Regeneration of protoplasts of Entomophthora egressa, a fungal pathogen of the eastern hemlock looper

Abstract

The regeneration patterns for protoplasts of Entomophthora egressa MacLeod and Tyrrell grown on modified Grace's insect tissue culture medium supplemented with fetal calf serum (a highly modified version of Grace's medium lacking serum and more closely approximating the hemolymph of the eastern hemlock looper), Müller-Kögler's coagulated egg yolk medium supplemented with Tristearin, and water agar are documented. Protoplasts on liquid and egg yolk media had varying degrees of common developmental patterns. On liquid media, development involving prophyphal spheres and fusion spheres predominated. Hyphae emerged from the prohyphal spheres; however, the fusion spheres did not develop further. On the coagulated egg yolk medium, the individual, non-chain, cells coalesced and developed into pleomorphs which later developed into osmotic shock-resistant postprotoplasts. A postprotoplast gave rise to a hypha which produced a terminal conidium. Later resting spores were detected. Water agar supported scant mycelial growth as compared with that on coagulated egg yolk but more than that on the liquid media. Solid substrates appeared to favor mycelial development. Regeneration could be avoided by the addition of fresh Grace's modified medium to either coagulated egg yolk plates or to cultures with Grace's modified medium. Obtaining protoplasts from hyphae and the reversion of these protoplasts to form hyphae appeared to be a fixed property of E. egressa.This report is believed to be the first for reversion of protoplasts initially produced by non-enzymic means.