Cultivation and preliminary chemical analyses of the sclerotium-forming, termite fungus garden-associated Xylaria minima

Xylaria nigripes and its close relatives, specialized for their associations with termite fungus combs, are capable of producing massive sclerotia within the chambers of dead termite nests. These sclerotia, traditionally referred to in Chinese medicine as “Wulingshen,” exhibit a wide range of pharmacological activities. Massive Xylaria sclerotia are typically harvested from natural habitats, and their formation under laboratory conditions has rarely been reported. This study induced the formation of small sclerotia in Xylaria minima by incubating mature Macrotermes fungus combs. Sclerotium formation on the comb was characterized by an inconspicuous vegetative growth phase, rapid sclerotial development, early dormancy, and temporally delayed reproduction. Sclerotia formed more sporadically on potato dextrose agar (PDA), water agar (WA), and oat-based media. In contrast, growth on comb leachate agar (CLA) was faster, and sclerotia developed readily. The sclerotia of X. minima show strong potential for cultivation using comb materials and agricultural substrates. Exploratory chemical analyses revealed that sclerotia were enriched in crude polysaccharides, alkaloids, and terpenoids, as well as amino acids and amino acid derivatives such as L-glutamic acid and γ-aminobutyric acid (GABA), compared with mycelia. These findings indicate that X. minima does not exhibit strict substrate specificity for sclerotium formation and highlight its potential for future studies on cultivation and bioactive compound discovery. This study additionally highlights that the sclerotium-forming habit in comb-associated Xylaria species has likely evolved independently multiple times, occurring in both the newly reported small-sclerotium-forming X. minima and the X. nigripes species complex.