Studies On The Endophytic Fungi From Illicium Difengpi And Their Antimicrobial Ingredients

The difengpi is Illicium plant of Magnoliaceae,which grows in the hills of Karst landforms and was III-class national protection of wild plants.Its dry stem bark is a kind of special medicinal material in Guangxi and has the curative effect of treating bruises and rheumatic bone pain.Due to the habitat was destroyed and excavated by human,the wild population of Illicium difengpi was reduced and its comprehensive utilization rate decreased.The isolation and purification of endophytic fungi from Illicium difengpi have not been reported at home and abroad.The isolation and purification of endophytic fungi from Illicium difengpi and its antibacterial active substances will be reported for the first time in this paper.In this paper,the endophytic fungi were isolated from the roots,stems and leaves of Illicium difengpi,and the active strains were identificated by morphological and molecular biological methods.Four of them were selected as research objects,and they were used to expand fermentation culture.The crude extract was obtained by organic solvent extraction.The 8 plant pathogenic fungi and 13 species of pathogenic bacteria were tested,and the antimicrobial activity of crude extract was determined by the mycelial growth rate method and the toxic tablet method.According to the activity tracking,the antibacterial active components were isolated and purified by silica gel column chromatography,thin layer chromatography,solvent recrystallization and high performance liquid chromatography,and the structure of monomer compounds was identified by nuclear magnetic resonance spectroscopy.At the same time,the biological characteristics of two endophytic fungi were studied.The results of the study are as follows:Thirty endophytic fungi were isolated from the roots,stems and leaves of the Illicium difengpi of featured herbal plant by tissue culture in Guang Xi.The antibacterial activity of8 kinds of plant pathogens was detected by the tablet countermeasure and it was found that DFP-G-5,DFP-G-7,DFP-G-9,DFP-P-7.1 had strong antibacterial activity.DFP-G-5,DFP-G-7,DFP-G-9 and DFP-P-7.1 were identified by morphological and molecular biological methods,the results show that DFP-G-5 was Chaetomium sp.fungal C.globosum,DFP-G-7 was Fusarium sp.fungal F.nematophilum,DFP-G-9 was Cylindrocarpon sp.fungal C.olidum,however,the species relationship of DFP-P-7.1 has not been confirmed.Four strains of endophytic fungi were cultured by using rice solid fermentation medium.The ethyl acetate extracts were used to extract the fermented material and four ethyl acetate crude extracts of endophytic fungi fermentation products were obtained.Eight phytopathogenic fungi and thirteen animal pathogens were used as test organisms to determine the antibacterial activity of 4 ethyl acetate crude extracts of endophytic fungi fermentation products.When the concentration of the extract was 5g/L,the ethyl acetate extracts from the fermentation products of endophytic fungi from four strains of Illicium difengpi had inhibitory activities against 8 strains of plant pathogenic fungi.Ethyl acetate extracts of DFP-G-7 fermentation products had the highest inhibitory activity on mycelium growth of Pestalotiopsis theae,Cochliobolus miyabeanus,Ceratocystis paradoxa,Phytophthora parasitica var.Nicotianae.The inhibition rate was 100%and the inhibitory activity against the other four plant pathogenic fungi was higher too.The inhibition rate was 67.07%⁸6.80%.Ethyl acetate extract of DFP-G-9 fermentation products had the highest inhibitory activity against Alternaria oleracea,Cochliobolus miyabeanus,Exserohilum turcicum,Ceratocystis paradoxa,Phytophthora parasitica var.Nicotianae at the concentration of 5g/L,and the inhibition rate was 100%.The inhibitory activity against Alternaria citri was ordinary,and the inhibition rate was 67.65%.The inhibition effect on Bacillus anthraci and Colletotrichum capsici was poor,and the inhibition rate was 47.33%and 29.16%respectively.Ethyl acetate extract of DFP-G-5 fermentation products had the highest inhibitory activity against Cochliobolus miyabeanus,Exserohilum turcicum,Alternaria citri,Colletotrichum capsici and Phytophthora parasitica var.Nicotianae at the concentration of 5g/L.The inhibition rate was 100%and the inhibitory activity against the other three plant pathogenic fungi was also higher.The inhibition rate was 84.56%and 95.13%respectively.Ethyl acetate extracts of DFP-P-7.1 fermentation products had the highest inhibitory activity against Phytophthora parasitica var.Nicotianae,Ceratocystis paradoxa,Cochliobolus miyabeanus,Exserohilum turcicum,Alternaria citri and Alternaria oleracea,and the inhibition rates were 97.62%¹00%.The inhibitory activity against Pestalotiopsis theae and Colletotrichum capsici was poor,and the inhibitory rate was 30.19%and 44.99%respectively.The toxicity of ethyl acetate crude extract of 4endophytic fungi to the growth of fungal mycelia against 8 plant pathogens was further determined.The highest toxicity of ethyl acetate extract of DFP-G-5 to Ceratocystis paradoxa was 0.0095g/L,EC₅₀ of the lowest toxicity to Alternaria citri was 0.8927g/L,and the EC₅₀ to other six plant pathogenic fungi was 0.5191g/L⁰.1644g/L.The EC₅₀ of ethyl acetate extract of DFP-G-7 fermentation products to mycelium of four plant pathogenic fungi was 0.0015g/L².0005g/L.The EC₅₀ of ethyl acetate extract of DFP-G-9 fermentation products to mycelium of five plant pathogenic fungi was0.0026g/L⁰.1292g/L.The EC₅₀ of ethyl acetate extract of DFP-P-7.1 fermentation products to mycelium of six plant pathogenic fungi was 0.0217⁰.7884g/L.Poison plate method was used to determinate the antibacterial activity of ethyl acetate extract of the fermentation products of four endophytic fungi against thirteen kinds of animal pathogens.The results showed that the ethyl acetate extract of the fermentation products of DFP-G-7had higher antibacterial activity against Proteusbacillus vulgaris,Bacillus anthraci,Bacillus subtilis,Escherichia coli,Micrococcus luteus and Bacterium paratyphosum B,the MIC was 0.3125 g/L⁰.625g/L,while the inhibitory activity against Candida albicans,Enterobacter aerogenes,Bacillus megaterium,Bacillus cereus,and Pseudomonas aeruginosa was low,the MIC was 1.25 g/L².5 g/L.The ethyl acetate extract of the fermentation products of DFP-G-9 showed high inhibitory activity against Escherichia coli,Micrococcus luteus,Bacillus cereus,Paratyxobacterium serotype B,Bacillus anthracis,Micrococcus stavise,Bacillus megaterium,and the MIC was 0.039g/L⁰.156g/L.The inhibitory activity against Candida albicans,Bacillus subtilis,Staphylococcus aureus,Proteus vulgaris,and Pseudomonas aeruginosa was poor.The MIC was 0.313 g/L¹.25g/L.The inhibitory activity against Enterobacter aerogenes was the worst,with an MIC was 5 g/L.When the ethyl acetate extract concentration of DFP-G-5 fermentation products was 2.5 g/L,it had no inhibitory activity against Pseudomonas aeruginosa and Enterobacter aerogenes,and had inhibitory activity against other 11 animal pathogens,and against Bacillus megaterium,Escherichia coli and Proteus vulgaris had the best inhibitory activity,the MIC was 0.0098 g/L.The inhibitory activity against Micrococcus lysodeikticus,Candida albicans,and Bacillus anthracis was good.The MIC was 0.0195 g/L.While the inhibitory activity against Bacillus cereus,Bacillus subtilis,Micrococcus luteus,Staphylococcus aureus,Bacterium paratyphosum B were low,and the MIC was 0.0391⁰.1563g/L.When the concentration of ethyl acetate extract of DFP-P-7.1 fermentation products was 2.5 g/L,it had inhibitory activity for 12 animal pathogenic bacteria for 72hours and no inhibitory activity against Enterobacter aerogenes.Ethyl acetate extract of DFP-P-7.1 fermentation products against eight species of Bacillus anthracis,Candida albicans,Bacillus megaterium,Bacillus cereus,Micrococcus luteus,Micrococcus lysodeikticus,Bacterium paratyphosum B and Escherichia coli had the best inhibitory activity of animal pathogens,and the MIC was 0.313g/L.The inhibitory activity against Gram-positive bacteria of Staphylococcus aureus and Bacillus subtilis was good and the MIC was 1.25 g/L.And the inhibitory activity against two strains of Gram-negative bacteria of Proteusbacillus vulgaris and Pseudomonas aeruginosa were poor,and the MIC was 2.5g/L.The inhibitory activity against Enterobacter aerogenes the worst and the MIC was 5g/L.The ethyl acetate extract of the fermentation products of DFP-G-9 was subjected to silica gel column chromatography,thin layer chromatography and solvent recrystallization to isolate and purify the antibacterial active monomer compounds.Two compounds were isolated and named for:DFP-9b?DFP-9c.The inhibitory rates of the two compounds against the Ceratocystis paradoxa were 97.14%and 96.17%respectively,at a concentration of 0.1 mg/ml.DFP-9b and DFP-9c were identified by NMR spectra as7-chloro-1-O-methylemodin and 7-chloroemodin,respectively.The biological characteristics of DFP-G-7 and DFP-G-9 were studied,and the diameters of the colonies of two endophytic fungi were used as evaluation methods to determine colony growth of DFP-G-7 and DFP-G-9 under the different carbons sources,different nitrogen sources,different temperatures,and different pH values.The results show that DFP-G-7 of the optimum carbon source is mannitol;the optimum nitrogen source is calcium nitrate;the optimal temperature is 25?;the optimum pH value is 7.That the optimal carbon sources of DFP-G-9 is soluble starch,and the optimal nitrogen source is ammonium sulfate,the optimal temperature is 25?,and the optimum pH value is 7.