Teliospores of Entorrhiza casparyana (Ustilaginales): a correlated thin-sectioning and freeze–fracture study of endogenously dormant spores

Abstract

A correlated thin-sectioning and freeze–fracturing approach was used to reveal the ultrastructure of endogenously dormant teliospores in the smut fungus Entorrhiza casparyana (Magn.) Lagerh. Conventional fixation and embedding methods yielded poor preservation of the wall and protoplasm. Successful preservation was achieved by fixing frozen and cryosectioned spores in glutaraldehyde and subsequently processing by standard procedures for transmission electron microscopy. Freeze–fracturing provided cross- and contour-fractured views of the protoplasm and the different layers of the wall. The wall is thick, consisting of three main layers: outer, middle, and inner, with the outer and inner layers further differentiated into zones. The warty zone dominates the outer layer and consists of radial protuberances (warts) with the regions between filled to varying degrees with similar wall material containing electron-transparent lamellae. The extent of differentiation of the warty zone is reflected in the surface morphology of the spores, which ranges from verrucose to almost smooth. At the base of the outer layer is an electron-translucent irregular zone. The middle and inner layers are regular in thickness around the spore, with the middle layer being the most electron dense. The inner layer is differentiated into three zones. The most distinctive is zone 2 which in freeze–fractured walls has an unique mosaic of striae. Cytochemical staining of the wall for polysaccharide material gives a positive reaction only for the warty zone. The protoplasm contains a single nucleus and is dominated by numerous spheroidal storage lipid bodies. Squeezed among the lipid bodies are organelles, believed to be microbodies, containing a granular matrix and often electron-transparent areas. These organelles failed to show catalase activity with the 3,3′-diaminobenzidine method. Occasional short profiles of endoplasmic reticulum cisternae, a few mitochondria with sparse cristae, dispersed small clusters of glycogen, and sometimes scattered ribosomes are also present in the cytoplasm. All these features are typical of dormant spores with a low metabolic activity.