Localization of closteroviruses on grapevine thin sections and their identification by immunogold labelling.

Different fixation and embedding procedures have been tested in order to facilitate closterovirus identification on thin sections of leafroll-affected grapevine plants. Standard fixation with glutaraldehyde followed by osmium tetroxide proved to be the most reliable for cytopathological studies while simultaneous fixation with glutaraldehyde, picric acid and osmium tetroxide facilitated the discrimination between aggregates of P-proteins and virus particles in routine e. m. work. However the identification of every single virion among P-protein filaments was only possible by means of post-embedding immunogold labelling carried out on non-osmicated tissues embedded in London Resin White. This technique has also been applied to both section sides thus allowing the identification of different closteroviruses contemporaneously present in the same phloem tissue. Numerous ultrastructural observations of different grapevine cultivars infected with either GVA, or GLRaV-I or GLRaV-III evidentiated the presence of some recurrent and peculiar cytopathic effects that can be used as diagnostic parameters for at least two of the above viruses.