EXPLOITATION OF ZOOSPORE TAXIS IN THE DEVELOPMENT OF A NOVEL DIPSTICKIMMUNOASSAY FOR THE SPECIFIC DETECTION OF PHYTOPHTHORA-CINNAMOMI

Species-specific monoclonal antibodies (MAbs) were used to develop a d ipstick immunoassay for the detection of Phytophthora cinnamomi. The a ssay is based on the phenomena of chemotaxis and electrotaxis to attra ct zoospores in suspension to a membrane where they encyst and are det ected by immunoassay. A chemotaxis assay was used to identify compound s that could be incorporated into the dipstick. The most attractive co mpounds included several amino acids, alcohols, phenolics, and isovale raldehyde. Some compounds, such as pectin, aspartic acid, and the phyt ohormone abscisic acid, caused encystment in addition to attraction. Z oospores also exhibited strong electrotaxis to a positively charged ny lon membrane. As few as 40 zoospores per milliliter were detected with the dipstick assay in an assay time of less than 45 min. Immunolabele d cysts attached to the membrane were observed with the naked eye or w ith low-power magnification after silver enhancement of a gold-labeled secondary probe or after an enzyme color reaction. Screening of 24 MA bs raised to aldehyde-fixed zoospores demonstrated that there were nin e MAbs that reacted strongly with P. cinnamomi cysts. Two MAbs were re vealed to be species specific from an extensive screening of isolates of P. cinnamomi (15 isolates of mating type A1 and 29 of mating type A 2), of other Phytophthora species (21 species or varieties of Phytopht hora comprising 73 isolates), and of other oomycetous genera (11 speci es of Pythium encompassing 14 isolates and one isolate each of three s pecies of Saprolegnia).