A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis.

Phytophthora hibernalis, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation and identification by DNA sequencing. However, symptoms caused by P. hibernalis can be confused with those by other Phytophthora and fungal species. Moreover, pathogen isolation, PCR amplification and sequencing are time-consuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the Ypt1 gene and 5-min visualization using lateral flow dipsticks was developed for detecting P. hibernalis. This assay was able to detect 0.2 ng of P. hibernalis genomic DNA in a 50-µL reaction system. It was specific to P. hibernalis without detection of other tested species including P. citrophthora, P. nicotianae, P. palmivora and P. syringae, four other important citrus pathogens. Using this assay, P. hibernalis was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect P. hibernalis at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.,© 2019 Dai et al.