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

Barbara Hočevar1, Darja Kušar2, Igor Gruntar2

  • 1Institute of Pathology, Forensic Veterinary Medicine, Wildlife, Bees and Aquaculture, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.

Journal of Fungi (Basel, Switzerland)
|May 26, 2026
PubMed

Insights

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a rapid and reliable method for identifying Ascosphaera apis, the fungus causing chalkbrood disease in honeybees.

Area of Science:

  • Mycology
  • Apiculture
  • Analytical Chemistry

Background:

  • Ascosphaera apis causes chalkbrood disease, weakening honeybee colonies and increasing susceptibility to other stressors.
  • Effective management of chalkbrood disease relies on accurate and timely identification of A. apis.

Purpose of the Study:

  • 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.
  • To assess the efficacy of different protein extraction methods for A. apis identification using MALDI-TOF MS.

Main Methods:

  • Construction of a MALDI-TOF MS spectral library using reference Ascosphaera species.
  • Validation using whole-genome sequencing of 31 clinical A. apis isolates.
  • Comparison of three protein extraction methods: liquid cultivation, formic acid-ethanol extraction, and extended direct transfer.

Main Results:

  • MALDI-TOF MS proved to be a rapid and reliable tool for identifying A. apis within the tested strain set.
  • Liquid cultivation and formic acid-ethanol extraction methods showed no misidentifications.
  • Extended direct mycelium transfer method demonstrated high sensitivity (83.9%) but was slightly less effective.

Conclusions:

  • MALDI-TOF MS, particularly with liquid cultivation or formic acid-ethanol extraction, provides an effective method for A. apis identification.
  • This study lays the groundwork for improved diagnostic strategies in managing honeybee fungal diseases like chalkbrood.
  • Accurate identification of A. apis is crucial for effective disease management and maintaining honeybee colony health.
Keywords:
Ascosphaera apishoneybee fungal diseasesin-house librarymatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)species identificationwhole-genome sequencing (WGS)

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