Probiotic Potential of Bacillus Subtilis Strain I3: Antagonistic Activity Against Chalkbrood Pathogen and Pesticide Degradation for Enhancing Honeybee Health

To explore the potential of probiotic candidates beneficial for honeybee health through the modulation of the gut microbiome, bee gut microbes were isolated from bumblebee (Bombus terrestris) and honeybee (Apis mellifera) using diverse media and cultural conditions. A total of 77 bee gut bacteria, classified under the phyla Proteobacteria, Firmicutes, and Actinobacteria, were identified. The antagonistic activity of the isolates against Ascosphaera apis, a fungal pathogen responsible for chalkbrood disease in honeybee larvae, was investigated. The highest growth inhibition percentage against A. apis was demonstrated by Bacillus subtilis strain I3 among the bacterial strains. The presence of antimicrobial peptide genes in the I3 strain was detected using PCR amplification of gene fragments encoding surfactin and fengycin utilizing specific primers. The export of antimicrobial peptides by the I3 strain into growth medium was verified using liquid chromatography coupled with mass spectroscopy. Furthermore, the strain's capabilities for degrading pesticides, used for controlling varroa mites, and its spent growth medium antioxidant activity were substantiated. The survival rate of honeybees infected with (A) apis was investigated after feeding larvae with only medium (fructose + glucose + yeast extract + royal jelly), (B) subtilis I3 strain, A. apis with medium and I3 strain + A. apis with medium. Honeybees receiving the I3 strain + A. apis exhibited a 50% reduction in mortality rate due to I3 strain supplementation under experimental conditions, compared to the control group. In silico molecular docking revealed that fengycin hydrolase from I3 strain effectively interacted with tau-fluvalinate, suggesting its potential in bee health and environmental protection. Further studies are needed to confirm the effects of the I3 strain in different populations of honey bees across several regions to account for genetic and environmental variations.