Abstract
A substrate made of two media, one for initiation of vegetative growth, the other for induction of spore formation, was developed to culture Ascosphaera aggregata, a fungal pathogen of the alfalfa leafcutting bee Megachile rotundata. Plugs from the first medium were placed in voids made in the second medium. Components of both media included low-sodium V8 juice, sucrose, yeast extract, vegetable shortening, agar, thiamine, biotin, magnesium sulfate, and Grace's insect medium without hemolymph. Both media were buffered to pH 6 with potassium phosphate buffer. The composite substrate (referred to as the plug/base medium) supported growth and development of A. aggregata from spore to spore. Koch's postulates were verified. Larvae infected with the in vitro produced spores exhibited the typical characteristics of chalkbrood disease. There was no significant difference ( P = 0.6818) in infection frequency between first instar larvae challenged with either in vitro cultured spores or inoculum from field-collected cadavers.
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