A Genome-Anchored TaqMan™ qPCR Assay for the Specific Detection of an Aggressive Genotype of Pseudomonas syringae pv. aptata

Bacterial leaf spot of table beet and Swiss chard caused by Pseudomonas syringae pv. aptata is a seedborne threat lacking specific methods for detection from seed, plant, or soil samples. To address this, we developed a genotype-specific, genome-informed TaqMan™ qPCR assay targeting TIGR02646, a marker uniquely conserved in the aggressive MLST3 genotype, a highly virulent and predominant genotype, identified through a k-mer inclusion/exclusion screen of 421 P. syringae draft genomes. The assay showed 100% inclusivity for MLST3 (48/48 target isolates), including the P. syringae pv. aptata pathotype (ICMP459), and 100% exclusivity across 220 nontarget isolates under a predefined decision rule with Cq at or below 30 scored as positive. Analytical performance was consistent across relevant sample types; in pure culture, the assay was log-linear from 10 ⁸ to 10 ⁶ CFU/mL with 104% efficiency and a limit of detection of 10 ³ CFU/reaction. In bacteria-spiked leaf extracts and seed extracts (1:10 dilution), the operational limit of detection was 10 ³ and 10 ⁴ CFU/reaction, respectively. In non-sterile soil, the limit of detection was 10 ² CFU/reaction with excellent linearity and acceptable efficiency despite typical inhibitors. Greenhouse spray-inoculation trials confirmed that the assay can track dose-responsive population dynamics and detect targets before the onset of severe symptoms. These results establish a genome-anchored, highly specific qPCR tool suitable for seed health testing, pre-plant risk assessment, and environmental surveillance of the aggressive P. syringae pv. aptata MLST3 genotype.