Use of RAPD analysis for in situ identification of Ascosphaera aggregata and Ascosphaera larvis in larval cadavers of the alfalfa leafcutting bee, Megachile rotundata.

Chalkbrood of the alfalfa leafcutting bee, Megachile rotundata, is caused by the fungus Ascosphaera aggregata. We used random amplified polymorphic DNA (RAPD) analysis for the in situ identification of A. aggregata and a related species, Ascosphaera larvis, in larval cadavers of M. rotundata. A simple DNA extraction method was developed to preferentially isolate DNA from fungal spores on the cadaver surface, or from ascocysts beneath the cuticle. Similar banding patterns were obtained in A. aggregata-infected larval cadavers from different sources and geographic areas. The RAPD banding pattern of cadavers infected with A. aggregata differed from that of healthy leafcutting bee prepupae. RAPD analyses of cadavers infected with A. aggregata and A. larvis resulted in similar banding profiles as those obtained from corresponding pure fungal cultures of the two species. This suggests that the RAPD bands of infected cadavers were amplified from fungal DNA, rather than from other DNA associated with the leafcutting bee cadaver. The banding patterns of "sporulating" and "non-sporulating" chalkbrood cadavers exhibited no differences; this provides the first definitive evidence that both forms of the disease result from infection with A. aggregata.