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Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity. [ABSTRACT FROM AUTHOR]