Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica)

Oospores of Peronospora manshurica, the causal agent of soybean downy mildew, were stained by a variety of techniques. TTC (tetrazolium chloride) and NBT (nitroblue tetrazolium chloride) primarily stained oospores which were cytologically abnormal and appeared degenerating. Cytological normal oospores were not stained by these compounds presumably because the dyes were excluded from the oospore cytoplasm by the oospore wall or the plasmalemma. Strong autofluorescence of dead/degenerating oospores in the FDA test (fluorescein diacetate) made scoring of the oospore viability by this technique unreliable. Phloxine B was found in a consistent way to stain the degenerating oospores and a small proportion of the oospores which by light microscopic, observations could not be scored cytologically abnormal. Control experiments with live and dead cells of yeast (Saccharomyces cerevisiae) confirm that phloxine B is excluded from live cells and dead cells become stained. The presumed mode of action is that the semipermeability of the plasma membrane of live cells excludes the stain. The phloxine B test described here appears a promising technique for the determination of oospore viability of P. manshurica.