Pathogenicity assays

For pathogenicity tests, conidia were harvested from 7-day-old cultures grown in liquid CM by filtration through two layers of Miracloth and resuspended at 10⁶ conidia/ml in sterile distilled water. The roots of the 1-year-old smoke tree seedlings were inoculated with conidial suspensions for 10 min. Control plants were mock-inoculated with sterile distilled water. All inoculated smoke trees were then replanted into the soil. Ten smoke tree seedlings were tested per strain, and the experiment was repeated three times. For better observation, 1-month-old tobaccos were also inoculated with 10⁶ conidia/ml conidial suspensions for 1–3 days, and the GFP-expressing strains of wild-type and ΔVdMcm1-5 used for tobacco infection tests were named as WT-GFP and ΔVdMcm1-5-GFP. For scanning electron microscopy, the freshly isolated roots of the smoke trees were inoculated with 10⁷ conidia/ml conidial suspensions for 24 h at room temperature, the water was sopped up with filter papers, then the dried roots were sputtered with Au and used for observation. To examine the ability of conidial adhesion, 10⁵ conidia of each strain were spread over the Hybond™-N⁺ membranes (GE Healthcare, UK), which were overlaid onto PDA plates at room temperature, and these membranes were then gently washed with 1 ml sterile distilled water after 18 h incubation. Approximately 750 μl conidial suspensions were obtained after gentle washing, and 50 μl conidial suspensions were used to coat the PDA plate. The number of colonies was determined after 5 days of growth on PDA at room temperature. The experiments were repeated three times.