| Plant virus disease is a serious diseases on agricultural production, which has broughtsevere losses to the output of crops. In recent years, Microbial source antiviral substances byits unique advantage have become a research focus in the screening of antiviral substance.Streptomyces ZX01was isolated from xinjiang kanas lake wetland by Northwest A&FUniversity Research&Development Center of Biorational Pesticide. Through the preliminarystudy found, ZX01fermentation broth had the obvious activity against TMV. For clearing theantiviral substances of Streptomyces ZX01secondary metabolites, we taking Nicotianaglutinosaã€tobacco-102and tobacco mosaic virus (TMV) as the host material and theexperiment virus, combining with the active tracking and modern separation technology, theantiviral substance in the ZX01fermentation broth were preliminarily researched. The mainresults were shown as follows:First, the antiviral substance of ZX01fermentation broth was enriched by macroporousresin, and50%methanol elution segment had the best activity against TMV. At theconcentration of10mg/mL, the inhibiting activity on TMV in vitro was95.94%. Then,50%methanol elution segment separated by liquid-liquid extraction, was founded that the activitysubstance was concentrated on the water phase. The water phase be analyzed by using TLCã€chromatographyã€dialyzing and proton nuclear magnetic resonance, we founded that themolecular weight which have high anti-TMV activity was more than3000, and hydrogenproton resonance peaks in spectra fitted the characteristics of glycoconjugates, so the mainingredient against TMV was natural macromolecule carbohydrate.First, using ethanol precipitation and sevage method were got a pure extracellularpolysaccharide (EPS). Then the EPS was separated by DEAE ion column chromatography,receiving four segments EPS-0ã€EPS-1ã€EPS-2and EPS-3. Through antiviral activity, whenthe concentration of the EPS was at0.5mg/mL, the inhibiting activity on TMV in vitro was81.32%, the inhibiting infectivity activity was63.77%, and the inhibiting multiplication onTMV was73.46%. When the four segments were at5mg/mL, EPS-0had no anti-TMVactivity; The activity of EPS-2was better than others, the inhibiting activity on TMV in vitro66.36%and the inhibiting infectivity activity48.26. Through the phenol-sulfuric acid method and coomassie brilliant blue method, the sugarcontent of EPS was51.83%, and the binding-protein content of EPS was12.19%; the sugarcontent of EPS-2was69.83%, and the binding-protein content of EPS-2was5.34%. Byultraviolet spectrum analysis, the nucleic acid and dissociative protein absorption were notseen under260nm and280nm in the EPS and EPS-2. The EPS-2presented a single andsymmetric peak by high performance liquid chromatography (HPLC) testing. By infraredspectrum and1H NMR analysis, EPS-2had the obvious absorption of polysaccharidecharacteristics, and the main glycosidic was alpha type.Field efficacy test of ZX01fermentation broth was performed in sprayingã€pouring rootand spraying+pouring root. The results showed that the efficacy of spraying+pouring rootwas the best of three ways, the preventive effect of31.81%after41days and the therapeuticeffect of26.18after30days, which were all higher than the control effect. When theconcentration of EPS was at200mg/mL, the efficacy of spraying was33.53%as the same asthe efficacy of ZX01fermentation broth spraying+pouring root after41days. |
