Study On Secondary Metabolites Including Mycotoxins Of Alternaria Panax And Antifungal Activity Evaluation Of Plant Essential Oils

Panax ginseng C.A.Meyer,one of the precious Traditional Chinese Medicines(TCMs)of our country.In the process of cultivation,ginseng is susceptible to be infected by many kinds of pathogens,up to now,more than 30 kinds of diseases have been reported.Among them,Alternaria panax,a host-specific fungus causing black spot symptoms on the above-ground parts of ginseng plants,is one of the most common and important diseases of ginseng.This disease accounts more than 20%to 30%incidence,and lead to severe yield loss annually.Therefore,studying the phytotoxin of A.panax and exploring safe,economical and efficient biological control methods,which are of great significance for the prevention of A.panax,so as to the comprehensive control,yield and quality of cultivated ginseng.This thesis mainly conducted the following researches:(1)Targeted isolation of mycotoxins from A.panax.It is well-known that mycotoxins produced by phytopathogens Alternaria genus play an important role in the process of infection.One of the previous studies ever reported that dibutyl phthalate(DBP)was the major mycotoxin produced by A.panax.However,more evidence supported that DBP is the constituent of plasticizers,which is quite common in chemical industry.In this part,two compounds were isolated by bioassay-guided method,they were identified as tyrosol(1)and 3-hydroxy-3-(4-methoxyphenyl)propanoic acid(2)by 1H-NMR analysis,both compounds 1 and 2 were isolated from A.panax as mycotoxins for the first time.Compound 1 showed strong phytotoxic activity on ginseng leaves,and compound 2 played weaker activity relatively.UPLC-MS/MS technique was first used in this study to analyze the origin of DBP in the extraction of A.panax,which revealed that DBP came from the residue of ethyl acetate,instead of A.panax.(2)Other secondary metabolites of A.panax.To establish an analytical method for rapid analysis of diketopiperazines,UPLC-Q-TOF-MS/MS was used to analyze the chemical constituents,molecular formula and fragment peak of crude extract of A.panax.Nine diketopiperazines were identified,and their fragmentation patterns were analyzed.To verify the correctness of these structures,Semi-preparative high-performance liquid chromatography(HPLC)was employed to further purify the target compounds,and 3 diketopiperazines were isolated:cyclo(Phe-hydroxy-Pro),cyclo(Hyp-Leu)and cyclo(ProPhe).The structures of the compounds were identified by nuclear magnetic resonance(NMR),mass spectrometry(MS),and comparison with literature data.UPLC-Q-TOFMS/MS combined with fragmentation patterns can rapidly and efficiently identify the target components in an unknown sample,which can guide the targeted isolation as well.In addition,to explore the chemical composition of A.panax,8 compounds were further isolated from the crude extract of A.panax fermentation broth.These include 4 diketopiperazines(3,5,6 and 10),one daidzein(7),one phenol compound(4)and two bases(8,9).(3)Antibacterial activity of plant essential oils against A.panax and mechanism of the main components.To explore the biocontrol substances of A.panax,in this part,disc diffusion assay was used to evaluate the antifungal activity.Two medicinal plant essential oils,Verbena officinalis essential oil and Cinnamomum camphora essential oil,both shown strong growth inhibitory effect against A.panax were identified.GC-MS were employed to analyze the volatile oil components of V officinalis and C.camphora,in which citral was the highest content of V.officinalis,accounting for 48.89%;Eucalyptol and linalool were the most abundant components in C.camphora,accounting for 46.33%and 16.65%,respectively.Antifungal activity of pure volatile compounds against A.panax was measured by contact phase method,the EC50 of these three compounds were 1.185 mg/g(citral),81.441 mg/g(linalool)and 35.534 mg/g(eucalyptol),respectively.Cell membrane permeability assay reults shown that,citral at 1.185 mg/g showed strong activity through destroy the cell membrane of A.panax mycelium.In addition,citral at the same concentration could cause morphological variation,inhibit sporulation and induce apoptosis of hyphal cells.