Early Detection of the Spinach Downy Mildew Pathogen in Leaves by Recombinase Polymerase Amplification.

Downy mildew of spinach, caused by , is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the mitochondrial genome to develop an RPA assay for the early detection of in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the specificity of the locus across spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, . TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.