17 Ghazala Muzafar Muhammad Asif Aziz Javaid Iqbal Mazhar Qayyum Shah Alam Farva Siddique Junaid Ali Siddiqui Journal of Animal and Plant Sciences JAPS 20262026 36 6 1018-7081 https://doi.org/10.36899/JAPS.2026.6.0134 <p style="text-align: justify;">Infection by the fungal pathogen&nbsp;<em>Ascosphaera apis</em>&nbsp;causes chalkbrood, a major larval disease that threatens colony health and productivity of honey bee (<em>Apis mellifera&nbsp;</em>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,&nbsp;<em>A. apis</em>&nbsp;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&nbsp;<em>Ascosphaera</em>&nbsp;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&nbsp;<em>A. apis</em>&nbsp;in the samples. Sequence analysis confirmed the pathogen&rsquo;s identity, and the sequences of&nbsp;<em>A. apis</em>&nbsp;(PQ199405 and PQ282636)<em>&nbsp;</em>were deposited in GenBank. BLAST and phylogenetic analyses of the ITS region sequences showed close similarity (99.12%) with&nbsp;<em>A. apis</em>&nbsp;isolates reported worldwide. This study provides the first molecular confirmation of&nbsp;<em>A. apis&nbsp;</em>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.</p> Chalkbrood disease, disease prevalence, honey bee, microscopic analyses, phylogenetic analysis Pakistan Agricultural Scientists Forum https://thejaps.org.pk/AbstractView.aspx?mid=2025-JAPS-1178