An ultrastructural study of Olpidium brassicae and its transmission of tobacco necrosis virus

This thesis concerns transmission of tobacco necrosis virus (TNV) by zoospores of Olpidiumbrassicae. Electron microscopic observations were made on: a. the fungus, the virus, and the outer layers of seedling roots of two host species (part I); b. ultrastructural aspects of the mode of virus transmission and specificity of transmission (part II). Summarized results of part I are:1. Uniflagellate zoospores of O.brassicae have a body plasmalemma that is continuous with the axonemal sheath (section 3.1).2. Upon encystment of the zoospore, the axoneme is withdrawn inside the body. This probably happens by a 'reeling-in' mode of withdrawal and later a structure that could be the axonemal sheath is present inside the cyst body (section 3.2).3. After encystment the cyst cytoplasm penetrates into the host cytoplasm through a hole in the host cell wall and through the papillum that develops between the host wall and the host plasmalemma at the attachment site. During the penetration the cyst plasmalemma and tonoplast remain behind in the empty cyst wall, but the young thallus is immediately surrounded by a new outer membrane (section 3.2).4. Up to 24h after penetration the young thallus is separated from the host cytoplasm by the thallus membrane only. Older thalli develop a wall (section 3.3).5. The epidermal cells in the zone of elongation of lettuce seedling roots rapidly change with increasing age. The relative amount of cytoplasm decreases and in the region between 5 and 15mm from the root tip very little cytoplasm remains and many cells seem to have died (section 4. 1).6. Epidermal cells of tomato roots undergo deterioration with increasing age, but at a slightly slower pace than lettuce. These cells are often covered with calyptra cells in the zone of elongation (section 4.2).7. Particles in sections of a TNV pellet have an apparent size of approximately 20nm after conventional double staining. For this reason TNV particles may be difficult to identify if present in cells that also contain ribosomes (chapter 5).Results published in part II of this thesis are also based partially on data obtained in part I. Summarized results of part II are:1. Zoospores of the lettuce isolate of O.brassicae, that transmit TNV after exposure to the virus, acquire TNV in vitro by a tight adsorption of the virus particles to their body plasmalemma and the axonemal sheath (section 7.1).2. Part of the adsorbed virus is taken into the encysting zoospore cytoplasm and this virus is eventually transmitted. Uptake of the virus probably takes place when the axoneme with its sheath is withdrawn. The fate of TNV not taken into the cyst by axonemal withdrawal is discussed and considered unimportant for transmission (section 7.2).3. TNV taken into the infecting zoospores is transmitted into the cytoplasm of the root epidermal cell by release from the young thallus. A possible mechanism for this virus release is proposed (section 7.3).4. Adsorption of TNV to zoospores of the lettuce isolate of O.brassicae is very specific in the sense that particles of other polyhedral viruses like cucumber necrosis virus (CNV), tomato bushy stunt virus (TBSV), and turnip yellow mosaic virus (TYMV) are not adsorbed under similar circumstances (section 8.1.1).5. Adsorption of TNV is also very specific in the sense that zoospores of the lettuce and tomato Olpidium adsorb many TNV particles, those of the oat Olpidium adsorb many fewer TNV particles, and those of the mustard Olpidium apparently do not adsorb any TNV under similar experimental conditions (section 8.1.1).6. A form of specificity of adsorption comparable to that between TNV and the lettuce Olpidium was encountered in the case of zoospores of O.cucurbitacearum. Zoospores of this species adsorb many particles of CNV, but they do not adsorb TNV when mixed in vitro with these viruses (section 8.1.2).7. Zoospores of lettuce Olpidium adsorb particles of satellite virus (SV) when mixed with a suspension of SV alone or in combination with either of two different isolates of TNV (NZ-TNV and AC-36) (section 8.1.2).8. Incompatibility in the fungus - host relationship is sometimes due to the absence or low incidence of encystment of a particular fungus isolate on that host plant, or to a break-down of the developing fungal thallus after a normal appearing infection process. The first form of incompatibility certainly influences transmission of TNV (section 8.2).9. Tomato roots seem to be good hosts for TNV inasmuch as they allow the build- up of a high TNV titer after infection and do not react necrotically but lettuce roots react with necrosis 18 h after TNV infection. Also, lettuce roots seem to be less favorable hosts for TNV in the sense that many virus particles have to be present at a given infection site in order for the infection to take place (sections 8.3 and 8.4).10. In epidermal cells of lettuce roots infected by TNV, thalli of O. brassicae do not mature. No virus was detected in lettuce epidermal cells that were not infected by Olpidium nor in the cortical cells. Nevertheless these cells often were necrotic. TNV-infected epidermal cells of tomato roots often contain growing thalli of O.brassicae but virus was not found in the thalli. These host cells also sometimes have granular or crystalline material between the cisternae of the endoplasmic reticulum. Virus could be detected in epidermal and cortical cells of tomato that had not been infected by Olpidium (section 8.3).11. It is possible to explain some forms of specificity of TNV transmission just by differences in fungus - virus relationship. Other examples of specificity of TNV transmission, however, can only be explained by taking into account simultaneously the combined effect of differences in fungus - virus, host fungus, and virus - host relationship (section 8.4).In conclusion, transmission of TNV by O.brassicae is explained by a sequence of events, all of which were shown to take place during exposure of zoospores to TNV and subsequent exposure of host roots to those zoospores. Furthermore, all known forms of specificity of TNV-transmission by different isolates of O.brassicae can be understood on the basis of specific fungus - virus, host fungus, and virus - host relationships.