Jem 1400 flash

Foc TR4 was inoculated on the PDA medium with 500 μg/ml of strain 5–10 extracts at 28°C for 5 days. The Foc TR4 mycelia were fixed with glutaraldehyde (2.5%, v/v) overnight at 4°C and postfixed using osmium tetroxide (1%, v/v). After washing three times with PBS (0.1 mol/L, pH 7.0), the samples were dehydrated with different gradients of ethanol solution and embedded in the Epon 812 resin at 37°C for 12 h, 45°C for 12 h, and 60°C for 24 h, respectively (Xing et al., 2014 ). The Foc TR4 mycelia were sliced by an ultra microtome (Leica, UC6 CM1950, Germany) and stained with uranyl acetate and citric acid for 30 min, respectively. The ultrastructure of transverse Foc TR4 mycelia was detected by TEM (JEM-1400 Flash, Hitachi Limited, Japan).

The morphology of Foc TR4 cells was detected after treatment with crude extracts of the isolate using a scanning electron microscopy (SEM, model S-4800, Hitachi Limited, Japan). One milliliter of Foc TR4 (1.0 × 10⁵ spores mL^–1) was inoculated with 25 μg mL^–1 of crude extracts for 24 h. The spores were fixed with 2.5% (v/v) of glutaraldehyde in a phosphate-buffered saline solution (0.1 mol L^–1, pH 7.0, PBS) for 2 h and dehydrated using a series of increasing concentrations of ethyl alcohol (30–100%, v/v) for 10 min. Samples coated with a film of gold-palladium alloy under vacuum were detected by SEM (Ruiz et al., 2016 (link)). To evaluate effects of crude extracts on cell ultrastructures, the Foc TR4 sample was fixed with OsO₄ (1%, w/v) in 0.1 mol L^–1 of PBS for 1 h at room temperature, then dehydrated by a gradient solution of methanol (50 to 100%) for 10 min (Lou et al., 2011 (link)). The samples were embedded in a spurr resin and cut with an Ultracut Ultramicrotom (EM UC6, Leica, Germany). These sections were stained with the saturated uranyl acetate and the lead citrate and observed by a Transmission Electron Microscope (TEM, JEM-1400 Flash, Hitachi Limited, Tokyo, Japan).