Deep Targeted Inter‐Simple Sequence Repeat Sequencing for Detection of Quarantine Seedborne Bacteria: Application to <i>Xanthomonas Euvesicatoria</i> pv. <i>Perforans</i> and <i>Ralstonia Solanacearum</i>

ABSTRACT Seedborne bacterial pathogens such as Xanthomonas euvesicatoria pv. perforans (XE) and Ralstonia solanacearum (RS) threaten tomato production and international seed trade. Conventional PCR assays are highly specific but typically limited to single‐target, qualitative detection in complex seed‐derived DNA backgrounds. In this study, we developed and evaluated a deep‐targeted inter‐simple sequence repeat (ISSR)‐ NGS workflow for the simultaneous detection of XE and RS in tomato seeds. The method incorporated a pre‐extraction incubation step and non‐destructive sampling to reduce host DNA carryover prior to sequencing. Primer screening using signal separation metrics identified a GC‐rich primer combination ((GTG)₃ + (GC)₄) with balanced cross‐pathogen performance. Dilution‐series experiments demonstrated strong dose‐dependent relationships between log₁₀ inoculum levels and classified read proportions for both pathogens (R² ≥ 0.92, p &lt; 0.01), supporting semi‐quantitative responsiveness across a broad dynamic range. Comparison with pathogen‐specific PCR showed high but incomplete agreement, with most discordant cases occurring near predefined detection thresholds. Low and stable background read proportions in negative controls supported discrimination between infected and non‐infected seed lots. These findings indicate that ISSR‐NGS offers a scalable approach for multi‐pathogen detection in seed health testing and quarantine surveillance.