Hydrogen peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON) production by Fusarium graminearum

Abstract

Background: Fusarium head blight is a very important disease of small grain cereals with F. graminearum as one of the most important causal agents. It not only causes reduction in yield and quality but from a human and animal healthcare point of view, it produces mycotoxins such as deoxynivalenol (DON) which can accumulate to toxic levels. Little is known about external triggers influencing DON production.

Results: In the present work, a combined in vivo/in vitro approach was used to test the effect of sub lethal fungicide treatments on DON production. Using a dilution series of prothioconazole, azoxystrobin and prothioconazole + fluoxastrobin, we demonstrated that sub lethal doses of prothioconazole coincide with an increase in DON production 48 h after fungicide treatment. In an artificial infection trial using wheat plants, the in vitro results of increased DON levels upon sub lethal prothioconazole application were confirmed illustrating the significance of these results from a practical point of view. In addition, further in vitro experiments revealed a timely hyperinduction of H2O2 production as fast as 4 h after amending cultures with prothioconazole. When applying H2O2 directly to germinating conidia, a similar induction of DON-production by F. graminearum was observed. The effect of sub lethal prothioconazole concentrations on DON production completely disappeared when applying catalase together with the fungicide.

Conclusions: These cumulative results suggest that H2O2 induced by sub lethal doses of the triazole fungicide prothioconazole acts as a trigger of DON biosynthesis. In a broader framework, this work clearly shows that DON production by the plant pathogen F. graminearum is the result of the interaction of fungal genomics and external environmental triggers.

Figure 1

Effect of prothioconazole + fluoxastrobin (a), prothioconazole (b) and azoxystrobin (c) on conidial germination of F. graminearum. 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/l and 0.67 g/l. For each treatment and repetition 50 conidia were scored for their germination and percentage of conidial germination was calculated at 4 h (solid line), 24 h (dashed line) and 48 h (point dashed line) after staining with 0.02% of cotton blue in lactic acid. Experiment consisted of two repetitions for each treatment and the experiment was repeated three times. Graphs represent the average of all three experiments. Different letters at each data point indicate differences from the control treatment at 4 h (**), 24 h (*) and 48 h after analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons.

Figure 2

Effect of prothioconazole + fluoxastrobin (a), prothioconazole (b) and azoxystrobin (c) alone or in combination with catalase (d,e,f) on production of deoxynivalenol (DON) by F. graminearum. 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/l and 0.67 g/l in absence (a,b,c) or presence (e,f,g) of 1000 U/ml catalase. DON content in the medium was determined using a competitive ELISA approach 48 h after start of the experiments. Each bar is the result of two pooled samples to reduce variance. The experiment was repeated twice in time of which one representative experiment is shown in the figure. Different letters above bars indicate significant differences after analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons.

Figure 3

Effect of prothioconazole + fluoxastrobin (a), prothioconazole (b) and azoxystrobin (c) on extracellular H₂O₂ concentrations. 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/l and 0.67 g/l. H₂O₂ was measured at 4 h (solid line), 24 h (dashed line) and 48 h (point dashed line) using TMB (trimethylbenzidine) as a substrate in the presence of an overdose of peroxidase. The H₂O₂ concentrations were calculated based on a 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 at 4 h (**), 24 h (*) and 48 h after analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons.

Figure 4

Effect of prothioconazole + fluoxastrobin (a, c) and prothioconazole (b, d) in absence (dashed line) or presence (solid line) of exogenous catalase on the germination of F. graminearum conidia after 4 h (a, b) and 48 h (c,d). Conidia at a concentration of 10e⁶ 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. 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 of catalase and respectively prothioconazole + fluoxastrobin (a) and prothioconazole (b) on extracellular H₂O₂ 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 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.

Figure 6

Effect of exogenously applied H₂O₂ on germination (a, b, c) of F. graminearum and DON production (d,e,f) after 4 h (a and d), 24 h (b and e) and 48 h (c and f). Conidia at a concentration of 10⁶ conidia/ml were challenged with a tenfold dilution series of H₂O₂. 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. DON content in the medium was determined using a competitive ELISA approach. Each treatment was measured in duplicate and the experiment was repeated twice in time (dashed and solid line represent the two experiments).

Figure 7

In vivo effect of prothioconazole + fluoxastrobin on symptoms of F. graminearum (a) and DON content (b) after point inoculation of wheat ears 14 days after infection. Wheat ears (variety Cadenza) were inoculated with two droplets of 20 μl of conidia at a concentration of 10e⁶ conidia/ml. Infection spots were indicated with a marker. Ears were subsequently treated with a tenfold dilution series of fluoxastrobin + prothioconazole starting from 0.5 g/l + 0.5 g/l. For each treatment, 10 plants were assessed for Fusarium symptoms. This experiment was repeated twice in time with analogous results. The figure represents one representative experiment. 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.