Biocontrol Of The Cucumber Fusarium Wilt By The Vinegar Residue Substrate-derived Pgpr NSY50

Cucumber Fusarium wilt is a severe vascular disease caused by Fusarium oxysporum f.sp.cucumerinum(FOC),a stubborn soil-borne fungal pathogen.It can survive in the soil for an extended period and repeatedly infect plants,seriously affecting crop quality and yield.Chemical methods to control the disease often cause environmental pollution and may result in hazardous levels of pesticide residues in the harvest,posing a health risk to the consumers.Therefore,biological control methods are favored for being safe and environmentally friendly.Vinegar residue substrates(VRSs)are horticulture organic substrates made from waste residues during vinegar production.Recent studies have shown that VRSs confer resistance to the cucumber Fusarium wilt pathogen.However,it is unclear what physiological responses induced by the VRSs in cucumber are responsible for such resistance.In this study,we evaluated the effect of VRSs on plant growth and pathogen suppression,and isolated the potential biocontrol agent Paenibacillus polymyxa NSY50(P.polymyxa NSY50).We studied the induction of host defense responses against Fusarium wilt by NSY50 and explored the effect of this strain on the microenvironment of cucumber rhizosphere.We also examined how NSY50 mediates the interaction between long noncoding RNAs(lncRNAs)and mRNA to improve resistance.The main research results are as follows:1.Based on the results of antagonistic activity assay and pot experiment we found that(?)VRS extracts exhibited higher biocontrol activity than control against FOC and significantly improved the growth inhibition of FOC with values of 48.3%and a relatively higher microbe quantity;(?)in response to challenge with FOC,compared with control substrate,antioxidant enzyme activities(SOD?POD?CAT and APX)and key enzymes of phenylpropanoid metabolism(PAL and TAL)were significantly increased,promoting the expression of pathogens inoculated protein(CHT and GLU)activities and stress-related genes,(?)Paraffin section staining observations found that the addition of VRS treatment significantly reduced the colonization of pathogenic mycelia on the vascular bundles of cucumber plants.These results showed that the cultivation of cucumber in VRSs can significantly increase the defense enzyme activity,disease-related protein activity,and stress-related gene expression,and effectively control cucumber Fusarium wilt.2.Next-generation sequencing of the rhizospheric soil microbial community revealed that NSY50 can effectively control cucumber Fusarium wilt by altering soil physical-chemical properties and the composition of the microbial community.These includes:(i)changing the structure and composition of rhizosphere microorganisms.increasing soil enzymes activties and improving soil physical and chemical characteristics(e.g.,Corg,Cmic,total N and pH)to maintain a healthy and stable microenvironment;(ii)reduce the richness of Fusarium and promoted potentially beneficial groups,such as Actinobacteria,Pachyphyte(Bacillus and Pseudomonas),Basidiomycota.These beneficial strain can effectively inhibit the growth of Fusarium oxysporum.3.We studied the role of lncRNAs in NSY50-induced cucumber disease resistance by using strand-specific RNA-Seq.The results showed that 504,302,354 Clean reads were obtained from the eight transcriptome databases.NSY50 induced the differentially expression level of 998 mRNAs and 167 lncRNAs in cucumber roots upon challenge with FOC.Quantitative RT-PCR results were consistent with the microarray analysis results of six lncRNAs and six mRNAs.Moreover,Gene category and KEGG pathway analysis found that NSY50-responsive genes were involved in phenylpropane biosynthesis,phenylalanine metabolism,plant-pathogen interactions,and plant hormone signal transduction.These metabolic pathways are closely related to the immune response of plants.Finally,the interaction analysis of differentially expressed lncRNA and mRNA produced during the resistance process of cucumber induced by NSY50,we found a total of 1471 lncRNAs adjacent mRNA and 684 antisense lncRNAs(IncNATs)and their sense strand mRNA.4.In order to study the regulatory effect of antisense lncRNA on gene expression in cucumber plants,the genes of CsPI4,CsHSP20 and CsPR-1,which are significantly differentially expressed in the NSY50+FOC treatment,were selected as candidate genes,then cloned in cucumber and functionally analyzed in Arabidopsis thaliana.The results showed that the Arabidopsis transgenic CsPI4,CsHSP20 and CsPR-1 genes increased resistance to FOC.It was suggested that P.polymyxa NSY50 regulate the expression of CsPI4,CsHSP20 and CsPR-1 genes in the sense chain by inducing the antisense chains TCONS₀0023628,TCONS₀0011014,and TCONS₀002685,therefore,increasing the resistance of cucumber plants to FOC.