MORPHOLOGICAL AND MOLECULAR CONFIRMATION OF THE CHALKBROOD (Ascosphaera apis) INFECTION IN HONEY BEE COLONIES Authors: Ghazala Muzafar, Muhammad Asif Aziz, Javaid Iqbal, Mazhar Qayyum, Shah Alam, Farva Siddique, Junaid Ali Siddiqui Journal: Journal of Animal and Plant Sciences (JAPS) ISSN: 1018-7081 (Print), 2309-8694 (Online) Volume: 36 Issue: 6 Year: 2026 DOI: https://doi.org/10.36899/JAPS.2026.6.0134 URL: https://doi.org/https://doi.org/10.36899/JAPS.2026.6.0134 Publisher: Pakistan Agricultural Scientists Forum Abstract:

Infection by the fungal pathogen Ascosphaera apis causes chalkbrood, a major larval disease that threatens colony health and productivity of honey bee (Apis mellifera L.) globally. This study aimed to determine the prevalence and molecular identity of the pathogen and to access its phylogenetic relationship with internationally reported isolates. In this study, A. apis was identified using morphological and molecular approaches, and the prevalence of chalkbrood infection was assessed in 140 bee colonies. Analyses of chalkbrood mummies revealed that 40 colonies (28.57%) were infected with Ascosphaera spores. Pure isolates cultured on Potato Dextrose Agar (PDA), and PCR-based amplification using ITS gene-specific primers produced a specific 485 bp band, confirming A. apis in the samples. Sequence analysis confirmed the pathogen’s identity, and the sequences of A. apis (PQ199405 and PQ282636) were deposited in GenBank. BLAST and phylogenetic analyses of the ITS region sequences showed close similarity (99.12%) with A. apis isolates reported worldwide. This study provides the first molecular confirmation of A. apis in Pakistan and establishes its phylogenetic relationship with global chalkbrood isolates. A baseline dataset generated in this study is essential for future epidemiological studies and supports the development of effective management strategies against chalkbrood disease in regional apiculture.

Keywords: Chalkbrood disease, disease prevalence, honey bee, microscopic analyses, phylogenetic analysis