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

Species-specific monoclonal antibodies (MAbs) were used to develop a dipstick immunoassay for the detection of Phytophthora cinnamomi. The assay is based on the phenomena of chemotaxis and electrotaxis to attract zoospores in suspension to a membrane where they encyst and are detected by immunoassay. A chemotaxis assay was used to identify compounds that could be incorporated into the dipstick. The most attractive compounds included several amino acids, alcohols, phenolics, and isovaleraldehyde. Some compounds, such as pectin, aspartic acid, and the phytohormone abscisic acid, caused encystment in addition to attraction. Zoospores also exhibited strong electrotaxis to a positively charged nylon membrane. As few as 40 zoospores per milliliter were detected with the dipstick assay in an assay time of less than 45 min. Immunolabeled cysts attached to the membrane were observed with the naked eye or with low-power magnification after silver enhancement of a gold-labeled secondary probe or after an enzyme color reaction. Screening of 24 MAbs raised to aldehyde-fixed zoospores demonstrated that there were nine MAbs that reacted strongly with P. cinnamomi cysts. Two MAbs were revealed to be species specific from an extensive screening of isolates of P. cinnamomi (15 isolates of mating type A1 and 29 of mating type A2), of other Phytophthora species (21 species or varieties of Phytophthora comprising 73 isolates), and of other oomycetous genera (11 species of Pythium encompassing 14 isolates and one isolate each of three species of Saprolegnia).