Mechanism Of Bacillus Velezensis CC09 In Controlling Pathogen Gaeumannomyces Graminis Var.tritici

Take-all disease is one of the wheat root diseases caused by pathogen Gaeumannomyces graminis var.tritici(Ggt),which lead to 20%-50%yield loss in disease region.However,until now,there is no effective resistant varieties available;the rotation measures are difficult to implement;extensive use of fungicides leads to the environmental health,pathogen resistance,and escalating costs.So far the use of biocontrol strain,such as Bacillus spp.and Psdeuomnoda fluorescens,have better control effects on take-all.However,since the colonization ability of Pseudomonas fluorescens is severely affected by the surrounded environment,which is difficult to maintain a high bacteria population density in the soil.Recently,more studies have shown that Bacillus spp.has a great advantage over Pseudomonas fluorescens in controlling take-all.Especially,Bacillus velezensis not only produces a variety of antibacterial substances and induces systemic resistance,but also has an excellent growth-promoting effect.Bacillus velezensis has the ability to produce spores,which brings strong resistance to external stresses,and therefore considered as an ideal biocontrol strain for take-all.In the course of this study,a strain identified as Bacillus velezensis CC09(Bv)is used as biomaterial,isolated from the leaf of Camphora officinarum.Using the methods of recombinant DNA technology/genetic engineering,histology,transcriptomics and proteomic,we studied Bv’s colonization,transmission,population growth dynamics and biocontrol effects on take-all and leaf spot diseases.The response of wheat roots to Bv colonization,pathogenic fungus Ggt infection and the combination of Bv and Ggt(Bv+Ggt)infection was further analyzed.The possible mechanism of Bv regulating Ggt gene expression in wheat plants was discussed.The main results are as follows:1.A Bv strain carrying green fluorescent protein(Bv-GFP)was constructed.The growth and antibacterial activity of Bv-GFP strain and wild-type Bv strain were evaluated.The results showed that Bv-GFP strain can replace the wild-type Bv strain for the study of colonization,transmission,population growth and disease prevention of Bv in wheat plants.2.Through the technology of confocal laser scanning microscopy(CLSM),the observation on wheat roots colonized by Bv strain showed that Bv-GFP strain can colonize and migrate in different wheat tissues including roots,stem and leaf.One day after inoculation,biofilm can be formed on the surface of wheat roots;10 days after inoculation,it has been widely distributed in the epidermis,cortex and xylem vessels of wheat roots;15 days after inoculation,Bv not only colonized in wheat root tissue but also migrated to stem and leaf tissue.3.One day after inoculation with Bv-GFP strain,followed by the Ggt infection Bipolaris sorokiniana(Bs)on wheat roots and leaf respectively,and then the biocontrol efficacity in controlling take-all and leaf spot is investigated.The results showed that Bv had a function similar to "vaccine".Once the wheat roots were inoculated,and the control effects on root disease and leaf blight were as high as 66.67%and 21.64%,respectively,at 20 days.4.The Bv-itu-GFP strain,containing GFP under the Pitu promoter that is responsible for regulating the iturin A synthease coding-gene,is inoculated on the wheat roots.After 15 days,the signal of green fluorescent is detected in wheat roots,stem and leaf,indicating that the promoter Pitu can initiate the expression of the iturin A synthase gene in wheat.Using qRT-PCR technology,it was further confirmed that iturin A synthetase coding-gene(itu A-D)also express in the Bv strain locating in the almost part of tissue.5.Using RNA-seq and iTRAQ techniques,the gene expression level of wheat root inoculation with Bv,Ggt and Bv+Ggt is studied.The results showed that inoculation of Bv in wheat roots resulted in significant differential expression changes of 3,127 genes and 171 proteins;inoculation of Ggt in wheat roots resulted in significant differential expression changes of 5,678 genes and 332 proteins;and the combination of Bv and Ggt inoculation resulted in significant differential expression changes in 10,780 genes and 360 proteins.Three treatments’ condition induce the expression of resistance genes or proteins associated with the wheat local defense system(PTI and ETI),including genes encoding PR and PRR proteins,calcium-regulated genes,plant hormone regulatory genes,and respiratory bursts,gene,mitogen-activated protein kinase(MAPK)cascade gene and gene encoding R protein.However,the expression of resistance genes of proteins are higher induced by combination oof Bv and Ggt than single microbial inoculation(Bv or Ggt).Additionally,the Bv colonization on wheat roots can induce the expression of PR1 resistance coding-genes involved in the SA-mediated defense pathway,and thus trigger an induction system resistance(ISR)similar to SAR.Ggt infection induce the systemic acquired resistance(SAR),also mediated by SA,However,the SAR induced by Ggt infection shows more drastic than ISR induced by Bv colonization.Whereas,the combination of Bv and Ggt inhibits JA synthesis and the expression of marker gene PDF 1.2,but it induces SA synthesis and the expression marker gene PR1,thereby increasing the SAR in wheat plants.6.The level of lignin synthesis can reflect the degree of interaction between wheat and Bv or Ggt.In this study,these three conditions,under the Bv colonization Ggt infection and the combination of Bv and Ggt,increased the content of wheat lignin,but the level of lignin content was:Bv+Ggt>Ggt>Bv.It indicates that the Bv pre-inoculation on wheat roots can further promote the lignin accumulation and strengthen the defense strength of wheat cell wall to resist the subsequent infection of pathogen Ggt.7.Using the comparative transcriptomics analysis,we next conducted a comparative transcriptomic analysis of the "take-all" pathogenic fungus Gaeumannomyces graminis var.tritici,by examining Ggt-infected wheat roots in the presence or absence of the biocontrol agent Bacillus velezensis CC09(Bv)compared with Ggt grown on potato dextrose agar(PDA)plates.Compared with the Ggt grown on PDA plates,a total of 4,134 differentially expressed genes(DEGs),of which 2,142 genes were up-regulated and 1,992 genes were down-regulated,were identified in Ggt-infected wheat roots;while,2,011 DEGs,of which 957 genes were up-regulated and 1,054 genes were down-regulated,were detected in Bv+Ggt-infected roots.Moreover,31 DEGs were identified between wheat roots respectively infected with Ggt and Bv+Ggt,consisting of 29 down-regulated genes coding for potential Ggt pathogenicity factors,e.g.,para-nitrobenzyl esterase,cutinase 1,ABA3,papain inhibitor,and catalase-3,and two up-regulated genes coding for tyrosinase and a hypothetical protein in the Bv+Ggt-infected roots when compared with the Ggt-infected roots.In particular,the expression of one gene,encoding the ABA3 involved in the production of Ggt’s hormone abscisic acid,was 4.11-fold lower in Ggt-infected roots with Bv than without Bv.This is the first experimental study to analyze the activity of Ggt transcriptomes in wheat roots exposed or not to a biocontrol bacterium.Our results,therefore,suggest the presence of Bv directly and/or indirectly impairs the pathogenicity of Ggt in wheat roots through complex regulatory mechanisms,such as hyphopodia formation,cell wall hydrolase,expression of a papain inhibitor,among others,all which merit further investigation.8.Using the method of biochemical analysis,Bv biocontrol mechanism of improving wheat growth and inhibit pathogen Ggt infection was further analyzed.The results showed that Bv not only possesses the genetic characteristics of synthesizing IAA,dephosphorization,and degrading cellulose,but also can regulate the synthesis of vitamins C and endogenous IAA in wheat,which in turn changes the structure and growth of the root system.In addition,Bv may not rely on the regulation of PAL,POD and PPO defense enzyme activities,soluble sugar and flavonoid content in wheat roots to inhibit pathogen Ggt infection.However,it can inhibit the degradation rate of wheat cell wall by inhibiting the polygalacturonase and 3-glucosidase activities,and indirectly enhance the wheat resistance ability against Ggt infection.The above results show that the multitrophic interactions between biocontrol strain Bv,wheat and pathogenic fungus Ggt are complex,and the biocontrol strain Bv can directly or indirectly regulate the pathogenicity of pathogenic fungi,the growth and defense response of wheat,and then reflect its biocontrol activity.Besides,Bv has the characteristics of stable colonization,transmission,synthesis of antibacterial substances and activation of host immune system in plants.Therefore,it can be used as a ecologically biocontrol means to control crop diseases for modern sustainable agriculture.