Rapid detection of Fusarium fujikuroi in rice seeds and soaking water samples based on recombinase polymerase amplification-lateral flow dipstick

Bakanae disease is a rice seedborne disease caused by the Fusarium (Gibberella) fujikuroi species complex (FFSC), among which F. fujikuroi is the dominant pathogen. Pathogens usually hide inside or on the surface of seeds, and infection occurs mainly at the germination stage. In this study, a method for the detection of F. fujikuroi in rice seeds and seed soaking water samples was established using recombinase polymerase amplification (RPA) technology with lateral flow device (LFD) chromatography test strips. A pair of specific primers and probes based on the cyp51c gene were screened. RPA-LFD was used to detect 10 F. fujikuroi strains, and the results showed that all of them tested positive and there was no cross-reaction with other Fusarium or non-Fusarium species. The target production of the RPA-LFD assay was obtained at 35-45(degrees)C for 8-14 min, and optimal reaction conditions of amplification at 39(degrees)C for 8 min is recommended. The sensitivity test showed that the detection limit of the RPA-LFD test for F. fujikuroi genomic DNA in rice seeds was 100 fg/mu L, and the detection limit for F. fujikuroi spores in submerged water samples was 100 spores/mL. In the assay for field samples, it successfully detected F. fujikuroi carried in the seeds of three out of five rice varieties. In addition, the whole RPA-LFD assay can specifically detect F. fujikuroi within 30 min. This method is expected to become an early field monitoring tool for rice bakanae disease.