Transmission electron microscopy of plunge-frozen and freeze-substituted samples was used to examine germinating conidia of Alternaria cassiae, a plant pathogenic fungus used as a biological control agent for sicklepod (Cassia obtusifolia). Hydrated conidia on small pieces of dialysis membrane were incubated for 1, 2, or 3 h on the surface of corn meal agar prior to fixation. Conidia were large, darkly pigmented, and surrounded by a thick, two-layered wall. Each conidium was divided by transverse and longitudinal septa into multiple cells, a few of which sometimes appeared necrotic. Each septum tapered to a small central pore region with which Woronin bodies were associated. Each healthy cell of a conidium contained a typical complement of cellular organelles including multiple nuclei. With the exception of lipid bodies, all the various organelles were well preserved by plunge freezing and freeze substitution. Evidence of germ tube development was visible by 2 h post-incubation and well-developed germ tubes were present by 3 h. Two modes of germ tube development were observed. In the less common mode germ tubes developed inside conidia and grew internally through one or more adjacent cells before emerging from the conidium surface. Cells penetrated by internal germ tubes appeared necrotic. In the more common mode of germination, germ tubes developed directly from the conidium surface. Multiple germ tubes usually arose from each conidium and grew out in all directions. Germ tubes that contacted the underlying dialysis membrane continued to grow along its surface. Extracellular material was produced in association with developing germ tubes and coated the sides of germinated conidia and covered germ tubes growing along membranes. Key words: transmission electron microscopy, cryofixation, freeze substitution, germ tube development.
Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures