Isolation,Purification And Biological Activity Of Toxin From Walnut Branch Blight Pathogen

Walnut（Juglans regia L.）is an essential economic tree species in the southwest region.which is widely planted due to its high economic and ecological value.However,in recent years,walnut branch blight in a large area has seriously harmed the economic benefits of walnut.The disease is caused by fungal infection and mainly damages branches and trunks.In severe cases,the disease susceptibility rate of walnut plants reaches over 80%,directly affecting walnut trees’healthy growth,thereby affecting walnut yield and quality.Because mycotoxins have high utilization value and are an essential factor affecting plant susceptibility,this study took Phomopsis capsici,the pathogen of walnut branch blight,as the research object to isolate and purify the toxicosis component of its metabolic product.Based on exploring its molecular structure with the help of HPLC,MS,NMR and other technologies,the effect of P.capsici toxin on the physiological metabolism and tissue structure of walnut plants was studied.From the perspective of toxins,it provides a particular reference for the prevention and control of walnut wilt disease,and the main results are as follows:（1）Through bioassay of the pathogenic substances in the original liquid of the walnut stem spot mold pathogen,it was found that macromolecular substances such as proteins did not have pathogenic activity.In contrast,nonprotein parts retained the pathogenic action of the pathogen culture filtrate.（2）Studies on the fundamental properties of crude extract of P.capici from walnut showed that UV irradiation at different times had a slight impact on its activity;Temperature has a significant impact on its activity,with 0-45℃having a small effect.When the temperature exceeds 45℃,the action will significantly decrease,and when it reaches 100℃,it will lose its activity;Using petroleum ether,ethyl acetate,and ether as extractants with three different polarities can extract toxin components,but the extraction effects are different.Among them,ethyl acetate has the best extraction effect,which is closest to the polarity of the toxin substance.（3）According to thin layer chromatography（TLC）results,petroleum ether:ethyl acetate=10:1 is selected as the initial eluent,and petroleum ether:ethyl acetate=2:1 is the proportion of the terminating developing agent.Column chromatography was performed on the crude toxin of P.capici,and HPLC purified the separated toxin components.Finally,PC toxins（PC-1 and PC-2）were isolated,and the structures of the two substances were identified.The molecular weight of the PC-1 compound was 192,with the molecular formula of C₁₁H₁₂O₃ and the chemical name of 5-methylmellin;PC-2 is p-hydroxyphenylethanol,with a molecular weight of 138 and a molecular formula of C₈H₁₀O_2.（4）Bioassays of the isolated PC toxins（PC-1 and PC-2）revealed that the PC-1 toxin component exhibited the same pathogenic symptoms as the pathogenic bacteria and had strong pathogenicity.The PC-2 toxin component could only cause localized disease,and it was determined that the PC-1 toxin component was the main viral component of Walnut stem spot mold.（5）A study on the defense enzyme system of walnut plants infected with PC toxin found that the two main defense enzymes,SOD and POD enzyme activities in walnut plants showed a significant change of first increasing and then decreasing after being infected with PC toxin,and the POD enzyme activity changed more violently than SOD enzyme,making them more sensitive to PC toxin.（6）After PC toxin infection,the ultrastructure of walnut histiocyte changed significantly:the host cell wall and cell membrane were damaged in a large area;Some intracellular organelle was hydrolyzed,the matrix was concentrated,the swelling structure of chloroplast grana lamella was blurred,the mitochondrial ridge disappeared,and vacuolization appeared.