| Endophytes are all microorganisms grow in plant organs,although they survive in the internal tissues,at the same time do not cause significant damage to the host plant.endophytes are widespread.The biological roles of endophytes cover a wide range of areas,including promoting host growth,increasing host tolerance,facilitating the production of host secondary metabolites and even having applications in plant biocontrol.Numerous studies have shown that endophytes have potential to produce the same or similar compounds as the host plant as well as novel compounds.Coptis chinensis Franch.is a traditional Chinese medicinal herb with a wide range of pharmacological activities,mainly include antibacterial,anti-inflammatory,antiviral,antitumor and hypoglycemic,growing in Sichuan,Guizhou,Yunnan,Hubei,Hunan and the southern part of Shaanxi Province,with diverse species and abundant resources in Sichuan,and the Emei region has its own unique variety.There are no studies on the species diversity of endophytes of Coptis chinensis Franch.in Sichuan,and there is a lack of specificity and systematization in the screening of berberine producing strains,as well as no studies on the inhibition mechanism of endophytes strains of Coptis chinensis Franch.In this study,the endophytes of Coptis chinensis Frauch.were isolated and purified by surface sterilization of plant tissues and mycelial tip inoculation,combined the book<Fungal Identification Manual>and modern molecular biology method,identified the endophytes.The results showed that isolated 14 endophytes,belonged to 12 species,including Paraboeremia sp.,Ilyonectria sp.,Clonostachys sp.,Volutella sp.,Fusarium avenaceum,Cylidrocladiella sp.,Cercospora sp.,Dendryphion nanum,Phomatospora biseriata,Colletotrichum sp.,Didymellaceae sp.Twelve strains were isolated for the first time,which complemented the species diversity of endophytes of Coptis chinensis Franch.in Sichuan;the perforation method was used to screen The strains with inhibition ability were further fermented and expanded to enrich alkaloids,and the presence of berberine in the products of the strains was determined by high performance liquid chromatography,while the reliability and yield of the method were tested by internal standard method.The results showed that 14 strains of endophytes had good antibacterial effect on Staphylococcus aureus,7 strains had antibacterial effect on Escherichia coli,and 3 strains had antibacterial effect on methicillin-resistant Staphylococcus aureus.The metabolites of Cop stem 01 and the standard berberine were detected by high performance liquid chromatography with peak area of 473.84747 mAU*S at 10 min,and the yield of Cop stem 01 was 6.49 mg/L,which belonged to Cercospora sp.The internal standard assay test method is reliable,and this method can effectively recover the alkaloid products in solution with a yield of 91.67%.Cop stem 01 is a new berberine producing endophyte strain;the paper diffusion method was used to further determine the inhibition of the fermentation products of other strains and to initially investigate the mechanism,and the dilution coating method was used to determine the MIC and MBC values of the product inhibition.The growth curves and extracellular macromolecule contents of the bacteria before and after the administration were measured by the UV spectrophotometric method,and the morphological changes of the bacteria were observed by scanning electron microscopy.The MIC value of the metabolite was 10 mg/mL and the MBC value was 11 mg/mL.Inhibition occurred after the addition of the product,and it was also found that the Cop leaf 01 fermentation product damaged the bacterial cell membrane and cell wall,the content of extracellular macromolecules increased with the treatment time,leading to bacterial death by damage.In conclusion,this thesis was conducted on Coptis chinensis Franch.in Sichuan,and successfully isolated 14 endophytes,12 strains were isolated for the first time;the berberine producing strain Cop stem 01 was successfully screened with a yield of 6.49 mg/L;the strain Cop leaf 01 with good inhibition ability was screened,and the preliminary study found that its metabolites damaged the integrity of the cell membrane and cell wall,leading to bacterial death. |
