Development and application of species-specific qPCR and RPA assays for the lettuce wilt pathogen, Globisporangium uncinulatum (syn. Pythium uncinulatum)

Authors: MCCOY, AUSTIN - Michigan State University; Martin, Frank; Matson, Michael; KOIKE, STEVE - Trical Inc; MILES, LAURA - Michigan State University; Hasegawa, Daniel; CAMELO, VIVIANA - Forest Service (FS); MILES, TIMOTHY - Michigan State University; CHILVERS, MARTY - Michigan State University

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2025
Publication Date: 5/13/2025
Citation: McCoy, A.G., Martin, F.N., Matson, M.E., Koike, S., Miles, L.A., Hasegawa, D.K., Camelo, V., Miles, T.D., Chilvers, M.I. 2025. Development and application of species-specific qPCR and RPA assays for the lettuce wilt pathogen, Globisporangium uncinulatum (syn. Pythium uncinulatum). PhytoFrontiers. 5(2):209-219. https://doi.org/10.1094/PHYTOFR-10-24-0112-FI.
DOI: https://doi.org/10.1094/PHYTOFR-10-24-0112-FI

Interpretive Summary: This submission describes the development of a diagnostic assay specific for detection of Globisporangium uncinulatum (syn. Pythium uncinulatum), the causative agent of pythium wilt, an emerging soilborne pathogen of lettuce in California. It is available in both a PCR format and an isothermal technology called recombinant Polymerase Amplification, the latter of which can be used directly in the field without the need for DNA extraction and provide results in as little as 20 minutes.

Technical Abstract: Pythium wilt of lettuce, caused by Globisporangium uncinulatum (syn. Pythium uncinulatum), has become one of the more important diseases of lettuce in the Salinas Valley, California since its identification in 2011. Losses attributed to G. uncinulatum in 2022 were estimated at $58 million dollars alone. Little is currently known of the causal organism, G. uncinulatum, and no molecular methods are currently available for detecting and quantifying the pathogen in plant or soil materials, limiting our ability to study this pathosystem. Therefore, qPCR assays for plant and soil samples were developed that incorporate a plant internal control and an exogenous DNA control for soil samples to enable quantification of G. uncinulatum. This assay was then transferred to a recombinase polymerase amplification (RPA) platform to enable rapid detection of G. uncinulatum in the lab or field. These assays target a unique mitochondrial locus of G. uncinulatum, putative orf368, providing added sensitivity of a multiple copy target and species specificity. The assays were determined to be highly specific against a diverse panel of 41 oomycete taxa including closely related species and spanning phytopathogenic clades of Pythium sensu latu, Phytopythium, Phytophthora, and Saprolegnia. The sensitivity of the plant and soil qPCR assays was 10-100 femtograms of extracted G. uncinulatum DNA as determined through a multi-laboratory validation. The RPA assay was able to detect as little as 5,000 femtograms (0.005 nanograms) G. uncinulatum from a crude plant extract. These assays were validated on lettuce plants and field soils from the Salinas Valley, California, and provide valuable diagnostic tools for the lettuce industry.