PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO VERTICILLIUM-DAHLIAE AND DEVELOPMENT OF A QUANTITATIVE IMMUNOASSAY FOR FUNGAL BIOMASS

Monoclonal antibodies (MAbs) were prepared against a purified mycelial protein from Verticillium dahliae, a predominant fungus species in th e potato early dying complex. The fungal protein was rendered immunoge nic by conjugating it to keyhole limpet hemocyanin. Three hybridomas w ere cloned from a mouse immunized with the conjugated protein. All thr ee cell lines produced immunoglobulin G1-type MAbs that recognized a 6 0-kDa protein. The specificity of the MAbs was tested using indirect e nzyme-linked immunosorbent assay (ELISA), immunoblots, and indirect co mpetitive ELISA. For two of the MAbs, no cross-reactivity was observed when tested against several isolates representing six species of fung i associated with potato. Cross-reactivity was observed to some extent with K albo-atrum. A quantitative immunoassay was developed using an indirect competitive ELISA format that could detect as little as 4 ng of total protein of the fungus and showed a linear relationship betwee n total protein (4 to 500 ng) and absorbance measured at 405 nm. The a ssay was used to detect and quantify K dahliae colonization in greenho use-grown potato plants. The immunoassay accurately distinguished colo nized from noncolonized plants and quantitively differentiated the sus ceptible cultivar (Kennebec) from the resistant cultivar (Reddale).