Subterranean biocontrol: Interactions between Bacillus and beneficial nematodes modulate larval mortality and recruitment of native soil biota

Subterranean larvae infected with pathogens can serve as ecological hotspots, mediating interactions between microbial biocontrol agents and native soil fauna. However, the extent to which microbial infection influences these community-level interactions remains poorly understood. Here, we investigated how Bacillus thuringiensis subsp. tenebrionis (Btt), applied as a soil inoculant, affects larval mortality of Diaprepes and the belowground recruitment of entomopathogenic nematodes (EPNs) and free-living nematodes (FLNs). A multi-tiered experimental approach was employed, including behavioral assays with adult, toxicological evaluations at the larval stage, and efficacy evaluations under greenhouse and field conditions. Btt reduced neonate weevil survival and disrupted larval development, with limited efficacy in older larval stages. Notably, when Steinernema riobrave was applied 14 days after larval exposure to Btt, larval mortality was enhanced, and root and shoot biomass increased in the greenhouse. In field plots, Btt-treated weevil larvae attracted significantly more EPNs and FLNs than controls. Soil samples were collected for metabarcoding to assess bacterial, fungal, nematode, and arthropod community shifts. Results suggest that Btt-infected larvae may function as biological signals that modify larval cues and promote recruitment of native natural enemies. Our findings highlight how combining microbial agents may contribute to pest suppression and the engineering of soil food web responses, providing a foundation for future studies in functional soil ecology.