Isolation And Purification Of Active Substances From Fermentation Broth Of Valsa Mali Wild-type Strain 03-8

Apple canker,caused by Valsa mali,is a serious disease of apple in China,known as the ?cancer? of apple tree.Currently,there were no effective prevention and control measures against this disease,mainly because of the lack of related knowledge about its pathogenic active substances.To reveal the pathogenic active substances of this fungus,the wild type Valsa mali 03-8 was cultured and its active substances were studied in this research.The results obtained were as follows:1.Fermentation broth of 03-8 was dealt with macroporous resin XAD-2 and eluted with different ratio(v/v)of methanol-water solutions,which obtained five segments.All the segments were detected with tobacco bioassay.It showed that segment ?(75% methanol-water as eluent)had the best activity.2.To determine the more sensitive material,biological activity assays were set with section ? as the sample,and amaranth,lettuce and radish seeds,tobacco leaves and apple leaves as the biological test objects,respectively.The results showed that section ? was insensitive to radicles and shoots of amaranth,lettuce and radishes,while exhibited activity on tobacco leaves and apple leaves.Tobacco leaves were determined to be the bioassay materials for they were easier to get than apple leaves.3.Lethal activity of various solvents on tobacco leaves were tested by the method of injection inoculation.It showed that 20% acetone,and 10% methanol and ethanol would not cause necrosis on tobacco leaves.So these three solvents showed above would act as the solvents in the active measurement in this study.4.Fungus 03-8 was fermented in MS culture medium with sucrose,starch,maltose and glucose as the carbon sources,respectively.Biological activity test of fraction ? in different carbon sources showed that the activity was highest when the carbon source was sucrose.In addition,pH of MS culture medium was adjusted to 2,3,4,5,6,7,8,9 and 10,respectively,to determine the optimum pH.The results showed that fraction ? had the best activity under pH 6.5.Cultural broth,obtained under the optimum conditions(carbon sources and pH),was adsorbed to the macroporous resin,and then the eluate fractions were extracted by dichloromethane and separated by thin layer chromatography and HPLC.One compound was obtained.Its structure was elucidated by 1H-NMR,13C-NMR,DEPT135 and HMQC spectra,which was identified as 1-(3-ethenylphenyl)-1,2-ethanediol.6.Biological activity test indicated that the compound 1-(3-ethenylphenyl)-1,2-ethanediol showed insignificant activity on tobacco leaves and apple leaves.The mass growth of bacillus subtilis cultivated in the culture medium contained this compound was 50% lower than in normal medium after 12 h,which indicated that compound 1-(3-ethenylphenyl)-1,2-ethanediol had antibacterial activity to bacillus subtilis,who was antagonistic to Valsa mali.7.The alkaloid constituents in the fermentation broth were detected using the standard alkaloid detection reagent such as iodine-potassium iodide reagent,iodide potassium bismuth reagent,picric acid,phosphomolybdic acid-ethanol solution,ammonium molybdate-sulfuric acid,modified bismuth potassium iodide and concentrated nitric acid.It indicated that the fermentation broth of 03-8 contained alkaloids.