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

Abstract

<i>Phytophthora hibernalis</i>, 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 <i>P. hibernalis</i> can be confused with those by other <i>Phytophthora</i> 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 <i>Ypt1</i> gene and 5-min visualization using lateral flow dipsticks was developed for detecting <i>P. hibernalis</i>. This assay was able to detect 0.2 ng of <i>P. hibernalis</i> genomic DNA in a 50-µL reaction system. It was specific to <i>P. hibernalis</i> without detection of other tested species including <i>P. citrophthora</i>, <i>P. nicotianae</i>, <i>P. palmivora</i> and <i>P. syringae</i>, four other important citrus pathogens. Using this assay, <i>P. hibernalis</i> was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect <i>P. hibernalis</i> at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.