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

Abstract

The difficulties in long-term storage of cultures of Entomophthorales create a barrier to working with these entomopathogenic fungi. Relatively few laboratories have access to controlled cooling apparatus and storage in liquid nitrogen, but a simpler, more affordable technique to store cultures at –80°C is available. We compared viability among three entomophthoraleans and pathogenicity for one species for both storage techniques over 10 months. Fluorescent staining for viability demonstrated that there was no statistically significant difference by storage treatment for all three fungi. Although cells of Entomophaga aulicae (Reichardt in Bail) Humber decreased in viability after 8 and 10 months of storage, similar declines were not seen with Entomophaga maimaiga Humber, Shimazu & Soper or Zoophthora radicans (Brefeld) Batko. Bioassays of E. maimaiga against gypsy moth larvae, Lymantria dispar (L.), demonstrated no differences in time to death or percent mortality after 10 months of storage by either method. However, after 10 months, fewer cadavers of larvae injected with cultures stored at –80°C abundantly produced conidia. Our findings suggest that for these isolates from three species of Entomophthorales, storage at –80°C after a simple freezing protocol had a minor effect compared with storage at –196°C, but some cultures were more sensitive to prolonged freezing than others.Key words: Entomophaga aulicae, Entomophaga maimaiga, Zoophthora radicans, storage, in vitro culture, entomopathogenic fungi.