【作者】 石志琦；

【导师】 范永坚；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 博士

【摘要】 蛇床子素（Osthol,7-甲氧基-8-异戊烯基香豆素）为来源于植物的天然化合物,被证实具有抗心律失常、增强免疫功能、抗炎、抗病毒、抗肝损伤等广泛的药理活性,对交链孢菌（Alternaria alternata）、新型隐球菌（Cryptococcusneoformans）及曲霉菌（Aspergillus sp.）等真菌具有抑制作用。论文作者发现蛇床子素对常见植物病原真菌具有广谱抑菌活性。本论文以专性寄生真菌南瓜白粉病菌（Sphaerotheca fuhginea）和腐生真菌小麦赤霉病菌（Fusarium graminearum）为材料,较为系统地研究了蛇床子素的抑菌与防病机制和其仿生合成衍生物抑菌谱,目的在于研究其作为农用杀菌剂的活性及前景。主要研究结果如下:1抑菌谱与农药活性测定离体条件下测得蛇床子素对多种常见植物病原真菌具有抑制活性,抑制中浓度(EC₅₀值)大多在20μg·mL^-1～60μg·mL^-1之间。采用小株喷雾法和叶盘沾药法测定蛇床子素对南瓜白粉病菌EC₅₀值分别为42.28和30.19μg·mL^-1,且兼具良好的预防和治疗效果:100μg·mL^-1蛇床子素（未加任何农药助剂）其预防效果为77.41%,治疗效果为68.72%。笔者所研制的1%蛇床子素水乳剂用量为33.75g.ai·hm^-2时,对黄瓜白粉病防效分别达83.45～94.59%,高于对照药剂15%三唑酮可湿性粉剂,与进口杀菌剂10%世高水分散粒剂防效相当。2防病机理2.1对病原真菌作用显微观察发现蛇床子素对南瓜白粉病菌生长的各个时期都有影响:采用水琼脂平板和离体叶盘法统计发现100μg·mL^-1蛇床子素在48h后对该菌分生孢子萌发抑制率90%以上并显著降低产孢量;采用DAB（二氨基联苯胺）染色法检测植物与病原菌互作过程中的细胞学定位,发现蛇床子素对分生孢子入侵的抑制率达75%,从而阻止分生孢子在寄主上定殖。显微观察表明该化合物可有效抑制南瓜白粉病菌菌丝生长,接菌48h后,对照菌丝平均长度接近400μm,而蛇床子素处理组菌丝平均长度仅有122μm。蛇床子素可能通过降低胞外酯酶的分泌来影响白粉病菌在寄主上的黏附而阻碍白粉病菌对营养的吸收。扫描电镜和透射电镜观察发现,蛇床子素能够引起白粉病菌菌丝体皱缩凹陷、细胞壁消融、细胞器溶解并伴随细胞内容物外泄,最终导致细胞死亡。但采用流式细胞仪检测和荧光显微观察,在短时间内未发现细胞膜通透性受到损伤,因此推断细胞膜可能并非蛇床子素最初的作用部位。采用对蛇床子素敏感性相当、也表现孢子萌发被抑制但可离体培养的腐生菌小麦赤霉病菌为材料研究抑菌的生化机制。结果表明液体培养条件下,以100μg·mL^-1蛇床子素处理小麦赤霉菌24h,造成菌丝大量断裂。对细胞壁形成所需底物和细胞壁水解相关酶测定,结果表明蛇床子素处理后小麦赤霉菌体内几丁质酶活性、N-乙酰葡萄糖胺含量均升高,而β-1.6-葡聚糖酶活性与对照相比无显著变化。在培养基中加入¹⁴C标记葡萄糖,以100μg·mL^-1蛇床子素处理小麦赤霉菌,结果表明菌体内葡萄糖含量迅速下降,3h时达到最低,为对照的51%,同时几丁质酶活性迅速升高为对照的3倍;由此推测可能由于糖饥饿诱导几丁质水解酶表达,引起几丁质前体水解,细胞壁形成受阻。以100μg·mL^-1蛇床子素处理小麦赤霉菌后12h,处理菌体内钙含量仅为对照的60%并改变菌丝顶端钙离子梯度,表明蛇床子素能抑制菌体对钙的吸收,推测其可能影响到Ca²⁺依赖泡囊内含物运输释放到细胞壁过程,从而影响到菌丝顶端生长。2.2对寄主的作用除直接抑菌外,蛇床子素能够引起南瓜叶片内抗性相关酶类如几丁质酶、过氧化物酶和苯丙氨酸解氨酶活性的增强。通过反转录聚合酶链式反应（RT-PCR）的方法研究了南瓜叶片内几丁质酶基因的转录情况,显示该化合物上调了叶片内几丁质酶基因的转录水平。其防病机理不仅表现在杀死白粉病菌、抑制其入侵过程的直接影响,而且可能诱导寄主的抗病能力提高。3其仿生合成衍生物的活性实验室合成了5个含吡喃环的蛇床子素衍生物,对蔬菜、果树、粮食上7种常见的病原真菌表现出一定活性,其中化合物JS-B对卵菌纲真菌辣椒疫霉病菌（Phytophthora capsici）活性最高(其EC₅₀值为41.78μg·mL^-1),与蛇床子素对该菌的活性相当。化合物JS-B对辣椒疫霉病菌茵丝生长、孢子囊形成和释放游动孢子三种不同生长发育阶段都表现抑制作用。显微观察发现:JS-B能够引起辣椒疫霉菌丝形态发生异常,而蛇床子素处理的菌丝形态则无明显变化。更多还原

【Abstract】 Osthol[7-methoxy-8-3-methyl-（2-buetenyl）]coumarin],extracted from a Chinese herb Cnidium monnieri（L.） Cuss,is reported having anti-arrhythmia effects,immuno enhancement effects,anti-inflammatory effects,anti-virus effects and antagonizing effects against hepatic damage in pharmacology.Its antifungal activity was proven on Alternaria alternate,A.ergillus sp.,Cryptococuus neoformans,etc.In this study, osthol displayed a wide antifungal activity and its potential as biopesticides.Therefore, Sphaerotheca fuliginea and Fusarium graminearum were used in this dissertation to investigate the bioactivity of osthol as a fungicide.And the antifungal mechanism of action of osthol was further studied.In addition,osthol derivatives were synthesized and studied by bioassays.Results were showed as the following:1.Antifungal Spectrum and Pesticide ActivityIn vitro studies,osthol showed a wide antifungal activity,with the EC₅₀ value ranging from 21μg·mL^-1 to 60μg·mL^-1.EC₅₀ values of osthol against S.fuliginea were respectively 42.28 and 30.19μg·mL^-1 using seedlings bioassays and detached leaves bioassays.100μg·mL^-1osthol（without additives） was proven having good performance preventing and controlling pumpkin powdery mildew with the EC₅₀ values 77.41%and 68.72%.1%osthol emulsion in water developed in writer’s lab showed higher protection efficiency（83.45-94.59%） against cucumber powdery mildew than 15%triadimefon wettable powder when used in the dosage of 33.75g.ai/hm²,which was similar to the protection efficiency of 10%difenoconazole water dispersible granule.2.Antifungal Mechanism of Action2.1 Effects on pathogensMorphology observation revealed that osthol could affect all life stages of S. fuliginea.In water agar plates and detached leaves,germination rates of S.fuliginea could be decreased 90%after treated with 100μg·mL^-1 osthol for 48h.And the same osthol treatment also led to observably decreasing of conidia amounts.By local cells DAB（3,4-diaminobenzidine） staining of host-pathogen interaction system,the penetration rate of germinating spores can be analyzed and the results indicated that osthol could prevent 75%spores invadation avoiding the further adhesive growth of S. fuliginea on host cuticles.By microscope observation,we found osthol could also inhibit the growth of S.fuliginea hyphae.At 48h after inoculation,the average colony length of S.fuliginea was about 400μm in the control,while it was only 122μm in 100μg·mL^-1 osthol treatment.Moreover,osthol impaired the adhesion of S.fuliginea to pumpkin cuticles by inhibiting the secretion of non-specific esterases from S. fuliginea cells,which hampered the pathogen’s taking in.Scanning and transmission electron microscope photos showed that the morphology of osthol-treated S.fuliginea cells became shrinking and wizened with melting cell walls and lysine cell organelles accompanying by the releasing of homogeneous cell contents.By FACS fluorescence microscope observation,we discovered that short-term treatment with osthol could not change the cytoplasmic membrane penetrability of S.fuliginea cells,which showed that cell membrane could not be the direct action target of osthol.Compared with S.fuliginea,F.graminearum,an ascomycotina fungus,had similar sensitivity to osthol and showed low germination rate after treated with osthol. With its vitro culture characteristic,F.graminearum was then used to study osthol’s biochemical mechanism.Results showed that the hyphae of F.graminearum broke into fragments in aliquous culture after treated with 100μg·mL^-1osthol for 24 hour.In the study of cell wall synthesis-and lysis-related substances and enzymes,N-GlcNAc contents and chitinase activity in osthol-treated F.graminearum hyphae increased whereasβ-1.6-glucanase activity remained unchanged.¹⁴C glucose was added into the culture to detect the hyphal sugar absorption changes.When F.graminearum was treated with 100μg·mL^-1 osthol for 3h,glucose content decreased to the lowest level, only 51%of the control,while at the same time chitinase activity was 3 times more than the control,which suggested glucose starvation might induce the expression of chitinase.And it was proposed that osthol treatment increased chitinase activity, which led to the degradation of chitins required for biosynthesis of fungus cell wall and led to obstacle of hyphal cell wall synthesis.When F.graminearum was treated with 100μg·mL^-1osthol for 12h,calcium in F.graminearum decreased by 40%,which suggested that osthol inhibited uptake of calcium required for Ca²⁺ dependent vacuole transportation and growing hyphae tip.2.2 Effects on pumpkin seedlingsBesides its direct antifungal activity to S.fuliginea,osthol could induce the activity increasing of chitinase peroxidase and phenylalanineammonialyase in pumpkin leaves,which conduced to the defence reactions of host plants.Results of reverse transcription-PCR indicated that osthol could increase the expression of pumpkin chitinase gene in transcript level.In conclusion,osthol had a multiple mode of action against pumpkin powdery mildew fungi not only for its direct antifungal activity to pathogen and its penetration but also for its resistance induction to pumpkin seedlings.3.Bioactivity of Synthesised DerivativesFive osthol derivatives with pyran ring（JS-A,B,C,D,E） were synthesised in lab and tested to show antifungal activity against seven phytopathgens on vegetables, fruite trees and crops.Among these phytopathgens,Phytophthora capsici showed the most sensitivity to compound JS-B with its EC₅₀ value 41.78μg·mL^-1,which was similar to that of osthol.JS-B could inhibit hyphal growth,restrain the formation of sporangium and decrease the release of zoosporangiospores of P.capsici.Microscope observation showed that JS-B could change morphology of P.capsici hyphae,while the osthol-treated P.capsici hyphae had no changes.更多还原