Screening And Antiviral Mechanism Research Of Biocontrol Bacteria Against Tobacco Mosaic Virus

Tobacco mosaic virus is one of the most common diseases in tobacco growing season.In this study,TMV was used as the research object to isolate and screen biocontrol strains from cake fertilizers and rhizosphere soils,and to conduct physiological and biochemical characteristics and molecular biological identification to determine their taxonomic status;explore fermentation conditions;and simultaneously conduct virus antagonism experiments and genome sequencing analysis To get a preliminary understanding of its antiviral mechanism.The results are as follows:(1)Through a series of separation and purification and screening,82 bacterial strains were successfully isolated,and 10 strains with obvious antagonistic effects on TMV were obtained by the semi-leaf blight screening method,of which BV1’s blight suppression rate reached 93.22%;follow-up The morphological identification,physiological and biochemical identification of BV1 and 16 s DNA sequencing were compared.The results showed that the BV1 strain was Bacillus velezensis.(2)A single factor experiment was used to optimize the fermentation conditions of BV1,and it was found that the best fermentation condition of the BV1 strain for TMV antagonistic activity was LB medium,p H 7.0,p H 2.5,optimal inoculation volume 2.5%,optimal fermentation temperature 30℃,speed 220r/ min,the best fermentation time is 54 h.(3)Using field plot experiments to verify the biological control effect of BV1,artificial inoculation of TMV,the incidence rate and disease index of blank control area were 90.06% and 40.03 respectively,and the incidence rate and disease index of BV1 fermentation broth treatment area were 60.20% and 17.90,BV1 respectively The relative control effect of fermentation broth is 55.28%.(4)The prevention and treatment effect of BV1 fermentation broth on TMV was tested by the half leaf blight method,and it was found that the prevention effect was71.43% and the treatment effect was 59.87%;BV1 fermentation broth was mixed with TMV-GFP to inoculate tobacco,and fluorescent expression The amount is far lower than the control group,indicating that BV1 has a passivating effect on TMV;real-time fluorescence quantitative PCR experiments found that the relative expression of PR genes in tobacco plants treated with BV1 fermentation broth was much higher than the control group,while the relative cumulative amount of TMV It is far lower than the control group,indicating that BV1 can induce tobacco plants to develop systemic resistance and inhibit TMV infection and proliferation.(5)Genomic sequencing and analysis of the BV1 strain found that the BV1 strain was on the same branch as Bacillus bellecii SCGB＿1 and Bacillus beiziensis SCDB＿291,with very close genetic relationships.The BV1 gene is combined into 12 secondary metabolite gene clusters.In addition to the gene clusters containing antibacterial substances such as bacteriocins,surfactants,Fengyuansu and macrolides,there is a peptide natural product type III wool in BV1 Thiopeptide may be related to anti-virus.