Influence of Substrate Stiffness on Growth, Morphology, and Mechanical Behavior of Penicillium rubens

The ability of filamentous fungi to adapt to the mechanical properties of their surrounding environment is critical for their ecological success, pathogenicity, and applications in biofabrication. Substrate stiffness regulates multiple aspects of fungal growth, including colony expansion, hyphal extension dynamics, and internal morphology, yet comprehensive quantitative analyses remain limited. Penicillium rubens, a model filamentous fungus, provides an excellent system to systematically dissect the biomechanical coupling between substrate stiffness and fungal morphogenesis. In this study, we investigated how substrate viscoelasticity modulates radial growth, vertical architecture, hyphal extension rates, and mechanical properties of mature colonies. Potato Dextrose Agar (PDA) substrates were prepared at concentrations of 1%, 2%, and 5% to generate a stiffness gradient. Rheological characterization showed that storage modulus (G') increased from ~10³ Pa for 1% PDA to ~10⁵ Pa for 5% PDA. Colonies were monitored over 21 days for radial growth, and short-term hyphal dynamics were captured through 24-hour time-lapse imaging. Vertical architecture was analyzed through cross-sectional imaging and validated with compression rheology under constant force. By combining morphological, kinetic, and mechanical measurements, we established a detailed quantitative framework linking substrate stiffness to fungal colony structure and growth behavior.