Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification-lateral flow strip assay

Abstract

Phytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

Supplementary information: The online version contains supplementary material available at 10.1186/s42483-021-00089-8.

Keywords: Alkaline lysis extraction; Lateral flow assay; Phytophthora cactorum; Rapid diagnosis; Recombinase polymerase amplification.

Fig. 1

Optimization of a lateral flow recombinase polymerase amplification (LF-RPA) assay for the detection of Phytophthora cactorum. a Optimization of the RPA amplification temperature (top). b Evaluation of the RPA reaction time (top)

Fig. 2

Comparison of the sensitivity of the LF-RPA assay (upper panel) and conventional polymerase chain reaction (PCR; lower panel) for P. cactorum detection. Serial dilutions of P. cactorum genomic DNA were used to evaluate the detection limit. NTC, no-template control. At least three replicate tests were performed

Fig. 3

Workflow of the entire equipment-free detection process, including simplified DNA extraction and the LF-RPA assay

Fig. 4

Evaluation of LF-RPA detection of P. cactorum in infected plant tissues. a P. cactorum-inoculated leaves at 5 days post-inoculation (dpi). Mycelia were visualized using trypan blue staining. b Representative disease symptoms of Phytophthora fruit rot in strawberry caused by P. cactorum. c LF-RPA and conventional PCR assays of genomic DNA extracted from healthy and infected strawberry tissues