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

## TL;DR

This study improves the production and effectiveness of a biocontrol agent against cotton wilt disease using optimized fermentation with insect frass.

## Contribution

A novel solid-state fermentation method using insect frass enhances myxospore yield and biocontrol efficacy of Myxococcus fulvus KS01.

## Key findings

- Optimized fermentation increased myxospore concentration 131.2-fold to 6.61 × 10⁷ CFU/g.
- The biocontrol agent achieved 71.9% control efficacy in greenhouse trials and outperformed commercial fungicides in field trials.
- Application of the agent reduced cotton yield loss by 50.4% compared to untreated controls.

## Abstract

Cotton Verticillium wilt, caused by Verticillium dahliae, is a devastating soil-borne disease that severely limits global cotton production. While Myxococcus fulvus KS01 has demonstrated potent antagonistic activity and multi-functional biocontrol effects against V. dahliae, 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 Protaetia brevitarsis 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 (w/w) 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 P. brevitarsis frass through optimized SSF significantly enhances the production and field performance of M. fulvus 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.