A fluorescence microscopy method for determining the viability of entomophthoralean fungal spores

Conidial viability of two species of entomophthoralean fungi was assessed using parallel vital fluorochrome staining and germination studies. Fluorescein diacetate (FDA) indicates viability when cells fluoresce yellow-green while propidium iodide (PI) indicates nonviability when cells fluoresce red. Various methods were used to induce Entomophaga maimaiga and Zoophthora radicans conidial mortality, including exposure to electromagnetic radiation, ethanol, or high temperatures. Following each treatment, half of the conidia were either stained with FDA and PI or held at ideal conditions for assessment of germination. In separate experiments, resting spores of Entomophaga grylli and E. maimaiga were treated either by autoclaving or with ethanol. Vital staining was used to assess the viability of the resting spores. FDA and PI were found to assess the viability of E. maimaiga and Z. radicans conidia accurately and precisely when compared to standard germination tests. No differences in comparative test results were found whether conidia were killed with ethanol, electromagnetic radiation, or high temperatures. When assessing resting spore viability, however, FDA and PI were not effective due to limited penetration of these stains through the thick walls of the spores and also due to the autofluorescence of E. maimaiga and E. grylli resting spores.