Promotion Of Tobacco Rhizosphere Colonization And Disease Prevention Mechanism Of Bacillus Amyloliquefaciens By Ulva Prolifera Polysaccharide

Tobacco bacterial wilt is a worldwide destructive soil-borne bacterial disease caused by Ralstonia solanacearum,which has a great impact on the yield and quality of tobacco and has brought huge economic losses to tobacco farmers.Plant growth-promoting rhizobacteria（PGPR）play an important role in agricultural production for crop health and high yield.The agricultural application of rhizosphere microorganisms is of positive significance for reducing the application of chemical fertilizers and pesticides.However,there are many problems in the practical application of biocontrol bacteria,such as large differences between indoor and field control effects,and unstable field control effects,which hinder their practical application in agricultural.This study selected the interaction system of tobacco-R.solanacearum as the research object,took Ulva prolifera polysaccharide and Bacillus amyloliquefaciens strain Cas02 as the test material,combined with bioinformatics,biochemistry,molecular biology and other experimental analysis technologies,preliminarily revealed the promotion of tobacco rhizosphere colonization and disease prevention mechanism of strain Cas02 by Ulva prolifera polysaccharide.The experimental results include the following aspects:1.In this study,B.amyloliquefaciens strain Cas02 was isolated from the rhizosphere soil of healthy tobacco plants and evaluated for its effect on plant growth promotion and bacterial wilt suppression.Strain Cas02 exhibited the plant growth-promoting-related features including siderophore production,cellulase activity,protease activity,ammonia production and catalase activity.Moreover,strain Cas02showed a significant inhibitory growth effect on R.solanacearum,and its active substances were separated and identified to be macrolactin A and macrolactin W by HPLC-ESI-MS/MS.To explore the underlying genetic mechanisms,complete genome sequencing was performed and the gene clusters responsible for antibacterial metabolites expression were identified.At the same time,greenhouse and field experiments demonstrated a good performance of Cas02 in plant growth promotion and bacterial wilt suppression.2.Ulva prolifera polysaccharide promotes root colonisation by B.amyloliquefaciens strain Cas02.Ulva prolifera polysaccharide serves as an environmental signal for strain Cas02 biofilm formation and its glucose residue is used as a carbon source for the synthesis of the exopolysaccharides and polygamma-glutamate present in biofilm matrix.The transcriptome also confirmed this result,and the genes related to carbohydrate transport and metabolism,cell wall/membrane/capsule bioformation and bacterial extracellular polymeric substances（EPS）synthesis were significantly upregulated in strain Cas02 after Ulva prolifera polysaccharide treatment.Greenhouse experiments showed that the combined application of Ulva prolifera polysaccharide and strain Cas02 could significantly promote the growth of tobacco seedlings and increase the content of NO₃^--N,NH₄⁺-N and other nutrients in the soil.This may be due to the increase in the population of Bacillus spp.,which led to the improvement of biological nitrogen fixation,and the formation of biofilm was crucial to the nitrogen fixation of rhizospheric bacteria.Ulva prolifera polysaccharide can enhance its rhizosphere colonization ability by increasing the population number and survival time of strain Cas02 under hydroponics and natural semiarid soil conditions.Furthermore,the microbiome analysis also indicated when the Ulva prolifera polysaccharide and strain Cas02 were jointly treated,the promoted rhizosphere colonisation of strain Cas02,as well as the improved bacterial rhizosphere community structure,the abundance of soil beneficial bacteria such as Burkholderia-Caballeronia-Paraburkholderia,Sphingomonas and Sinomonas was increased,which indirectly proved that after the combined application of Ulva prolifera polysaccharide and strain Cas02 had a synergistic effect in promoting plant growth.3.R.solanacearum RS10 has limited influence on the biofilm formation of strain Cas02 induced by Ulva prolifera polysaccharides,and Ulva prolifera polysaccharides selectively serve as a carbon source for strain Cas02 rather than RS10.Pot experiment showed that the combined application of Ulva prolifera polysaccharide and Cas02 could increase the abundance of Bacillus,and both of them significantly reduced the abundance of R.solanacearum in the rhizosphere soil of tobacco seedlings when inoculated with R.solanacearum after treatment.Analyzing the structure and diversity of soil microbial community,it was found that the abundance of Bacillus increased significantly after the combined application of Ulva prolifera polysaccharide and strain Cas02,and the prevention and control of R.solanacearum were focused on prevention rather than control.The prevention effect when bacterial wilt did not occur was better than the control effect when bacterial wilt broke out,which was mutually confirmed by soil q RT-PCR data.In addition,the abundance of Sphingobium,Dyadobacter,Shinella and Burkholderia-Caballeronia-Paraburkholderia increased significantly.To sum up,this study systematically understood the physiological characteristics and genetic background of B.amyloliquefaciens Cas02,and analyzed the mechanism of Ulva prolifera polysaccharide enhancing the rhizosphere colonization of strain Cas02 and selectively promoting the competitive colonization of strain Cas02 to RS10,which not only provided candidate strains for the development of new microbial agents,but also provided theoretical basis and practical reference for the biological control of tobacco bacterial wilt.