Towards a Novel Biocontrol Strategy: High Performance of Optimised Cell Wall‐Degrading Enzymes Secreted by Escovopsis primorosea LBM 277

Fungi of the genus Escovopsis are specialised mycoparasites of the mutualistic fungus Leucoagaricus gongylophorus, cultivated by leaf-cutter ants. Harnessing this natural antagonism represents a promising and environmentally friendly strategy for the indirect biological control of these agroforestry pests. This study optimised and characterised the production of cell wall-degrading enzymes (CWDEs), key effectors in the degradation of the host, by the Escovopsis primorosea LBM 277 strain. Using submerged fermentation and Box-Behnken response surface methodology (RSM-BBD), we identified optimal culture conditions, including the concentrations of carbon and nitrogen sources, initial pH and inoculum size. Under optimised conditions, protease, β-1,3-glucanase and chitinase activities increased by 3.5-, 6.7- and 6.8-fold, respectively. The enzymes remained active for at least 30 days at room temperature and in the pH range 4-6, resembling the microenvironment of ant fungal gardens. Zymographic analysis revealed one isoform of protease, one of β-1,3-glucanase and two chitinase isoenzymes. This is the first report combining RSM-based optimization with biochemical profiling of CWDEs in Escovopsis. These findings highlight the need for further functional assays to validate the role of these enzymes in the ant-symbiont fungus-mycoparasite interaction.