Exploration Of The Antibacterial Effect And Mechanism Of Bacillus And Root Exudates On Ralstonia Solanacearum In Mulberry

Mulberry bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum,which poses a serious challenge to the development of sericulture industry.At present,physical and chemical measures are mainly used to control mulberry bacterial wilt.However,due to the complex pathogenic mechanism of R.solanacearum and the pollution and harmfulness of traditional chemicals,there is still a lack of effective and environmentally friendly control strategies.Therefore,it is of great significance to explore new biocontrol agents for the prevention and control of mulberry bacterial wilt.Using antagonistic bacteria and plant secondary metabolites as biocontrol agents is a good strategy to protect plants from soil-borne diseases.In this study,Bacillus strains with good antibacterial activity against R.solanacearum were isolated from the rhizosphere soil of healthy mulberry,and their classification and antibacterial activity were determined.In addition,the effective antibacterial active substances in mulberry root exudates were identified by GC-MS analysis,and the antibacterial mechanism was preliminarily explored,in order to obtain the ecological microbial source and plant source new agents for controlling mulberry bacterial wilt.The main research contents are as follows:1.Isolation and identification of biocontrol Bacillus BM-7 against mulberry R.solanacearum and determination of its bacteriostatic effect.Using the method of colony morphology observation and PCR detection technology,a strain with strong pathogenicity to mulberry was isolated and identified,which was named MRS-5,and the strain belonged to PhylotypeⅠ,Sequevar 12,Biovars 5.Eight strains of Bacillus were isolated from the rhizosphere soil samples of healthy mulberry by gradient dilution method.Seven strains of Bacillus with antagonistic effect on MRS-5 were further screened by plate confrontation experiment.Among them,BM-7 strain had the best antagonistic effect.The diameter of the inhibition zone was 22.05±0.07 mm,and the inhibition bandwidth was 11.30±0.03 mm.Combined with the colony morphology,physiological and biochemical characteristics and16S r DNA sequence analysis of BM-7,it was found that the colony of the strain was white,with folds around,and Gram staining was positive.The physiological and biochemical characteristics were basically consistent with the Bacillus model strain.Sequence alignment showed that the strain had high homology with Bacillus pumilus and had an inhibitory effect on the growth of MRS-5,which could be used as a resource for the development of biocontrol agents for mulberry bacterial wilt.2.Identification of root exudates of mulberry seedlings and analysis of antibacterial properties of active substances.In order to clarify the interaction between mulberry root exudates and soil-borne disease-mulberry bacterial wilt,this study started with the root exudates of mulberry seedlings（Fengchi）,and determined the material composition of root exudates of hydroponic mulberry seedlings under different p H extraction conditions.The results showed that the root exudates of neutral acid components,alkaline components and neutral components were highly similar,mainly including hydrocarbons,alcohols,esters,organic acids,aldehydes,amides and other substances.The active substances with relatively high content were further screened.Amidates（（Z）-13-Docosenamide and（Z）-9-Octadecenamide）had the largest relative content of the three components,with accounted of 42.6%and the（endo）-（-）-3-Bromocamphor accounted of 18.33%.The antibacterial effect on mulberry R.solanacearum MRS-5 was determined that these three active substances had obvious bacteriostatic effect on MRS-5,and the bacteriostatic effect was concentration dependent.When the concentration of（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor was 1.5 mg·m L^-1,the inhibition rate reached more than 90%,and the inhibition rate of（Z）-13-Docosenamide(1.5 mg·m L^-1)reached more than 80%.Scanning electron microscopy showed that the three active substances significantly destroyed the morphology of MRS-5 at this concentration.Therefore,（Z）-13-Docosenamide,（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor have potential biocontrol value in the prevention and control of mulberry bacterial wilt.3.Preliminary study on the antibacterial mechanism of the main active substances of mulberry seedling root exudates.In order to further clarify the bacteriostatic mechanism of（Z）-13-Docosenamide,（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor,the effects of three active substances on the virulence factors of R.solanacearum at a concentration of1.5 mg·m L^-1were studied.The results showed that（Z）-13-Docosenamide,（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor significantly inhibited the formation and motility of biofilm of MRS-5,and the treatment of the three active substances induced the burst of reactive oxygen species in MRS-5,causing oxidative stress and inhibiting the growth activity of MRS-5.What’s more,（endo）-（-）-3-Bromocamphor changed the permeability of cell membrane of MRS-5 and significantly increased the content of extracellular polysaccharide.Based on the expression pattern analysis of MRS-5virulence-related genes,it was found that（Z）-13-Docosenamide,（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor significantly decreased the expression of hrp B,hrp X and hrp F in the hrp genes cluster,and inhibited the expression of type III effector protein-related genes rip AW,rip AE,Rs5-4819 and Rs5-4374 and other virulence-related genes ace,egl3and peh B.In addition,the expression of global regulator phc A gene decreased significantly after（endo）-（-）-3-Bromocamphor treatment.The above results show that these three active substances can achieve bacteriostatic effect by inhibiting the virulence factors of MRS-5.Among them,（Z）-13-Docosenamide can reduce the pathogenicity of MRS-5 and has no obvious inhibitory effect on the growth of mulberry seedlings which can be used as a new botanical agent to control mulberry bacterial wilt.In summary,this study successfully screened out the development resources that can be used as potential microbial sources and plant-derived biocontrol agents for mulberry bacterial wilt.Bacillus pumilus BM-7 had good antibacterial effect on MRS-5 and significantly inhibited the growth of R.solanacearum MRS-5.At the same time,three active substances identified from the root exudates of mulberry seedlings,（Z）-13-Docosenamide,（Z）-9-Octadecenamide and（endo）-（-）-3-Bromocamphor,inhibit the growth activity of MRS-5 strain by affecting the cell morphology,biofilm formation,motility,extracellular polysaccharide content,reactive oxygen species burst and the expression of related virulence genes,so as to achieve bacteriostatic action.In which,（Z）-13-Docosenamide can reduce the pathogenicity of MRS-5 and is friendly to mulberry seedlings.This study laid a theoretical foundation for the development and application of biocontrol agents against mulberry bacterial wilt.