A Novel Gene IrXyn01 Coding β-Xylanase Is Essential for Ilyonectria robusta Pathogenicity in Early Ginseng Infection

Ginseng (Panax ginseng), a traditional and valuable medicinal plant, is severely affected by rusty root rot caused by Ilyonectria robusta. Xylanase is hypothesized to play a key role in the pathogenicity of I. robusta. Through transcriptomic analysis during ginseng infection, the highly expressed xylanase gene IrXyn01 was identified. A PEG-mediated genetic transformation system for I. robusta was established in this study, with optimized protoplast preparation yielding 1.4 × 107 protoplasts/mL and 36% transformation efficiency. IrXyn01 was successfully knocked out by using the CRISPR/Cas9 method. No significant differences in colony morphology, growth rate, sporulation, and spore germination were observed between mutants and the wild-type stain, indicating IrXyn01 is unrelated to vegetative growth. Under salt stress (KCl and NaCl), mutant inhibition reached 37.2% and 48.2% compared with the wild-type, respectively. The gene also regulated nitrogen source utilization, particularly for ammonium sulfate (29.3% inhibition of the knockout). When xylan was the sole carbon source, ΔIrXyn01 showed 12.8% growth inhibition. Neutral xylanase activity in mutants remained low (35–46 nmol/min/mL), compared to 138–142 nmol/min/mL in the wild-type strain and complemented strains. Pathogenicity assays confirmed that the IrXyn01 knockout significantly reduced disease severity, with lower rusty root rot severity in ginseng inoculated with mutants. This study identified IrXyn01 as a virulence gene in I. robusta and improves our understanding of its molecular pathogenesis.