{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:14:36Z","timestamp":1785428076515,"version":"3.56.0"},"reference-count":30,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004329","name":"The Slovenian Research and Innovation Agency","doi-asserted-by":"crossref","award":["56947"],"award-info":[{"award-number":["56947"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"crossref"}]},{"award":["56947"],"award-info":[{"award-number":["56947"]}],"id":[{"id":"https:\/\/ror.org\/059bp8k51","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"\u2018Animal Health, Environment and Food Safety\u2019","doi-asserted-by":"publisher","award":["P4-0092"],"award-info":[{"award-number":["P4-0092"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"\u2018Molecular Biology of Microorganisms\u2019","doi-asserted-by":"publisher","award":["P1-0198"],"award-info":[{"award-number":["P1-0198"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>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\u2013ethanol 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\u2013ethanol 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.<\/jats:p>","DOI":"10.3390\/jof12050311","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T17:04:59Z","timestamp":1776963899000},"page":"311","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid MALDI-TOF Mass Spectrometry Identification of the Chalkbrood Pathogen Ascosphaera apis"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7816-2456","authenticated-orcid":false,"given":"Barbara","family":"Ho\u010devar","sequence":"first","affiliation":[{"name":"Institute of Pathology, Forensic Veterinary Medicine, Wildlife, Bees and Aquaculture, Veterinary Faculty, University of Ljubljana, Gerbi\u010deva 60, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8583-5901","authenticated-orcid":false,"given":"Darja","family":"Ku\u0161ar","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbi\u010deva 60, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4862-2090","authenticated-orcid":false,"given":"Igor","family":"Gruntar","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbi\u010deva 60, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0149-3674","authenticated-orcid":false,"given":"Cene","family":"Gostin\u010dar","sequence":"additional","affiliation":[{"name":"Chair of Molecular Genetics and Biology of Microorganisms, Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5419-2779","authenticated-orcid":false,"given":"Irena","family":"Zdovc","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbi\u010deva 60, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wynns, A.A., Jensen, A.B., and Eilenberg, J. (2013). Ascosphaera callicarpa, a new species of bee-loving fungus, with a key to the genus for Europe. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0073419"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1080\/00275514.1955.12024448","article-title":"A reclassification of the genus Pericystis Betts","volume":"47","author":"Spiltoir","year":"1955","journal-title":"Mycologia"},{"key":"ref_3","first-page":"703","article-title":"Assessment of losses in honey yield due to chalkbrood disease, with reference to the determination of its economic injury levels in Egypt","volume":"70","author":"Zaghloul","year":"2005","journal-title":"Commun. Agric. Appl. Biol. Sci."},{"key":"ref_4","first-page":"1","article-title":"Die Krankheit der Bienenbrut, hervorgerufen durch Pericystis apis","volume":"11","author":"Maassen","year":"1913","journal-title":"Arb. Kaiserl. Biol. Anst. Land-Forstwirtsch."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3896\/IBRA.1.52.1.13","article-title":"Standard methods for fungal brood disease research","volume":"52","author":"Jensen","year":"2013","journal-title":"J. Apic. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1002\/j.1537-2197.1955.tb11154.x","article-title":"Life Cycle of Ascosphaera apis (Pericystis apis)","volume":"42","author":"Spiltoir","year":"1955","journal-title":"Am. J. Bot."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2541","DOI":"10.1139\/b88-346","article-title":"Contribution toward a monograph of the genus Ascosphaera","volume":"66","author":"Bissett","year":"1988","journal-title":"Can. J. Bot."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.jip.2005.08.004","article-title":"PCR diagnostic methods for Ascosphaera infections in bees","volume":"90","author":"James","year":"2005","journal-title":"J. Invertebr. Pathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"826","DOI":"10.1093\/mmy\/myu064","article-title":"Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: Clinical evaluation of an extended reference spectra library","volume":"52","author":"Becker","year":"2014","journal-title":"Med. Mycol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1016\/j.jfda.2019.01.001","article-title":"Current status of MALDI-TOF mass spectrometry in clinical microbiology","volume":"27","author":"Hou","year":"2019","journal-title":"J. Food Drug Anal."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"G\u00f3mez-Vel\u00e1squez, J.C., Loaiza-D\u00edaz, N., Hern\u00e1ndez, G.N., Lima, N., and Mesa-Arango, A.C. (2020). Development and validation of an in-house library for filamentous fungi identification by MALDI-TOF MS in a Clinical Laboratory in Medellin (Colombia). Microorganisms, 8.","DOI":"10.3390\/microorganisms8091362"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1002\/(SICI)1097-0231(20000315)14:5<307::AID-RCM823>3.0.CO;2-3","article-title":"Characterization of fungal spores by laser desorption\/ionization time-of-flight mass spectrometry","volume":"14","author":"Welham","year":"2000","journal-title":"Rapid Commun. Mass Spectrom."},{"key":"ref_13","unstructured":"Bruker Daltonics (2019). Standard Operating Procedure: Cultivation and Sample Preparation for Filamentous Fungi, Bruker Daltonics. Revision E."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1007\/s10096-023-04652-3","article-title":"A comparative study for optimization of MALDI-TOF MS identification of filamentous fungi","volume":"42","author":"Jeraldine","year":"2023","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e107","DOI":"10.1002\/imt2.107","article-title":"Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp","volume":"2","author":"Chen","year":"2023","journal-title":"iMeta"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e102","DOI":"10.1002\/cpbi.102","article-title":"Using SPAdes de novo assembler","volume":"70","author":"Prjibelski","year":"2020","journal-title":"Curr. Protoc. Bioinform."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4647","DOI":"10.1093\/molbev\/msab199","article-title":"BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes","volume":"38","author":"Manni","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"W601","DOI":"10.1093\/nar\/gkad406","article-title":"WebQUAST: Online evaluation of genome assemblies","volume":"51","author":"Mikheenko","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e1660","DOI":"10.7717\/peerj.1660","article-title":"AMAS: A fast tool for alignment manipulation and computing of summary statistics","volume":"4","author":"Borowiec","year":"2016","journal-title":"PeerJ"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1530","DOI":"10.1093\/molbev\/msaa015","article-title":"IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era","volume":"37","author":"Minh","year":"2020","journal-title":"Mol. Biol. Evol."},{"key":"ref_21","unstructured":"R Core Team (2023). R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing. Available online: https:\/\/www.R-project.org\/."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Cassagne, C., Ranque, S., Normand, A.-C., Fourquet, P., Thiebault, S., Planard, C., Hendrickx, M., and Piarroux, R. (2011). Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0028425"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Normand, A.-C., Cassagne, C., Ranque, S., L\u2019Ollivier, C., Fourquet, P., Roesems, S., Hendrickx, M., and Piarroux, R. (2013). Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi. BMC Microbiol., 13.","DOI":"10.1186\/1471-2180-13-76"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1631\/jzus.B2100718","article-title":"Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis","volume":"23","author":"Li","year":"2022","journal-title":"J. Zhejiang Univ. Sci. B"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.ab.2010.11.025","article-title":"Fungal pigments inhibit the matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi","volume":"411","author":"Buskirk","year":"2011","journal-title":"Anal. Biochem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1080\/15572536.2007.11832549","article-title":"High mobility group (HMG-box) genes in the honeybee fungal pathogen Ascosphaera apis","volume":"99","author":"Aronstein","year":"2007","journal-title":"Mycologia"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1128\/JCM.02852-12","article-title":"Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionization time-of-flight mass spectrometry","volume":"51","author":"Lau","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","first-page":"621","article-title":"Pseudallescheria\/Scedosporium complex species identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry","volume":"49","author":"Coulibaly","year":"2011","journal-title":"Med. Mycol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"bpz003","DOI":"10.1093\/biomethods\/bpz003","article-title":"A method for filamentous fungal growth and sample preparation aimed at more consistent MALDI-TOF MS spectra despite variations in growth rates and\/or incubation times","volume":"4","author":"Reeve","year":"2019","journal-title":"Biol. Methods Protoc."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Honsig, C., Selitsch, B., Hollenstein, M., Vossen, M.G., Spettel, K., and Willinger, B. (2022). Identification of filamentous fungi by MALDI-TOF mass spectrometry: Evaluation of three different sample preparation methods and validation of an in-house species cutoff. J. Fungi, 8.","DOI":"10.3390\/jof8040383"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/5\/311\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T04:19:28Z","timestamp":1778559568000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/5\/311"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,23]]},"references-count":30,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["jof12050311"],"URL":"https:\/\/doi.org\/10.3390\/jof12050311","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,23]]}}}