A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA

Ascosphaera spp. fungi are associated with social and solitary bees, in some cases as pathogens causing chalkbrood disease. As a supplement to morphological identification, we developed a simple PCR-based method for selected Ascosphaera species. We exploited sequence differences in the internal transcribed spacer regions of rDNA to design species-specific primers. Analysis involves simply scoring the presence or absence of a single band for a given pair of primers. The method can distinguish the four Ascosphaera species known to be associated with honey bees. It also distinguishes Ascosphaera aggregata, the chalkbrood pathogen of the alfalfa leafcutting bee, from other Ascosphaera species associated with this bee. We expect the method will be useful for determining purity of Ascosphaera cultures, and may be a first step toward development of an early detection method of chalkbrood infection in honey bees and leafcutting bees. We also present a new, quick and reliable method for preparing fungal DNA suitable for PCR amplification from mycelia grown in liquid or on solid media.