| Plant endophytic fungi are a kind of diverse microbes and they can produce a variety of bioactive metabolites,which were one of the important sources of natural active substances.In order to obtain new strains that produce activitive antibacterial metabolites,the endophytic fungi in Paulownia tomentosawere isolated,screened,and the strains with activity were identified.The metabolites of the strain KLBMP-Pt686 were isolated and identified.The results were as below.92 strains of endophytic fungi were isolated from Henan Paulownia tomentosa using 5 kinds of selective media.Seven plant pathogenic fungi and 4 pathogenic bacteria were used as indicators to screen the endophytic fungi with antagonistic activity by plate confrontation and agar fast method.There were 10 endophytic fungus strains with antifungal effect against at least one plant pathogenic fungi,and 9 strains endogenic fungi with antibacterial effect against at least one pathogenic bacteria.Using Ceratocystis fimbriata Ellis et Halsted as the indicator bacteria,the antifungal activity of fermentation liquid was screened by the oxford cup method.The results showed that there were 7 endophytic fungi against Ceratocystis fimbriata Ellis et Halsted.,among them,the inhibition zone diameters of the strains KLBMP-Pt630,KLBMP-Pt675 and KLBMP-Pt686 were greater than 25 mm.Ethyl acetate crude extract of the 3 strains endophytic fungi was rescreened by filter paper diffusion method.These results suggested that the strains KLBMP-Pt675 and KLBMP-Pt686 had inhibition effects against 4 pathogenic bacteria.The strains KLBMP-Pt630,KLBMP-Pt675 and KLBMP-Pt686 were identified by morphological identification and ITS sequencing analysis.The results showed that KLBMP-Pt630 was Fusarium tricinctum,KLBMP-Pt675 was Aspergillus clavatus,KLBMP-Pt686 was Hypocreales sp..KLBMP-Pt686 was selected as the target strain for the separation of metabolites according to the activity of the strain and the species.The carbon nitrogen source and fermentation condition of KLBMP-Pt686 were optimized using a single factor method The results showed that the optimal carbon nitrogen source were glucose,the optimal time was 9 d,the culture temperature was 26 ?,the culture speed was 160 r/min,and the inoculation amount was 4%.The inhibition zone diameter was increased to 37.67 mm,which was about 15.3% higher than other methods.The isolation and purification of the KLBMP-Pt686 secondary metabolites were performed by silica gel column chromatography,Sephadex LH-20 column chromatography and high performance liquid chromatography.Six compounds were isolated respectively: 2-(1-oxhydryl-propan-2-ol,acetate)-7-oxabicyclo[4.1.0] hepta-2,4-diene,4-oxhydryl-5-propenyl-7-oxabicyclo [4.1.0] heptane-2-yl acetate,16-paullinic acid,11-oxhydryl-24-hydro-hexagonin C,dibutyl phthalate and methyl octadecadienoate.Compounds 2-(1-oxhydryl-propan-2-ol,acetate)-7-oxabicyclo [4.1.0] hepta-2,4-diene and 4-oxhydryl-5-propenyl-7-oxabicyclo[4.1.0] heptane-2-yl acetate didn't been found in the Sci-Finder database.And 2-(1-oxhydryl-propan-2-ol,acetate)-7-oxabicyclo[4.1.0] hepta-2,4-diene had inhibitory activity against Ceratocystis fimbriata Ellis et Halsted.The exopolysaccharides of strain KLBMP-Pt686 was extracted by alcohol-precipitation method and purified by gel column chromatography.At last,two kinds of exopolysaccharides were obtained.The exopolysaccharides 1 was a light brown floc solid,and the exopolysaccharides 2 was a white floc solid.The sugar content of the exopolysaccharides 1 was 94.12%,the molecular weight was 53.0 kDa,and monosaccharide compositions were rhamnose:D-arabinose:glucose = 7:12:427.The sugar content of the exopolysaccharides 2 was 92.17%,the molecular weight was 9.4 kDa,and monosaccharide composition was single glucose.The protect effect of polysaccharide on the damage of hydrogen peroxide in cells was measured by alamar blue method.The results showed that the exopolysaccharides 1 could protect cell damage caused by hydrogen peroxide. |
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