Low Nitrogen Concentration and Acidic pH Enhance the Antifungal Activity Against Botrytis cinerea of an Endophytic Alternaria sp. Isolated from Opuntia ficus-indica

Endophytic fungi from the Cactaceae family are an underexplored source of bioactive secondary metabolites with potential applications in sustainable agriculture. This study investigated an endophytic fungus obtained from healthy cladodes of <i>Opuntia ficus-indica</i> growing in the Chilean Andean Precordillera. The influence of culture conditions, specifically pH and nitrogen concentration, on the production of diffusible and volatile antifungal compounds against the phytopathogen <i>Botrytis cinerea</i> was evaluated using dual-culture (confrontation) and sandwiched Petri dish assays. Morphological characteristics and molecular analyses confirmed that the isolate belongs to the genus <i>Alternaria</i>. Antifungal activity increased significantly under acidic conditions and limited nitrogen availability. The strongest inhibition by volatile compounds occurred at pH 4.5 and the lowest concentration of ammonium tartrate. Furthermore, ethyl acetate extracts at 40 mg/L obtained from <i>Alternaria</i> sp. cultures grown at pH 4.5 with 2.3 g/L ammonium tartrate inhibited <i>B. cinerea</i> mycelial growth by 60%. The study provides a framework for improving the yield of antifungal metabolites produced by <i>Alternaria</i>, contributing to the development of biofungicides for gray mold control.