Chiral Mandelic Acid Derivatives as Inhibitors Targeting the Type III Secretion System of <i>Xanthomonas citri</i> Subsp. <i>citri</i>

<i>Xanthomonas citri</i> subsp. <i>citri</i> (<i>Xcc</i>), the pathogen responsible for citrus canker, causes major economic damage worldwide. The type III secretion system (T3SS) is essential for <i>Xcc</i> pathogenicity. Here, 39 chiral 1,3,4-oxadiazole sulfide derivatives of mandelic acid were synthesized, with 17 compounds exhibiting significant inhibitory activity (>70% inhibition) against the T3SS of <i>Xcc</i> jx-6 at 40 μg/mL. Among these, the <i>R</i>-configuration W_R4 significantly suppressed the symptoms of citrus canker in an <i>in vivo</i> bioassay, whereas neither the <i>S</i>-configuration W_S4 nor the racemic mixture W_RS4 showed inhibitory effects. Mechanistically, W_R4 suppressed T3SS gene transcription and Hpa1 protein secretion, and counteracted pathogen-induced suppression of host immunity, without affecting other virulence factors. From an environmental safety perspective, W_R4 exhibited low toxicity against nontarget organisms, including earthworms, silkworms, and zebrafish. These findings reveal the potential of the <i>R</i>-configuration derivatives as sustainable T3SS inhibitors.