| Fusarium headling blight(FHB)caused by Fusarium graminearum is an important disease in wheat production,which not only leads to severe reduction of wheat yield,but also produces mycotoxins that affect food safety.Biological control of wheat FHB,which can reduce the use of chemical fungicides,is considered to be a promising way to prevent and control wheat FHB.Bacillus amyloliquefaciens S76 and its lipopeptides can effectively inhibit F.graminearum and can be developed as a biological agent to control wheat FHB.However,there is no systematic study on the molecular mechanism of Bacillus and its produced lipopeptides that interact with Fusarium pathogens and wheat.At the same time,during the storage,the variation of microbiome and mycotoxins in wheat grains that are infected with Fusarium head blight in the field is not clear.In this study,the molecular mechanisms of the interactions between S76 strain together with its antagonistic compounds Iturin(Itu)and Fengycin(Fen)and F.graminearum and its host wheat were studied by using tools of molecular biology,genetics and plant pathology.In addition metagenomic and chemistry techniques were used to study the variation of microbiome and mycotoxins in storage wheat grains.The main results are as follows:1.Iturin,Fengycin and an antagonist strain S76 interact with many targets of F.graminearum,which participate in the different pathways in this fungal pathogen.The genechip expression profile showed that Iturin,Fengycin and the strain S76 can significantly affect the ergosterol synthesis genes,membrane lipids synthesis genes,signal transduction genes,cell wall related genes,glucan synthesis and degraded genes,and GPI anchored structure synthesis genes and GPI anchored proteins genes expression.We kockouted these genes,and analyzed the gene-deficient mutants during their interaction with two lioppeptides,by using chemical analysis,phosphorus immunoassay and biomass assays;the results consistently showed that Iturin and Fengycin have multiple targets in F.graminearum,such as membrane lipids,GPI anchor structures,ANK membrane proteins and intercellar proteins.Through inducing the HOG and CWI pathways,Iturin and Fengycin could lead to cell swelling and structure changes in F.graminearum,especially at the growth top point and branch point of the mycelia.Furthermore,chemical analysis and phosphorus immunoassay revealed that Iturin and Fengycin regulated fungal glycerol biosynthesis through induing the level of the Fg Hog1 protein phosphorylation in the HOG pathway which leaded to an increase of the osmolarity in F.graminearum,and regulated the chitin biosynthesis through inducing the level of the Fg Mgv1 protein phosphorylation in the CWI pathway which leaded to a decrease of the protection in F.graminearum.2.Iturin and Fengycin can indue wheat systemic resistance to F.graminearum,an increase of wheat growth,a reduction of the germination of wheat seeds,and inhibition of fungal DON biosynthesis in wheat plants.RNAseq revealed that Iturin and Fengycin can activate ROS,hypersensitivity reaction,stomatal closure,ubinqutin-mediated proteolysis,phytoalexin accumulation,salicylic acid,and ethane resistence genes,as well as homore regulation genes.And Iturin and Fengycin can also induce callose deposition and H2O2 accumulation in wheat,thus enhancing the resistance to F.graminearum.Expression analysis of DON biosynthesis genes of F.graminearum that infected wheat and chemical detection of DON contents in wheat spikelets showed that Iturin and Fengycin can significantly inhibit DON biosynthesis genes expression and reduce the DON accumulation in wheat.3.Antagonist strain S76 can significantly induce hydrolase genes expression in F.graminearum during the process of infecting wheat.The transcripts based on RNAseq showed that antagonist strain S76 can significantly induce expression of cellulase,hemicellulase,and glucoside hydrolase genes in F.graminearum that infect wheat.Deletion of the hydrolase genes in F.graminearum together with supplementation of carbon sources in medium and pathogenicity assays showed that cellulase(Fg01596)and glucoside hydrolase(Fg06873)can utilize the cellulose of wheat cell walls to provide carbon sources for mycelial growth,as well as to enhance the pathogenicity of F.graminearum to infect wheat.4.The microbiome and mycotoxin contents of wheat grains from different silo positions(top,middle and bottom)and storage times(3,6,9,and 12 months)have significant variations.The fungal community in wheat at time 0 included 105 classified species(81 genera)and 41 unclassified species,and four species had over 10% of the relative abundance: Alternaria alternata(12%),Filobasidium floriforme(27%),Fusarium graminearum(12),and Wallemia sebi(12%).The fungal diversity and relative abundance of Fusarium in wheat from top silo positions were significantly lower than at other silo positions during storage.Nivalenol and deoxynivalenol in wheat were 13%-34% higher in all positions at 3 months compared to time 0,and mycotoxins in wheat from middle and bottom positions at 6 to 12 months were 24%-57% higher than at time 0.The relative abundance of toxigenic Aspergillus and aflatoxins were low at time 0 and during storage.These results provide information and may serve as a basis for revealing the molecular mechanism of S76 strain controlling F.graminearum and developing new stratigies for controlling FHB based on S76 strain.This study also provide information and methods for reducing Fusarium mycotoxin contamination during wheat storage. |
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