| Summary: | Leaf-blight disease caused by the <i>Fusarium oxysporum</i> is an emerging problem in <i>Dendrobium chrysotoxum</i> production in China. Symptoms of leaf blight were observed on seedlings of <i>D. chrysotoxum</i> cultivated in a nursery in Ruili City, Yunnan Province, China. In this study, we isolated the <i>Fusarium</i> sp. associated with leaf-blight disease of <i>D. chrysotoxum</i> from the diseased seedlings. A pathogenicity test was performed to fulfill Koch’s postulates to confirm the pathogenicity of isolated strains and identified using morphological and molecular techniques. The results revealed that all four isolated <i>Fusarium</i> sp. isolates (DHRL-01~04) produced typical blight symptoms followed by marginal necrosis of leaves on the <i>D. chrysotoxum</i> plants. On the PDA medium, the fungal colony appeared as a white to purplish color with cottony mycelium growth. Microconidia are oval-shaped, whereas macroconidia are sickle-shaped, tapering at both ends with 2–4 septations. The phylogenetic trees were construed based on internal transcribed spacer (<i>ITS</i>), translation elongation factor (<i>EF-1α</i>), and RNA polymerase subunit genes <i>RPB1</i> and <i>RPB2</i> genes, respectively, and blasted against the NCBI database for species confirmation. Based on the NCBI database’s blast results, the isolates showed that more than 99% identify with <i>Fusarium oxysporum</i>. To our knowledge, this is the first comprehensive report on the identification of <i>Fusarium oxysporum</i> as the causal agent of <i>Dendrobium chrysotoxum</i> leaf blight in Yunnan Province, China, based on morphological and molecular characteristics.
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