Sterilized cellophane membrane

For the penetration assays, sterilized cellophane membrane (DINGGUO, Beijing, China) was overlaid onto minimal medium (glucose at 2 g/liter, NaNO₃ at 2 g/liter, KH₂PO₄ at 1 g/liter, MgSO₄-7H₂O at 0.5 g/liter, KCl at 0.5 g/liter, citrate at 10 mg/liter, ZnSO₄-7H₂O at 10 mg/liter, FeSO₄-7H₂O at 10 mg/liter, NH₄Fe(SO₄)₂-12H₂O at 2.6 mg/liter, CuSO₄-5H₂O at 0.5 mg/liter, MnSO₄-H₂O at 0.1 mg/liter, H₃BO₃ at 0.1 mg/liter, Na₂MoO₄-2H₂O at 0.1 mg/liter and agar at 20 g/liter). The cultures were incubated on the cellophane membrane for 3 days. The membranes were removed, and hyphae were observed in the underlying medium to determine if there were any breaches in the cellophane.

For penetration assays, sterilized cellophane membrane (DINGGUO, Beijing, China) was overlaid onto MM medium. The cultures were incubated on the cellophane membrane for various lengths of time. The membranes were removed, and hyphae were observed in the underlying medium to determine if there were any breaches of the cellophane. For DPI treatment, fungi were grown on MM medium overlaid with cellophane membrane for 1 day. Then, the membrane was transferred to MM medium with 25 μM DPI (Sigma) followed by another 2 days incubation and removed for penetration detection. The experiments were repeated independently at least three times.
The fungus was recovered from infected cotton as follows: the stem sections above cotyledons of cotton plants were taken at 30 days after inoculation and surface-sterilized for 1 min in 70% ethanol, followed by 60 min in 10% hydrogen peroxide. The samples were then rinsed three times with sterile water, cut into 1 cm slices and cultured at 26°C on PDA medium.
Upland cotton was inoculated with V592 and transformants for the infection assays, using our laboratory unimpaired root-dip inoculation method [36 (link)]. Disease severity was counted by the percentage of cottons that showed wilting symptom at 30 dpi after inoculation. The infection assays for each mutant colony were repeated at least three times.

Sterilized cellophane membrane (DINGGUO, Beijing, China) was overlaid onto MM. Equal amounts of conidia collected from V. dahliae strains as indicated were inoculated on the cellophane membrane and grown for 3 days. The cellophane membrane was then removed and further cultured for an additional 3 days.