Tracking the infection dynamics of in based on GFP labelling.

Introduction: root rot, caused by , has caused severe damage to the industry. Due to the unclear pathogenic mechanisms of on , the effective implementation of control measures has been greatly restricted.

Methods: An efficient protoplast preparation and genetic transformation system was established for FO-1, enabling real-time tracking of fungal colonization in . 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 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 and offers guidance on protective measures to maintain rhizome integrity during cultivation.