Vibrio harveyi is a prominent bacterial pathogen of marine aquaculture. Vibrio harveyi has not previously been reported in starry flounder. In this study, phenotypic and genotypic characterizations and pathogenicity assessments of V. harveyi isolated from starry flounder in South Korea were conducted. Phylogenetic analysis identified strains VH‐SF‐1, VH‐SF‐2, VH‐SF‐4 and VH‐SF‐5 as V. harveyi . Motility assays showed that VH‐SF‐2 displayed the highest activity at 27°C. In pathogenicity tests, VH‐SF‐2 caused 100% mortality within 16 and 48 h at doses of 3 × 10 8 and 3 × 10 7 CFU/fish, respectively, whereas a 10% mortality rate was observed at 3 × 10 6 CFU/fish. The lethal dose (LD 50 ) was estimated to range from 3 × 10 6 to 3 × 10 7 CFU/fish, providing the first evidence of the pathogenicity of V. harveyi in starry flounder. These findings suggest that V. harveyi may emerge as a novel pathogen of this species. To investigate genomic characteristics and virulence factors, whole‐genome sequencing (WGS) was conducted. Strain VH‐SF‐2 harboured multiple virulence‐related mechanisms, including outer membrane proteins, motility, quorum sensing, bacterial secretion systems, iron uptake and regulation and virulence regulators. The study aimed to elucidate the biological and pathogenic characteristics of V. harveyi isolated from starry flounder.
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