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<div>Seed infestation is an important factor contributing to the dissemination of <i>Pseudomonas syringae</i> in vegetable crops. In recent years, disease reports identifying the bacterial species as a leaf spot pathogen of watermelon and squash have increased in frequency in the United States and Europe, indicating a need for improved diagnostic methods. Previous studies have shown the strains associated with disease outbreaks to be members of <i>P. syringae</i> phylogroup 2, however genotypic heterogeneity coupled with an unresolved taxonomy pose a serious problem for the precise identification of pathogenic strains. We used comparative genomic approaches to design several molecular markers and screened them against an international collection of over 300 <i>P. syringae</i> strains isolated from contaminated seeds and diseased fields over various years. A phylogenetic analysis of <i>syl</i>C involved in the biosynthesis of the virulence factor syringolin A revealed two divergent groups (<i>syl</i>C_a and <i>syl</i>C_b) within <i>P. syringae</i> phylogroup 2, and a TaqMan probe showed 93% of the collected strains to possess <i>syl</i>C_b. Preliminary pathogenicity testing indicated that the <i>syl</i>C_a strains were weakly virulent to non-pathogenic on squash (cv. Spineless Beauty) whereas all <i>syl</i>C_bstrains tested were moderately to highly virulent.<i> In-silico</i> analysis suggests <i>syl</i>C_b to be semi-specific for <i>P. syringae</i> strains isolated as pathogens of watermelon and squash, and therefore increased specificity can be achieved with additional strain specific markers.</div>