Identification And Mechanism Of Biocontrol Streptomyces Anginst Tobacco Root Rot & Tobacco Black Shank Disease

In this study,a strain SN53 was screened for tobacco root rot and tobacco black shank,which has a high inhibition effect on both Fusarium oxysporum and Phytophthora nicotianae.Strain identification,optimization of fermentation conditions,determination of antimicrobial spectrum and extraction of active ingredients were studied.The active substances in crude extracts were detected by untargeted metabolomics and verified by high performance liquid chromatography.The mechanism of SN53 metabolite anisomycin in inhibiting F.oxysporum and P.nicotianae were studied.The main results were as follows:1.The biocontrol strain SN53 has been identified as Streptomyces diastatochromogenes,which has inhibitory effects on many pathogenic fungi.Through the plate confrontation experiment and the inhibitory effect assay of the fermentation broth,a Streptomyces strain SN53 with high inhibitory effect on both F.oxysporum and P.nicotianae.Combined with the morphological observation,culture characteristics,physiological and biochemical characteristics and molecular biology identification,SN53 was identified as Streptomyces diastatochromogenes.The antimicrobial spectrum of SN53 was determined,it had different degrees of inhibitory effect on 10 pathogenic fungi,showing a broad spectrum effect.In addition,two biocontrol bacteria with good biocontrol effect on F.oxysporum,which were identified as Bacillus subtilis and Paenibacillus polymyxa.2.Strain SN53 physicochemical properties,active ingredient extraction and analysis and validation of untargeted metabolome showed that the fermentation broth contained two fungicidal active substances,anisomycin and cyclo-（phe-pro）.By measuring the inhibitory effect of SN53 fermentation broth at different fermentation times,the optimal fermentation time was determined to be 6d.The physical and chemical properties of SN53 fermentation broth were studied,and the results showed that the active substances in the fermentation broth had biological activity under neutral or weak alkaline conditions,and were more resistant to high temperature and storage,and had good stability.The best preservation effect is under the conditions of low temperature and dark.The active substances in the fermentation broth of strain SN53 were isolated and purified,and the untargeted metabolomics detection of crude extracts based on ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry（UHPLC-Q-EXACTIVE-MS）was carried out.The detected anisomycin and cyclo-（phe-pro）were verified by HPLC.The standard curves and linear equations were made according to the peak area at different concentrations.3.The metabolite of strain SN53,anisomycin,has high biological activity,with EC₅₀ of1556.36μg/ml and 40.74μg/ml against F.oxysporum and P.nicotianae,respectively;and its mechanism was investigated by transcriptome analysis.Anisomycin had good inhibition effect on F.oxysporum and P.nicotianae,which indicated that anisomycin was one of the active substances of SN53.The hyphae of F.oxysporum and P.nicotianae were deformed and distorted treated by anisomycin.The mycelial growth rate method was used to determine the inhibitory activity of anisomycin against F.oxysporum and P.nicotianae,and the EC₅₀ were 1556.36μg/ml and 40.74μg/ml,respectively.The antimicrobial spectrum of anisomycin was measured,it had inhibitory effect on 7 pathogenic fungi tested.Transcriptome analysis were performed and revealed various differentially expressed genes treated by anisomycin of F.oxysporum and P.nicotianae.The analysis found that there were1453 differentially expressed genes of F.oxysporum,735 up-regulated genes and 718 down-regulated genes.Gene Ontology（GO）enrichment analysis indicated that the significantly differential expressed genes were mainly enriched in the biological process,molecular function and cellular component.There were 1913 differentially expressed genes of P.nicotiana,861 up-regulated genes and 1052 down-regulated genes.GO enrichment analysis indicated that the significantly differential expressed genes were mainly enriched in the biological process,molecular function and cellular component.Further analysis of differentially expressed genes showed that anisomycin could damage the cell wall of F.oxysporum,resulting in a decrease in pathogenicity,while the effect on the cell wall of P.nicotiana was a complex process.