Effects And Mechanisms Of Antagonistic Rhizobacterial Bacteria On Biological Control Of Bacterial Wilt In Tobacco

Tobacco bacterial wilt is one of the most important soil borne diseases in tobacco production in the main tobacco producing areas of China,which can cause devastating impact on tobacco production.At present,chemical control is the main control measure of Tobacco bacterial wilt,but the continuous use of chemical agents not only caused the problems such as the resistance of pathogenic bacteria,but also was not conducive to the protection of ecological environment and the sustainable development of agriculture.Using plant rhizosphere bacteria to control plant diseases was increasingly concerned.In this study,the antagonistic bacteria were isolated,screened and identified from healthy tobacco soil,and the control effect of antagonistic bacteria on tobacco bacterial wilt and its effect on tobacco growth were studied by pot experiment.On this basis,the effects of root exudates and root tissue components on the colonization of antagonistic bacteria were studied by chemotaxis and biofilm forming experiments,and the biological control mechanism and colonization characteristics of antagonistic bacteria in tobacco plants were preliminarily clarified.This study can further enrich the biocontrol resources of tobacco bacterial wilt and provide theoretical basis for further development and utilization of excellent biocontrol strains.The main results are as follows:1.The rhizosphere bacteria GZYCT-4 and GZYCT-9 with diameter of 16.64 mm and18.90 mm were isolated and purified from the rhizosphere soil of healthy tobacco plants in Huangshan and Xuancheng tobacco areas of Anhui Province by dilution coating method.it was determined that GZYCT-4 and GZYCT-9 were Bacillus sp.by molecular identification,both of them had the ability to synthesize lipopeptide.2.The results showed that different tobacco varieties treated with GZYCT-4 and GZYCT-9 could effectively control tobacco bacterial wilt.The control effect of red flower dajinyuan tobacco strain treated with GZYCT-4 and GZYCT-9 on bacterial wilt was 51.83%and that of K326 tobacco strain treated with GZYCT-9 on bacterial wilt was 58.62%.In addition,they can promote the growth of tobacco seedlings,and the growth rate of the plants treated with GZYCT-9 on the 10th,25th and 45th day was 34.29%,27.69%and 20.03%respectively,the growth rate of the plants treated with GZYCT-4 on the 10th,25th and 45th day was 11.43%%,22.5%and 17.43%respectively.3.The effects of root exudates of Honghuadajinyuan and K326 tobacco varieties on.the colonization of antagonistic bacteria and R.solanacearum were studied by chemotaxis and biofilm forming experiments.The results showed that oxalic acid,malic acid,citric acid and succinic acid could be produced from the root exudates of Honghuadajinyuan and K326.Among them,oxalic acid has the largest attraction to GZYCT-4,reaching 61.50×10⁴ CFU/ml^-1;citric acid has the largest attraction to GZYCT-9,reaching 34.00×10⁴ CFU/ml^-1,but none of the four organic acids has the chemotaxis to R.solanacearum.The results of biofilm assay showed that the root exudates of K326 could promote the biofilm formation of GZYCT-4,GZYCT-9 and R.solanacearum more than those of Honghuadajinyuan.Further study showed that malic acid,oxalic acid and citric acid can effectively promote the biofilm formation of GZYCT-4 and GZYCT-9,while citric acid can promote the biofilm formation of R.solanacearum4.The effects of root tissue extraction of Honghuadajinyuan and K326 on antagonistic bacteria colonization were studied by chemotaxis and biofilm formation experiments.Among them,GZYCT-4 and GZYCT-9 had obvious chemotaxis to the root extraction of K326 tobacco plant,and the chemotaxis of the GZYCT-4 and GZYCT-9 to the root extraction of K326 were 18 times and 4 times their chemotaxis to the root extraction of Rhododendron respectively.Further carbohydrate chemotaxis test showed that six carbohydrates had little attraction to GZYCT-4 and GZYCT-9,which was significantly lower than that of the control group.The biofilm formation test showed that the root extraction of K326 tobacco plant could more promote the biofilm formation of antagonistic bacteria,and the lactose and glycerin could promote the formation of antagonistic GZYCT-4 biofilm by 1.3%and 0.53%respectively compared to the control group,and the sucrose and glycerin could promote the formation of antagonistic GZYCT-9 biofilm by 0.8%and0.54%respectively.The results of plate culture showed that the inhibitory effects of lipopeptides of GZYCT-4 with sucrose and GZYCT-9 with xylan on tobacco bacterial wilt were significantly higher than that of the control groups,and the inhibitory diameter were 21.14mm and 20.54mm respectively.The Real-Time Fluorescent Quantitative PCR results showed that lactose and the root extraction of K326 could increase the expression of epsD,yqxM and kinC genes of GZYCT-4,while sucrose and the root extraction of K326 could promote the expression of eps D,kin C or yqx M genes of GZYCT-9.5.The colonization of antagonistic bacteria GZYCT-4 and GZYCT-9 in tobacco seedling were detected by antibiotic labeling method.It was found that the mutant strains GZYCT-4^kan and GZYCT-9^kan could be stably colonized in the root,stem and leaf tissues of tobacco seedling.The colonization dynamics of the two antagonistic bacteria in tobacco plant first increased and then decreased,and then tended to be stable.The two antagonistic bacteria could form symbiotic relationship with tobacco seedling...