【作者】 武晓颖；

【导师】 金幼菊；

【作者基本信息】 北京林业大学 ， 植物学， 2009， 博士

【摘要】 双条杉天牛Semanotus bifasciatus（Motschulsky）是我国荒山造林和城市绿化的重要树种侧柏Platycladus orientalis（L.）Franco的毁灭性蛀干害虫,对我国的林木生产和生态环境构成严重威胁和巨大损失。本文采用4种提取方法提取出侧柏有效成分后,对双条杉天牛进行生物测定,筛选出有效的植物源引诱剂。同时建立了吹扫捕集-气质联用法（PTI-GC/MS）分析侧柏挥发物的最佳方法;研究了衰弱侧柏体内非挥发性次生代谢物及营养成分的变化以及含水量、树体温度和叶绿素荧光动力学等生理指标与植物树势之间的相关性,旨在为害虫的综合治理提供理论基础和技术方法,深入揭示衰弱侧柏诱虫的机理。采用改装简易蒸馏法、超临界CO₂萃取法、索氏提取法和并时蒸馏萃取法4种方法提取侧柏成分。GC-MS分析结果表明:侧柏树干提取物中主要成分为单萜、倍半萜及其氧化物和高分子量的树脂酸类物质。改装简易蒸馏样品和超临界CO₂萃取物以及并时蒸馏萃取物中含量最高的组分是罗汉柏烯和柏木脑,但是超临界CO₂萃取物中未检测出单萜;索氏提取法样品中含量最高的组分为非挥发性的树脂酸类物质。建立了吹扫捕集-气质联用法（PTI-GC/MS）分析侧柏挥发物的方法条件:取样量为0.2g,吹扫时间为5min,预反吹2min。该方法对单萜类化合物的分析效果优于常用的热脱附-气质联用法（TCT-GC/MS）。触角电位测定结果表明,双条杉天牛雌雄成虫对样品的EAG反应无显著差异。1/10浓度的3-蒈烯、柠檬烯、萜品烯和罗汉柏烯的EAG值最大。Y型嗅觉行为生测结果表明,1/100浓度的柠檬烯、3-蒈烯和罗汉柏烯对天牛有明显引诱效果,萜品烯则是原液和1/10浓度的引诱效果最好;α-蒎烯和β-蒎烯无明显引诱效果。4种提取方法中,改装简易蒸馏法样品（A2）和超临界CO₂萃取样品（D1）对天牛具有明显引诱作用;索氏提取物和并时蒸馏萃取物无引诱效果。野外诱捕结果验证了EAG和室内嗅觉行为实验的结论,样品A2与1%浓度的单萜混合物（α-Pinene:β-pinene:3-Carene:Limonene:Thujopsene:α-Terpinene=1:1:10:1:1:1）共同使用后的引诱效果最佳,其有效期可持续10d以上。而且十字形铁皮诱捕器的诱捕效果优于三角形诱捕器。采用吹扫捕集-气质联用法分析了不同生理状态的侧柏挥发物的组成及含量。结果表明,与健康侧柏相比,衰弱侧柏树干中没有检测到新的化学组分产生,同样是由苧烯、α-蒎烯、莰烯、β-蒎烯、β-水芹烯、3-蒈烯、柠檬烯、萜品烯、p-薄荷-1,4（8）二烯共9种单萜和雪松烯、罗汉柏烯和石竹烯3种倍半萜烯组成。但是衰弱侧柏中3-蒈烯和罗汉柏烯含量明显升高,α-蒎烯、β-蒎烯、β-水芹烯和柠檬烯含量减低。侧柏树势衰弱后树干挥发性化学组分含量的变化是导致衰弱侧柏诱虫的主要原因之一。采用紫外分光光度法分析不同生理状态侧柏的木质素、高效液相色谱法分析酚酸和氨基酸的含量变化。与健康对照相比,衰弱侧柏中次生代谢物质木质素、酚酸含量均有所下降,营养物质亮氨酸、丝氨酸含量升高,苯丙氨酸和精氨酸含量降低,表明衰弱侧柏受到胁迫后抗性物质含量降低,同时营养物质含量增高,为害虫的取食和生长发育提供了条件。此外,衰弱侧柏的含水量显著低于对照,树体温度的日变化幅度较大,早晚温度低,11:00-14:00时高于健康木,该时间段是天牛的频繁活动期。叶绿素荧光动力学研究表明,侧柏衰弱木的光系统Ⅱ发生了严重的光破坏,天线系统和反应中心的运转速率均大幅降低。生理指标与植物树势的相关性研究既从植物生理水平上揭示了衰弱侧柏诱虫的原因,又可以作为判断植物树势生理状况的指标。更多还原

【Abstract】 Semanotus bifasciatus(Motschulsky) is one of the most destructive borers of Platycladus orientalis(L.) Franco.The ecological environment and forest resources have been threaten seriously by S.bifasciatus in China.The dissertation has emphasized on extracting and analyzing active ingredient of P. orientalis,bioassay and field tests of S.bifasciatus for filter plant attractants,as well as the new method for volatiles analysis by using Purge & Trap Injection combined with GC/MS (PTI-GC/MS),the changes of secondary metabolites and some related physiological indicators of the weak P.orientalis,which can be taken as a theoretical basis for an effective management of S.bifasciatus and may lay the foundation in understanding the mechanism of insects hurts weak host-plant.The chemical compounds of P.orientalis were extracted by four kinds of methods respectively,Modified simple distillation(MSD),Supercritical fluid extraction(SFE), Soxhlet extraction(SE) and Simultaneous distillation extraction(SDE).The results indicated that monoterpenes,sesquiterpene and oxygenated compounds of larger molecule were the dominant contents in P.orientalis.The relative contents of thujopsene and cedrol were higher in P.orientalis extracted by SFE and MSD methods.No monoterpenes was found in the compounds extracted by SFE and SDE,and the wood resin acids were the major compounds in the compounds extracted by SE.The analytial conditions of PTI-GC/MS were optimized for the analysis of volatiles: sample 0.2g,purge time 5 min and pre-back flashing 2 min.The results of monoterpenes analysis by PTI-GC/MS were better than by TCT-GC/MS.The EAG results showed that no significant difference between female and male S.bifasciatus.The EAG responses of S.bifasciatus to 3-Carene,Limonene,Terpinene and Thujopsene were increased with the concentration respecitively within the range of 1/1000-1/10.The results of "Y" tube olfactory bioassay showed the pests can be attracted by 1/100 Limonene,3-Carene and Thujopsene.Original and 1/10 Terpinene was the best. In addition,the extracts of A2(by MSD) and D1(by SFE) also caused positive attraction to S.bifasciatus,while the extracts by SE and SDE were non-attractive.Above results were proved further by field tests.Extract(A2) mixed with monoterpenes with a certain ratio (α-Pinene:β-pinene:3-Carene:Limonene:Thujopsene:α-Terpinene=1:1:10:1:1:1) was the best attractant which could trap S.bifasciatus and lasted for more than ten days.Moreover, the effect in cross-trapping was better than in triangle-trapping.The volatiles emitted from P.orientalis in different physiological state were analyzed by PTI-GC/MS.The results indicated that there were 9 kinds of monoterpenes and 3 kinds of sesquiterpenes were identified both in healthy and weak trees.The components included Thujene,α-Pinene,β-Phellandrene,β-Pinene,Camphene,3-Carene,Limonene, Terpinene,p-Mentha-1,4(8)-diene,Cedrene,Thujopsene andα-Caryophyllene.Compared with the control,the relative contents of 3-Carene and thujopsene increased significantly and that ofα-Pinene、β-phellandrene、β-Pinene and Limonene decreased in weak trees.The differences between healthy and weak tree only related to the relative contents of above volatiles.The contents of lignin,phenolic acid and two amino acids(Phe and Arg) decreased, Comparing with the control,whereas some other amino acids(Leu、Ser and Pro) increased in weak trees.This indicated that the plant resistance reduced when they suffered from environmental stress or biological damage.These changes were more beneficial to the insects to feed,grow and develop.This is one of the main evidences for insects damage weak P.oriental.Furthermore,the moisture content of weak P.orientalis was lower than that of healthy one.The temperature of weak tree body fluctuated frequently within one day.It was lower in the morning and night and was higher during 11:00 to 14:00 than the control.The result of chlorophyⅡfluorescence kinetics showed that photosystemⅡin weak P.orientalis was destroyed seriously and the transport rates of antenna system and reaction centers were reduced considerably.The correlation between them not only indicates the mechanism which caused insects damage weak tree,but also indicates the different physiological status of the tree as indicators.更多还原