Effects of two cryopreservation techniques on viability and pathogenicity of entomophthoralean fungi

The difficulties in long-term storage of cultures of Entomophthorales creat e a barrier to working with these entomopathogenic fungi. Relatively few la boratories have access to controlled cooling apparatus and storage in liqui d nitrogen, but a simpler, more affordable technique to store cultures at - 80 degreesC is available. We compared viability among three entomophthorale ans and pathogenicity for one species for both storage techniques over 10 m onths. Fluorescent staining for viability demonstrated that there was no st atistically significant difference by storage treatment for all three fungi . Although cells of Entomophaga aulicae (Reichardt in Bail) Humber decrease d in viability after 8 and 10 months of storage, similar declines were not seen with Entomophaga maimaiga Humber, Shimazu & Soper or Zoophthora radica ns (Brefeld) Batko. Bioassays of E. maimaiga against gypsy moth larvae, Lym antria dispar (L.), demonstrated no differences in time to death or percent mortality after 10 months of storage by either method. However, after 10 m onths, fewer cadavers of larvae injected with cultures stored at -80 degree sC abundantly produced conidia. Our findings suggest that for these isolate s from three species of Entomophthorales, storage at -80 degreesC after a s imple freezing protocol had a minor effect compared with storage at -196 de greesC, but some cultures were more sensitive to prolonged freezing than ot hers.