Figure 5.

Effect of the peptides on the membrane permeability of phytopathogenic fungi. The membrane permeability was analyzed after the peptide treatments using SYTOX Green staining. The fungi Botrytis cinerea (A,B) and Colletotricum gloeosporioides (C) were cultured on optimal rich (PD) agar media for 7–10 days, and then, their spores were recovered and resuspended in sterile water. (A,C) Five microliters of the fungal spores (10⁶ spores/ml for (A) and 10⁷ spores/ml for (C) was mixed with 10μl of SYTOX Green (2μM) and 5μl of water, 4% Triton X-100, or pepD2M (64μg/ml). (B) Twenty-five microliters of the fungal spores (10⁵ spores/ml) was mixed with 25μl of PD broth (pH=6.36–6.37), after which 5μl of the spore mixture was transferred to a glass slide and kept moist at room temperature for 24h. Then, 10 microliters of SYTOX Green (2μM) and 5μl of water, 4% Triton X-100, or pepD2M (64μg/ml) were added to the mixture. Crude peptides that were not purified via HPLC were used. The SYTOX-Green-stained cells were observed via confocal microscopy after they were incubated for 2h in the dark (A–C). In (A,C), the ratios of the spores with SYTOX Green fluorescence are from a single experiment that was repeated at least three times with similar results. Bc, Botrytis cinerea; Cgl, Colletotricum gloeosporioides. Bar=20μm.