Study On The Mechanism Of Action Of Beta-asarone On Brown Planthopper (Nilaparvata Lugens) And Its Synergistic Effect With Chemical Insecticides

Nilaparvata lugens is one of the major pests endangering rice production in China.The current control of N.lugens relies heavily on chemical control.However,the irrational use of chemical insecticides has induced serious resistance problems.It is urgent to explore new control methods that are efficient,low toxic and environmentally friendly,and can reduce or replace the use of chemical insecticides while effectively controlling N.lugens.Theβ-asarone is main insecticidal active ingredient of Acorus calamus essential oil,and previous studied have shown that this ingredient has great potential for application in pest control,but its mechanism of action on N.lugens has not been reported.This thesis investigated the biological activity ofβ-asarone on N.lugens;the effect of sublethal dose ofβ-asarone on the life table and transcriptome of N.lugens population;the detoxification effect of N.lugens cytochrome P450 monooxygenase（P450）onβ-asarone;and the synergistic effect ofβ-asarone on nitenpyram,buprofezin and pymetrozine was studied by inindoor toxicity,greenhouse toxicity and field test.The main findings were as follows:1.Biological activity ofβ-asarone against N.lugensThe LC₅₀values and 95%confidence intervals were 44.465μg/L（39.199-50.356μg/L）and 112.506μg/L（94.292-134.548μg/L）for 30 min after fumigation withβ-asarone for 3^rd-instar nymphs and brachypterous adult females,respectively.The LC₅₀values and 95%confidence intervals were 4.803 mg/m L（4.169～5.508 mg/m L）and 14.448 mg/m L（12.276～16.800 mg/m L）after spotting withβ-asarone for3^rd-instar nymphs and brachypterous adult females,respectively.After spraying rice with different concentrations ofβ-asarone,the egg egg production of N.lugens on rice plants of different treated rice was significantly lower than that of the control.This indicates thatβ-asarone has yield significant fumigant activity,contact activity and oviposition deterrent activity against N.lugens.2.Effect of sublethal dose ofβ-asarone on the life table of N.lugens populationsThe females and males adult longevity of F₀generation of N.lugens in the LD₄₅treatment group were significantly shorter than that of the control group,and significantly reduced egg production.Compared with the control group,the LD₃₀and LD₄₅treatment groups significantly prolonged the egg-laying period and 1^st-instar stage of F₁generation of N.lugens,but significantly reduced the adult longevity and hatching rate.In addition,the intrinsic rate of increase（r）,finite rate of increase（λ）and net reproductive rate（R₀）of F₁generation of N.lugens in LD₃₀and LD₄₅treatment groups were significantly lower than those in the control group.The egg production of F₁generation N.lugens in LD₃₀and LD₄₅treatment groups were significantly lower than that of the control group.3.Effect of sublethal dose ofβ-asarone on the transcriptome of N.lugensCompared with the control,a tatal of 126 differentially expressed genes were obtained from the LD₃₀dose ofβ-asarone treatng the 3^rd-instar nymphs for 24 h,of which 72 showed significant up-regulated and 54 were significant down-regulated;while 1771 differentially expressed genes were obtained from the treatment for 72 h,of which 882 were significant up-regulated and 889 were significant down-regulated.These differential genes were mainly enriched to GO functional terms such as structural constituent of cuticle,extracellular region,carbohydrate biosynthetic process,chitin binding and chitin metabolic process and KEGG pathways such as tyrosine metabolism,ribosome biogenesis in eukaryote and entose and glucuronate interconversions.We selected 18 P450 differential genes for fluorescence quantitative PCR verification,and the results were consistent with the transcriptome results.4.CYP301B1 and CYP6AX1v2 are involved in the detoxification ofβ-asaroneThe relative expression of CYP301B1 was highest in egg stage,while CYP6AX1v2 was highest in macropterous female.The relative expression of CYP301B1 was highest in head of the N.lugens,while CYP6AX1v2 was highest in fat body.After silencing CYP301B1 in 3^rd-instar nymphs of N.lugens,the mortality of nymphs after 72 h of treatment with LD₃₀dose ofβ-asarone was 53.33%,which was obviously higher than that of the control at 39.17%.Likewise,the mortality of nymphs of CYP6AX1v2 after silencing was 69.17%,which was significantly higher than that 42.50%of control.It indicates that silencing CYP301B1 and CYP6AX1v2respectively,both significantly increase the sensitivity of N.lugens toβ-asarone.5.Synergistic effect ofβ-asarone on nitenpyram,buprofezin and pymetrozineThe resistance ratios of Nanchang,Ga’an and Taihe N.lugens populations to nitenpyram were 22.55,17.19 and 53.59 times higher than those of the indoor populations;the resistance ratios to buprofezin was 115.81,182.41 and 253.03 times higher than that of the indoor populations;the resistance ratios to pyrazinon was 94.59,64.00 and 86.22 times higher than that of the indoor population,respectively.The best synergistic effect was achieved whenβ-asarone was mixed with nitenpyram at a mass retio of 1:1 or with pymetrozineor at a mass retio of 1:4,with co-poisoning coefficients of 202.18 and 159.72,respectively.The greenhouse test showed that the mortality of N.lugens was above 70%after spraying 6.2500 mg/Lβ-asarone mixed with nitenpyram and 20 mg/Lβ-asarone mixed with pymetrozine for 5 days.The field control effects of 6.2500 mg/L ofβ-asarone mixed with nitenpyram and 20 mg/L ofβ-asarone mixed with pymetrozine were comparable to those of 50%nitenpyram aqueous dispersible granules and 40%pymetrozine wettable powder respectively,and the control effect were above 70%after 5 days,which could reduce the dosage of nitenpyram or pymetrozine by more than 90%or more than 80%,respectively.In summary,the study found thatβ-asarone had strong contact activity against N.lugens,and also significantly reduced the egg production and hatching rate,and significantly inhibited the growth of N.lugens population.It was clarified that silencing CYP301B1 and CYP6AX1v2 alone both significantly increased the susceptibility of N.lugens toβ-asarone.In addition,it was confirmed thatβ-asarone has s significant synergistic effect on either nitenpyram or pymetrozine.Mixing with them can significantly reduce the amount of chemical insecticides while achieving the same control effect.The study indicate thatβ-asarone has great potential for the control of N.lugens,and provide a theoretical basis for the development of new environmentally friendly mixes usingβ-asarone.