Laccase-IN-3 (Compound 2b) is a laccase inhibitor with an IC50 of 1.02 μM, demonstrating significant antifungal activity. It effectively inhibits Botryosphaeria dothidea with an EC50 of 0.17 mg/L. By binding to the active site of laccase, Laccase-IN-3 disrupts its catalytic function and also compromises the integrity of the pathogen's cell membrane while increasing reactive oxygen species (ROS).

Laccase-IN-3 at a concentration of 25 mg/L exhibits inhibition rates against various fungi species, achieving 98.2% against B. dothidea, 92% against S. sclerotiorum, 93.9% against F. graminearum, 53.1% against B. cinerea, 59.9% against P. capsici, 22.0% against P. infestans, and 81.5% against P. nicotianae. Its EC50 values for different fungi are 0.17 mg/L (B. dothidea), 2.55 mg/L (S. sclerotiorum), and 2.03 mg/L (F. graminearum). Laccase-IN-3 concentrations ranging from 0.79 to 6.25 mg/L inhibit fungal growth by disrupting the morphology of B. dothidea mycelia. At concentrations between 10 and 25 mg/L over 120 minutes, Laccase-IN-3 compromises the integrity of B. dothidea mycelial cell membranes, increasing membrane permeability, lowering extracellular pH, and destabilizing the intracellular environment.

Laccase-IN-3, at concentrations of 100 and 200 mg/L over 6 days, exhibits significant protective activity (92.6% and 86.8%) and therapeutic activity (85.0% and 78.9%) against B. dothidea infection in apples, outperforming Boscalid.