Screening,identification And Resistance Mechanism Of Rhizosphere Keystone Taxa Against Tobacco Bacterial Wilt

As a bridge between plants and soil ecosystem,rhizosphere microorganisms are the first line of defense for plants to resist pathogen infection,which is closely related to plant health.In fact,a few key microbial groups,namely keystone taxa,may really play a role in rhizosphere microorganisms.Therefore,finding the rhizosphere keystone taxa and revealing the mechanisms of enhancing plant disease resistance are expected to alleviate the problems of current biocontrol agents and provide new strategies for the prevention and control of soil-borne diseases in tobacco.Based on Ralstonia solanacearum-tobacco(Nicotiana tabacum L.)interaction system,this thesis studied the response of tobacco plants and rhizosphere microorganisms to the infection of R.solanacearum,comprehensively analyzed the community characteristics and functions of tobacco rhizosphere microorganisms in different types of soil(disease-suppressive and disease-conducive),and obtained the rhizosphere keystone taxa of tobacco in disease-suppressive soil.Finally,the mechanisms of controlling tobacco bacterial wilt by tobacco rhizosphere keystone taxa was revealed at the molecular level.The following results were obtained:1.The infection of R.solanacearum had multiple effects on tobacco plants and rhizosphere microorganisms.R.solanacearum were inoculated into the rhizosphere of tobacco seedlings,and the rhizosphere microbiome and leaf transcriptome were sequenced.The integrative analysis of microbiome and transcriptome showed that,compared with the control group,the treatment inoculated with R.solanacearum would increase the number of pathogens in tobacco rhizosphere,affect the structure and function of rhizosphere microbial community,destroy the nutritional composition of cultivated soil,inhibit the relative expressions of structure genes in phenylpropane metabolic pathway related to plant system disease resistance,and lead to the incidence of bacterial wilt exceeding 80%.2.There were significant differences in the structure and function of rhizosphere microbial community between disease-suppression and disease-conducive tobacco fields.The rhizosphere soil and root samples of tobacco in disease-suppression and disease-conducive tobacco fields were collected and sequenced by 16 S r RNA gene.The results showed that there were great differences in rhizosphere bacterial and fungal communities in different samples,and the disease-suppression soil had a more complex bacterial network structure than the disease-conducive soil,and the key groups to maintain the stability of the bacterial network in the disease-suppression soil were identified.By means of microbial isolation and culture and phylogenetic tree analysis,three keystone taxa strains of tobacco rhizosphere were obtained: Pseudomonas koreensis HCH2-3,Pseudomonas rhodesiae MTD4-1,and Pseudomonas lurida FGD5-2.3.Tobacco rhizosphere keystone taxa had good growth-promoting effects on tobacco.Three tobacco rhizosphere keystone taxa isolates were tested in vitro and pot experiments.The results showed that the three Pseudomonas strains had good growth-promoting characteristics in vitro,such as phosphorus dissolution,synthesis of iron carrier,and synthesis of plant auxin(IAA),and could significantly improve the agronomic characteristics of the height,effective leaf number,maximum leaf area,and biomass of tobacco,so as to promote the rapid growth of tobacco plants.4.Tobacco rhizosphere keystone taxa could effectively reduce the incidence of bacterial wilt.Under greenhouse conditions,the obtained rhizosphere keystone taxa were inoculated into tobacco,and then the host was infected with the pathogen of bacterial wilt to determine the control effects of tobacco rhizosphere keystone taxa on bacterial wilt.The results showed that compared with the control group,the three strains of Pseudomonas significantly reduced the number of R.solanacearum in rhizosphere and root,and reduced the incidence of tobacco bacterial wilt,and changed the bacterial community structure,in which the relative abundance of Azospira increased significantly,and the absolute abundance of nitrogen fixing gene nif H also increased significantly.The inoculation treatments of three Pseudomonas strains significantly improved the physical and chemical properties of soil,and increased the contents of total nitrogen,ammonium nitrogen,and nitrate nitrogen in soil.In addition,it enhanced the activity of plant-related defense enzymes and activated the structural genes of phenylpropane metabolic pathway and the disease-resistance genes EFE26,ACC Oxidase,and H1N1 related to ethylene(ET)and hypersensitive response(HR)pathways.To sum up,this thesis explored the impact of bacterial wilt infection on tobacco plants and rhizosphere microorganisms,studied the characteristics of tobacco rhizosphere keystone taxa dominated by Pseudomonas and their role in tobacco growth promotion and disease resistance,and speculated that tobacco rhizosphere keystone taxa provide defense responses for host plants through a multiple network,mainly including direct sterilization,improving the structure and function of rhizosphere microbial community,promoting the release of soil nutrients,especially nitrogen,and inducing plant systemic disease resistance.The above research results are helpful to guide the field control of tobacco bacterial wilt and promote the research and development of a new generation of biocontrol agents.