CLEO induced ROS production in F. graminearum
In the present study to examine the cell death, the production of reactive oxygen species (ROS) was assessed in CLEO treated mycelium using DCFH-DA staining followed by van der Weerden, Lay, and Anderson (2008). The hydrophobic non-fluorescent molecule (DCFH-DA) enters quickly into the cell and hydrolyses to DCFH molecule by intracellular esterases, which further oxidized to fluorescent molecule 2-electron product 2′,7′dichlorofluorescein (DCF). The percentage of release of reactive oxygen species increased with increasing the concentration of CLEO in a dose-dependent manner (Fig. 4a). A significant difference in fluorescence intensity was observed upon DCF-DA staining to CLEO treated and untreated samples under a fluorescent microscope (Fig. 4b). The results of the present study indicated that, the accumulation of free radicals in CLEO treated mycelium may be responsible for the apoptotic cell death and altered toxin metabolic pathway gene regulation thus to decrease in toxin production. In this context, further experiments are needed to conclude these effects at the cellular level.