| Nowadays,along with the rising of living quality,people's demanding on agricultural production has changed from the need of basical living to heathy,environmentally friendly and high quality product.Biological control has become one of the most important ways of controlling plant dsease as it is from the nature.However,biological control apllication still meets problems like unstable control effect and unfavorable control effect of single strain.To solve these problems,our lab selected a three-strain consortium,including Bacillus cereus AR156,B.subtilis SM21 and Serratia sp.XY21,called 'Ning shield' to promote biocontrol effiency and stability by synergistic activity of the strains.Bacillus sp.bacteria have been an important part of biocontrol microbes as its series of advantages such as its outstanding capacity of surviving in natural environment,unique spore forming system for stress resistance and strong antibiotics production systems.Biofilm is the main form of Bacillus in the environment.Studies have shown that biofilm formation is important in the biological control process of Baillus sp.bacteria.In this study,B.cereus AR156,B.subtilis SM21 are studied for the mechanism of single strain biofilm formation and the effect of interactions of the two bacteria on biofilm formation.1.Applied strategy for selecting stale biocontrol agents to control rice sheath blight caused by Rhizoctonia solaniWith continuously rising culture conditions,fertilizer input as well as demand for better taste,rice sheath blight caused by Rhizoctonia solani Kuhn is increasingly threatening rice production in China.We assessed the values of potential biocontrol abilities of biocontrol agents(BCAs)according to their inhibition zones against R.solani,extracellular enzymes activity and siderophores production.Fourteen strains,including 10 with values above 6.0 as potential biocontrol agents and 4 below 3.0 as control,were tested for their biological control efficacy against rice sheath blight caused by genetically diverse pathogen formerly collected in 11 province in China.Biocontrol efficacy of a single BCA differed significantly(-36.23%?88.24%)in controlling disease caused by various R.solani strains.However,Pseudomonas fluorescens 4aYN11 had a relatively stable control efficacy of 32.26%-78.79%and growth promotion of 18.43%.B.cereus AR156 and B.subtilis 3ReF17 had an average control efficiency of 56.78%and 24.81%respectively.The correlation coefficient between assessment of each BCAs and their biocontrol efficacy is 0.717.In the present study,the result provides us one promising biocontrol strain 4aYN11.Moreover,based on an assessment system,growth promotion potential and phylogenetic diversity of pathogen R.solani,we improved the strategy for screening stable biocontrol agents and for future screen of novel BCAs.2.Poly-y-glutamic acids contribute to biofilm formation and plant root colonization in selected environmental isolates of Bacillus subtilisThe soil-dwelling bacterium Bacillus subtilis is known to produce poly-y-glutamic acids(y-PGA)as one of the major secreted polymeric substance.Production of y-PGA confers B.subtilis a mucoid colony phenotype on nutrient agar media.In B.subtilis,the role of ?-PGA in bacterial physiology and its possible involvement in biofilm formation and bacteria-host interactions are unclear.In this study,we show that various environmental isolates of B.subtilis exhibit diverse y-PGA production,which is possibly due to differential regulation of y-PGA biosynthesis gene.In many of those isolates,y-PGA production plays a significant role in robustness and morphology of the colony biofilms.We presented genetic evidence for a co-regulation on y-PGA biosynthesis genes and genes for biofilm matrix production,but in an opposing fashion.We also investigated the role of?-PGA in bacteria-plant interactions.Our results show that y-PGA plays a notable role in root colonization by bacteria and biological control of the tomato plant,pinpointing an important function of y-PGA in B.subtilis-plant interactions.Lastly we proposed an integrated model based on the inverse regulation between genes for y-PGA biosynthesis and those for biofilm matrix production,in which we highlighted an sequential roles of y-PGA and biofilm matrix in facilitating B.subtilis cells attach and colonize the root surface,and forming root surface-associated bacterial multicellular communities in rhizosphere.3.Bacillus subtilis induces biofilm formation upon sensing extrapolysaccharide from Bacillus cereusThe use of microbial consortium inoculants is an important strategy to achieve efficient and stable biological control of plant diseases.Understanding the interaction mechanism between microorganisms is very important for the preparation and application of compound microbial agents.In the field study,it was found that a composite microbial inoculum containing Bacillus cereus AR156 and Bacillus subtilis SM21 could effectively control many plant diseases.In this study,a co-culture system of Bacillus cereus AR156 and Bacillus subtilis SM21 was developed and the biofilm formation ability of co-culture was studied.It is found that in the co-culture,Bacillus cereus AR156 can induce the expression of biofilm-related genes epsA,tasA and sdpC of Bacillus subtilis SM21,increase the microbial biomass and enhance the biofilm formation.The kinC deletion mutant of Bacillus subtilis did not respond to Bacillus cereus,indicating that KinC was involved in the induction of Bacillus subtilis biofilm formation by Bacillus cereus.The induced phenomenon of the biofilm induction is common in co-culture of Bacillus subtilis and Bacillus cereus.Further nuclease and protease treatment and purification showed that Bacillus subtilis induces biofilm formation upon sensing extracellular polysaccharide from Bacillus cereus.4.Functional study of Bacillus cereus secreted protein BC1280 on biofilm formation and interaction with Bacillus subtilisBacillus cereus is widely known as an agent in plant disease control.The ability to form biofilm is considered one important trait for B.cereus to successively survive in natural environment and subsequently play its role in disease control.Two genomic loci,sipW-tasA and calY,encoding two orthologs of the amyloid-like protein TasA of B.subtilis as well as a signal peptidase SipW are characterized recently to be involved in biofilm formation in B.cereus.In this study,we show that,along with the two loci,expression of a genomically adjacent protein,formerly hypothetical BC1280,is also under control of biofilm master regulator SinR.SinR protein represses the three operons by directly binding to their promoter regions.We found that BC1280 is required for formation of matrix amyloid protein.We further showed that expression of BC1280 rescue biofilm formation in B.subtilis tapA mutant.Interestingly,little similarity in protein sequence could be observed between TapA and BC1280.Instead,BC1280 contains unique KE rich peptide repeats in its C terminal.Truncated BC1280 with partial KE rich peptide repeats remains functional in supporting biofilm formation,while complete loss of the repeat domain leads to a defect in biofilm.We also found that the predicted(?-helix of BC1280 also plays a role in the process of biofilm formation.Thus,we propose that these domains are important for the functionality of BC1280,perhaps by mediating the interaction between BC1280 or BC1280 and TasA molecules.This study on one side analysed the cause of unstable biocontrol effect in a perspective of genetic diversity of pathogen and set a system for selecting stable biocontrol bacteria against rice sheath blight;on the other side,find poly-y-glutamic acids play an important role in B.subtilis biofilm formation and are co-regulated with biofilm-related genes.In further study,extracellular polysaccharides of B.cereus is able to stimulate the biofilm formation of B.subtilis,and hypothetical protein BC1280 is capable of rescue the biofilm defect phenotype caused by the loss of the biofilm matrix protein BC1280 in B.subtilis.The study on the machanisms of multi-strain biocontrol consortium will provide new aspects of thinking on mechanism study of biological control and material and theoretical basis. |
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