Study On The Rhizosphere Microbial Community Of Tobacco-growing Soil Under Continuous Cropping Conditions

Tobacco is one of the most important economic crops in our country,expecially in southwest China.Large-scale continuous cropping and unscientific application of chemical fertilizers are common in tobacco production,which causes the imbalance of soil microorganisms and frequent occurrence of tobacco bacterial wilt.Tobacco-planted field was gradually developed into conduced soil and suppressive soil.It is found that the main substances of tobacco root exudates from different resistant varietyies were diverse.And the effect of root exudates on Ralstonia solanacearum(Rs)was significant different.Starting from this phenomenonon,high-throughput sequencing technology was employed to investigate the difference in rhizosphere microbe of disease conduced and suppressive soil mediated by tobacco root exudates.On this basis,the stable isotope nucleic acid probe(DNA-SIP)technology was used to clarify the nitrogen-related microbes in disease-conduced and suppressive soil.After the study finished,the characteristics and succesion of rhizosphere soil microorganism sill be revealed,and the control mechaniss of various resistant tobacco on tobacco bacterial wil will be illustrated,as well as the microbial mechanisms of soil continuouse cropping obstacle.The results will provide theoretical significance for biological control of tobacco bacterial wilt.The main results were showed as follows.1.The root exudates of different resistant varieties of tobacco was collected.Variety K326 with medium resistance and Honghua Dajinyuan(HD)high susceptible to tobacco bacterial wilt were provided.More than 60 kinds of substances from K326 and HD were identified by HPLC-MS.The main substances with significant differences in root exudateswere benzoic acid,palmitic acid,fumaric acid and arabinose.The main culturable microorganisms in the tobacco rhizosphere were screened and identified.The bacteria isolated from K326 rhizosphere were K2 and K3,identified as Pseudacidovorax intermedius and Rhizobium gallicum.While H2,H3 and H4 isolated from HD rhizosphere were identified as Microbacterium saperdae,Mycobacterium neoaurum and Nocardioides aromaticivorans.Strains K2 and K3 had the highest inhibitory rates against Rs,with 52.97%and 46.89%respectively.The isolated strains showed obvious differences in their chemotactic response to the main substances in root exudates.The chemotactic effect of fumaric acid on K2 and K3 was significantly higher than that of Rs.The utilization rate of benzoic acid by Rs was significantly higher than that of H2,H3,H4,K2 and K3,and was 0.21,0.23,0.26,3.24 and 2.84 timeshigher,respectively.2.A ready-to-use dynamic dialysis tube device was used to simulate the slow release of root exudates in soil in order to study the differences in the rhizosphere soil microbes of disease-conduced and suppressive soil mediated by different resistant tobacco root exudates.Throughput sequencing technology was employed to study the difference of soil microbial community structure and functional genes.The correlation analysis related soil nutrient indicators was performed.At the phylum level,the dominant phylums in disease-conduced soil were Proteobacteria and Actinobacteria,while the dominant phylums in disease-suppressive soil were Proteobacteria and Acidobacteria.The principal component analysis(PCA)and principal axis(PCo A)analysis showed that the disease-conduced soil mediated by K326 root exudate-(KCST),disease-conduced soil mediated by HD root exudate-(HDCST)and the disease-induced soil control(CSCK)were closely distributedin the map,indicating the community structure was similar.However,the disease-suppressive soil mediated by K326 root exudate-(KSST),the disease-suppressive soil mediated by HD root exudate(HDSST)and the disease-suppressive soil control(SSCK)were weperated in the map,suggesting there were significant differences in community structure.The Metastat analysis of the different species between the groups and the LEf Se analysis of the different species between the groups were used to further verify that tobacco root exudates have a significant impact on soil microorganisms:Honghua Dajinyuan root exudates significantly increase Actinobacteria,Streptomyces and Solirubrobacter significantly reduced Nitrospirae.Root exudates of K326 significantly increased Agromyces in disease-causing tobacco-growing soils.At the level of phylum,except for Planctomycetes,all the microbial species in disease-conduced soil were significantly related to environmental factors,apart from total phosphorus(P)and p H.There are 10 species that were significantly negatively correlated with total nitrogen(N),alkali-hydrolyzable nitrogen(AN),available phosphorus(AP),total potassium(K),available potassium(AK)and organic matter(OM).20 species were extremely significantly positively correlated with total nitrogen(N),alkali hydrolyzed nitrogen(AN),available phosphorus(AP),total potassium(K),available potassium(AK)and organic matter(OM).3.¹⁵N-DNA-SIP technology was used to study the succession of microbial communities in disease-conduced and disease-suppressive soil.It was found that the dominant bacterium involved in the nitrogen cycle in disease-conduced soil is Proteobacteria,in which Ralstonia solanacearum was included.The Proteobacteria involved in the nitrogen cycle in disease-conduced soils were mainly affected by root exudates,indicating that the disease-conduced soils were more affected by crops and are prone to disease.The bacteria involved in nitrogen cycle were actinomycetes in disease-suppressive soils accounted for a relatively high proportion,indicating that disease-suppressive soil is beneficial to exert resistance for the pathogen.Mediated by root exudates,Proteobacteria involved in the ¹⁵N cycle were positively correlated with disease-conduced soils,while Actinobacteria were positively correlated with disease-suppressive soils.Conlusion:1.The chemotaxis of root exudates on rhizosphere microorganisms is the foremost reason for the difference resistance of different resistant varieties to bacterial wilt.Benzoic acid may be the inducing substance of tobacco bacterial wilt occurrence for HD.While fumaric acid may be an auxiliary substance that inhibites bacterial wilt occurrence for K326.2.Under the mediation of root exudates,the microbial community structure and gene expression in disease-conduced and disease-suppressive soils are significantly different.Variety HD and K326 have different effects on the microbial community structure and regulation influence in disease-conduced and disease-suppressive soils.3.There are differences in microorganisms involved in the nitrogen cycle between disease-conduced and disease-suppressive soils.The nitrogen-cycling microorganisms in the disease-conduced soil are mainly affected by root exudates,while the nitrogen-cycling microorganisms in the disease-suppressive soil are mainly affected by the soil itself.In conclusion,the disease-conduced soil is affected by crops,thus it is easy to cause diseases.