| Summary: | <i>Fusarium oxysporum</i> f. sp. <i>radicis-lycopersici</i> (Forl) causes crown and root rot disease in tomato, effecting severe economic losses. However, research on the pathogenicity genes and infection strategy of Forl is limited compared to that on <i>F. oxysporum</i> f. sp. <i>lycopersici</i> (Fol). In this study, we characterized <i>FoMC69</i> gene in Forl as a homolog of <i>MC69</i> required for pathogenicity in rice blast pathogen—<i>Magnaporthe oryzae</i>. Gene expression analysis revealed that <i>FoMC69</i> expressionin Forl is higher than that in Fol<i>in planta</i>. <i>FoMC69</i>-knockout mutant of Forl had significantly reduced root rot symptoms compared to the wild-type strain, and full pathogenicity was restored by complementation. By contrast, <i>ΔFoMC69</i> mutant of Fol presented the same symptoms as the wild type, suggesting that <i>FoMC69</i> of Forl, but not of Fol, was essential for full virulence in tomato plants. Morphological differences between the Forl and <i>ΔFoMC69</i> in the roots were observed by fluorescent labeling using WGA-FITC. Chlamydospores of the <i>ΔFoMC69</i> mutant of Forlcontinuously increased during infection and were three times higher than that of the wild type at 21 days post-inoculation. These observations suggest that <i>FoMC69</i> of Forl is required for virulence to tomato plants by involving the normal development and germination of chlamydospores.
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