Plasma membrane integrity of the P. digitatum cells with citral (0 or 1/2MIC) were analyzed by propidium iodide (PI) staining coupled with fluorescence microscopy (Liu et al., 2010 (link)) with minor modifications. The 2-day-old mycelia from 50 mL PDB were collected and centrifuged at 4,000 g for 10 min. The collected mycelia were stained with 10 μg/mL of PI for 15 min at 30°C. Residual dyes were removed by washing twice with PBS (pH 7.0). Samples were observed with an ECLIPSE TS100 microscope (Nikon, Japan), and the fluorescence value was determined by a F97 PRO fluorescence spectrophotometer (Lengguang Technology, Shanghai, China). These experiments were also performed with antioxidant Cys at a concentration of 10 μM.
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