Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study.

Abstract

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.