Alginate-calcium encapsulation of root-associated fungi: viability, storage stability and functional recovery

The formulation and preservation of fungal inocula remain critical challenges for their effective use in forestry and ecological restoration. In this study, a calcium-alginate bead formulation was developed for two root-associated fungal species, the ectomycorrhizal fungus Hebeloma crustuliniforme and the dark septate endophyte Phialocephala fortinii, and its biological performance was evaluated in terms of encapsulation load, short-term storage stability, and post-storage functional recovery. Encapsulation resulted in reproducible and quantifiable inoculum loads per bead, with high initial viability of the entrapped propagules. Storage temperature strongly influenced fungal viability: refrigerated storage at 4 °C largely preserved viability for 30 days, whereas storage at 25 °C led to a progressive loss of viable propagules in both species. Functional recovery following storage was confirmed through hyphal outgrowth assays, demonstrating rapid reactivation and extensive mycelial growth from the alginate beads upon transfer to a nutrient medium, with radial growth comparable to non-encapsulated agar plug controls. Preliminary growth assays on glycine-loaded starch-based aerogels revealed species-dependent substrate colonization behavior. P. fortinii exhibited visible hyphal colonization of the porous biomaterial, whereas H. crustuliniforme showed limited or no detectable growth under the same conditions. Together, these results suggest that alginate encapsulation preserves the viability and functional integrity of root-associated fungi during short-term refrigerated storage. This formulation provides a standardized and biologically compatible carrier for fungal inocula and supports the development of integrated bio-based delivery systems for forestry and ecological restoration applications.