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

Author
item MURRAY, K - TEXAS A&M EXP. STATION
item Aronstein, Katherine
item Jones, Walker

Submitted to: Journal of Apicultural Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2005
Publication Date: 7/28/2005
Citation: Murray, K.D., Aronstein, K.A., Jones, W.A. 2005. A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA. Journal of Apicultural Research. 44(2):61-64.

Interpretive Summary: Several species of fungi in the genus Ascosphaera are found in nature associated with the honeybee and the alfalfa leafcutting bee. Some of those fungal species cause chalkbrood disease in those bees. Owing to the difficulty of identifying the various fungi by visual inspection, we have developed a method which identifies many of these species using a rapid laboratory technique which relies on differences in the DNA of the fungi. Our method is able to distinguish the four Ascosphaera fungal species associated with honeybee colonies. Preliminary results suggest that our method will also be able to distinguish the fungi causing chalkbrood in leafcutting bees from all the other related fungal species found in those bees. In the laboratory, this method will be useful for ensuring purity of fungal cultures being studied. Although not yet tested on honey, pollen, or other samples from apiaries or managed leafcutting bee populations, we envision this method could be easily adaptable for such samples. This could make the method useful for early detection of chalkbrood in honeybees or leafcutting bees. Our work also describes a new quick, safe, and reliable method for preparation of DNA from fungi.

Technical Abstract: Fungi of the genus Ascosphaera are associated with social or solitary bees in nature, in some cases as pathogens causing chalkbrood disease. Both the honeybee (Apis mellifera) and the alfalfa leafcutting bee (Megachile rotundata) encounter several different Ascosphaera species. Morphological means of identification of fungi have some serious drawbacks. Therefore, in this study, we describe a simple PCR-based method for identification of selected Ascosphaera species. We show that our method is able to distinguish the four Ascosphaera species associated with honeybee colonies. In addition, we present evidence supporting the likelihood that this method will also be able to distinguish Ascosphaera aggregata, the chalkbrood pathogen of the leafcutting bee, from all other Ascosphaera species associated with M. rotundata. The method exploits sequence differences in the internal transcribed spacer regions of rDNA in order to distinguish the species. We foresee the method as useful for determining purity of Ascosphaera cultures, as a first step toward an early detection method of chalkbrood infection in honeybee colonies, and expect it will be adaptable to various field collected biological samples from honeybees and leafcutting bees. The study also describes a new quick, safe, and reliable method for preparation of fungal DNA which is suitable for PCR amplification.