Use of polyclonal antibodies in the diagnosis of Phytophthora cinnamomi

A polyclonal antiserum against water soluble proteins from Phytophthora cinnamomi mycelium was produced in rabbit. Bleedings were carried out after 25, 65, and 180 days. The obtained antisera gave positive reactions in western-blot analyses against 9 species of Phytophthora and Pythium sp., but not against Rhizoctonia solani, a binucleate Rhizoctonia, Verticilllium dahliae, Fusarium oxysporum and Cryphonectria parasitica. The antiserum obtained 25 days after the injection was less avid, but more specific than the one from the 180 days bleeding. All Phytophthora species showed common epitopes on proteins of a molecular mass of 77, 66, 51, and 48 kD. On the other hand, a species specific protein of 55 kD was immunodecorated only in P. cinnamomin samples. When used in ELISA assays, the 1:10,000 diluted antiserum revealed only the Phytophthora isolates, giving the strongest reaction with P. cinnamomi protein extracts (150-175% higher absorbance than with other Phytophthora species). The antiserum is active at dilutions ranging from 1:10,000 to 1:40,000 with a sensitivity threshold of 2 mg of total fungal proteins. In western-blot, the antiserum positively identified P. cinnamomi in infected chestnut, blueberry and azalea tissues.