How temperature reshapes interactions in mixed cultures of mycotoxigenic fungi

Climate change is shifting the distribution of mycotoxigenic fungi, which may alter patterns of co-occurrence in staple crops. Yet, quantitative, reproducible in vitro approaches to characterise multispecies growth and interaction outcomes under environmentally relevant conditions remain limited. Here, we developed a small-scale workflow to quantify temperature-dependent interaction outcomes among three major cereal-associated fungi, Aspergillus flavus, Fusarium verticillioides, and F. graminearum. High-frequency optical density time series were collected in 96-well liquid cultures across 10 °C–45 °C. Optical density trajectories were summarised with three-parameter logistic fits used as empirical descriptors of culture-level dynamics (carrying capacity, growth rate, and inflexion point). Species-specific quantitative PCR was then used to quantify endpoint DNA-based species contributions in mixed cultures. Monoculture responses were consistent with established thermal preferences. In co-cultures, optical density-derived interaction patterns indicated a shift in mixed-culture optical performance towards warmer conditions. Quantitative PCR endpoint composition supported an increasing contribution of A. flavus at elevated temperatures, with reduced contributions of both Fusarium species. These results show that temperature reshapes interaction outcomes through changes in culture-level growth kinetics and endpoint community composition. The workflow provides data suitable for predictive modelling of climate-driven mycotoxin risk and supports interpretation of fungal interaction outcomes in warming agroecosystems.