Adaptation of Fusarium graminearum to Tebuconazole Yielded Descendants Diverging for Levels of Fitness, Fungicide Resistance, Virulence, and Mycotoxin Production

Abstract

Azole fungicides play a prominent role for reliable plant disease management. However, quantitative azole resistance has been shown to develop in fungal pathogens, including Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Due to widespread application of azole fungicides, resistance may accumulate to higher degrees in fungal field populations over time. Although azole fungicides are prominent components in FHB control, little effort has been made to investigate azole resistance in F. graminearum. We allowed F. graminearum strain NRRL 13383 to adapt to an azole fungicide in vitro, applying a strongly growth-reducing but sublethal dose of tebuconazole. Two morphologically distinguishable azole-resistant phenotypes were recovered that differed with regard to levels of fitness, fungicide resistance, virulence, and mycotoxin production. Isolates of the adapted "phenotype 1" exhibited azole-specific cross-resistance, whereas "phenotype 2" isolates displayed the phenomenon of multidrug resistance because the sensitivity to amine fungicides was also affected. Assessment of individual infected spikelets for mycotoxin contents by high-performance liquid chromatography mass spectrometry and for Fusarium DNA by quantitative polymerase chain reaction indicated that some of the adapted isolates produced significantly higher levels of nivalenol per fungal biomass than the NRRL 13383 strain.

Sensitivity of Fusarium graminearum causing head blight of wheat in Brazil to tebuconazole and metconazole fungicides

“…This agrees with results of Becher et al (2010) who did not observe changes in both mycelial growth and sporulation between resistant (induced by successive cultures on media amended with 10 mg/L of tebuconazole) and the respective wild isolate. These results are supported by the recent knowledge that sensitivity to triazoles is under the control of three paralogous genes (cyp51A, cyp51B e cyp51C), which are not related to mycelial growth (Liu et al, 2011).…”

“…In Europe and Asia, the increasing usage of triazoles in FHB management is suspected to have led to selection of less sensitive isolates of F. graminearum (Klix et al, 2007;Yin et al, 2009;Becher et al, 2010). Fungal resistance to triazoles is associated with mutations in the 14-α-demethylase (CYP51) gene and overexpression of the ATPbinding cassette transporters (Ma and Michailides, 2005;Liu et al, 2010).…”

“…Fungicide sensitivity was quantified by measuring the radial growth of the cultures on PDA (potato dextrose agar) media (Becher et al, 2010;Tateishi et al, 2010) amended with increasing concentrations of tebuconazole (Tebuconazole Nortox 200 EC, Nortox S.A.) or metconazole (Caramba 90, BASF S.A.) (0.0; 0.01; 0.05; 0.1; 1 and 10 mg/L). The fungicides were added to PDA media in three replicates each.…”

“…Effective concentration (mg/L) leading to a 50% reduction (EC 50 ) of mycelial growth was calculated on the basis of linear regression analysis of log-transformed fungicide efficiency and the log10 transformed fungicide concentrations (Becher et al, 2010). Bartlett test was used to assess variance homoscedasticity and the data from the two experiments were combined (P < 0.05) for analysis (Chen et al, 2012).…”

“…Significant difference between the two groups of ten isolates (the more and less sensitive groups) was assessed using t-test (Becher et al, 2010). Cross-resistance was evaluated by the significance of the Spearman correlation coefficient (r s ) between the log transformed EC 50 values of tebuconazole and metconazole.…”