Study On The Mechanism Of High Nitrogen Fertilizer Application Promoting Outbreak Of Tobacco Bacterial Wilt

Ralstonia solanacearum is a Gram-negative bacterium that can spread through soil and infect more than 450 plant species in more than 50 families,causing catastrophic blight disease.This bacterium is widely distributed around the world and is one of the ten most harmful,widely distributed and most difficult to control pathogens in plant bacterial diseases.R.solanacearum enters through the root system of the host plant,and then migrates into the vascular bundle ducts,grows rapidly and secretes a large amount of viscous exopolysaccharides,causing the ducts to be blocked,affecting the transportation of nutrients,and leading to rapid wilting of plants die.Rhizosphere microorganisms play an important role in plant growth and development,stress resistance,and disease defense.For example,some beneficial microorganisms reduce the occurrence of diseases by inducing plant systemic defense responses;some antagonistic microorganisms reduce the incidence of disease by inhibiting the colonization of pathogenic microorganisms.Disease occurs.Our previous studies have shown that excessive application of nitrogen fertilizer can increase the expression level of R.solanacearum host infection-related genes,promote the erosion of R.solanacearum on the root tip of tobacco,and lead to the outbreak of tobacco bacterial wilt disease,but whether excessive nitrogen application affects the rhizosphere Microorganisms that cause disease prone to occur still need to be further revealed.This paper takes this as a starting point to study the changes of tobacco rhizosphere microorganisms under different nitrogen application levels and R.solanacearum infection,in order to reveal the rhizosphere microecological process that high nitrogen input promotes the outbreak of R.solanacearum.The specific results are as follows:(1)Tobacco rhizosphere microorganisms were sequenced using Pacbio’s 16 S rDNA full-length diversity.The results showed that the α-diversity of microbial communities was significantly reduced under high nitrogen levels,regardless of whether R.solanacearum was inoculated or not.The results ofβ-diversity analysis showed that the amount of nitrogen application was the most important factor for the variation of microbiome.The results of rhizosphere microbial species composition analysis showed that nitrogen application rate affected the relative abundance of Proteobacteria,while inoculation with R.solanacearum mainly affected the relative abundance of Bacteroidetes and Acidobacteria.Differential OTU analysis showed that the composition of soil microbial dominant flora was different at different nitrogen application rates,and there were 31 OTUs with significant changes in high nitrogen level(N3)compared with no nitrogen application(N0),and N3 compared with low nitrogen level(N1)There were 70 OTUs with significant changes.The results of co-occurrence network analysis showed that when R.solanacearum was inoculated,relative to the N3 level,the difference OUT between N0 and N1 experimental treatments was mainly Bacillus.It was speculated that Bacillus may antagonize the occurrence of R.solanacearum.Metabolic characteristics and functions of rhizosphere microorganisms.(2)Low nitrogen levels(N0,N1)tobacco disease was mild,but the plant growth was slow.Inoculation of nitrogen-fixing growth-promoting bacteria(Bacillus Velez XD-S1)can significantly increase soil total nitrogen content at N0 and N1 levels by 20.51% and 18.60%,respectively,and significantly promote the growth of tobacco plants at N0 and N1 levels: Maximum The leaf area increased by 138.06% and 130.07%,the plant height increased by 37.26% and 37.71%,and the stem diameter increased by 61.16% and 34.66%,respectively.The occurrence degree of bacterial wilt disease shows that the speed of plant growth is not an important factor for the occurrence of bacterial wilt disease.(3)The research on the gene cluster of R.solanacearum can reveal its metabolic pathway,and further understand its interaction mechanism with plants.According to research,the rsc2037-41 gene cluster of R.solanacearum may be related to nitrate nitrogen metabolism,the rsc0379-82 gene cluster may be related to urea metabolism,and the rsc0343 gene cluster may be related to ammonium nitrogen metabolism.Construction of reporter gene results show that,the expression of this part of the gene is low in rich culture medium,but it will be specifically up-regulated under nutrient-poor conditions.However,the knockout of nitrogen-related genes related to nitrate nitrogen and urea metabolism did not affect the infection of tobacco by R.solanacearum at different nitrogen levels.At N0,N1,and N3 levels,the mutants exhibited the same pathogenicity as the wild type,indicating that genes related to nitrogen metabolism in R.solanacearum may not be directly related to nitrogen levels in the environment.