Studies On The Influence Of Different Microbial Inoculants On Ensiling Quality For Sweet Sorghum Based On Microbiome And Metabolomics

Tomato（Solanum lycopersicum L.）originated in South America,when it was introduced to China as an ornamental plant,because of its rich carotene,vitamin C and B vitamins,high nutritional value,is an important cash crop in the world.Tomato bacterial wilt caused by Ralstonia solanacearum is one of the main soil-borne diseases of tomato,which has caused huge economic losses all over the world.Due to the special pathogenic mechanism of the pathogen,the current control measures cannot fundamentally inhibit the occurrence of tomato bacterial wilt.Therefore,the purpose of this study was to explore the biocontrol effect and bacteriostatic mechanism by screening and constructing efficient biocontrol strains.In order to obtain rhizosphere bacteria with antagonistic function,rhizosphere soil samples of healthy tomato plants in the affected fields were collected,and antagonistic bacteria with potential biocontrol ability against pathogenic bacterial Ralstonia solanacearum were initially screened,and ARTP mutagenesis and genome rearrangement techniques were used to improve their bacteriostatic ability,and efficient antagonistic strains were obtained.The constructed biocontrol bacteria were combined with chemical control,and the control effect was evaluated from the phenotypic shape and physiological characteristics of tomato.Microbial diversity and metabolomics were used to explore the control mechanism of Bacillus against tomato bacterial wilt.The main research results are as follows:Based on the current situation that microbial antagonists have important application value and potential to control plant diseases,the rhizosphere microorganisms of tomato were isolated and screened,and antagonistic strains for biological control of tomato bacterial wilt were dug up,and a screening system of antagonistic strains was established to provide strain resources for biological control agents.（1）In this study,121 strains of tomato rhizosphere bacteria were initially isolated and screened by the plate confrontation method,and 16 strains of antagonistic bacteria were obtained,which had the ability to inhibit bacterial wilt.Then,the established root extraction method was used to rapidly evaluate the biocontrol potential of antagonistic bacteria against tomato bacterial wilt in a short period of time,and the pot control efficiency of strain WJB0802 was up to 68.25%.WJB0802 was identified as Bacillus subtilis by the whole genome sequencing analysis,which had the ability to secrete a variety of antibacterial substances,produce a variety of antagonistic cell active substances and antibacterial proteins,so as to achieve the control of tomato bacterial wilt.（2）Using whole genome sequencing and resequencing,a total of 38211 bases of GSJB-1210 had been modified with the reference gene of the starting strain,which had a certain genetic distance from the starting strain.By predicting metabolite synthesis gene clusters,it was found that the strain had 10 antibiotic gene clusters,and more than 200 genes related to biocontrol ability increased significantly,and its antibacterial activity was significantly higher than that of the original strain.Therefore,it is feasible to mutagenize and rearrange the antagonist strains by using ARTP mutagenesis and genome rearrangement through molecular biotechnology to obtain the model strains with high inhibitory capacity.（3）In order to minimize the use of chemical agents,antagonist strain GSJB-1210 was combined with chemical bactericides to study its control effect on tomato bacterial wilt.The bacteriostatic zone method was used to determine the virulence of six bactericides against Ralstonia solanacearum,and their EC₅₀values were calculated.UV spectrophotometry was used to determine the compatibility of bactericide and GSJB-1210,the most suitable bactericide was selected and combined with strains for pot experiment,and the optimal compound ratio was determined by Horsfall method.The results showed that the EC₅₀of ningnanmycin was 3.66 mg/L,which had good biocompatibility with GSJB-1210 strain.When the volume ratio of ningnanmycin（3.66 mg/L）to GSJB-1210（10⁸CFU/m L）was 4:6,the control effect on tomato bacterial wilt reached 77.02%,and the biocontrol effect was significantly higher than that of single agent.Ningnanmycin can be combined with GSJB-1210 to control tomato bacterial wilt,and the synergistic effect is significant.（4）Application of biocontrol agents and chemical bactericides significantly affected the composition and diversity of rhizosphere bacterial community of tomato.Proteobacteria,Actinomyces,Bacteroidetes,Patescibacteria,chlorobacterium and Firmicutes are fine Bacteria dominated the bacterial communities in all soil samples.RA of Firmicutes and Actinomycetes increased significantly at phylum level after the application of single microbiocontrol agent GSJB-1210.At the genus level,RA of Bacillus and Pseudomonas were higher than CK.Compared with CK,the abundance of microorganism in F0 treatment group was significantly reduced,which had a certain negative effect on the abundance of rhizosphere microbial community and would lead to the destruction of rhizosphere microbial community structure.The combination of ningnanmycin and biocontrol strain GSJB-1210 reduced the negative effects of chemical fungicides on the abundance of microbial community,significantly inhibited the occurrence of bacterial bacterial wilt,and made the microbial community in a healthier state.（5）Based on metabolomics analysis,the effects of microbial and chemical bactericides on the composition of metabolites in tomato rhizosphere were revealed.The results of KEGG enrichment in metabolome analysis showed that ABC transport,phenyl-C biosynthesis and phenyl-propane biosynthesis were significantly enriched in the three treatment groups.The metabolite composition of tomato rhizosphere could be significantly changed after treatment with bactericide,chemical bactericides,biocontrol bactericide and chemical bactericides.The metabolite composition of tomato rhizosphere could be significantly changed after treatment with bactericide,chemical fungicide,biocontrol bactericide and chemical fungicide.After biocontrol bacteria treatment,metabolites with antibacterial activity such as indolealdehyde,tryptophan,ferulic acid,methyl salicylate,erucinic acid,protocatechuic acid,2-hydroxycinnamic acid,artemisia,chlorogenic acid and bamboo amycin were significantly enriched.These significant differences in metabolites and their enriched pathways were related to the ability of plants to resist pathogen invasion.