人参（Panax ginseng C. A. Meyer）是我国传统名贵中药材，具有大补元气、复脉固脱、补脾益肺、生津养血、安神益智等功效。人参栽培过程中易受到多种病原菌侵染。已报道的人参病害多达30余种，其中，由人参链格孢菌（Alternaria panax）引发的人参黑斑病是人参生产上常见病害之一，每年发病率达20%～30%，常造成严重减产和经济损失。已有研究发现，真菌毒素是链格孢属病原菌引发病斑形成的重要物质基础，研究A. panax真菌毒素类物质，探索安全、经济、高效的防治方法，对于人参黑斑病防治及人参产量、品质提升等具有重要意义。本文主要进行了以下研究：

（1）人参链格孢菌中真菌毒素的靶向分离。植物病原体产生的真菌毒素在Alternaria spp.病菌侵染过程中起重要作用。先前曾有报道称邻苯二甲酸丁酯（Dibutyl phthalate，DBP）是一种源自A. panax的真菌毒素，但更多证据表明，DBP是一种化工行业常见的增塑剂。本实验采用活性追踪方法首次从A. panax中分离到真菌毒素类物质tyrosol（1）和3-hydroxy-3-(4-methoxyphenyl) propanoic acid（2），通过¹H-NMR对其化学结构进行了鉴定。研究发现，化合物1对人参叶片具有较强的植物毒性，化合物2对人参叶片毒性较弱。首次采用UPLC-MS/MS技术对A. panax提取物中的DBP来源进行分析，发现该物质来源于溶剂乙酸乙酯残渣，并非先前报道的A. panax的次生代谢产物。

（2）人参链格孢菌其余次生代谢产物。为建立一种快速挖掘二酮哌嗪类化合物资源的分析方法，利用超高效液相色谱和串联四级杆飞行时间质谱仪联用技术（UPLC-Q-TOF-MS/MS）分析了A. panax乙酸乙酯部粗提物的化学成分及其分子式与裂解碎片，共鉴定出9个二酮哌嗪类化合物，并总结了它们的质谱裂解规律。3为验证结构推测的准确性，采用半制备高效液相色谱方法（HPLC）进一步分离纯化，靶向分离鉴定了3个二酮哌嗪类化合物，分别为cyclo(Phe-hydroxy-Pro)、cyclo(Hyp-Leu)和cyclo(Pro-Phe)，结合NMR数据、高分辨质谱数据及文献数据比对，靶向分离到的化合物结构与质谱分析结构一致，证实了该方法的准确性。UPLC-Q-TOF-MS/MS技术结合质谱裂解规律能快速、高效地完成样品中目标成分的分析，并指导化合物的靶向分离。此外，为探究人参链格孢菌的化学成分组成，分离得到除真菌毒素外，A. panax发酵液粗提物中其余8个化合物，包括4个二酮哌嗪类化合物（3，5，6和10）、1个大豆苷元（7）、1个苯酚类化合物（4）和2个碱基（8，9）。

（3）植物精油对人参链格孢菌的抑菌活性及主效成分的抑菌机理。通过圆盘扩散法对70余种药用植物精油的抑菌活性进行评价，发现马鞭草（Verbena officinalis）精油和罗文莎（Cinnamomum camphora）精油对A. panax有较强的抑菌活性，且性价比较高。利用GC-MS分析了两种精油的挥发油组分，马鞭草精油中柠檬醛（citral）含量较高（48.49%）；罗文莎精油中桉树脑（eucalyptol）和芳樟醇（linalool）含量较高（46.33%和16.65%）。通过接触抑制法测定了柠檬醛、芳樟醇及桉树脑对人参链格孢菌菌丝生长的抑菌活性，EC₅₀值分别为1.185 mg/g、81.441 mg/g和35.534 mg/g。细胞膜渗透实验结果表明，1.185 mg/g的柠檬醛能破坏A. panax菌丝细胞膜结构，表现出较强的抑菌活性；与对照相比，处理组菌丝形态发生明显变异，产孢能力受到显著抑制。此外，1.185 mg/g的柠檬醛能诱导病原菌菌丝细胞凋亡。

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(Pro-Phe). The structures of the compounds were identified by nuclear magnetic resonance (NMR), mass spectrometry (MS), and comparison with literature data. UPLC-Q-TOF-MS/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.