【作者】 关丽杰；

【导师】 吴元华；

【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2008， 博士

【摘要】 通过对植物病原菌具有抑菌活性植物的筛选,得到广谱、高效的目标植物-补骨脂（Psoralea corylifolia L.）。采用活性示踪的方法对补骨脂抑菌活性成分系统深入研究,得到5种活性物质,5种活性物质的农用活性均为首次报道。初步探讨补骨脂提取物的作用机理,将补骨脂提取物分别加工成0.4%水乳剂和0.4%可溶液剂,并进行田间应用研究,为植物源杀菌剂的研制与开发提供理论依据。具体研究内容如下:1.采用孢子萌发抑制率法、菌丝生长速率法、室内盆栽试验等生物测定方法,对105种药用植物的粗提物抑菌活性进行筛选,得到目标植物-补骨脂。试验结果表明,补骨脂粗提物在1000mg/L浓度下,对苹果腐烂病菌（Valsa mali Miygabe et Yamada.）、水稻稻瘟病菌（Pyricularia grisea Sacc.）、黄瓜炭疽病菌（Colletotrichum orbiculare（Berk.et mont.）Arx）、禾谷镰刀病菌（Fusarium graminearum Schw.）、番茄炭疽病菌（Colletotrichumcoccodes（Wallr.）Hughes）等15种植物病原菌菌丝生长的抑制率均在50%以上,抑制中浓度(EC₅₀)值分别为23.027mg/L,40.55mg/L,69.729mg/L,80.244 mg/L and 91.699mg/L。其中对苹果腐烂病菌的抑制效果最好,其次为水稻稻瘟病菌、黄瓜炭疽病菌、禾谷镰刀菌和番茄炭疽病菌。2.通过单因素试验,得出补骨脂中有效抑菌成分的最佳提取工艺条件:提取溶剂为70%乙醇、固液比1∶5g/mL、提取温度为室温、每次提取3h、合并滤液2次以上。试管预试法与圆形滤纸法相结合,对补骨脂粗提物的化学成分进行了预试,结果表明,补骨脂中含有酚性成分、黄酮类、内酯、香豆素、甾体、萜类以及挥发油类等类化学成分。3.以苹果腐烂病菌为指示菌,对补骨脂粗提物的活性成分进行系统的分离,最终得到5种活性物质,经鉴定5种化合物分别为补骨脂酚（Bakuchiol）、补骨脂查尔酮（Bavachalcone）、补骨脂二氢黄酮（Bavachin）、呋喃香豆素精（Bakuchicin）和补骨脂定（Psoralidin）。5种化合物对苹果腐烂病菌均具有很高的抑制活性,其EC₅₀分别为:6.117mg/L、3.420mg/L、6.144mg/L、60.441mg/L、36.815mg/L,EC₉₀分别为:204.480mg/L、15.334mg/L、89.631mg/L、97.795mg/L、221.860 mg/L。说明补骨脂查尔酮和补骨脂二氢黄酮为补骨脂的主要抑菌活性成分,其次为补骨脂酚、呋喃香豆素精和补骨脂定。4.显微观察结果表明,补骨脂提取物对固体培养的苹果腐烂病菌、黄瓜炭疽病菌的菌丝形态具有一定的影响。经补骨脂提取物处理的苹果腐烂病菌菌丝顶端细胞质物质凝缩、轮廓模糊、分支增加;黄瓜炭疽病菌菌丝较对照更为密集,菌丝顶端产生大量的泡囊。在液体培养条件下,补骨脂提取物对苹果腐烂病菌菌丝干重的抑制率随含药浓度的升高而增大,在80mg/L浓度下、处理3d时抑制率达98.3%。对细胞壁形成相关底物和水解相关酶测定,结果表明50mg/L补骨脂提取物处理后3h,苹果腐烂病菌菌体内的几丁质酶活性、β-1,3葡聚糖酶活性明显高于对照,分别是对照的3.62、9.98倍,几丁质酶和β-1,3葡聚糖酶这2种细胞壁相关水解酶活性的升高,使菌体细胞壁降解而破坏菌体结构,导致菌体细胞自溶;可溶性蛋白质、还原糖含量一直显著低于对照,说明病原菌代谢速度减慢,从而抑制菌体的生长。5.补骨脂粗提物处理两叶一心生长期的黄瓜幼苗,补骨脂提取物在40mg/L和400mg/L浓度下,黄瓜体内过氧化物酶（POD）、多酚氧化酶（PPO）、苯丙氨酸解氨酶（PAL）等一些防御酶活性均在处理后7d～9d达最高。几丁质酶酶活性在处理后2、3d时达到高峰,分别是对照的1.64、1.5倍,然后逐渐下降,分别在7、9d基本回到对照水平;β-1,3-葡聚糖酶活性在处理后2、5d达到最大值,酶活性是对照的2.02、1.34倍,然后迅速下降,处理后分别在5、9d基本回到对照水平。从所测定的几种酶活性变化时间来看,补骨脂提取物处理黄瓜幼苗后,首先,使黄瓜体内的几丁质酶和β-1,3-葡聚糖酶等病程相关蛋白活性升高,然后在两者的作用下,诱导了黄瓜体内过氧化物酶（POD）、多酚氧化酶（PPO）、苯丙氨酸解氨酶（PAL）等一些防御酶活性的提高。因此,补骨脂提取物可以诱导黄瓜产生抗病性。6.通过配方筛选试验得到0.4%补骨脂水乳剂配方（质量浓度）:补骨脂查尔酮≥0.4%,苯乙基苯酚聚氧乙烯聚氧丙烯醚7%,聚氧乙烯山梨醇酐单月桂酸酯2%,山梨醇酐单月桂酸酯1%,环己酮3%,乙二醇5%,水加至100%。0.4%补骨脂可溶液剂配方（质量浓度）:补骨脂查尔酮≥0.4%,0201B（苯乙烯基苯酚聚氧乙烯醚、烷基酚甲醛树脂聚氧乙烯醚）5%,0203B（苯乙烯基苯酚聚氧乙烯醚、烷基酚甲醛树脂聚氧乙烯醚）3.3%,十二烷基苯磺酸钙1.7%,水10%,乙二醇10%,二甲基甲酰胺补足至100%。试验表明,0.4%补骨脂水乳剂和可溶液剂均达到控制项目指标。田间试验表明,0.4%补骨脂水乳剂对苹果腐烂病具有很好的防治效果,刮治处理的效果好于割条处理,刮治涂抹处理田间推荐剂量100倍液,割条涂抹0.4%补骨脂水乳剂推荐剂量50倍液。更多还原

【Abstract】 In this dissertation,Psoralea corylifolia L.was selected as plant material through antifungal activity screening.The active ingredients in Psoralea corylifolia L.extract were systemically isolated and 5 components were achieved,and their fungicidal activities was firstly reported in this dissertation.The mechanism of action was also preliminarily studied. The optimized formulation of 0.4%Buguzhi emulsion and soluble was prepared,and 0.4% Buguzhi emulsion was applying in field.This research provides theoretical proofs for the development of Psoralea corylifolia L.as a botanical fungicide.1.Psoralea corylifolia L.was selected as target plants for further studies through antifungal tests on 105 herbal plant extracts.Spore germinating inhibition test,hypha growth inhibition test and potted plant test were used for the bio-activity evaluations.Test results demonstrated that Psoralea corylifolia L.extract showed＞50%hyphae growth inhibition against 15 pathogens at 1g/L.It has specific inhibition activities against Valsa mali Miygabe et Yamada.、Pyricularia grisea Sacc.、Colletotrichum orbiculare（Berk.et mont.）、Fusarium graminearum Schw.、Colletotrichum coccodes（Wallr.）Hughes with EC50 values of 23.027mg/L,40.55mg/L,69.729mg/L,80.244 mg/L and 91.699mg/L respectively。2.An optimized preparation process of Psoralea corylifolia L.extract was get through single factor trials as follows:70%ethanol was selected as extracting solvent,the ratio between plant material and solvent was 1:5（w/v）,extracted in room temperature for 3 h,and combine filtered solution for more than 2 times.Through test-tube test and circle filter paper method,the chemical components were preliminarily determined that phenols,flavones, lactones,coumarins,sterols terpenes and essential oils may exist in Psoralea corylifolia L. extract.3.The active ingredients in Psoralea corylifolia L.extract were systemically isolated and 5 components were achieved and identified as bakuchiol,bavachalcone,bavachin,bakuchicin, and psoralidin.Their fungicidal activity were tested against Valsa mali Miygabe et Yamada. and the result showed that these 5 compounds have good inhibition activity,with EC₅₀values of 6.117 mg/L,3.420 mg/L,6.144 mg/L,60.441 mg/L and 36.815 mg/L respectively,with EC₉₀values of 204.480mg、15.334mg/L、89.631mg/L、97.795mg/L、221.860 mg/L respectively.It suggested that bavachalcone and bavachin are the main active ingredients in Psoralea corylifolia L..4.Microscopic observations showed that Psoralea corylifolia L.extract has obvious effects on the hyphae morphorlogy of plant pathogens on PDA growth media.Deformed growth of hyphae of Valsa mali with agglomerated protoplasm,faint outlines and ramulose was induced.For Colletotrichum orbiculare,its hyphae was condensed and yielded a large number of cystids.In liquid culture condition,the inhibition on hyphal dry weight of Valsa mali was increased with the enhancement of test concentration and inhibition ratio was 98.3% under 80 mg/L after 3 hours treatment with Psoralea corylifolia L.extract.Tests on the cell wall formation related substrates and hydrolysis related enzymes in Valsa mali showed that after treatment with 50 mg/L Psoralea corylifolia L.extract for 3 hours,activities of chitinase andβ-1,3-glucanase were significantly increased to 3.62 and 9.98 folds of the check,resulted in the degradation of cell walls and the deformation and autolysis of hyphal cells.The content of soluble protein and reductive glucose kept at lower level than check resulted in the decrease of metabolism and growth inhibition.5.Effects of Psoralea corylifolia L.extract on the defensive enzymes on cucumber were studied and the results showed that activities of peroxidase（POD）,polyphenol oxidase（PPO）, phenylalanine ammonialyase（PAL）of cucumber leaves were increased to highest level after 7 to 9 days at 40 mg/L and 400 mg/L.Activities of chitinase achieved at peak level after 2 to 3 days and were 1.64 and 1.5 fold of check,then deceased gradually and resumed to normal level after 7 to 9 days.Forβ-l,3-glucanase,its activity increased to peak level after 2 to 5 days and was 2.02 and 1.34 folds of check,then decreased rapidly and resumed to normal level after 5 and 9 days.Generally Psoralea corylifolia L.extract may enhance the activities of chitinase andβ-l,3-glucanase in cucumber leaves,then induce the enhancement of activities of defensive enzymes.So it can be deduced that Psoralea corylifolia L.can induce the resistance of cucumber against the plant diseases.6.An optimized formulation of 0.4%Buguzhi emulsion（EW）was prepared as follows （w/w）:Bavachalcone 0.4%,Triton-1600 7%,Tween-20 2%,Emulsifier S-20 1%, cyclohexanone 3%;ethylene glycol 5%and add water to 100%.An optimized formulation of 0.4%Buguzhi soluble（SL）was prepared as follows（w/w）:Bavachalcone 0.4%,emulsifier 0201B 5%,emulsifier 0203B 3.3%,Triton-500 1.7%,water 10%,ethylene glycol 10%and add N,N-dimetylformamide to 100%。Quality assessment tests showed that these two formulations had good dispersibility,suspensibility storage properties.After stored under 54±2℃for 14 days,the degradation ratio of active ingredients in these two formulation were both less than 5%.Field try indicate that 0.4%Buguzhi emulsion have good control effect on Apple rot,scraping effect is better than cutting effect,scraping method should dilute 100 times when applying in field,cutting metod should dilute 50 times when applying in field.更多还原