| Fusarium head blight,a worldwidely-distributed and destructive disease on wheat,is mainly caused by Fusarium graminearum.In our country,MBC and other benzimidazole fungicides have been used to control FHB since 70s.However,the control effect of benzimidazole fungicides have dropped due to the increasingly prominent drug resistance in recent years.Our study found that tubulin beta 2 mutations at different amino acid positions were the major causes of resistance to benzimidazole resistance.Mutation at codon 167 on ?2-tub of Fusarium graminearum results in resistance to MBC and enhancement of toxin production,inhibiting protein synthesis in eukaryotic cells and threatening human and livestock health.Studies have shown that low doses of fungicide might trigger intracellular metabolic mechanism in the pathogen,resulting in higher trichothecenes.Early research has shown that tubulin beta 2 mutations at different amino acid positions regulate DON indirectly,which is associated with Fghxk hexokinase gene expression in Fusarium graminearum.In addition,the concentration of pyruvic acid in MBC-resistant strain 167 was significantly higher than that in sensitive strain 2021,which is consistent with DON toxin production.Therefore,in order to investigate whether pyruvic acid takes part in pathway that tubulin beta2 mutations at different amino acid position regulating DON toxin,we explore the relationship with synthesis and metabolism of pyruvic acid and DON toxin.First,we explore whether pyruvic acid affect the synthesis of DON toxin and virulent in Fusarium graminearum.We select wild-type sensitive strains,the MBC-resistant strains induced by laboratory and Fghxk knockout mutant as research materials.The determination of pyruvate show that the concentration of pyruvic acid in MBC-resistant strains induced by laboratory was significantly higher than that in sensitive strain 2021,which is consistent with DON toxin production.We found that Exogenous pyruvic acid could increase the production of DON toxin significantly as well as gene expression of Tri5 and Tri6.Pathogencity assays showed that exogenous pyruvate could encourage the infection of wheat in Fusarium graminearum,indicated that the increaseed trichothecene production may be related with this.All these results conclude that Exogenous pyruvic acid can affect the synthesis of DON toxin and pathogenicity in Fusarium graminearum.Exploring the relationship between exogenous pyruvic acid and DON toxin,then we strengthening pyruvic acid synthesis for further research in Fusarium graminearum.Pyruvate was produced in the pathway of glycolysis that catalysis by hexokinase which is controlled by Fghxk mainly.So we overexpressed the hexokinase in Fusarium graminearum to explore the relationship between pyruvic acid and DON-producing.Our research showed that mutant overexpressing Fghxk grow slower,and their aerial hyphae significantly reduced,indicating that Fghxk overexpression can affect the growth of hypha.The sensitivity of the cell walls of various agents and MBC have no change while the resistance of ion osmotic pressure has been increased in the Fghxk overexpression mutant.Gene expression analysis showed that the expression of Tri family gene has been enhanced significantly when overexpressing Fghxk.In addition,Fghxk overexpression could enhance the synthesis of DON toxin.Carbon source utilization measurement show that overexpression of Fghxk gene could weak the use of fructose in Fusarium graminearum.In conclusion,Fghxk overexpression can influence morphological phenotypes and the synthesis of DON toxin in Fusarium graminearum.Pyruvic acid is mainly catabolised through the pyruvate dehydrogenase complex which plays an important role in the organism.Gene expression analysis showed that the expression of pyruvate dehydrogenase complex component Fglatl has increased significantly in the carbendazim resistant strains of the fields,therefore our research knockout Fglatl protein to further explore the relationship between the pyruvic acid and DON production.Our studies have shown that deletion of Fglatl could significantly affect the growth of hypha in both wild type 2021 strains and field resistance strain 167.In terms of osmotic pressure sensitivity,Fglatl knockout mutants were more sensitive to KC1 and LiC while performing NaCl resistance,incating that the Fglatl may be associated with the transportion of K+ and Li+.However,the resistance of fungicide carbendazim and cell wall toxics has been increased in Fglatl knockout mutant.the expression of Tri family gene has been decreased significantly as well as DON production in the Fglatl knockout mutant.Carbon source utilization measurement show that deletion of Fglatl gene could weak the use of fructose,sugar and glucose in Fusarium graminearum.All these results conclude that Fglatl genes has been involved in the regulation of morphology phenotype and DON toxins synthesis in Fusarium graminearum. |
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