Ultrastructure and cytochemistry of extramural substances associated with intercellular hyphae of several rust fungi

Abstract

Several types of extramural substance(s) associated with rust fungal intercellular hyphae were identified using a variety of tissue processing techniques. With conventional glutaraldehyde–OsO4 fixing and uranyl acetate – lead citrate staining, little material could be discerned on the hyphal surfaces in nonsporulating areas except at locations of cell–cell contact, where a lightly staining fibrous or darker staining amorphous material was apparent. Freeze-substitution or freeze-fracturing preserved greater amounts of coating material, which could be distinguished from the outer fungal wall layers. In freeze-substituted samples the extramural material was amorphous in nonsporulating areas, whereas near sporulating zones it had a fibrous consistency, with the fibrils oriented perpendicularly to the fungal wall. At locations of cell–cell contact there was additional extramural material that was composed of randomly oriented fibrils or was amorphous and densely staining. All types of extramural material stained positively with the periodate – thiocarbahydrazide – silver proteinate or periodate – chromate–phosphotungstate stains, but Concanavalin A bound only to some of the dense amorphous material. Sodium ethoxide etching and platinum–carbon shadowing also revealed the extramural material. When rust-infected wheat leaves were flooded with suspensions of colloidal gold, adhesion of gold particles occurred outside the hyphal walls and coincided with the location of extramural material.