Study On Pathogenic Metabolites Of Valsa Mali

Apple canker,caused by valsa mali,not only result in the branch rot of apple trees,but also seriously affects the development of apple industry,which is also called apple's cancer.Presently,there was little understanding about the secondary metabolites produced by valsa mali.Previously,it has been reported that the protein substances produced by valsa mali had pathogenic activity.However,the active ingredient has not been isolated and purified.In addition,the volatile substances produced by valsa mali has not been reported.Therefore,the active proteins and the volatile active substances in the secondary metabolites of valsa mali were isolated and extracted in this experiment.The following results were obtained:1.According to the relative content of proteins,clarity of electrophoresis band and the pathogenic activity on apple leaves,the protein substances from the 1% malt extract fermentation of 03-8 were analyzed,which extracted by four protein precipitation methods including acetone precipitation method,TCA precipitation method,ammonium sulfate precipitation method and pH grading method.The following results were obtained:(1)The saturation of ammonium sulfate was studied.It was found that the precipitates of 50% saturation ammonium sulfate had the highest relative content of proteins and the most obvious pathogenic activity.So 50% saturation was used in ammonium sulfate precipitation method.(2)The precipitates under different pH were analyzed,which showed that the relative contents and the activity of proteins in pH 2 were less than that in pH 5 and pH 6,but it had an obvious activity.More importantly,the protein bands of pH 2 precipitates in the SDS-PAGE electrophoresis pattern were clear,and the number was small,which might be easier to be purified.Thus,the optimum pH in pH grading precipitation was pH 2.(3)Four kinds of protein precipitation methods were compared.The result showed following orders.The relative content of proteins was pH precipitation method > ammonium sulfate precipitation > TCA precipitation > acetone precipitation method;the clarity of electrophoresis band was ammonium sulfate precipitation > pH precipitation method > TCA precipitation and acetone precipitation method,the number of electrophoresis bands was ammonium sulfate precipitation > pH precipitation method;and the biological activity was pH precipitation method > ammonium sulfate precipitation > acetone precipitation > TCA precipitation method.In summary,protein substances precipitated by pH precipitation had the highest relative content of proteins and obvious pathogenic activity on apple leaves,whose protein bands in the SDS-PAGE electrophoresis pattern were clear.Therefore,pH precipitation method was selected as the crude protein extraction method in this experiment.2.The crude proteins extracted by pH precipitation method were further purified by AKTA protein purification chromatography system.The protein purified had obvious pathogenic activity on apple leaves,but no activity on the tobacco leaves.Its protein band were identified by LC-MS-MS and three peptides were obtained,according to the reliability evaluation and molecular weight,which were AGFAGDDAPR,AGFAGDDVPK and QEYEGALRR,respectively.3.The volatile metabolites from the culture filtrate of 03-8 were collected by ordinary distillation method with ethyl acetate as solvent,their structures were identified by GC-MS and compared with the database standard substances' mass spectrometry,and the pathogenicity was determined with apple branches.Two compounds had been identified,the structures of which were isoamyl alcohol and 2-phenylethyl alcohol.Both of these substances were pathogenic to apple branches.Pathogenicity of isoamyl alcohol was weak and 2-phenylethyl alcohol was relatively strong.