Study On The Pathogenicity Of Biocontrol Strains In Dioryctria Abietella

Dioryctria abietella is one of the most harmful forest borers,and its entire larval stage feeds on the pith tissue and nuts of young cones,resulting in deforms cone development and decreases seed yield,causing great economic losses.Biological control has become an environment-friendly way to control pests due to its advantages of no pollution and residue to the environment as well as no resistance of insects.In order to effectively control the D.abietella,this study combines indoor and field biocontrol experiments to test the insecticidal activity of eight biocontrol strains in the D.abietella larvae.The indoor study determined Bt05041 as a highly pathogenic strain,and studied the effects of this strain on the gut bacterial community and midgut epithelial cells of D.abietella at different times after treatment.Based on transcriptome data,the differential genes related to immunity were screened out,and the spatiotemporal expression profiles of Bt receptors and defense enzyme genes were analyzed by real-time PCR.The specific results were showed as follows:（1）In the laboratory determination of the virulence of eight strains,Bt05041 had the best virulence at a concentration of 1×10⁸ CFU/m L,90%of D.abietella was died cumulatively within10 days.At the same time,we found that the corrected mortality and LC₅₀ at 3^rd day after treatment reached 45.00%and 3.15×10⁸ CFU/m L,respectively.Five strains（Bt223176,Bb01,Bb122077,LF01 and LN01）were applied for biological control in a forest plantation,and the results showed it was 82.74%mature cones and 15.87%damaged cones after spraying Bt223176（1×10⁸ CFU/m L）during the flowering phase of Korean pine,and this treatment was effectively promoted seed quality and seed yield.We also found that the number of D.abietella larvae was significantly reduced in dissected cones after spraying bacterial or fungal solutions.（2）The changes of protective enzymes and detoxification enzymes in the D.abietella larvae were determined,and the results showed that Bb01,Bb122077,Bt2913 and Bt05041 induced the stronger activity of protective enzymes under the treatment of eight strains.Among the detoxification enzymes,Bb01 and Bt2913 had higher activity.（3）The gut microbiome compared the normal diet with Bt treatment in D.abietella,it was found that the abundance of unclassified Enterobacteriaceae,Wolbachia sp.and Bacillus sp.had significantly improved after 6 h of Bt05041 treatment,but the relative abundance of Enterococcus sp.decreased from 76.94%to 32.14%.Serratia sp.was only detected 24 h after Bt treatment.（4）It was observed that the normal epithelial cells of D.abietella larvae had a large number of compactly arranged microvilli at the apical region.The median region of epithelial cells had typical nuclei,dense chromatin and prominent nucleoli,the structure of septal junction was intact between cells,and there were a large number of rough endoplasmic reticulum and mitochondria in the cytoplasm.The basal area of epithelial cells displayed a highly in-folded basement plasma membrane,which outer region was surrounded by well-structured microtrachea and annular fibers.There were obvious pathological changes in the midgut epithelial cells of D.abietella larvae after Bt05041 treatment.In the apical region of the midgut epithelial cells,the tissue was rapidly expanded from several small vacuoles to large autophagic vacuoles,the microvilli were deteriorated from scattered deformation to a large number of shedding.In the median region of the midgut epithelial cells,the nuclei were deteriorated from swelling and deformation to complete rupture,a variety of organelles in the cytoplasm were deformed to dissolution,and the structure of septate junction was also disrupted between digestive cells.At the same time,the basement plasma membrane was broken at the basal area of epithelial cells,and the epithelial cells were gradual apoptosis.（5）Based on the larval transcriptome of D.abietella,there were 1870 upregulated genes and1861 downregulated genes after 2 h of Bt05041 treatment,and the differential genes in pathways showed that Bt-feeding mainly activated the c AMP signaling pathway in D.abietella.In the differential genes,we identified 29 immunity related genes,including 2 cadherin genes,8 membrane alanyl aminopeptidase genes,7 ATP-binding cassette sub-family genes,1 glycolipid gene,2superoxide dismutase genes,2 phenol oxidase genes,and 7 glutathione S-transferase genes.The spatiotemporal expression profiles were found that Bt receptors（Dabi ABCs,Dabi APNs,Dabi Cad1）were mostly highly expressed at the early stage of Bt treatment,while the high expression of defense enzyme genes（Dabi GSTs,Dabi SODs）occurred in the middle phase of Bt treatment,and the coding sequences of Dabi APN4,Dabi APN8 and Dabi ABCG3 genes were cloned.In summary,Bt05041 has well gastric toxicity for the D.abietella larvae,destroys the structure of midgut epithelial cells,and rapidly proliferates of some bacterial communities,resulting in larval death.At the same time,the high expression of Bt receptors in the short term was analysed using RT-q PCR technology.The research results clarify the action of Bt toxic protein in the D.abietella midgut,and further improve the mechanism of Bt-triggered insect pathogenesis,provide new foundations for forest pest control.