Quantitative evaluation of early fungal morphogenesis identifies markers of colony establishment

Quantitative methods to identify when a fungal colony becomes an established, radially expanding structure remain limited, particularly during early growth on solid media. Here, we present an image-based framework to evaluate early colony morphology in the filamentous fungus Trichoderma atroviride for different inoculum sizes. Two-dimensional colonies initiated either from a single conidium or from thousands of spatially confined conidia were imaged at defined time points following germination and analyzed using digital image processing to extract morphology metrics based on skeleton and tip structure. The framework quantifies colony local density, geometry, angular symmetry of biomass distribution, hyphal tip orientation, and the structure and roughness of the expanding growth front. Multiple conidia rapidly produced colonies with stable circular geometry, uniform angular biomass distributions, radially oriented hyphae, and smooth growth fronts, indicating early coordinated expansion. In contrast, colonies from single conidia showed prolonged anisotropic and highly variable growth before converging to the same morphological state. Overall, sustained circularity, reduced angular variability, and growth front smoothing emerged as reproducible markers of colony establishment. Our approach provides a reproducible method to quantify early microbial colony development.