Isolation,Identification And Biological Control Of Soil-Borne Strain B56 Against Tobacco Brown Spot

Tobacco brown spot is a serious leaf disease in the mature period of tobacco,caused by the fungus Alternaria alternata（Fries）Keissler,which has a short incubation period,spreads rapidly and can infect large areas of tobacco.It is mainly controlled by chemical pesticide fungicides at present,however,chemical control tends to deteriorate soil conditions,cause environmental pollution and increase pathogen resistance,and in recent years,these fungicides have become less effective in controlling tobacco brown spot.There is an increasing amount of research into the biological control of tobacco brown spot,and the screening of antagonistic bacteria has become one of the hotspots.In this thesis,an active strain of Bacillus velezensis B56 was isolated from tobacco rhizosphere soil samples,which provided a theoretical basis for its field application.The main research results are as follows:1.A total of 216 strains were isolated from 65 tobacco rhizosphere soil samples from different regions by dilution coating method and temperature screening method,A.alternata was used as an indicator bacteria to re-screen the most inhibitory strain with the plate confrontation method.The experimental results showed that strain B56 has the strongest inhibitory effect on A.alternata with an inhibitory bandwidth of 11.5 mm,which was identified as Bacillus velezensis by cellular fatty acid characteristics,biochemical characteristics and molecular biology.2.The fermentation medium and conditions of B.velezensis B56 were optimized through single factor optimization experiments.The results showed that the best medium carbon source,nitrogen source and inorganic salt were glucose,yeast extract and KH₂PO₄,And the orthogonal experiment results show that for each effect level yeast>KH₂PO₄>glucose>sucrose,the optimal combination should be yeast 1.5%:KH₂PO₄1.5%:glucose 1%.Based on this medium,the initial p H,culture temperature,filling volume and inoculum volume of the fermentation broth of strain B56 were optimized.The results showed that the optimal fermentation conditions were as follows:incubation p H of 7,incubation temperature of 30°C,filling volume of 50 m L/250 m L,and inoculum level of 1.5%.Comparing the antibacterial effects of the aseptic filtrate before and after optimization,the results show that the optimized aseptic filtrate can increase the antibacterial bandwidth by 2 mm.3.The biocontrol mechanism of B.velezensis B56 was further explored,and the results showed that the hyphae of A.alternata after being treated with the fermentation broth of B.velezensis B56 had irregular hyphae,increased breakage,deformed swelling,and slower growth.The inhibition rate of spore germination of A.alternata is about 37%.The secondary metabolite of strain B56 has an inhibition rate of 46.88%against A.alternata.It has a good inhibitory effect on the eight kinds of pathogens including Bipolaris sorokiniana,and the overall bacteriostatic bandwidth is between 3.87±0.25～15.10±0.10,showing a broad spectrum of bacteriostasis.The strain B56 can produce cellulase and protease,and has the ability to solubilize phosphorus and fix nitrogen,after inhibiting different bacteriostatic substances,the comparison of activity shows that protein is the most active bacteriostatic substance in B.velezensis B56.The optimal ammonium sulfate saturation of precipitated protein is 80%,and the concentration of the protein is 0.8422 mg/m L.which laid the foundation for further extraction and identification of antibacterial proteins.4.A mutant strain of B56^SMresistant to 300μg/m L streptomycin was screened by the method of pharmaceutical domestication.The strain has stable antibacterial activity and can colonize tobacco leaves for about 15 days with a trend of increasing and then decreasing bacterial load,which can help guide agricultural drugs to be supplemented after 15 days of application,the results of growth promotion experiments show that after 30 days of cultivation,the tobacco plants treated with the fermentation broth of strain B56 grow better than the control group,and the dry leaf weight increases the weight rate was 86.11%.In the pot control experiment,the control effect of 50%carbendazim positive control group was88.24%,and the control effect of the strain B56 treatment group was 77.78%;in the field control experiment,the control effect of treatment group was 29.67%after 7 d of the first application.and the positive control group was 60.07%,the control effect of the treatment group was 62.88%after 7 d of the third application,and the positive control group was80.56%.The results showed that with the increase of application days,the amount of bacterial strain B56 colonized on tobacco leaves gradually increases,and the effectiveness of prevention increased.It has been observed that bacterial strain B56 has a promoting effect on tobacco growth and has no phytotoxicity,which can replace some chemical pesticides and reduce the use of chemical pesticides,it provides theoretical guidance for the practical application of B.velezensis B56.