Study On The Relationship Between The Diversity Of Tobacco Bacterial Community And The Occurrence Of Tobacco Wildfire And Its Regulation Effect

Tobacco wildfire is an explosive,devastating bacterial leaf disease caused by Pseudomonas tabaci.The prevalence of tobacco wildfires often causes large-scale decay and shedding of tobacco leaves,seriously affecting the roasting of tobacco leaves,and causing huge economic losses to tobacco production.It is one of the key problems that restricts the sustainable development of tobacco production.In recent years,with the continuous in-depth research of plant micro-ecological environment by scientific researchers,the occurrence of plant diseases is considered to be largely inseparable from the imbalance of plant micro-ecological environment.Therefore,the occurrence of tobacco wildfire can be understood as the imbalance of leaf microecology.Inter-leaf microorganisms are important components in the inter-leaf micro-ecological environment.Adjusting the composition of inter-leaf microorganisms and increasing the abundance of antagonistic microorganisms are considered to be an important means of biological control of leaf diseases.However,the current research on the influencing factors of tobacco wildfires mainly focuses on environmental factors such as climate,temperature and humidity,and the research on the use of antagonistic microorganisms for disease prevention in leaves mainly focuses on inducing systemic resistance,promoting plant growth or interfering with pathogenic bacteria.Factors,etc.,have not yet clearly explained the changes in the microbial community structure caused by antagonistic microorganisms in the leafy micro-ecological environment and their relationship with disease prevention and control.In addition,the phyllosphere is an extreme environment with high radiation,oligotrophic,and low moisture,and the colonization of antagonistic microorganisms on leaves also needs further study.Therefore,by exploring the characteristics of the inter-leaf micro-ecological characteristics of tobacco wildfire at different disease levels,to find the key factors affecting the incidence,reuse coordinated leaf nutrition,use antagonistic microorganisms to regulate the inter-micro-ecological balance,and play a key role in effectively controlling tobacco wild-fire disease.In this paper,by collecting tobacco leaf samples with different incidences of tobacco wildfire,and using high-throughput sequencing technology,the relationship between the diversity of tobacco leaf bacterial community and the occurrence of wildfire was studied.At the same time,in the field environment,the role of microbial inoculants in preventing and controlling tobacco wildfire was explored.And on this basis,using molecular biology technology,we studied the dynamic changes of beneficial bacteria in the tobacco leaf and how to regulate the diversity of bacterial communities in the leaf,in order to provide support for the research on the prevention and control of tobacco wildfires.The main research results of this dissertation are as follows:1.There is a close relationship between the composition of the inter-leaf bacterial community at different levels of tobacco wildfire disease.With the increase in the level of disease,the richness of the inter-leaf bacterial species decreases.The 16S rRNA high-throughput sequencing technology was used to analyze the community diversity of unfired and diseased tobacco plants and leaf-level microbial communities with different disease levels.The results showed that the most abundant bacterial phylums in leaf samples were Proteobacteria.Proteobacteria,the top three bacterial groups are Firmicutes,Bacteroidetes and Actinobacteria,and in different disease levels,the four bacterial groups are relative The abundance varies greatly.Analyze the microbial community structure of tobacco leaves in the unoccurred and different disease levels.Compared with the unoccurred samples,the abundance and diversity index of the bacteria is reduced.There is a trend of decreasing in turn,and the abundance and structure of inter-leaf microorganisms may be the key factors affecting the incidence of tobacco wildfire.Further analysis of the main difference groups between the unoccurred and the diseased phyllospheric microbes,52 predominantly bacterial groups with significant differences were selected from the unoccluded phyllosphere samples;seven significantly different bacterial predominant groups were selected from the diseased samples.At the genus level,the four bacterial genera,Xanthomonas,Neorhizobium,Massilia,and Phycicoccus,are the predominant groups in the disease samples;Acinetobacter,Methylobacterium,Sphingopyxis and other 22 genera are the predominant groups in the non-morbid samples.And through the difference test between groups,the relative abundance of Enterococcus and Acinetobacter in the unoccurring samples on the bacterial community was significantly higher than that of the onset samples.2.Through the qPCR method,it was verified that the beneficial microorganism Bacillus subtilis can effectively colonize tobacco leaves.Through molecular biology methods,we obtained plasmid quantitative standards of Bacillus subtilis and total bacteria,established a quantitative PCR system,and drawn quantitative PCR curves of the two.On this basis,a total of 26 tobacco samples were collected 58 days after treatment with 10~7cfu/mL Bacillus subtilis.The dynamic changes of the number of Bacillus subtilis and total bacterial cells on tobacco leaves were analyzed and plotted.Tests have shown that Bacillus subtilis can colonize tobacco leaves,and the number of Bacillus subtilis cells has been changing.Exogenously added microorganisms have a certain effect on the inter-microbial community,which is manifested by the large changes in the number of bacterial cells,but after a period of time,the total amount of bacteria in the inter-leaf region tends to stabilize.3.The application of leaves of Bacillus subtilis and Bacillus polymyxa can regulate the composition of the tobacco leaf bacterial community.At the genus level,the community composition between different treatments was analyzed.The relative abundance of Pseudomonas treated by Bacillus subtilis decreased by 27.37%,and the relative abundance of Acinetobacter increased.20.80%;the relative abundance of Pseudomonas treated by Bacillus polymyxa increased by 6.32%.After treatment with beneficial microorganisms,the richness index of phyllobacteria decreased,while the diversity index did not change much.Through predominant group analysis,after treatment with Bacillus subtilis,4 significantly different bacterial predominant bacterial groups were selected from Bacillus subtilis treatment;30significantly different bacterial predominant bacterial groups were selected from control treatment.After treatment with Bacillus polymyxa,2 significantly different bacterial predominant bacterial groups were selected from Bacillus polymyxa;46 significant bacterial predominant bacterial groups were selected from the control treatment.At the genus level,the three bacterial genera Xanthomonas,Paenibacillus,Massilia,etc.are the dominant groups in the treatment of Bacillus subtilis;Saccharibacillus,Xanthomonas and other two bacterial genera are the dominant groups in the treatment of Bacillus polymyxa.4.In the field treatment,Bacillus subtilis'field control effect on tobacco wildfire can reach56.62%.By balancing the nutrient balance of the leaves,spraying beneficial microbial inoculants and other micro-ecological control techniques,experiments were conducted in areas with high incidence of tobacco wildfire.Field results showed that beneficial microbial inoculants treatment significantly affected the occurrence of tobacco wildfires.The disease index and prevention and control effects are reflected in three aspects.The wildfire leaves appeared in the long-term period of the tobacco plant to the prosperous period,and reached the peak of the incidence in the prosperous period.The current bud period to the topping period is another high incidence period of tobacco wildfire disease.The results of three investigations found that Bacillus subtilis treated The diseased leaf rate was significantly lower than that of Bacillus polymyxa treatment and water control.From the perspective of prevention and control effects,in the three growth stages of tobacco plants,the chemical treatments reduced the occurrence of tobacco wildfires to varying degrees,and the control effects of Bacillus subtilis reached 46.95%,49.88%,and 56.62%,respectively.Obviously,it can be used as a biological agent to control tobacco wildfire in the field.