Enhanced Biocontrol of Cotton Verticillium Wilt Through Optimized Solid-State Fermentation of Myxococcus fulvus KS01 Using Insect Frass as a Matrix

Cotton Verticillium wilt, caused by <i>Verticillium dahliae</i>, is a devastating soil-borne disease that severely limits global cotton production. While <i>Myxococcus fulvus</i> KS01 has demonstrated potent antagonistic activity and multi-functional biocontrol effects against <i>V. dahliae</i>, its practical application has been hindered by low myxospore yields and inconsistent efficacy in initial solid-state fermentation (SSF). This study aimed to optimize the SSF process for strain KS01 to maximize myxospore production and systematically evaluate its biocontrol efficacy against Verticillium wilt. Using a mixture of wheat straw and <i>Protaetia brevitarsis</i> frass (an agricultural byproduct) as the base substrate, we utilized single factor experiments and Response Surface Methodology (RSM) to optimize nutritional supplements and fermentation parameters. The optimized SSF process was determined as follows: a 3:1 (<i>w</i>/<i>w</i>) frass-to-straw ratio, supplemented with 3.08% potato starch and 1.05% yeast powder, with a 15.03% inoculum size, 65.05% moisture content, and an initial pH of 7.0, fermented at 30 °C for 6 days. Under these conditions, the myxospore concentration reached 6.61 × 10⁷ CFU/g, representing a 131.2-fold increase compared to unoptimized conditions (5.0 × 10⁵ CFU/g). Greenhouse pot trials showed that the optimized KS01 solid agent achieved a control efficacy of 71.9%. In field trials conducted in heavily infested soil, the agent maintained control efficacies of 71.2% at the budding stage and 54.5% at the bolling stage, significantly outperforming the commercial fungicide Benziothiazolinone (51.4% and 41.4%, respectively) and the sterile substrate control. Furthermore, application of the KS01 agent significantly promoted cotton growth, with seed cotton yield reaching 5380.0 kg/ha, equating to a 50.4% reduction in yield loss compared to the untreated control. Our results demonstrate that the valorization of <i>P. brevitarsis</i> frass through optimized SSF significantly enhances the production and field performance of <i>M. fulvus</i> KS01. This study provides a novel technical framework and a robust microbial resource for the sustainable management of Verticillium wilt in saline alkali cotton production systems.