5 g/l + 0.5 g/l, 0.83 g and 0.67 g/l. At the beginning of the experiment, catalase (1000 U/ml) was added to the germinating conidia. For each treatment and repetition
50 conidia were scored for their germination after staining with 0.02% of cotton blue in lactic acid and percentage of conidial germination was calculated. This experiment was repeated twice in time. Different letters at each data point indicate differences from the control treatment after analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons. Figure 5 Effect of a combined application Dabrafenib supplier of catalase and respectively prothioconazole + fluoxastrobin (a) and prothioconazole (b) on extracellular H 2 O 2 concentrations at 4 h after fungicide application. Conidia at a concentration of 106 conidia/ml were challenged with a
tenfold dilution series of fluoxastrobin + prothioconazole, azoxystrobin and prothioconazole starting from 0.5 g/l + 0.5 g/l, 0.83 g and 0.67 g/l in the absence (dashed line) or presence of 1000 U/ml catalase (solid line). H2O2 was measured at 4 h using TMB (trimethylbenzidine) as a substrate in the presence of an overdose of peroxidase. The H2O2 concentrations were calculated based on a high throughput screening standard curve included in each experiment. Each data point is the result of three repetitions and the experiments were repeated twice in time. Different letters at each data point indicate differences from the control treatment after analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons. Stress-induced H2O2
accumulation upon fungicide application is necessary and sufficient as a trigger to induce DON To further decipher a direct link between H2O2 at one hand and the production of the mycotoxin DON at the other acetylcholine hand, the accumulation of DON was monitored upon exogenously single pulse application of H2O2ranging from 0.01 mM up to 100 mM. H2O2 influenced germination of F. graminearum conidia in a concentration-dependent manner (Figure 6). As early as 4 h after the start of the assay, exogenously application of H2O2 at concentrations from 1 mM up to 100 mM retarded or stopped conidial germination. The sub lethal concentration of 10 mM H2O2 induced DON production as fast as 4 h after application of H2O2 in one of the experiments. In the other experiment, 4 h was probably just too early to observe the increased DON production and in this experiment, the increment in DON was observed at 24 h. The ability of 10 mM H2O2 to initiate DON production is in concordance with H2O2 concentrations induced by sub lethal prothioconazole concentrations (Figure 3A). At later time points, DON did not further accumulate and concentration remained the same for the subsequent 24 and 48 h time points.