Tracking the infection dynamics of Fusarium oxysporum in Codonopsis pilosula based on GFP labelling

Introduction Codonopsis pilosula root rot, caused by Fusarium oxysporum , has caused severe damage to the C. pilosula industry. Due to the unclear pathogenic mechanisms of F. oxysporum on C. pilosula , the effective implementation of control measures has been greatly restricted. Methods An efficient protoplast preparation and genetic transformation system was established for F. oxysporum FO-1, enabling real-time tracking of fungal colonization in C. pilosula . Single-factor experiments were conducted to determine optimal conditions, followed by response surface methodology to further optimize enzymatic parameters. PEG-mediated transformation was performed to generate GFP-tagged strains for infection tracking. Results Single-factor experiments identified the optimal conditions as 12-hour-old mycelia treated with 0.7 M NaCl and 20 mg/mL driselase at 28°C and 180 rpm for 4 h. Response surface methodology optimized parameters to 188.24 rpm, 4.51 h, and 27.5°C, yielding 1.44 × 10 8 CFU/mL protoplasts, representing a 30-fold improvement over single-factor optimization. PEG-mediated transformation produced 11 GFP-tagged strains, with FO-GFP-7 retaining wild-type morphology, growth rate, and pathogenicity. Microscopic observation revealed infection dynamics: conidia aggregated at the rhizome by 2 days post-inoculation (dpi), followed by phloem colonization at 4 dpi and vascular invasion at 6 dpi. Wound inoculation at the rhizome accelerated infection, consistent with field disease patterns linked to soil microfauna-induced injuries. Discussion This study provides a robust platform for investigating F. oxysporum pathogenicity in C. pilosula and offers guidance on protective measures to maintain rhizome integrity during cultivation.