Sustained-release essential oil delivery system based on M-β-CD/CS-encapsulated honokiol nanocomposites for controlling rot disease in Phallus rubrovolvatus

Red rot disease of Phallus rubrovolvatus , triggered by Trichoderma sulphureum , severely undermines both the safety and nutritional quality of this valuable mushroom, resulting in considerable economic losses across its production chain. The search for effective, low-risk botanical fungicides has thus become an urgent priority. Although honokiol is a potent natural antifungal compound, its therapeutic potential is largely constrained by poor hydrophilicity and susceptibility to oxidation. In response to these challenges, we designed a honokiol-loaded M-β-CD/CS composite delivery system (HK@CS), rigorously characterized its in vitro antifungal performance against T. sulphureum, and further deciphered the molecular events contributing to its antifungal action. In this study, a HK@CS was developed by encapsulating honokiol in methyl-β-cyclodextrin and incorporating the inclusion complex into a chitosan matrix. The film showed high encapsulation efficiency (87.64%) and, in dissolution tests, markedly enhanced the apparent water solubility of honokiol in this composite system by over 1300‑fold. This marked increase, arising from the combined effect of cyclodextrin encapsulation and chitosan-based dispersion rather than from honokiol’s intrinsic solubility, underpins the sustained-release behavior of the formulation. HK@CS exhibited strong antifungal activity against T. sulphureum with a minimum inhibitory concentration of 19 μg·mL ⁻1 . Mechanistic dissection indicated that HK@CS compromises cellular membrane integrity through dysregulation of phospholipid metabolism, which in turn triggers membrane permeabilization and extravasation of intracellular solutes. The treatment also induced reactive oxygen species accumulation, suppressed antioxidant enzyme activity, and impaired energy metabolism. Transcriptomic and metabolomic analyses further demonstrated that HK@CS downregulated key genes involved in membrane lipid biosynthesis, including ned1 , DGAT1 , and Etnk1 , and reduced levels of critical membrane-associated metabolites such as phosphorylcholine and phytosphingosine. These molecular changes collectively compromised membrane integrity and disrupted cellular homeostasis. Overall, the HK@CS sustained‑release composite film exhibits promising in‑vitro antifungal activity against T. sulphureum by disturbing membrane lipid homeostasis, showing potential to be developed as an environmentally benign botanical biocontrol agent. Its desirable physicochemical properties and sustained‑release performance provide a theoretical basis for the future eco‑friendly control of Phallus rubrovolvatus red rot disease, though practical application still requires further validation.