Development of a Novel Pseudoperonospora cubensis-Specific LAMP Assay for Rapid Field Detection and Surveillance of Cucurbit Downy Mildew

Abstract

Cucurbit downy mildew, caused by the oomycete Pseudoperonospora cubensis, is a devastating disease of cucurbit crops requiring rapid detection for effective management. Current molecular diagnostics often rely on complex laboratory infrastructure, limiting their on-site applicability. To address this limitation, we developed a highly specific, field-deployable loop-mediated isothermal amplification (LAMP) assay targeting the P. cubensis-specific c2555.2e1 marker gene. The assay’s optimal reaction conditions were determined, and its specificity was evaluated using closely related oomycetes, including P. humuli and P. belbahrii, alongside other fungal species. Assay sensitivity was compared to conventional PCR using a pathogen DNA dilution series, and diagnostic efficacy was validated using field-collected winter squash samples suspected of infection and environmental spore-trap samples. The optimal LAMP reaction conditions were 71 °C for 60 min. The assay accurately amplified P. cubensis with no observed cross-reactivity. Sensitivity analysis demonstrated a detection limit of 100 pg/µL, and was at least 10-fold more sensitive than conventional PCR. Furthermore, the LAMP assay exhibited greater detection sensitivity than conventional PCR when analyzing environmental and field samples. This rapid and sensitive LAMP assay provides a rapid and highly sensitive tool for on-site diagnostics, offering a crucial advancement for the early detection and management of cucurbit downy mildew.