Study On The Combined Insecticidal Activities Of Beauveria Bassiana And Pyemotes Zhonghuajia On The Larvae Of Dioryctria Abietella

Dioryctria abietella is a destructive pest of coniferous forests.In recent years,this pest has occurred in a large area in Northeast China,seriously endangering Pinus koraiensis cone seed.Beauveria bassiana and Pyemotes zhonghuajia are widely used as important biological control methods.In this study,the toxicity（insecticidal）power of B.bassiana and P.zhonghuajia to D.abietella larvae was determined.Changes of enzyme activity and nutrient content in the larvae of D.abietella after treatment with B bassiana and P.zhonghuajia.Scanning electron microscopic observation of the body surface and digestive tract of B.bassiana infecting D.abietella larvae.Transcriptome analysis of the larvae of D.abietella after treatment with B.bassiana and P.zhonghuajia.It lays a theoretical basis for the biological control of D.abietella larvae.The main findings are as follows:（1）Determination of toxicity（insecticidal）power of B.bassiana and P.zhonghuajia to larvae of D.abietellaThe virulence of TS370911,TS313128,CFCC81428,CFCC83116 and CFCC83486 were determined,it was found that CFCC81428 was more virulent to larvae,its LC₂₅ and LC₅₀ at the 10^th day after treatment were 2.04×10⁴ and 7.24×10⁵spores/m L,respectively.The LT₅₀ of CFCC81428 strain infected with 1×10⁸ spores/m L was 5.36 d.The cumulative calibrated mortality of larvae infected with strain CFCC81428 with a concentration of 1×10⁸ spores/m L for 10 days was 77.30%.The spore germination rate was higher than 90%.At the 3^rd days after treatments,the LD₂₅ and LD₅₀ of the parasitic larvae of P.zhonghuajia were 16.60 and 25.12 individuals.The cumulative calibrated mortality of larvae after combined treatment was higher than that after being parasitized by P.zhonghuajia alone.The cadaver rate of larvae after combined treatment was higher than that of larvae infected with B.bassiana alone.（2）Changes in enzyme activity and nutrients in D.abietella larvae after treatment with B.bassiana and P.zhonghuajiaIt was found that the activities of GSTs and ACh E in the larvae after B.bassiana infection were higher than those in the control group and were induced.The activities of GSTs and ACh E in the larvae parasitized by P.zhonghuajia were higher than those in the control group before 60 h and were induced.After 72 h,they were lower than those in the control group and were inhibited.The detoxification enzyme activity of larvae after combined treatment showed a further downward trend compared with the other two treatment groups.The activities of PPO,SOD and CAT in the larvae were higher than those in the control group at the rest of the sampling time,except that the activities of PPO and CAT were lower than those in the control group at 12 h.After the parasitism of P.zhonghuajia,the activities of these three protective enzymes in the larvae increased first and then decreased.After combined treatment,the activities of PPO and CAT in larvae decreased in advance compared with the other two treatment groups.Compared with the control group,the total sugar and protein content of D.abietella larvae decreased gradually after the parasitism of P.zhonghuajia.（3）Scanning electron microscopic observation of the body surface and digestive tract of B.bassiana infecting D.abietella larvaeConidia were found to attach to different areas of the larval body wall 12 h after inoculation,after 36 h of inoculation,B.bassiana invaded the larval body wall in the form of hyphae or germ tubes,after 72 hours of inoculation,the outer wall of the digestive tract was folded and broken,rigid and fragile,after 96 h of inoculation,hyphae penetrated the larval body wall and multiplied on the surface,after 120 h of inoculation,new conidia grew on the mycelium.（4）Transcriptome analysis of D.abietella larvae after treatment with B.bassiana and P.zhonghuajiaIn this study,the transcriptome c DNA libraries of 12 samples were sequenced by Illumina high-throughput sequencing platform,and 75.92 Gb Clean Data were obtained.Through the annotation of major databases,25636 annotated genes were finally obtained.Through BLAST comparison of NR database,a total of 24087 Unigenes were matched to the NR database.It was found that 11172 Unigenes were matched to Amyelois transitella,which was the species with the highest matching rate,accounting for 46.38%of the total NR annotations.GO functional classification showed that a total of 18517 Unigenes were annotated to biological processes,cellular components,and molecular functions,the successfully annotated Unigenes were further classified into 59 functional subgroups.Through the screening of stress-resistant anti-oxidant genes in larvae,it was found that GST-2,ACh E-1,SOD2-2,GST-23 genes were significantly correlated with detoxification enzymes and protective enzymes in larvae.A total of 749 different genes were obtained in the treatment group of B.bassiana,including 115 up-regulated genes and 634 down-regulated genes.Through GO annotation,the differentially expressed genes were annotated to 37 GO terms.The differential genes were aligned to the COG database for annotation.In the COG database,these sequences were annotated into 17 functional categories.KEGG pathway library enrichment analysis showed that the differential genes could be compared to137 metabolic pathways.The immune-related genes controlling the expression of peptidoglycan recognition protein,heat shock 70 kda protein 1/2/6/8 and autophagy-related protein 5 in the larvae were activated,which showed that the gene expression was up-regulated.A total of 79 differentially expressed genes was identified in the P.zhonghuajia treated group,of which 33 were up-regulated and 46 were down-regulated.Through GO annotation,the differentially expressed genes were annotated to 33 GO terms.The differential genes were aligned to the COG database for annotation.In the COG database,these sequences were annotated into 9 functional categories.KEGG pathway library enrichment analysis of differentially expressed genes can be compared to 33 metabolic pathways.Immune-related genes that control the expression of CD63antigen and spermine oxidase were activated,showing up-regulation of gene expression.The analysis of the combined treatment group of B.bassiana and P.zhonghuajia showed that 242 differentially expressed genes were obtained,including 132 up-regulated genes and 110 down-regulated genes.Through GO annotation,the differentially expressed genes were annotated to 36 GO terms.The differential genes were aligned to the COG database for annotation.In the COG database,these sequences were annotated into 12 functional categories.KEGG pathway library enrichment analysis of differentially expressed genes can be compared to 74 metabolic pathways.Immune-related genes that control the expression of cytochrome c oxidase subunit 1/2 were activated,which showed up-regulated gene expression.