Synergistic Action of the Essential Oils of Elionorus muticus and Lippia junelliana Against the Causative Agent of Chalkbrood in Bees (Ascosphaera apis)

Ascosphaera apis is the fungus that causes chalkbrood in Apis mellifera. Traditionally, its control has relied on synthetic fungicides, which can be harmful to bees and ecosystems. An alternative treatment has explored safer alternatives, such as the antimicrobial properties of plant-based essential oils. This study evaluated the combined effect of the essential oils of Elionorus muticus and Lippia junelliana on 10 isolates of A. apis using a “chessboard” experimental design. Minimum inhibitory concentrations, fungal growth rates and inhibition percentages were determined. Analyses were performed with ANOVA and post-hoc tests. The results showed that the combination of 200 µL/L of E. muticus and 25 µL/L of L. junelliana (C5) completely inhibited fungal growth after 144 h, with MIC50 significantly more effective than controls. The combined use of E. muticus and L. junelliana shows synergistic activity (FICI ≤0.5) in controlling A. apis and achieves full inhibition of the fungus under controlled conditions. These findings suggest that essential oils may be a viable alternative for the sustainable management of bee diseases. Highlights: The fractional inhibitory concentration index (FICI) for combination C5 was 0.5, demonstrating a significant synergistic interaction between both essential oils. The combining of junelliana(25 µL/L) and E. muticus (200 µL/L) inhibited A. apis growth. Combinations C1 (12.5–400 µL/L), C2 (25–200 µL/L), C3 (50–100 µL/L), and C4 (200–25 µL/L) of the Eos showed a progressive decrease in inhibitory capacity over time, whereas combination C5 maintained a consistent inhibition above 50% throughout the study period, indicating greater temporal stability.