Detection of Tilletia caries, causal agent of common bunt of wheat, by ELISA and PCR

The paper reports about the development and evaluation of two methods, a PCR-based assay and an enzyme-linked immunosorbent assay (ELISA), for the detection of the common bunt fungus Tilletia caries (syn. T. tritici) in young wheat plants. Using the published primer pair Tcar2A/Tcar2B for polymerase chain reaction (PCR) with DNA from axenic cultures of T. caries or from T. caries-infected plants, we obtained a single band after electrophoresis of the amplification products. By PCR the bunt pathogen could be detected in shoots (EC 12) as well as in leaves (EC 13-14) of infected plants. Immunological detection was performed using a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with biotinylated detection antibodies. The antibodies were obtained after injection of mycelial homogenates of axenic cultures of T. caries into rabbits. The detection limit was 16 pg DNA per 100 mg plant fresh weight for the PCR and 7 ng/ml fungal protein for the ELISA, respectively. Except for the closely related T. controversa, no cross-reactions with other fungi were observed with both methods. While it was possible to detect teliospores of T. caries by PCR, the ELISA did not react with spore extracts. Analysis by ELISA of shoots of individual plants grown from inoculated seeds revealed that at EC 10 all plants were infected. There was, however, a large variability in the amount of T. caries present in the plants. This observation and reports in the literature indicate quantitative differences in the degree of colonization of the tissue between individual plants even in a given variety. Regarding the use of modern diagnostics to assist in the development of resistant varieties we therefore suggest that for the wheat -T. caries pathosystem the non-quantitative PCR-assay employed here is less suited than the ELISA that allows precise quantification of the amount of fungal antigen present in the plant. However, to routinely employ the ELISA in resistance breeding further development work is needed.