Insecticidal Activity And Mechanism Of Ascendens Mucronatum Essential Oil Against Sitophilus Zeamais

The storage of grain pests has always been an important factor affecting grain storage.The prevention and control methods that rely on chemical fumigation are very restricted due to pest resistance,environmental pollution,and safety.Therefore,research and development of new green and environmentally friendly insecticides is imminent.The essential oil of Ascendens mucronatum extracted from the branches and leaves of A.mucronatum has the advantages of wide source and low cost.In order to explore its biological activity and improve its utilization value,the research group has established the extraction method of A.mucronatum essential oil.On this basis,this paper studies the insecticidal activity of A.mucronatum essential oil against stored grain pest Sitophilus zeamais,clarifies that its main insecticidal active ingredient is limonene,and facilitates transcriptomics sequencing and RNAi technology to explore the mechanism of S.zeamais to limonene.It provides a theoretical basis for the development and utilization of A.mucronatum essential oil.The main research results are as follows:1?Insecticidal activity of A.mucronatum essential oil against S.zeamaisUsing S.zeamais as test insects,the fumigation and contact activities of A.mucronatum essential oil on stored grain pests were studied.A.mucronatum essential oil has significant fumigation activity on S.zeamais and the activity has obvious time effect and dose effect.LC₅₀ values at 24,48 and 72 h were 22.90,17.25 and 15.39?L/L,and the adjusted mortality rate at the same concentration increased significantly with the extension of fumigation time.2?Identification and analysis of insecticidal active components in A.mucronatum essential oilThe chemical composition of A.mucronatum essential oil was analyzed and identified by GC-MS technique,and the relative content of each component was calculated by area normalization method.The main active ingredients include:?-Pinene,Limonene,Caryophyllene,cis-Thujopsene,Caryophyllene oxide,?-Pinene,Camphene,?-Muurolene,?-Terpineol,Bornyl acetate.Among them,the contents of?-Pinene and limonene are the most,with 35.38%and 28.38%respectively.The results showed that limonene had significant fumigation activity,and its LC₅₀ of 24,48 and 72 h were 10.17,7.79,and 5.43?L/L:Limonene should be the main insecticidal active ingredient of A.mucronatum essential oil.3?Transcriptional level analysis of S.zeamais in response to limonene and indentification of detoxification enzyme genesFunctional annotation and pathway analysis were carried out on transcriptome level of S.zeamais after limonene stress by RNA-seq technique.The results showed that 50129unigenes were obtained after assembly and matching,including 7049 differentially expressed genes,3982 up-regulated genes and 3067 down-regulated genes.The differentially expressed genes of several detoxifying metabolic enzymes were identified and analyzed,including 12 genes of Cytochrome P450?P450?,4 genes of Glutathione S-transferase?GST?,11 genes of Esterase?EST?and 11 genes of UDP-glucuronyl transferase?UGT?.All the differentially expressed P450 were up-regulated,most of which were from the CYP6 family related to exogenous metabolism and pesticide resistance,indicating that the P450 gene may play an important role in the metabolism of corn-elephant response to limonene.4?Study on the expression pattern of P450 genes induced by limoneneBased on the transcriptome sequencing results,the expression levels of 12 P450 genes were detected by q RT-PCR.The results showed that under different treatment times,the P450 gene expression levels reached the peak at 24h and then decreased to different degrees.The expression levels of most P450 genes increased significantly with the increase of concentration,while the expression levels of Unigene4757?All and CL4050.Contig1?All reached higher expression levels at low concentrations and had dose effect.5?RNAi was used to verify the function of four P450 genes in S.zeamais induced by limoneneFour ds RNA of P450 genes were synthesized in vitro for RNAi experiment of S.zeamais.The experimental results showed that,except the CYP9E2-like group,the sensitivity of S.zeamais to limonene in the other three groups increased to different degrees,among which the CYP6MS6 group was the most significant.The results suggest that the P450 genes may play an important role in the metabolism of limonene in S.zeamais.