Development and Application of Species-Specific qPCR and RPA Assays for the Lettuce Wilt Pathogen, <i>Globisporangium uncinulatum</i> (syn. <i>Pythium uncinulatum</i> )

Pythium wilt of lettuce ( Lactuca sativa L.), caused by Globisporangium uncinulatum (syn. Pythium uncinulatum), has become an important disease of lettuce in the Salinas Valley, California, since its identification in 2011. Losses attributed to G. uncinulatum in 2022 were estimated between 47 and 77 million USD. Little is known of G. uncinulatum, and no molecular methods are currently available for detecting and quantifying the pathogen in plant or soil materials. Therefore, qPCR assays for plant and soil samples were developed that incorporate a plant internal control and an exogenous DNA control for soil and environmental 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. These assays target a unique mitochondrial locus of G. uncinulatum, putative orf368, providing species specificity and added sensitivity of the target with a multiple-copy locus. The assays were determined to be highly specific against a diverse panel of 41 oomycete taxa, including closely related species and spanning clades of Pythium, Globisporangium, Elongisporangium, Phytopythium, Phytophthora, and Saprolegnia. The sensitivity of the plant and soil qPCR assays was 10 to 100 fg of extracted G. uncinulatum DNA as determined through a multi-laboratory validation. The RPA assay was able to detect down to 5,000 fg of G. uncinulatum DNA from a crude plant extract. These assays were validated on lettuce plants and field soils from the Salinas Valley, California, and provide valuable diagnostic and monitoring tools for the lettuce industry. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .