Study On The Antimicrobial Ingredients Of Two Endophytic Fungi From Sarcandra Glabra

Sarcandra glabra（Thunb.）Nakai is an important medicinal plant in China,which has antibacterial,anti-inflammatory,anti-tumor,antioxidant and other pharmacological effects.Plant endophytic fungi can produce the same or similar metabolites as host plants.Based on the broad-spectrum antimicrobial activity of Sarcandra glabra,this study aims to search for antimicrobial active substances from its endophytic fungi,which is of great significance for the development of green,efficient,safe and environmentally friendly natural antimicrobial agents.In this paper,the antimicrobial activity of two endophytic fungi Sg G4 and Sg G5 from Sarcandra glabra was studied.The active fermentation products of endophytic fungi Sg G4 and Sg G5 were screened by mycelium growth rate method and drug-containing medium method,and the antimicrobial activities of the active fermentation products were determined by 13 plant pathogenic fungi and 9 animal pathogenic bacteria.The antimicrobial active substances from active fermentation products were isolated and purified by silica gel column chromatography,thin-layer chromatography and other separation techniques combined with activity tracking.The structure of the active substances was identified by nuclear magnetic resonance spectroscopy,mass spectrometry,gas chromatography mass spectrometry,and other identification techniques.The antimicrobial mechanism of the active substance was preliminarily explored,and the fermentation conditions of the active strain Sg G4 to produce the active substance were preliminarily studied.The main research results are as follows:1.The antimicrobial activity of different extracts from the fermentation products of five endophytic fungi from Sarcandra glabra against plant pathogenic fungi and animal pathogenic bacteria was determined by mycelial growth rate method and the drug-loaded medium method.It was found that the ethyl acetate extract from the fermentation products of the other four endophytic fungi,except for Sg J7 strain,have a 100%inhibitory rate against the three plant pathogenic fungi tested at a mass concentration of 5 mg/m L.The ethyl acetate extracts of Sg G4 and Sg G5 have inhibitory activity against two animal pathogenic bacteria tested,Escherichia coli and Staphylococcus aureus;The ethyl acetate extracts of Sg J8 and Sg J9 have inhibitory activity against Staphylococcus aureus,but have no inhibitory activity against Escherichia coli;The ethyl acetate extract of Sg J7 have no inhibitory activity against the two tested animal pathogenic bacteria.The results showed that strains Sg G4 and Sg G5 had strong antimicrobial activity and broad antimicrobial spectrum,and the antimicrobial active substances mainly existed in the ethyl acetate extract of the fermentation products.2.Selected ethyl acetate extracts from Sg G4 and Sg G5 fermentation products,and further determined their toxicity to 13 plant pathogenic fungi by mycelial growth rate method.The results showed that the EC₅₀ value of ethyl acetate extract of Sg G4 against 13plant pathogenic fungi ranged from 0.0002 mg/m L to 2.4757 mg/m L,the extract have the strongest toxicity to Ceratocystis paradoxa and have the weakest toxicity to Fusarium verticillioide;The EC₅₀ value of ethyl acetate extract of Sg G5 against 13 plant pathogenic fungi ranged from 0.2394 to 2.9701 mg/m L,the extract have the strongest toxicity to Alternaria oleracea and have the weakest toxicity to Athelia rolfsii.3.The broth dilution method was used to determine the minimum inhibitory concentration（MIC）of ethyl acetate extract of Sg G4 and Sg G5 against 9 animal pathogenic bacteria.The results showed that the two ethyl acetate extracts have different degrees of antibacterial activity against 9 animal pathogenic bacteria,with MIC values of0.125 mg/m L to 1 mg/m L and 0.125 mg/m L to 0.5 mg/m L,respectively.4.By silica gel column chromatography,thin layer chromatography,and other separation techniques combined with activity tracking,a pure antimicrobial active compound and a group of oily active substances were isolated from the ethyl acetate extract of the fermentation product of strain Sg G4.The structure of the active compound was studied by nuclear magnetic resonance and mass spectrometry techniques,and its structure was identified as Trichoderma acid;The structure of a group of oily active substances was analyzed by gas chromatography-mass spectrometry（GC-MS）technology,and four compounds were identified,namely 5,6-dimethyl-1H-Benzimidazole,methyl palmitate,methyl stearate and 3.5-bis（1,1-dimethylethyl）-4-hydroxy-Benzenepropanoic acid methyl ester.An antimicrobial active compound named Sg G5-A was isolated from the ethyl acetate extract of the fermentation product of strain Sg G5.Its structure is still under further identification.5.The toxicity of Trichoderma acid to Pestalotiopsis theae was determined,the EC₅₀value was 0.0042 mg/m L,which have significant inhibitory activity.The toxicity of Sg G5-A to 13 plant pathogenic fungi and the minimum inhibitory concentration to 9animal pathogenic bacteria were determined.The results showed that the EC₅₀ value of Sg G5-A against 13 plant pathogenic fungi ranged from 0.3509 mg/m L to 1.4852 mg/m L,and the MIC value against 9 animal pathogenic bacteria ranged from 0.0625 mg/m L to0.25 mg/m L.6.The antimicrobial mechanism of Trichoderma acid was preliminarily explored with Pestalotiopsis theae as the test strain.The results showed that the permeability of the cell membrane of the mycelium was changed,the relative permeability was increased,and the content of soluble protein was decreased after the treatment with Trichoderma acid.It is indicated that Trichoderma acid can inhibit mycelial growth by disrupting the structure of cell membrane,extravasating the cell contents and affecting protein synthesis,and so on,which eventually lead to death.7.After morphological and molecular biological identification,the strain Sg G4 was identified as Trichoderma spirale,and the strain Sg G5 was identified as Fomitopsis palustris.Through research on biological characteristics,it was found that Trichoderma spirale is suitable for growing in environments of full darkness,temperature of 28℃,and p H 6,while Fomitopsis palustris is suitable for growing in environments of full darkness,temperature of 35℃,and p H 6.8.With the mass concentration of Trichoderma acid as the index,the fermentation conditions of Trichoderma spirale producing Trichoderma acid were preliminarily explored.Through single factor test and orthogonal test,the optimal fermentation conditions for Trichoderma spirale to produce Trichoderma acid were determined as follows:the initial p H was 8,the fermentation time was 25 d,the filling volume was 130m L/250 m L,and the inoculum volume was 4 fungus cakes.The results of this study on the antimicrobial activity of endophytic fungi of Sarcandra glabra provide a basis for the research and development of related microbial fungicides.