| Apriona swainsoni is a highly destructive wood-boring pest that poses a significant threat to forestry in China.In recent years,the utilization of biological control methods has become increasingly important for managing A.swainsoni.Beauveria bassiana,a fungal entomopathogen,has emerged as a prominent bioinsecticide among these methods.Previous studies have indicated the involvement of insect symbiotic bacteria in the defense mechanisms exhibited by hosts against pathogenic fungi.However,the precise role played by symbiotic bacteria in the infection process of A.swainsoni by B.bassiana remains poorly understood.This study focused on the larvae of A.swainsoni and their symbiotic bacteria to explore whether symbiotic bacteria are involved in host defense against pathogenic fungi and to provide insights into the theoretical basis of"controlling pests with microbes"in biological pest control.Firstly,we assessed the pathogenicity of B.bassiana to A.swainsoni larvae.Subsequently,using microbial 16S rDNA sequencing technology,we elucidated the effects of B.bassiana infection on the abundance,composition,and structure of symbiotic bacterial communities in A.swainsoni larvae.In addition,through resistance tests,we determined the antagonistic effects of symbiotic bacteria strains of A.swainsoni on B.bassiana.Finally,we validated the role of symbiotic bacteria in defense against entomopathogenic fungi by reintroducing symbiotic bacteria to sterile A.swainsoni larvae and assessing their resistance to B.bassiana infection.The specific research findings were summarized as follows:（1）The entomopathogenic fungus B.bassiana exhibited strong pathogenicity to the larvae of A.swainsoni;its pathogenicity was positively correlated with the concentration.The mortality rates of A.swainsoni larvae exposed to different concentrations（1×106,1×107,1×108 spores/m L）of B.bassiana strain Bb3275 were 70.00%,76.67%,and 93.33%,respectively.The median lethal times(LT50)were 16.91 d,15.68 d,and 11.72 d,respectively.Overall,B.bassiana exhibited high toxicity against A.swainsoni larvae,and its pathogenicity increased with higher fungal concentrations.（2）The gut and cuticle of A.swainsoni larvae harbored rich bacterial communities,and B.bassiana had different effects on the gut and cuticle symbiotic bacteria of A.swainsoniThe 16S rDNA sequencing data revealed that there were 156 identified genera in the gut samples,with the dominant genus being Unclassified＿f＿Enterobacteriaceae.In the cuticle samples,there were 187 identified genera,with Corynebacterium being the dominant genus.The results obtained from the Alpha diversity analysis and Principal Coordinates Analysis（PCo A）indicated that there were no significant differences in the diversity and richness of bacterial flora in the gut and cuticle samples before and after infection.Moreover,the structure of the gut samples did not exhibit any significant differences,whereas the structure of symbiotic bacteria on the cuticle showed a significant change after infection.（3）The symbiotic bacteria of A.swainsoni larvae exerted a significant influence on the pathogenicity of B.bassiana.Antibiotics were employed to eliminate the symbiotic bacteria from the A.swainsoni larvae.Subsequently,the larvae were infected with B.bassiana strain Bb3275 at a concentration of 1×108 spores/m L.The results demonstrated that the LT50 of larvae with intact symbiotic bacteria was 11.17d,whereas the LT50 of sterilized larvae was reduced to8.75 d.These findings clearly indicated that symbiotic bacteria significantly influenced the pathogenicity of B.bassiana when infecting A.swainsoni larvae.（4）To Verify the antagonistic effect of symbiotic bacteria of A.swainsoni on B.bassianaWe isolated and purified several culturable gut and cuticle bacteria from A.swainsoni.In vitro confrontation,experiments were conducted using these purified bacteria and gut and cuticle homogenates against B.bassiana.The results revealed that Sphingobacterium cladoniae No.6,Enterococcus casseliflavus TSA-3,Raoultella terrigena 2020GS223,gut homogenate,and cuticle homogenate all exhibited antagonistic interactions with B.bassiana.Notably,Raoultella terrigena 2020GS223 demonstrated the strongest ability to inhibit the growth of the fungus.（5）Reintroduction of the symbiotic bacterial community significantly enhanced the antagonistic ability of A.swainsoni against B.bassianaSterilized A.swainsoni larvae were inoculated with the symbiotic bacterial community.Subsequently,the larvae were reinfected with B.bassiana strain Bb3275.The results showed that the presence of the cuticle and gut commensal bacteria delayed the time of death in the larvae,with an LT50 of 11.28 d and 11.64 d,respectively.In comparison,sterilized larvae had an LT50 of 8.97 d.This finding indicated a significant decrease in the lethal effect of B.bassiana on A.swainsoni larvae inoculated with symbiotic bacteria.Thus,the results clearly highlight the impact of the symbiotic bacteria of A.swainsoni on the pathogenicity of B.bassiana towards A.swainsoni. |
