Antifungal Metabolites Produced by the Coculture of <i>Clonostachys rosea</i> VDGF806 and <i>Alternaria brassicicola</i>

Co-culturing the endophytic fungus Clonostachys rosea VDGF806 from Vaccinium dunalianum Wight with Alternaria brassicicola on solid rice medium resulted in the production of two new compounds, altenarizine C (1) and clonbrasterol A (2), along with three known ones (3-5). Their structures were identified based on extensive spectroscopic analyses and electronic circular dichroism experiments. The isolated compounds were evaluated for in vitro antifungal activities against five phytopathogenic fungi (Fusarium oxysporum, Alternaria brassicae, F. solani, Botrytis cinerea, and F. graminearum). Clonbrasterol A (2) and nafuredin (3) showed antifungal activity, with minimum inhibitory concentration (MIC) values of 7.8-62.5 µg/mL. In comparison, the positive controls thiabendazole and carbendazim had MIC values of 0.98-3.9 and 1.95-3.9 µg/mL, respectively. Molecular docking analysis suggests that clonbrasterol A (2) targets CYP51, while nafuredin (3) inhibits fatty acid synthase to exert antifungal activity. These findings validate fungal co-cultivation as an effective strategy for discovering novel bioactive metabolites.