Screening,Identification And Biocontrol Effect Of Bacteria Against Tobacco Mosaic

Tobacco mosaic virus disease is a very representative of tobacco diseases,difficult to prevent and treat,seriously affecting tobacco production in China.When tobacco is infected by tobacco mosaic virus,the leaves show symptoms such as chlorosis,shrinkage and dwarfing,and the quality and yield of tobacco leaves decrease,resulting in serious economic losses.With the popularization of green prevention and control,biological control has gradually become an important way to control plant diseases and insect pests,reduce environmental pollution and pesticide residues.Among the microbial resources,Bacillus sp.has wide source channels,rich species,strong stress resistance and broad application prospects,which makes it the best choice for development of biocontrol agents.The aim of this paper is to enrich the resources of microorganisms resistant to tobacco mosaic virus,reduce the environmental pressure,and contribute to the promotion of green and sustainable development of agriculture.The main research contents and results are as follows:1.From 50 soil samples collected from Panzhihua tobacco planting base in Sichuan,235 strains with different forms were isolated and purified by high temperature water bath(70℃,30 min),the strain numbered SC-7 had the best anti-TMV effect after screening by the hemi-leaf blotch method;It was finally identified as Bacillus velezensis combining morphological characteristics,physiological and biochemical indexes,fatty acid content,16Sr RNA and gyr B gene sequences;Secondary metabolite production was measured and it was confirmed that strain SC-7 can secrete cellulase,protease,ferredoxin,and has the ability to dissolve phosphorus and produce ammonia.The results of the plate confrontation test showed that strain SC-7 produced inhibition bands against several pathogens,so lt has broad antibacterial spectrum.2.In order to improve the biomass of antagonistic bacteria and virus inhibition rate,the fermentation conditions of the strain were optimized by single factor experiment.The results showed that the optimal components of the medium were yeast,glucose and potassium chloride,and the optimal culture conditions were temperature 35℃,p H=7 and liquid volume80 m L(250 m L);The results of the orthogonal test showed that the optimal ratio of glucose,yeast and potassium chloride was 3:2:1,and the level of influence of each factor was glucose>yeast>potassium chloride.After optimization,the biomass of the strain increased by 22.94%compared with the basic medium,and the virus inhibition rate did not significantly increase.At the same time,the stability of fermentation broth of strain SC-7was measured,and the results showed that it had certain ultraviolet resistance,drought resistance and salt tolerance3.The anti-TMV effect and mechanism of strain SC-7 were explored.The results of in vitro passivation test showed that the sterile fermentation broth,crude protein extract and deproteinized supernatant of strain SC-7 had remarkable passivation effects in vitro,the blight inhibition rate were 90.68%,83.86%and 78.41%respectively;The results of pot experiment showed that after spraying the fermentation broth of strain SC-7 for 24 hours and then inoculating the virus,the control effect on tobacco mosaic virus was 40.54%after 7days,and decreased to 28.38%after 14 days,which had little difference with the control drug ningnanmycin.After spraying the fermentation broth of the strain,the activities of SOD and POD in tobacco were increased,the content of MDA was inhibited,so the tobacco resistance was improved.4.The role of biocontrol bacteria in promoting growth is closely related to biofilm formation and rhizosphere colonization.It was found that the strain SC-7 had ability to produce biofilm,as time goes on,the biofilm became thicker and more density;The colonization pattern of the labeled strain SC-7′in tobacco roots and rhizosphere soil was explored by antibiotic labeling method,the results showed that the colonization of the labeled strain SC-7′in the root and rhizosphere soil increased at first and then decreased within 28 days,reaching the highest values on day 5 and day 3,respectively,and the colonization amounts were 4.40×10~5CFU/g and 1.27×10~6CFU/g after 28 days,which showed a good colonization effect in rhizosphere soil.The results of in-dish experiment showed that the root length and fresh weight of tobacco seeds increased by 68.03%and23.67%respectively after soaking in fermentation broth for 2 hours,the results of pot experiment showed that the plant height,stem circumference,fresh and dry weight of above-ground part,fresh and dry weight of root of tobacco increased by 38.30%,27.00%,71.31%,65.86%,96.25%and 90.80%respectively after irrigation with fermentation broth,which indicated that strain SC-7 had remarkable growth promoting effect.In summary,Bacillus velezensis SC-7 has a broad application prospect in controlling tobacco mosaic virus and promoting tobacco growth.