An immunofluorescence assay for the detection of wheat rust species using monoclonal antibody against urediniospores of Puccinia triticina

Abstract

Aims: Wheat (Triticum aestivum) is one of the most important crop species, but yields are often drastically reduced by rust epidemics. In this report, we describe a rapid and sensitive immunofluorescence method for the detection of urediniospores of the fungi Puccinia striiformis f. sp. tritici, Puccinia triticina and Puccinia graminis f. sp. tritici, which are causal agents of wheat rust. Methods and Results:The method uses monoclonal antibody LPT-2 against the urediniospores of P. triticina and PE-cy3 goat anti-mouse. Urediniospores of P. triticina or those of two species that are difficult to distinguish from P. triticina, P. striiformis f. sp. tritici or P. graminis f. sp. tritici were immobilized on a glass slide, and the sample was then treated with LPT-2. Thereafter, a second antibody, goat anti-mouse conjugated PE-cy3, was added, and the slide was observed in a fluoroscope. The fluorescent signal was strong with P. triticina urediniospores, weak with P. striiformis f. sp. tritici urediniospores and weakto-intermediate with P. graminis f. sp. tritici urediniospores. The detection limit of this method was 2 ng ml À1 of the monoclonal antibody LPT-2. Conclusions:In this article, we describe the production and diagnostic application of a novel mouse monoclonal antibody specific to urediniospores of P. triticina. Significance and Impact of the Study: After further technical development, this method may become a tool for on-site identification of P. triticina urediniospores and will therefore help in the selection and timing of fungicide applications for control of wheat rust outbreaks.

Detection of Tilletia controversa using immunofluorescent monoclonal antibodies

“…Monoclonal antibodies were produced according to the procedure of Gao et al . (), except that the suspension contained 0·5 mg ml −1 dry teliospores. As indicated in the Results, six monoclonal antibodies were selected for further study.…”

“…All protocols described in Gao et al . () were followed, except that the suspension included 100 μ l volume of a T. controversa teliospore suspension (0·5 mg ml −1 ) was incubated at 37°C for 2 h. Each well was read three times.…”

“…Following the procedures as previously described (Gao et al . ), briefly, the spore suspensions were dropped onto different glass slides and allowed to dry. The slides were then washed in PBS two times for 5 min each, treated with monoclonal antibody and washed with PBS three times.…”

“…Immunologic methods based on monoclonal antibodies can easily and rapidly produce detection results, and they do not require complex equipment or technical skills; therefore, such methods are the most suitable for on‐site detection (Gao et al . ). Previous attempts have been made to identify T. controversa and other closely related species using immunologic techniques; however, only unsatisfactory results were achieved.…”

“…tritici and Puccinia graminis (Gao et al . ). In the present study, we report the development of an immunofluorescence assay using a novel mouse monoclonal antibody to specifically detect teliospores of T. controversa .…”