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Vaccinium corymbosum L., commonly known as blueberry, is a valuable small fruit tree in terms of its economic significance and is primarily found in China within the regions of Heilongjiang and Jilin provinces. Additionally, it thrives in the areas spanning the middle and lower reaches of the Yangtze River. Beyond their economic value, blueberries play a crucial role within forest ecosystems, serving as a significant energy source and source of nutrients. Their presence contributes significantly to fostering stability, enhancing biodiversity, and acting as an indicator of environmental quality within forest ecosystems. Since August 2022, an unknown leaf disease has been found on a large scale in blueberry fields in Nanjing, Jiangsu Province, China. The disease causes leaf curling, wilting, and even early defoliation, severely reducing the yield and production value of blueberries. The pathogenicity test confirmed the virulence of the isolates (NG5-1, NG5-2, NG5-3, NG5-4, N2-1, and N2-2) against V. corymbosum. The two pathogens were identified as Colletotrichum fructicola and C. aenigma by observing the morphological characteristics of the isolates and combined with multilocus phylogenetic analyses (ITS, CAL, ACT, TUB2, ApMat, and GAPDH). Blueberry anthracnose, caused by C. aenigma, is the first report of this disease in China. The biological characteristics of C. aenigma were investigated under different conditions, including temperature, pH, light conditions, culture medium, and carbon and nitrogen sources. The optimal temperature for growth was determined to be within the range of 25–30 °C; C. aenigma exhibits optimal growth at a pH of 7–8. Mycelial growth is favored under conditions of partial light, whereas complete darkness promotes spore production. It was found that PDA medium was the most favorable medium for C. aenigma mycelial growth, and MM medium was the best medium for spore production; the most suitable carbon sources for colony growth and spore production were sorbitol and glucose, respectively, and the most suitable nitrogen source was peptone. This study furnishes a theoretical foundation for a more scientifically informed approach to the prevention and control of anthracnose on V. corymbosum. [ABSTRACT FROM AUTHOR]