Chitosan–κ-Carrageenan–Lysozyme Nanoparticles Disrupt Appressorium Formation and Cellular Architecture in Colletotrichum siamense with Low Sensitivity to Chitosan

Colletotrichum species are among the most destructive phytopathogens worldwide, with appressorium-mediated penetration representing a critical stage in host infection. Targeting this morphogenetic transition offers a promising strategy for sustainable disease control by interfering with the infection process rather than solely inhibiting fungal growth. In this study, chitosan–κ-carrageenan nanoparticles (CS–κ-CRG) without and with lysozyme (CS–κ-CRG/Lz) were synthesized, characterized, and evaluated for their ability to inhibit appressorium formation in Colletotrichum siamense, a strain exhibiting low sensitivity to chitosan. The nanoparticles showed monodisperse size distributions, with hydrodynamic diameters of 503 and 333 nm for CS–κ-CRG and CS–κ-CRG/Lz, respectively, positive surface charges of approximately +26 mV, spherical morphology, and a lysozyme encapsulation efficiency of 63%. Both formulations significantly reduced conidial viability and delayed germination, inducing morphological alterations such as conidial swelling, hyphal deformation, and vacuolization. Fluorescence microscopy using calcofluor white and propidium iodide revealed disturbances in cell wall organization and loss of membrane integrity. Both nanomaterials markedly affected appressorium development in a concentration- and formulation-dependent manner. Notably, CS–κ-CRG/Lz showed stronger suppression of appressorium formation, whereas at 200 µg·mL−1, CS–κ-CRG nanoparticles stimulated appressorium formation, suggesting that sublethal nanoparticle stress may trigger compensatory or hyper-pathogenic responses. These findings highlight the potential and complexity of utilizing chitosan-based nanomaterials for phytopathogen management and emphasize the importance of mechanistic and dose–response evaluations before field application.