Submitted to: Canadian Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 15, 2001
Publication Date: July 6, 2001
Citation: Lopez Lastra, C.C., Hajek, A.E., Humber, R.A. 2001. Effects of two cryopreservation techniques on viability and pathogenicity of entomophthoralean fungi. Canadian Journal of Botany. 79(7):861-864.
Interpretive Summary: There are many possible techniques for the long-term preservation of living cultures of fungi, with considerable variations in the overall cost and effectiveness of these various methods for any particular fungus. Storing cultures of some fungi pathogenic to insect pests can be a special problem because of a tendency to loose essential characters such as virulence, pathogenicity or the ability to sporulate after time in cryogenic storage. This study examined the effects of an inexpensive, passive freezing device and an expensive electronically controlled cell freezer on the viability and pathogenicity of several important fungal biocontrol agents. It was shown effectively for those fungi tested that the inexpensive and low-tech freezing device followed by storage in an electric freezer at -80C was very nearly as effective as using the electronic cell freezer and storage under liquid nitrogen at -196C. This study confirms that many labs with limited financial and technological can store cultures of a wide range of the most important fungal pathogens of insects (including even some species that are highly host specific and that may even have complex nutritional requirements) without having to depend on the more secure but also much more expensive approaches that are more routinely available in a dedicated microbial germplasm repository.
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 simple, more affordable technique to store cultures at -80C is available. We compared viability among three entomophthoraleans sand pathogenicity for one species for both storage techniques over ten months. Fluorescent staining for viability demonstrated that there was no statistically significant difference by storage treatment for all three fungi. Although cells of ENTOMOPHTHOA AULICAE decreased in viability after 8 and 10 months of storage, similar declines were not seen with E. MAIMAIGA or ZOOPHTHORA RADICANS. Bioasays of E. MAIMAIGA against gypsy moth larvae, LYMANTRIA DISPAR, demonstrated no differences in time to death or percent mortality after 10 mo of storage by either method. However, after 10 months, fewer cadavers of larvae injected with cultures stored at -80C abundantly produced conidia. Our findings suggest that for these isolates from three species of Entomophthorales, storage at -80C after a simple freezing protocol had a minor effect compared with storage at -196C but some cultures were more sensitive to prolonged freezing than others.