Immunodetection and Characterization of Antigens Expressed by Uncinula necator

Conidia from four genetically distinct isolates of Uncinula necator (Schw.) Burr. were used to raise a polyclonal antiserum. Immunofluorescent detection of the fungus on Vitis vinifera (cv. Chardonnay) indicated that the antiserum bound specifically to fungal antigens present on both conidia and hyphae, with no detection of underlying berry tissues. The antibody reacted with three antigens present on the conidia (Mr 21, 29 and > 250 kDa). Immunoreactivity with the 21 kDa antigen was dependent upon the preservation of at least one disulphide bond linkage. Evidence from immunoblot staining and enzyme immunoassay indicated that only a small proportion of the recognized epitopes contained carbohydrate moieties. The antibody detected homologous U. necator conidial antigens in a plate trapped antigen-enzyme-linked immunoabsorbent assay (PTA-ELISA), with a linear range of detection extending from 1000 to 9000 conidia/ml at a 1/5000 dilution of serum. Under these conditions the immunoassay also detected antigens from pooled heterologous U. necator isolates. The antiserum exhibited cross-reactivity with antigens present on Aspergillus, Pithomyces and Sporobolomyces species co-isolated from powdery mildew-infected grapes, which could not be removed by fractionation of the antiserum on antigen affinity columns. Monoclonal antibodies were subsequently produced to avoid the problems of cross-reactivity associated with the polyclonal antibody. Antibodies produced from two clonal lines exhibited specificity for U. necator and were shown to detect a 21 kDa conidial antigen. Use of either of these antibodies enabled the differentiation of grapes grouped on the basis of powdery mildew disease levels.