An enzyme system to liberate spore and mycelial protoplasts from a dimorphic fungal plant pathogen Ophiostoma ulmi (Buism.) Nannf.

Abstract

Viable protoplasts were successfully liberated from both the spore (yeast) and mycelial growth phases of a dimorphic fungal plant pathogen Ophiostoma ulmi (Buism.) Nannf. A high yield of spore protoplasts was obtained through treatment of spores with the lysing enzyme driselase prepared in 0·6 m mannitol, 0·1 m sodium citrate, 0·01 m EDTA, pH 58 and a high yield of mycelial protoplasts was obtained through treatment of mycelia with driselase prepared in 0·6 m mannitol, pH 58. When the enzyme treatments were interchanged the protoplast yield for each fungal growth phase was reduced approximately 50%. All protoplasts fluoresced in the presence of fluorescein diacetate when observed by fluorescence microscopy indicating that the protoplasts were viable. Approximately 50% of the spore and mycelial protoplast yield regenerated into actively growing fungal colonies when incubated on agar medium. The enzyme system developed provides a means whereby viable protoplasts from both the spore (yeast) and mycelial growth phases of O. ulmi can be obtained for studies focused on the elucidation of mechanism (s) that regulate dimorphism in O. ulmi.