Mechanism Of A Novel Fungicide JS399-19 Against Fusarium Graminearum

Fusarium head blight(FHB)is a devastating disease of cereal crops.In addition to severe yield losses,FHB leads to mycotoxin contamination in infested grains that poses a serious threat to food safety and human health.In spite of FHB can be effectively controlled by several fungicides recently,which application is frequently associated with greater DON concentrations in grain.Therefore,applying a novel antifungal and anti-mycotoxin inhibitor is urgently needed for sustained management of FHB.A novel cyanoacrylate fungicide JS399-19 which exhibits aggressively inhibitory activity towards Fusarium species,not only mycelial growth but also mycotoxins are effectively controlled in infested grains.JS399-19 has been extensively applied in controlling FHB and rice bakanae disease,but the mechanism of this compound is not clear.In this study,JS399-19 resistant strains of Fusarium graminearum was obtained under the dual selection pressure derived from drug screening and ultraviolet mutagenesis.Molecular biology and biochemistry experimental techniques were performed to explore the mode of action of this species-specific compound.Results of our study showed that:1)JS399-19 exhibits an excellent efficacy in inhibiting mycelial growth and DON production of F.graminearum.JS399-19 shows stronger inhibitory activity against F.granminearum compared to carbendazim and tebuconazole,which belong to benzimidazoles and demethylation inhibitors(DMIs)respectively,and have been applied widespreadly for FHB management.JS399-19 was still able to effectively control DON production even when grains were infected by F.graminearum.JS399-19 is highly active against most Fusarium species but shows a low or even no activity against mycelial growth of other fungi.2)A point mutation in myosin I confers F.graminearum resistance to JS399-19.we conducted a whole-genome transcript profiling together with resequencing assays,and discovered that a point mutation S217L or E420K in Fusarium graminearum myosin I(FgMyo1).The mutated FgMYO1 containing either S217L or E420K was introduced into the wild-type strain PH-1 and the resulting strains became resistant to JS399-19.These results strongly indicated that the point mutations in FgMyol were responsible for the resistance of F.graminearum to JS399-19.3)JS399-19 inhibits the ATPase activity of myosin I.JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1WT,but not the mutated FgMyolS217L/E420K,indicating that JS399-19 targets FgMyol resulting in highly activity against mycelial growth.The failure to obtain a FgMYO1 null mutant and phenotypic defects of JS-R2 mutants indicating that FgMyol is essential in F.graminearum.4)Divergences in myosin I amino acids leading to JS399-19 species-specific.Transformation of F.graminearum with the myosin I gene from M.grisea(MgMYO1)led to JS399-19 resistance indicating the divergences in myosin I amino acids are responsible for other fungal species resistance to JS399-19.5)JS399-19 suppresses toxisome formation.Deletion of FgAbp1,which encodes a vesicle formation protein,led to highly susceptible to JS399-19 and vacuole formation was blocked in mutant ?FgAbp1.Colocalization of FgAbpl with Tril,which is a key enzyme in DON biosynthesis,showed that they co-localized onto the toxisome membrane.The interactions of Tril with FgMyol and Tri4,which is another key enzyme in DON biosynthesis,were confirmed by co-immunoprecipitation(CoIP)assays.We suggested that JS399-19 suppresses toxisome formation leading to affect DON biosynthesis in Fusarium graminearum.