Geographic and seasonal variation of culturable bacteria associated with the diseased silkworm (Bombyx mori)

The domesticated silkworm, <i>Bombyx mori</i>, is critically impacted by bacterial pathogens, yet the environmental and ecological drivers of their spatiotemporal dynamics remain poorly defined. In this study, 514 bacterial strains were isolated from diseased and healthy silkworm larvae across major sericultural regions in China. Through 16S rRNA gene sequencing and multi-tier pathogenicity assays, 51 isolates were identified as potential novel insect pathogens. Fulfilling Koch's postulates via oral infection tests, the pathogenicity of nine strains belonging to the genera <i>Raoultella, Stenotrophomonas</i>, and <i>Citrobacter</i> were confirmed, while the remaining isolates are considered putative pathogens. All isolates were classified into 33 genera within the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, with <i>Enterobacter, Bacillus</i>, and <i>Serratia</i> being the most prevalent. Multivariate analysis indicated that geographic and climatic factors-specifically distance from the coastline and key thermal and radiative variables-collectively explained a significant though modest portion of the variance in bacterial community composition. Bacterial diversity correlated positively with cocoon yield. Guangxi presented the highest potential pathogen diversity and co-infection frequency, aligning with its intensive sericulture practices. Seasonal analyses indicated higher bacterial abundance and virulence in spring-summer compared to summer-autumn. Many isolates are taxonomically affiliated with genera containing insect gut microbiota, plant-associated bacteria, and human (opportunistic) pathogens, suggesting diverse environmental and anthropogenic origin. The detection of bacteria related to mulberry phyllosphere microbes underscore the role of host plant ecology in shaping the silkworm microbiome. These findings illuminate the ecological drivers of silkworm-associated bacterial communities and highlight the complex microbial connections within sericulture ecosystems, suggesting potential pathways relevant to a One Health perspective. Furthermore, the repository of 514 identified bacterial isolates from the model lepidopteran <i>B. mori</i> here provides a valuable resources for exploring novel biocontrol agents against other lepidopteran pests.