Establishment of Protoplast Preparation and Genetic Transformation Methods in Two Ilyonectria Species

Ilyonectria is a common soil-inhabiting fungal genus that comprises numerous plant phytopathogenic species capable of infecting a wide array of crops, medicinal herbs, and horticultural plants. However, the lack of a reliable and efficient genetic transformation method has severely hindered the elucidation of the pathogenic mechanisms of Ilyonectria pathogens. In this study, we established an efficient protoplast-mediated genetic transformation method for two dominant Panax root rot pathogens, I. robusta and I. vredehoekensis. Key parameters governing high-quality protoplast preparation, including mycelium culture time, enzyme composition, osmotic stabilizer type, digestion speed, and digestion time, were systematically optimized. Subsequently, orthogonal experiments were conducted to optimize the PEG-CaCl₂-mediated transformation conditions and to screen regeneration media for protoplasts. The optimal enzymatic system is composed of 20 mg/mL driselase and 10 mg/mL lysing enzyme, with 0.7 M NaCl as the osmotic stabilizer. Under these conditions, high-viability and high-quality protoplasts were obtained from I. vredehoekensis after 3 h of digestion at 150 rpm, and from I. robusta after 2 h of digestion at 100 rpm, yielding 5.52 × 10⁷ CFU/mL and 5.75 × 10⁷ CFU/mL protoplasts, respectively. Efficient transformation was achieved using a mannitol-prepared STC buffer mediated by 40% PEG4000. PCR and fluorescence microscopy verified positive transformants. Additionally, pathogenicity assays showed no significant differences in virulence between the transformed and wild-type strains, suggesting that the transformation procedure did not alter virulence. To the best of our knowledge, this is the first study to successfully establish genetic transformation methods for I. robusta and I. vredehoekensis, providing an essential technical platform for functional gene analysis, pathogenicity studies, and host–pathogen interaction research. In addition, the optimized transformation strategy may serve as a valuable reference for studies on other Ilyonectria species.