3.3.35
DEVELOPMENT OF A SPECIES-SPECIFIC MONOCLONAL ANTIBODY FOR THE DETECTION AND QUANTIFICATION OFBOTRYTIS CINEREA

UM MEYER and FM DEWEY

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OXI 3RB, UK

Background and objectives
Botrytis cinerea Pers. [teleomorph Botryotinia fuckeliana] causes considerable losses of vegetables, fruits and cutflowers. Grey mould of grapes is probably the most significant disease caused by B. cinerea. In the early stages of infection the pathogen commonly remains quiescent or symptomless and it does not become pathogenic and produce a necrotic rot until ripening or postharvest. Detection and quantification of the fungus at an early stage of infection is difficult. A Monoclonal antibody (MAb) immunoassay, that employs near-genus specific MAb, BC-KH4 was developed and has been used to detect, quantify and visualize B. cinerea and B. fabae in other host tissues [1], but the cross reactivity of the antibody with other post-harvest spoilage fungi, e.g. Aspergillus niger has limited its usefullness[l, 21. The aim of this project was to raise a more specific MAb, in order to both quantify the pathogen in mixed infections and to detect and visualize B. cinerea in the quiescent phase.

Materials and methods
Cell free surface washings of a solid culture of a grape isolate of B. cinerea(P-6g) to which Quil A had been added as an adjuvant, was used as the immunogen. Balb c mice were given eight intraperitonal injections at two week intervals. 300 Ál of the immunogen were used per injection.

Splenocytes from a immunized mouse, 3 days after the last injection, were fused with myeloma cells (Sp2/0-Ag 14 cell line), following the method of Dewey et al [31. The fusion products were diluted in 100 ml selective media (HAZA) and plated out in 96 well trays, 100 gl per well. Wells were fed after 7 days with selective medium, 100 Ál per well. After 9 days the wells were screened and hybridoma supernatants were tested by PTA-ELISA against surface washings of cultures of B.cinerea and other relevant fungi. Selected cell lines were recloned and preserved by slowly freezing in FBS/DMSO (92:2 v/v) and maintained in liquid nitrogen.

Results and conclusions
From one fusion a total of 785 cell lines were produced. Supernatants from 80 of these cell lines produced antibodies that recognized the immunogen. Out of these Cell lines one cell line, BC-HA4 proved to be highly specific. It did not cross-react with other fungi or plant tissues. It is an IgA antibody, that recognizes B. cinerea by both PTA-ELISA and immunofluorescence. Studies with periodate and heat-treatment have shown that BC-HA4 recognizes an epitope on a non-heat stable protein. Further characterization of the antibody is in progress to determine if the MAb is isolate and/or lifestage specific, and its ability to detect and quantify the fungus in both the quiescent and necrotrophic phases.

References
1. Bossi R, Dewey FM, 1992.Plant Pathology 41, 472-82.
2. Dewey FM, Cole L, 1997. In. Dehne HW, Adain G, Diekmann M, Frahm J, Muler-machnik A, van Halteren P (Eds.),Diagnosis and Identification of Plant Pathogens, 91-94. Kluwer Academic Publishers.
3. Dewey FM, MacDonald MM, Phillips SI, 1989. Journal of General Microbiology 135, 361-374.