reported by the predecessors,the resistance ratios were as follows:31.28,5.9,0.8,2.3,2.6.It was found that the phenylpyrazole insecticide ethiprole and the neonicotinoid insecticides imidacloprid and thiamethoxam had higher toxicity and L.striatellus are in the susceptible stage to these insecticides.The resistance of pyrethroid insecticide deltamethrin and the organophosphate insecticide chlorpyrifos are at moderate and low levels,respectively.The baselines of ethiprole and deltamethrin was used to determine the resistance of three populations of the L.striatellus in Jiangsu Dafeng,Liaoning Shenyang,and Hubei Xiaogan.It was found that there were little differences in the resistance to ethiprole of three L.striatellus populations,but the resistance to deltamethrin in Jiangsu Dafeng population was significantly higher than other two populations.These results have a guiding significance for the selection of insecticides for controlling L.striatellus in the fields.It has been reported that the GABA receptor Rdl subunit A2’N mutation is a resistance mutation of the L.striatellus to phenylpyrazole insecticides.In this paper,amplicon sequencing technology was used to analyze the mutation frequency of the GABA receptor Rdl subunit A2’N in three populations of the L.striatellus in China.The results showed that the A2’N mutation frequency of the Rdl subunit genes of the L.striatellus from different populations were as follows:Jiangsu Dafeng(18.90%),Liaoning Shenyang(14.48%)and Hubei Xiaogan(0.04%).The mutation frequency of A2’N in the Jiangsu Dafeng population was the highest and in Hubei Xiaogan was the lowest,which was not consistent with their resistance to ethiprole.However,after ethiprole treatment,the A2’N mutation frequency was increased,indicating that the A2’N mutation of the L.striatellus has some function in resistance.The discussion concluded that the resistance of the L.striatellus population in the field depends on a variety of resistant mechanisms,and the detection of this gene mutation alone may not predict the resistance of the L.striatellus to ethiprole accurately.In addition,we also compared the impact of the sampling volume on the detection results.The results showed that the sampling volume between 50-200 insects did not have a significant impact on the results of the amplicon sequencing to detect the A2’N mutation of the L.striatellus.In this research,we used PCR and molecular cloning techniques to clone the partial fragments of the 5’flanking region of the CYP6AY3v2 gene,the genomic DNA of the L.striatellus from Jiangsu Dafeng,Liaoning Shenyang,and Hubei Xiaogan were used as templates and the deletion was detected for resistance to deltamethrin.The results showed that the frequency of base deletions mutation in the 5’flanking region of the CYP6AY3v2 gene in the three populations of Jiangsu Dafeng,Liaoning Shenyang,and Hubei Xiaogan were very high,the frequencies were as follows:Hubei Xiaogan(93.5%),Jiangsu Dafeng(90.0%),Liaoning Shenyang(86.7%).Compared with the mutations detected in Jiangsu Jianhu population in 2009,it was significantly increased,indicating that the base deletion in the 5’flanking region of the CYP6AY3v2 gene is conducive to the reproduction of the L.striatellus in the field,and is also conducive to the reproduction of the L.striatellus in the field.It is also consistent with the change of resistance to deltamethrin in L.striatellus,but the detection of this gene mutation alone cannot accurately predict the resistance of L.striatellus to deltamethrin as well. |
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