Regulation Mechanism Of Succinate Dehydrogenase Inhibitors To The DON Biosynthesis In Fusarium Graminearum

Fusarium head blight（FHB）is a global economic disease.Fusarium graminearum and Fusarium asiaticum are the main pathogen in China,which is a typical climatic disease.In addition to causing loss of grain yield,F.graminearum produces a series of toxic secondary metabolites during the infestation of the wheat grain,which is dominated by DON.It not only threatens the health of humans and animals,but it is also an important virulence factor.At present,the control of FHB always depends on chemical fungicides,such as carbendazim.However,it is known that with the long-term use of carbendazim,not only the emergence of drug-resistant pathogens in production,but also the failure of prevention and treatment,and the prevention and control of wheat scab by carbendazim,will also stimulate the biosynthesis of DON,increasing DON pollution of wheatSuccinate dehydrogenase inhibitors（SDHIs）are respiratory inhibitors,whose target is succinate dehydrogenase（complex II,SDH）in the mitochondrial electron transport chain of pathogens.The most obvious structural feature of SDHIs fungicides is that they all contain "amide" groups.At present,this class of fungicides is widely used to control diseases such as rice sheath blight,wheat rust,wheat sheath blight and gray mold.Among them,carboxin is listed as the earliest product of SDHIs fungicide,and is also registered for the prevention and control of wheat scab in China.Therefore,this study selected five SDHIs fungicides:flutolanil,boscalid,fluxapyroxad,benzovindiflupyr and fluopyram,to explore their inhibitory activities against F.graminearum and regulation mechanism of DON biosynthesis.The results indicate that these five SDHIs fungicides had no significant inhibitory effect on the mycelial growth of F.graminearum except for fluopyram,but all had significant inhibitory effects on spore germination（EC50 values 0.39-0.74 μg/mL）.At the same time,they can significantly inhibit the DON production and TR15 gene expression in F.graminearum.Toxisomes are special spherical structures formed by F.graminearum under the conditions of toxin production.Under the treatment of these five SDHIs fungicides,we found that the structure of the toxisomes was destroyed,which indicated SDHIs fungicides could inhibit the DON biosynthesis in F graminearum.Succinate dehydrogenase is a common functional part of the tricarboxylic acid cycle（TCA）and the mitochondrial electron transport chain.To explore the regulation mechanism of SDHIs fungicides on the biosynthesis of DON from physiological and biochemical aspects,we determined the pyruvic acid content,acetyl-CoA content and the expression level of key genes in the glycolysis pathway in 2021 treated with these five SDHIs fungicides.The results showed that the expression of hexokinase and 6-phosphofrucose kinase was inhibited,and the content of pyruvic acid and acetyl-CoA were significantly decreased,revealing that SDHIs fungicides inhibit the expression of key genes in glycolysis pathway,leading to a decrease in the content of pyruvic acid and acetyl-CoA in the synthesis of DON,thereby inhibiting the mechanism of DON biosynthesis.In addition,in order to investigate the cause for the inhibition of the glycolysis pathway,the changes in succinate dehydrogenase（SDH）activity,isocitrate dehydrogenase（ICDHm）activity and citric acid content in 2021 were determined treated with SDHIs.We found that the activities of SDH and ICDHm were reduced,while the content of citric acid was increased.ICDHm is the rate-limiting enzyme in TCA,and the decrease in enzyme activity slows down the rate of TCA.When the citric acid content rises,it is possible to feedback to inhibit the glycolysis pathway.Meanwhile,we found that ATP content in 2021 decreased significantly treated with these five SDHIs fungicides,further indicating that SDHIs fungicides can effectively inhibit the energy synthesis of F,graminearum by inhibiting glycolysis pathway and TCA.To further investigate whether succinate dehydrogenase in F.graminearum is involved in the regulation of DON biosynthesis,we selected the succinate dehydrogenase C subunit deletion mutants（ΔFGSG₀1981 and ΔFGSG₀9012）as research target.The effects of succinate dehydrogenase C subunit knockout on DON production,TR15 gene expression and pyruvic acid content was determined.The results showed that two C subunits of succinate dehydrogenase regulated the biosynthesis of DON and pyruvic acid in F.graminearum.In addition,after the knockdown of the succinate dehydrogenase C subunit,the normal structure of toxinsomes could not be formed,indicating that succinate dehydrogenase in F.graminearum was involved in the regulation of DON biosynthesis.In summary,SDHIs fungicides effectively inhibit the biosynthesis of DON in F.graminearum by inhibiting SDH,interfering with TCAand the glycolysis pathway.In addition,succinate dehydrogenase in F.graminearum is involved in the regulation of DON biosynthesis.