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.

In situ PCR detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease

“…Because phytoplasmas are pleomorphic, possess few distinctive features and are present in host plant tissues often in low titer, it can be difficult to detect them by TEM (Thomas, 1979); or to differentiate them from other membrane‐bound bodies that occur in sieve elements of healthy plants or plants affected by virus diseases (McCoy, 1979). In such situations, positive identifications have been facilitated by combining microscopy with immunolabeling of phytoplasmas (Jiang et al ., 1989; Lherminier et al ., 1990). Because of difficulties in acquiring suitably purified phytoplasmas from palm tissues in sufficient quantity for use as antigen, antibodies have not been raised against the LY agent thus precluding immunocytochemistry to complement our TEM analysis of coconut embryo tissues.…”

Phytoplasmas: bacteria that manipulate plants and insects

“…In insects, phytoplasmas invade the guts and salivary glands and many other tissues where they can accumulate at great numbers inside and outside cells (Ammar and Hogenhout, 2006). Phytoplasmas have to traverse the gut and salivary gland cells in order to reach the saliva for subsequent introduction into the phloem during insect feeding (Lefol et al ., 1994; Lherminier et al ., 1990; Nakashima and Hayashi, 1995). The latent period, i.e.…”

Detection of Bacterial and Phytoplasmal Pathogens

“…The phytoplasma causing Flavescence dorée (FD) disease of grapevine was detected by ISEM technique in extracts of both infected grapevine and leafhopper vector Euscelidius variegatus (Caudwell et al 1982). The antiserum specific to FD-enriched material from inoculative leafhoppers was employed to detect the phytoplasma in plants, whereas the antiserum to material from infected Vicia faba plants was used to detect the phytoplasma in leafhoppers to prevent the effects of host-directed antibodies (Lherminier et al 1990). …”