DETECTION OF PHYTOPHTHORA FRAGARIAE IN STRAWBERRY PLANTS USING PTA-ELISA
B KYNEROVA, J KRATKA, A ZEMANOVA, M PODANA and M KUTIKOVA
Research Institute of Crop Production, Department of Mycology, Drnovski 507, 161 06 Praha 6-Ruzyn6, Czech Republic
Background and objectives
Phytophthora fragariae causes red core disease on strawberry.This pathogen is included in the List of quarantine Pests for the Czech Republic. The research on the detection of this pathogen in strawberry plants in the Czech Republic started in 1996. Over two years the polyclonal antibodies were prepared and the first experiments with artificially inoculated plants under greenhouse conditions were evaluated. The sucseptibility of ten cultivars of strawberry growing in the Czech Republic was tested. At present monoclonal antibodies are developed.
Materials and methods
Polyclonal antibodies (IgG class) were purified from antisera of laboratory rabbits intramuscularly immunized with purified and unpurified antigens extracted from mycelial mats of Phytophthora fragariae . The specifity of antibodies were tested in vitro with antigens prepared from various fungi. Monoclonal antibodies were purified from the antibody-containing cell culture supernatant. These antibodies were directed against unpurified antigen.
Strawberry plants were inoculated while sinking roots in water suspension of fungal spores and mycelium. The intensity of disease symptoms was evaluated visually. PTA-ELISA was used in detection of pathogen in root extremities, root and hypocotyl.
Results and conclusions
Reactions of polyclonal antibodies directed against purified antigen in PTA-ELISA were stronger than reaction of antibodies against unpurified antigen (A405nm was higher but of the same tendency). Both antibodies were found specific for the genus Phytophthora. However weak cross-reactions with Pythium ultimum were observed as well. The 1µg/ml concentration of antibodies was suitable for the detection of pathogen by means of PTA-ELISA.
Monoclonal antibodies (IgG class) were also genus specific without cross-reactions with other tested fungal pathogens.
All ten strawberry cultivars tested to this pathogen were susceptible.
Pathogen in artificially inoculated plants (cv. Eisanta) was detectable by PTA-ELISA in root extremities, root and hypocotyl only after the symptoms became visible. Unfortunately, latent infection was impossible to detect under test conditions.
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