Screening,Control Effect And Optimization Condition Of Antagonistic Bacterial Strains Against Alternaria Alternata In Vermicompost

As an important economic crop in China,tobacco is of great significance in promoting local economic structure.Tobacco brown spot is a common fungal disease that severely reduces tobacco yield and economic benefits.Therefore,biological measures to prevent tobacco brown spot disease have significance for flue-cured tobacco production.At present,chemical control is the primary method,but it has brought seriously environmental pollution.Biological control being safe,efficient and pollution-free has become the most potential method to prevent tobacco diseases.Earthworm cast is a kind of bio-organic fertilizer rich in organic matter,plant hormones and antagonistic microbial resources.Screening antagonistic bacteria from vermicompost to prevent tobacco fungal diseases can effectively solve ecological pollution and provide a new idea for the resource utilization of organic waste.In this study,A.alternata was used as an indicator bacterium to isolate and screen antagonistic bacteria from vermicompost,and identified and verified the control effect of antagonistic bacteria,optimized fermentation conditions,and clarified the suitable training program.The preliminary conclusions of this study were as follows;1.A total of 200 strains of bacteria were isolated from earthworm cast,of which 28strains had antagonistic activity against A.alternata.The results showed that the inhibition diameters of Q161 and Q96 were more than 20 mm,and the inhibition rates of Q161 and Q96 were 67.95%and 65.15%respectively.The results of morphological identification,physiological and biochemical index determination,and 16S r DNA sequencing showed that Q161 strain was B.velezensis and Q96 strain was B.paralicheniformis respectively.2.The results of control effects showed that Q161 and Q96 could inhibit the spore germination and mycelial growth of A.alternata through competitive inhibition and secretion of active antibacterial substances which has stronger inhibitory effects.The inhibited spores showed vesicular at the germinating site,and the germ tubes were twisted,deformed,and lost growth vigor.Under the antagonism of Q161 and Q96 strains,the color,texture and edge morphology of the A.alternata colony were changed.Experiments results showed that Q161 and Q96 strains could reduce the incidence rate and disease index of tobacco brown spot to varying degrees in vitro and potted plant.3.The optimization results of fermentation conditions showed that the best medium ratio for Q161 strain were 0.5%yeast extract,4.0%sucrose,3.0%tryptone,1.0%NaCl,0.15%CaCl₂,and 0.25%K₂HPO₄,and the optimal culture conditions were as culturing the seed solution for 48 hours,inoculating the seed solution with a p H of 5.5 according to 0.6%of the inoculum,and culturing at 34°C and 210 r/min.Similarly the best medium ratio for Q96 strain were yeast extract 0.5%,sucrose 3.5%,tryptone 1.5%,NaCl 1.0%,CaCl₂0.20%,Ca CO₃0.25%;and the optimal culture conditions were as follows:culturing the seed solution for 48 hours,inoculating the seed solution with a p H=5.5 according to 0.5%of the inoculum,and culturing at 31°C and 180 r/min.In this study,we have elucidated the taxonomic identification,antagonistic effects and fermentation conditions optimization of Q161 and Q96 strains provided a theoretical basis for the preparation of biological agents against Alternaria alternata.