Evidence for the physical integrity of flavescence dorée phytoplasmas purified by immunoaffinity from infected plants or leafhoppers and the plant pathogenicity of phytoplasmas from leafhoppers

Abstract

Phytoplasmas were extracted from flavescence dorée‐infected broadbean (Vicia faba) using a vacuole isolation medium, and were immunoaffinity purified from infected leafhoppers. Purified phytoplasmas from both host sources were immunolabelled and observed under the electron microscope. The infectivity of the purified phytoplasmas from leafhoppers was checked by injecting healthy leafhoppers which were then allowed to feed on healthy V. faba seedlings. The appearance of typical symptoms in these plants, and the positive results obtained in ELISA with extracts of some of the injected leafhoppers and with symptomatic V. faba, indicated that the purified phytoplasmas had retained their infectivity and had multiplied in the injected leafhoppers which had become infective. These results support a previous report that phytoplasmas purified by immunoaffinity chromatography are well preserved.

Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

“…Proteins were transferred from the polyacrylamide gel by electroblotting onto Immobilon-P TM (Millipore) membrane using a Millipore semi-dry transfer apparatus according to the manufacturer's instructions. After proteins being transferred, the membranes were treated with the immunological method described previously for the detection of FDphytoplasma antigens in broadbean (Seddas et al, 1995).…”

“…Specific monoclonal antibodies (K6hler and Milstein, 1975) have the advantage over polyclonal antibodies to avoid cross reactivity against host material. For this reason, monoclonal antibodies to FD-phytoplasma enriched preparations, which had been obtained in several fusions (Schwartz et al, 1989), were reacted on westem blot of proteins prepared from FD-infected leafhopper or broadbean (Seddas et al, 1993;Seddas et al, 1995). Labeling was obtained only on either of the two main components and not on any of the minor components revealed with polyclonal antibodies.…”

“…The immunoaffinity-purified phytoplasma cells are undamaged and the morphology of the phytoplasma structure following this procedure was reported well preserved (Seddas et al, 1995).…”

Establishment of a relationship between grapevine leafroll closteroviruses 1 and 3 by use of monoclonal antibodies

“…Purified virus preparations (100 g) were resuspended in 70 L protein solubilization buffer (0·2 M Tris-HCl pH 6·8, 0·5 M sucrose, 0·1 g L ¹1 bromophenol blue, 5 mM EDTA, 10 g L ¹1 methionine), 6 L 20% (w/v) SDS and 1 L 1 M DTT (Seddas et al, 1995). After electrophoresis, the gels were blotted onto Immobilon-P TM membrane (Millipore; Saint-Quentin-Yvelines, France) in the semi-dry transfer apparatus (Millipore), according to the manufacturer's procedure.…”

“…After electrophoresis, the gels were blotted onto Immobilon-P TM membrane (Millipore; Saint-Quentin-Yvelines, France) in the semi-dry transfer apparatus (Millipore), according to the manufacturer's procedure. The membranes were then treated using polyclonal antibodies (Pabs) or Mabs as previously described (Seddas et al, 1995).…”