Quantifying fungal growth in 3D: an ergosterol-based method to distinguish growth modes

Abstract

Mycelium colonization of fungi on solid substrates occurs in three dimensions: hyphal extension on the substrate surface, mycelium network densification, and invasive hyphal growth into the substrate. Quantifying fungal biomass in three dimensions presents a challenge, because current methods either require the separation of mycelium from the host material or rely on pure 2D optical density measurements. Here, we quantitatively assessed fungal growth of Ganoderma sessile by measuring ergosterol, a sterol specific to fungi that effectively represents biomass estimation. To investigate and quantify the global fungal growth in 3D, we focused on two primary growth profiles: extensive growth, describing lateral colonization of hyphae across the substrate surface, and local growth, reflecting invasive penetration into the substrate and mycelium network densification. Distinguishing between these regimes is critical, as they contribute differently to biomass distribution and substrate interaction, enabling a more accurate and functionally relevant assessment of fungal growth in 3D systems. By measuring local and global ergosterol accumulation, we estimated that extensive growth contributes around 300 times more to global biomass accumulation than local growth. By altering the nutrient density and stiffness of the host materials, we assessed whether global biomass accumulation is primarily driven by extensive or local growth increase. Our results demonstrate that the common assumption that radial extension corresponds to biomass increase is not correct and consequently, not a reliable method for comparing fungal strains or growth conditions when interested in fungal biomass. Therefore, using ergosterol to measure the local and global growth allows the quantification of the contribution of both growth profiles to the final global biomass accumulation, providing an approach that can quantify the effects of substrate morphology and nutrient density.