| Plant virus disease is known as"plant cancer"because of its wide distribution,rapid prevalence,serious harm and difficult prevention and control.Due to the excessive use of chemicals,the environment is overwhelmed.People pay more attention to the use of green biological control methods to control plant virus diseases.In this study,the classification,genome-wide analysis,optimization of fermentation conditions,analysis of antiviral active substances and antiviral mechanism of strain B55 with inhibitory effect of tobacco mosaic virus（TMV）were systematically studied,so as to provide available resources and theoretical basis for the research and development of antiviral microbial pesticides.The results of this study are as follows:1.Strain B55 was classified and identified.The morphological,physiological,biochemical characteristics of strain B55 were determined and 16S r DNA molecular identification was carried out.The results showed that strain B55 was Gram-positive and rod-shaped.It could produce spores.The spores were oval,mesophytic and cysts were expanded,the colony shape was irregular,the surface was wrinkled,and it had a variety of activities.The homology analysis of 16S r DNA sequence showed that strain B55 had high homology with Bacillus velezensis,up to 100%,Combined with the determination of morphological,physiological,biochemical characteristics,it was identified as Bacillus velezensis.2.The whole genome of Bacillus velezensis B55 was analyzed.The whole genome sequencing analysis showed that the total number of genes of strain B55 was 4018,the total genome sequence was 3929772 bp,the average GC content was 46.5%,and it contained 27r RNAs,86 t RNAs,70 tandem repeats,1 genome island and 1 prephage.By analyzing its metabolic system,genes related to a variety of carbohydrate active enzymes were annotated,12 gene clusters related to the synthesis of secondary metabolites were detected,and cog,go and KEGG were annotated with a variety of functional genes.3.The fermentation conditions of Bacillus velezensis B55 were optimized.The study was optimized from three nutritional conditions and four environmental conditions.The best substances in several carbon sources,nitrogen sources and inorganic salt components were determined through single factor test.The ratio was optimized by Box-Behnken response surface method.The best formula was 5 g soluble starch,5 g peptone,15 g Zn SO4and 1000ml distilled water.According to the optimized medium,the four environmental conditions of p H,temperature,time and rotating speed were screened.The optimal fermentation scheme was determined as p H 5,28℃,180 r/min and 48 h fermentation.It was verified that the inhibition effect was significantly better than that of the fermentation broth before optimization,and the yield of active substances was increased by 25%.4.The antiviral active substance of Bacillus velezensis B55 was analyzed and showed to be biosurfactin.The light yellow active crude extract was obtained by acid precipitation.After purification by Sephadex LH-20 column chromatography and thin layer chromatography,there were fluorescent bands under the irradiation of 365 nm ultraviolet light.After spraying ninhydrin,it did not develop color,and there were light purple bands after heating.The surface tension of the active substance is close to that of pure surfactins.It has good oil drainage activity and certain heat resistance.It still has activity after being treated at 120℃for 30 minutes.Through comprehensive test and analysis,it is preliminarily judged that the active substance is biosurfactant.Surfactins has inhibitory effect on Bacillus cereus when the concentration is 25μg/m L still has inhibitory effect.When the concentration of biosurfactin is200μg/m L,the inhibition rate of TMV was 69.4%,the preventive effect of TMV was 54.2%,and the therapeutic effect was 32.4%,which could promote the growth of tomato seedlings.5.The anti TMV mechanism of surfactins was preliminarily explored.Will 200μg/m L surfactins was mixed with TMV particles in the same amount.The morphology of TMV particles was observed by transmission electron microscope with negative staining method.The results showed that surfactins had no effect on the morphology of TMV particles.The accumulation of TMV was measured by real-time fluorescence quantitative PCR.The results showed that surfactins could down regulate the accumulation of TMV and inhibit the replication and proliferation of TMV in tobacco;The expression levels of several tobacco related defense genes were measured.The results showed that surfactins could induce the expression of related defense genes such as COIl,NPR1,PAL,PDF1.2 and PR1a.The expression levels of related defense genes were higher than those in the control group.The expression level of NPR1 gene was gradually increased within 5 days of treatment,and the expression level was 17 times higher than that in the control on the 5th day,indicating that surfactant induced plant system to produce disease resistance through salicylic acid signal pathway,So as to resist the infection of TMV. |
