Duplex real‐time PCR for routine detection of Pantoea stewartii subsp. stewartii in maize seeds

Pantoea stewartii subsp. stewartii is a phytopathogenic bacterium affecting maize, responsible for Stewart's wilt and leaf scorch along the veins. Native to North America and seed‐transmitted, this species is considered a quarantine pest and is under enhanced surveillance within the European Union. Despite the implementation of phytosanitary measures, no sufficiently reliable detection test is currently available. Existing tests—based on antigen recognition (ELISA) or DNA detection (PCR)—often fail to differentiate this pathogen from closely related subspecies, leading to false‐positive results. Recent studies have identified specific PCR primer sets that improve the specificity of P. stewartii subsp. stewartii detection. Building upon these findings, we present a diagnostic protocol on maize seeds using a quantitative duplex real‐time PCR test suitable for routine laboratory testing. The primer pair cpsAB2313F/cpsR was selected for specific detection of Pantoea stewartii subsp. stewartii , while the primer pair Wu‐F/Wu‐R was used as an internal amplification control. The test was able to detect a single P. stewartii subsp. stewartii contaminated seed within a subsample of 2000 healthy seeds but had only 83.3% repeatability with one contaminated seed in a subsample of 1000 seeds. Therefore, one contaminated seed in a subsample of 400 seeds was set as the detection limit. This batch size is suitable for routine seed testing programs.