Alternaria alternata is the main pathogenic species of various crops, including kiwifruit (Actinidia cinensis). In this study, an antagonistic fungus, J-1, with high antifungal activity against A. alternata was isolated from A. cinensis “Hongyang.” The strain J-1 was identified as Fusicolla violacea via morphological identification and DNA sequencing. This study aimed to evaluate the antifungal activity and potential mechanism of the strain J-1 against A. alternata. The strain J-1 exhibited antifungal activity against A. alternata, with an inhibition rate of 66.1% in vitro. Aseptic filtrate (AF) produced by the strain J-1 could suppress the mycelial growth and conidia germination of A. alternata at the inhibition rates of 66.8% and 80%, respectively, as well as suppress the spread of Alternaria rot in fresh kiwifruit. We observed that many clusters of spherical protrusions appeared at the mycelial tips of A. alternata after treatment with 200 mL L−1 AF of J-1. Scanning electron microscopy analysis results showed that the mycelial structures were bent and/or malformed and the surfaces were rough and protuberant. Variations in temperature, pH, and storage time had little effect on the antifungal activity of the AF. Moreover, the AF could damage the integrity of cell membranes and cause intracellular content leakage. Meanwhile, the chitinase and β-1,3-glucanase enzyme activities increased significantly, indicating that the function of A. alternata cell wall was seriously injured. Eleven antimicrobial metabolites were identified by gas chromatography–mass spectrometry (GC–MS). The strain J-I and its AF exhibited well broad-spectrum antifungal activity against Diaporthe eres, Epicoccum sorghinum, Fusarium graminearum, Phomopsis sp., and Botryosphaeria dothidea, with inhibition rates ranging from 34.4% to 75.1% and 42.7% to 75.2%, respectively. Fusicolla violacea J-1 is a potential biocontrol agent against A. alternata and other fungal phytopathogens.

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