Identification Of Anti-virus Streptomyces,Purification Of The Metabolites And Action Mechanism On TMV And PVY

Tobacco mosaic virus（TMV）and Potato virus Y（PVY）are devastating pathogens of many economic crops,but there are almost no chemical reagent to control TMV and PVY and the control effects are limited.Compared with chemical control,the biological control approachs on TMV and PVY has the characteristics of low toxicity,low residue and environmental friendliness,which can better meet the needs of agricultural production.Among them,microbial metabolites have become a hot spot in the control of the plant virus diseases.In this study,we conducted an in-depth study on the control of plant virus using Streptomyces.1.The Streptomyces spp.SN40 with antiviral activity was identified as Streptomyces albulus,which had broad-spectrum antifungal activity.Firstly,a Streptomyces spp.SN40 stored in our laboratory was identified as S.albulus by morphological observation,physiological and biochemical property characterization,and sequence and phylogenetic analysis of the 16S r DNA and Hsp70 gene.TMV was inoculated on Nicotiana glutinosa and PVY was inoculated on Nicotiana tabacum L.cv.NC89 using mechanical inoculation.It was found that the fermentation broth and fermentation filtrate of SN40 effectively inhibit TMV,and the inhibition rate against TMV ranged from 71.52%～81.52%;For the detection of anti-PVY activity on tobacco,PVY-Ros1 which can induce dark red pigmentation in infected tobacco tissue and real-time quantitative PCR（RT-q PCR）were used to detect the acculumation levels of PVY on tobacco.The results showed that SN40fermentation broth and fermentation filtrate exhibited robust inhibitory effect against PVY.In addition,the results showed that strain SN40 also had different degrees of inhibition on ten plant pathogens such as Alternaria solani,and the inhibition rate was between 25.07%～66.26%by using plate confrontation method.2.The crude extraction,metabolome analysis and validation of crude fermentation extract of Streptomyces SN40 showed that anisomycin and trans-3-indoleacrylic acid existed in the fermentation metabolites of strain SN40.The crude extract of strain SN40 metabolites was obtained by shaking table culture,resin adsorption and methanol elution and other methods,which contained a large number of active components by metabolomic analysis.Anisomycin(C₁₄H₁₉NO₄)and trans-3-indoleacrylic acid(C₁₁H₉NO₂)with antiviral potential were screened.In this paper,the crude extract was detected taking anisomycin with purity of 98%and trans-3-indoleacrylic acid with purity of 95%as the control by analytical high performance liquid chromatography.It was further clarified that anisomycin and trans-3-indoleacrylic acid did exist in the fermentation metabolites of strain SN40 combined with the ion flow diagram and MS/MS secondary mass spectrum in metabolomics.3.Study on antiviral activity and mechanism of anisomycin and trans-3-indoleacrylic acid.According to literatures,anisomycin with purity of 95%was isolated and purified from SN40 fermentation filtrate.The antiviral activity was studied using purified anisomycin and commercially purchased trans-3-indoleacrylic acid standard.The results showed that the inhibition rate of anisomycin on TMV was 32.94%～46.94%at the concentration of 1000?g/m L,and the inhibition rate of trans-3-indoleacrylic acid on TMV was 37.97%～43.97%at the concentration of 500?g/m L.Anisomycin had obvious inhibitory effect on PVY,and the inhibitory effect of 1000?g/m L anisomycin was greater than that of 500?g/m L.However,trans-3-indoleacrylic acid treatment did not show significant inhibitory effect on PVY.The mechanism of anti-virus action was studied by transcriptomic sequencing and RT-q PCR.The differentially expressed genes（DEGs）were analyzed by fermentation broth,fermentation filtrate,anisomycin and trans-3-indoleacrylic acid treatments on PVY-infected Nicotiana tabacum L.cv.NC89.The results indicated significant enrichment of DEGs in pathways such as plant hormone signal transduction,glutathione metabolism,α-linolenic acid metabolism,photosynthesis,cysteine and methionine metabolism,flavonoid biosynthesis,porphyrin and chlorophyll metabolism,and the biosynthesis pathway of various secondary metabolites;The possible binding sites of anisomycin and trans-3-indoleacrylic acid on TMV and PVY proteins were investigated by molecular docking and the results suggested that anisomycin may inhibit the multiplication or packging of TMV binding to TMV helicase domain as well as the coat protein.In addition,the results showed potential binding ability of anisomycin with PVY HC-Pro protein,that means it affect the aphid transmission of PVY as well as its replication or systemic infection in tobacco.