Rapid MALDI-TOF Mass Spectrometry Identification of the Chalkbrood Pathogen Ascosphaera apis

Ascosphaera apis is a fungal pathogen of honeybee larvae and the primary cause of chalkbrood disease, which weakens bee colonies, impairing their ability to function effectively and making them more susceptible to other pathogens and environmental stressors. This study aimed to develop and validate an in-house matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral library for A. apis. A new MALDI-TOF MS library was constructed using reference Ascosphaera species and validated through whole-genome-based confirmation of 31 clinical isolates of A. apis. Three different protein extraction methods were tested and compared: liquid cultivation, formic acid–ethanol extraction and extended direct transfer. Our findings demonstrate that MALDI-TOF MS is a rapid and reliable tool for identifying A. apis under the tested laboratory conditions and within the analyzed strain set, with no misidentifications observed for the liquid cultivation and formic acid–ethanol extraction methods. The extended direct mycelium transfer method was slightly less effective but still showed a high sensitivity of 83.9%. This study provides a foundation for improving diagnostic approaches in the management of honeybee fungal diseases.