When Ophiostoma ulmi or Ophiostoma novo-ulmi are grown on either 0.22- or 0.45-μm millipore filter membranes placed on impoverished agar medium, the fungus grows through these membranes and takes on various irregular pleomorphic growth forms (P-forms). Links of continuity between these forms and the more regular ones have been shown using light, confocal, and transmission electron microscopy. Tests with labelled probes, such as gold-complexed wheat germ agglutinin for chitin and β-exoglucanase for cellulosic β-1,4-glucans, have indicated that in P-forms deposition of chitin is much altered but is less so in the case of cellulosic glucan. The cytology of these forms compared with the regular fungal ones is also very different, particularly with reference to mitochondria and nuclei. Also, numerous vesiculate structures were noted in the rarely septate P-forms. Similar irregular forms with opaque contents were produced by these fungi when they were grown on sterilized elm wood sections. When these latter samples were fixed by high-pressure freezing, the following main features were noted: fungal cells with a very thin wall, slightly labelled for chitin but more intensely for cellulosic glucans; well-preserved structures, such as plasmalemma and endoplasmic reticulum; and a slightly opaque, fibril-containing extracellular sheath. Differences in labelling for galactose, whether of wall layers or cell contents, were also observed in regular and P-forms. Electron opaque bodies that labelled strongly for galactose were also numerous in P-forms in some samples.Key words: transmission electron microscopy, high-pressure freezing, gold labelling, extracellular sheaths, wall constituents.
Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy