Early detection of the spinach downy mildew pathogen in leaves by recombinase polymerase amplification

Authors: Clark, Kelley; Anchieta, Amy; DA SILVA, MYCHELE - University Of California; Kandel, Shyam; CHOI, YOUNG-JOON - Kunsan National University; Martin, Frank; CORRELL, JAMES - University Of Arkansas; VAN DENYZE, ALLEN - University Of California; BRUMMER, CHARLES - University Of California; Klosterman, Steven

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2022
Publication Date: 3/6/2022
Citation: Clark, K.J., Anchieta, A.G., da Silva, M.B., Kandel, S.L., Choi, Y., Martin, F.N., Correll, J.C., Van Denyze, A., Brummer, C.E., Klosterman, S.J. 2022. Early detection of the spinach downy mildew pathogen in leaves by recombinase polymerase amplification. Plant Disease. 106(7):1793-1802. https://doi.org/10.1094/PDIS-11-21-2398-RE.
DOI: https://doi.org/10.1094/PDIS-11-21-2398-RE

Interpretive Summary: Downy mildew is a major threat to spinach production, especially in organic sectors where there are no effective biopesticides. Downy mildew on spinach results in yellow diseased spots on leaves which render the leaves unmarketable. Early detection of the pathogen in the leaves is pertinent for management, as the information of whether the pathogen is present prior to symptom appearance. can affect decisions to harvest and/or treat fields with pesticides. Our research presents a solution to this issue via the use of a novel genetic marker for detection of Peronospora effusa paired with recombinase polymerase amplification (RPA) assay, which can be used directly in the field.

Technical Abstract: Downy mildew of spinach, caused by Peronospora effusa, is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic as symptoms can develop post-harvest. Therefore, early detection methods for P. effusa 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 P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative polymerase chain reaction (PCR) assay and used this assay to validate the P. effusa-specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, Peronospora schachtii. TaqMan qPCR has a detection threshold of 100 femtograms of DNA and RPA 900 femtograms. Both assays could detect P. effusa in pre-symptomatic leaves, with RPA-based detection of P. effusa occurring as early as five days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24hrs 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.