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<i>Fraxinus americana</i> L. (white ash), a native North American tree commonly cultivated for its ornamental qualities, displayed symptoms of leaf spot disease in a sentinel garden located in Nanjing, Jiangsu, China, in 2022. This disease led to premature leaf shedding, adversely affecting the plant’s growth and substantially diminishing its ornamental value. Potential fungal pathogens were isolated from the diseased leaves and the subsequent application of Koch’s postulates confirmed the pathogenicity of the fungal isolates (BL-1, BL-2). Through a combination of multi-locus phylogenetic analysis, including ITS, <i>ACT</i>, <i>ApMat</i>, <i>CAL</i>, <i>CHS-1</i>, <i>GAPDH</i>, and <i>TUB2</i>, alongside morphological assessments, the fungus was conclusively identified as <i>Colletotrichum jiangxiense</i>. This represents the first record of <i>C. jiangxiense</i> affecting white ash, highlighting the important role of sentinel gardens in uncovering novel pathogen–plant host interactions.