Study On The Mechanism Of Cinnamic Acid Affecting The Occurrence Of Tobacco Bacterial Wilt

Tobacco bacterial wilt caused by Ralstonia solanacearum is an important soil borne bacterial disease,which occurs widely and causes serious harm,and has become a focus of prevention and control.Currently,a large number of studies have confirmed that root exudates are closely related to the occurrence of tobacco bacterial wilt.They can serve as a nutrient carbon source,signal compounds to stimulate the growth of bacterial wilt,as well as biological activities such as chemotaxis and movement,causing early infection of bacterial wilt and causing the occurrence of bacterial wilt.Previous laboratory studies evaluated the effects of various organic acid root exudates on the occurrence of tobacco bacterial wilt,and found that cinnamic acid was an important root exudate factor inducing the occurrence of tobacco bacterial wilt.However,the mechanism by which cinnamic acid induces R.solanacearum to recognize its host and promote the occurrence of the disease is still unclear.Based on this,this paper systematically analyzed the metabolic differences and rules of cinnamic acid in different tobacco varieties resistant to and susceptible to bacterial wilt,and studied the effects of cinnamic acid on the infection characteristics of bacterial wilt;In addition,the molecular mechanism of cinnamic acid induced chemotaxis and pathogenicity of R.solanacearum was explored using transcriptome high throughput sequencing technology and functional verification of key genes;Based on the regulation of cinnamic acid,the effects of Trichoderma harzianum TMN-1 degrading cinnamic acid on the occurrence of tobacco bacterial wilt were investigated through indoor and field experiments.The main research results of this paper are as follows:1.Clarified the metabolic characteristics of cinnamic acid in tobacco bacterial wilt resistant and susceptible varieties,elucidated the chemotactic activity and infection activity of cinnamic acid induced bacterial wilt,and analyzed the relationship between cinnamic acid and the occurrence of bacterial wilt.The metabolites of the susceptible variety Honghuadajinyuan（HD）and resistant variety Yanyan 97（YY97）at different growth stages of tobacco bacterial wilt were detected by the LC-MS method.The results showed that the metabolites of cinnamic acid were the same in the growth process of the two varieties.Cinnamic acid was higher in the clumping stage,decreased gradually in the fast growing stage and the budding stage,and increased gradually at the maturing stage.The cinnamic acid content in root exudates of susceptible variety HD was higher than that of resistant variety YY97 at fast growing and maturing stage.The effects of cinnamic acid on the motility,chemotaxis and biofilm formation of R.solanacearum bacteria were evaluated by plate swimming method,capillary method and crystal violet staining.The results showed that cinnamic acid at concentrations of 100μM and 150μM significantly promoted the motility,chemotaxis and biofilm formation of R.solanacearum.In terms of motility,after 72h of culture,the motility diameters of R.solanacearum treated with 100μM and 150μM cinnamic acid were 2.33 times and 2.52 times of that of the control,respectively.In terms of chemotaxis,the number of R.solanacearum entering the capillary under 100μM and 150μM cinnamic acid treatment reached 3.75×10⁷CFU m L^-1 and 3.30×10⁷CFU m L^-1,respectively,which was significantly higher than that of the control(2.30×10⁷CFU m L^-1).In terms of biofilm formation,100μM and 150μM cinnamic acid treatment could significantly promote the formation of bacterial biofilm after incubation for 24 h,and the increase rates were 31.25%and 20.31%,respectively,compared with the control.In addition,150μM cinnamic acid treatment for 12 h to 48 h could induce R.solanacearum colonization in tobacco roots,accelerate the colonization rate and increase the colonization amount.2.Transcriptome sequencing analyzed the important signal pathway of R.solanacearum response to cinnamic acid induction,and screened the important response gene RSc1155 involved in cinnamic acid induced chemotaxis of R.solanacearum.Through transcriptome sequencing analysis,the effects of cinnamic acid treatment on transcription expression of related genes in R.solanacearum were investigated.The results showed that there were significant differences in the expression abundance of 360 genes in cinnamic acid treated bacterial solanacearum.GO classification and KEGG enrichment analysis were performed on the significantly different genes.It was found that the response of cinnamic acid induced transcriptional expression genes in R.solanacearum was mainly enriched in pathways such as bacterial chemotaxis,locomotion,flagellar assembly,and localization.26 chemotactic genes were selected,and RSc1155 was the most significantly expressed gene with log2 value of 4.13,which may be an important response gene for cinnamic acid induced chemotactic activity of R.solanacearum.The wild-type strain CQPS-1 RSc1155 gene deletion strain△RSc1155 was constructed by homologous recombination method and the functional verification was carried out.The results showed that compared with the wild type strain,the mutant strain△RSc1155decreased motility,chemotaxis,pathogenicity,and response to cinnamic acid induction.The motional diameter of the mutant strain△RSc1155 was 2.73 cm at 150μM cinnamic acid concentration,and the motional diameter of the mutant strain△RSc1155 was 22.88%lower than that of the wild strain in response to cinnamic acid induction,and the amount of chemotaxis was 41.16%lower.In addition,in terms of pathogenicity,the pathogenicity of the mutant strain decreased significantly.14 days after inoculation,the disease index of△RSc1155 was 31.65,which was significantly lower than 86.56 of the wild-type strain,and the addition of cinnamic acid could not improve the degree of bacterial wilt.3.The degradation activity of Trichoderma harzianum TMN-1 on cinnamic acid was tested and the effect of using T.harzianum TMN-1 to degrade cinnamic acid to alleviate the occurrence of tobacco bacterial wilt was evaluated.T.harzianum TMN-1 can degrade cinnamic acid efficiently.Under the condition of medium,the degradation rate of cinnamic acid at 5 mg/L～200 mg/L reached 100%,and the degradation rate of cinnamic acid at 400 mg/L was 92.82%.In the soil environment,after inoculation with T.harzianum TMN-1,the degradation rate of cinnamic acid in the sterilized soil reached 77.84%,and the degradation rate increased to 93.28%when cooperating with local microorganisms.The indoor application of T.harzianum TMN-1can degrade cinnamic acid,cut off the chemotaxis induction of cinnamic acid on R.solanacearum,and the colonization amount of R.solanacearum root was 73.53%lower than that induced by cinnamic acid.At the same time,it alleviated the occurrence of tobacco bacterial wilt,and reduced the incidence rate and disease index of tobacco bacterial wilt.The results of field experiments showed that cinnamic acid content of tobacco root exudates was positively correlated with bacterial blight content,bacterial wilt incidence and disease index in rhizosphere soil.Application of T.harzianum TMN-1 could regulate the content of cinnamic acid in root exudates,improve soil microbial community,and reduce the content of bacterial blight in rhizosphere soil,so as to effectively control tobacco bacterial wilt with the control effect of41.23%～54.16%.In summary,this study clarified the metabolic characteristics of cinnamic acid in different tobacco resistant and susceptible to bacterial wilt varieties and different growth stages.It was found that cinnamic acid can promote the movement,chemotactic activity,biofilm formation,and root colonization of bacterial wilt;It has been clarified that RSc1155 is a key gene in response to cinnamic acid in R.solanacearum;The degradation activity of T.harzianum TMN-1 on cinnamic acid was evaluated,and it was determined that the key mechanism of applying T.harzianum TMN-1 to control bacterial wilt was the degradation of cinnamic acid secreted and accumulated during tobacco growth.The research results have important theoretical significance and practical value in revealing the molecular mechanism of rhizosphere secretion regulating the occurrence of bacterial wilt and developing disease prevention and control technologies based on chemical ecology.