【作者】 刘勇；

【导师】 戴华国；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 小蠹虫是鞘翅目(Coleoptera)小蠹科(Scolytidae)昆虫的总称,全世界约有220属,6000种左右,是林木害虫中一个极重要类群。由于其在木材和树皮下危害,使得小蠹虫随木材进口和各种货物木质包装材料在国际间传播的机会大大增加,因此具有十分突出的检疫重要性。由于受通关时间、检疫工具的约束,目前对小蠹虫的检疫方法仍主要以“目测、手检”为主,方法比较落后,容易出现疏漏,因此,应用新技术进行检疫已是当务之急。此外,由于小蠹科昆虫种类繁多,目前对检疫截获小蠹虫的鉴定工作存在较大困难,且比较系统地反映我国进口货物中截获小蠹虫的专业资料较少,因此,急需建立针对我国进口木材中小蠹虫检疫用的形态图谱以加强检疫人员对其的识别;并通过分子生物学分类技术的研究,辅以DNA条形码技术以提高对其的鉴定水平。为解决上述木材检疫中的实际问题,本项目进行了信息化合物引诱技术在小蠹虫检疫中的应用,以及检疫性小蠹科昆虫形态与分子鉴定等一系列研究,取得了多项研究结果。1.信息化合物在小蠹虫检疫中的应用1.1小蠹虫对各种信息化合物的触角电位及嗅觉反应能诱发小蠹虫产生嗅觉反应,进而使之产生聚集行为的信息化合物有很多种,主要包括:树木的挥发性组分;小蠹虫自身产生的信息素和寄生后转化寄主化学物质合成的信息素(统称虫体信息素);以及树木被小蠹虫寄生后,其组织在微生物作用下释放的物质。本研究通过触角电位(EAG)技术,研究了3种具有代表性的小蠹虫对不同信息化合物及其不同浓度的EAG反应,并结合“Y”型嗅觉仪测定了3种小蠹虫对不同信息化合物及其组合的趋向反应。研究结果表明:(1)云杉大小蠹与光臀八齿小蠹均能对其较低浓度的虫体信息素成分产生嗅觉反应,说明其对虫体信息素的感受阈值较低;且随着虫体信息素浓度的增大,反应值增加。(2)3种小蠹虫均能对各种浓度的寄主树木挥发性组分产生嗅觉反应,但3种小蠹虫的EAG反应值随树木挥发性组分浓度的变化有所不同:云杉大小蠹与光臀八齿小蠹对树木挥发性组分中等浓度或较低浓度的EAG反应值最大,而随着浓度的升高,EAG反应值有所降低;对粒材小蠹的EAG反应值则随着树木挥发性组分浓度的升高而持续升高;高浓度时,EAG反应值最大。(3)对于α-蒎烯的两种同分异构体(1S)-(-)-α-pinene与R-(+)-α-pinene,测试的3种小蠹虫均对低浓度(0.0001μL/mL)的(1S)-(-)-α-pinene敏感,而对高浓度(10μL/mL)的R-(+)-α-pinene敏感。(4)通过“Y”型嗅觉仪测试3种小蠹虫对信息化合物及其不同组合趋向反应的结果可以看出:乙醇和单萜类化合物在引诱小蠹虫时具有相互增效作用;寄主挥发物能够增强小蠹虫对虫体信息素的反应。1.2引诱剂在木材检疫中的应用及环境因子对诱捕效果的影响利用寄主树木的挥发性组分、小蠹虫虫体信息素的某些成分、侵染寄主的微生物产生的成分等多种信息化合物配制了7种小蠹虫引诱剂,进行了进口木材上小蠹虫的诱捕试验。研究结果表明:(1)根据有关成分配制的引诱剂,在我国的主要进口木材上均能诱捕到小蠹虫。目前已在东南亚、非洲、大洋洲和俄罗斯进口木材上诱捕到4个科:小蠹科(Scolytidae),长小蠹科(Pkatypodidae),长蠹科(Bostychidae),郭公虫科(Cleridae)的昆虫;其中小蠹虫科有9个属,共18个种;长小蠹科4个种;长蠹科1个种。(2)有效成分较全面的BB06和BB07效果较好,与加拿大诱芯相比差异不显著,且成本仅为加拿大诱芯的1/5～1/8左右,是可以推广的诱捕剂。(3)温度、风和降雨对诱捕效果的影响均比较明显:有风条件下的诱捕效果明显差于无风条件;引诱剂在29-36℃温度下的诱导效果明显好于23-30℃;降雨导致木材湿润,可提高诱捕效果。(4)在木材检疫时,宜在木堆上层悬挂诱捕器,这样诱捕效果较好。悬挂时间以24h为宜,为检测更为准确,可以48h为标准。1.3引诱剂的缓释剂研制及缓释效果试验根据不同检疫对象,研制了4种小蠹虫的引诱缓释剂。通过诱捕试验,初步推断出4种缓释剂的药效释放速率为:多孔淀粉缓释剂＞缓释胶囊缓释剂≥微胶囊水剂缓释剂＞无纺布缓释剂。多孔淀粉缓释剂在放置初期诱捕效果最好,但由于其药效释放速率较快,在中、后期诱捕效果即显著降低,其“缓释”效果并不明显,此类缓释剂适合应用于进口集装箱木材等通关时间较短的木材检疫。而多孔淀粉缓释剂经过改进添加PVA作为阻隔材料后,持效期明显延长,可调整PVA外壳的厚度制成适应各种需要的缓释剂,有较好的应用前景。无纺布缓释剂药效的释放较为平缓,较好地达到了“缓释”的效果,且各个时段诱捕效果均较好,使用起来也较方便,适合应用于进口货轮木材等通关时间相对较长的木材检疫或小蠹虫的监测工作。缓释胶囊与微胶囊水剂缓释剂药效的释放速率适中,但持效期较短;且微胶囊水剂缓释剂添加低温黏合剂后,持效期延长也不明显,特别是其使用过程较为烦琐,因此不是理想的缓释剂类型。2.进口木材易携带小蠹虫形态鉴定数据库的初步构建从中科院动物研究所昆虫标本馆与江苏出入境检验检疫局木材检疫重点实验室收藏的众多小蠹虫标本中选择了进口木材及木质包装材料易携带的132种小蠹虫进行了形态描述和数码摄影,编写了形态鉴定数据库;其内容基本涵盖了我国口岸截获的小蠹虫常见种类,其中很多种类是国内首次进行中文描述。数据库通过准确的形态描述和大量的原色图片来展现小蠹虫的直观鉴定特征,弥补了以前资料中特征描述不足和黑白图片直观感觉不强的缺陷;同时,该数据库还包含了涉及小蠹虫的寄主植物和分布,为口岸检疫和昆虫研究提供了有实用价值的资料。上述材料已编辑出版了“进口木材小蠹虫鉴定图谱”(上海科技出版社,2007),将通过在木材小蠹虫检疫中的应用和验证,不断完善,并增加新的内容,以在木材检疫中发挥更大的作用。3.基于线粒体COⅠ基因的DNA条形码技术应用于小蠹科昆虫分子鉴定的可行性研究通过对采自不同地区、不同寄主的15个小蠹虫样本的线粒体COⅠ基因研究,对基于线粒体COⅠ基因的DNA条形码技术应用于小蠹科昆虫分子鉴定的可行性进行了探讨。研究结果表明:小蠹科15个样本的mtDNA COⅠ基因目的片段全部可用通用引物扩增出来,并且有足够的变异将物种区别开;其种内遗传距离均小于、等于0.019,无明显的地理差异及与寄主相关的差异,而种间遗传距离均大于等于0.040,其种间平均遗传距离为0.213。小蠹科昆虫15个样本基于mtDNA COⅠ序列构建的NJ树结果与小蠹科昆虫形态学分类的结果基本一致,且同一物种不同寄主及地理居群的最初分支自展值均达到99%,说明基于Kimura-2-Parameter双参数模型的NJ树可以很好地满足小蠹科昆虫种类鉴定的需要。该研究证明:基于线粒体COⅠ基因的DNA条形码技术应用于小蠹科昆虫分子鉴定是切实可行的。该研究为进境原木易携带小蠹虫DNA条形码数据库的构建提供了理论依据,可提高对截获小蠹虫的总体鉴定水平。本研究从解决进境木材及木质包装材料携带小蠹虫检疫的实际问题出发,进行了信息化合物引诱技术在小蠹虫检疫中的应用,以及检疫性小蠹科昆虫形态与分子鉴定等一系列研究,为小蠹虫整体检疫水平的提高奠定了理论与实践基础。更多还原

【Abstract】 Bark beetle is one of approximately 220 genera with 6,000 species of beetles in the family Scolytidae(Coleoptera),which is the most common and important pest infesting forest.As the bark beetle damages in the trunk or the inner bark of trees,it can be easily transmitted during international trade in timber and wooden packing material,so the quarantine of bark beetle is very important.At present,restricted by customs clearance time and quarantine facility,the main method of bark beetle quarantine is to check the cargo with eyes and hand.It is hard to avoid oversight with the backward technology.So it is time to apply new technology to quarantine.On the other hand,the number of bark beetle species is very large and the identification of intercepted and captured bark beetle during quarantine is very difficult.Further more,at present,there are tess systemic professional references about intercepted and captured bark beetle during quarantine in China,so it’s time to establish a database of morphologic characteristics of bark beetle for quarantine in order to help quarantine inspection officer correctly identify them.Together with DNA bar-coding technique,the database will greatly promote the ability in identifying intercepted and captured bark beetle during quarantine.In order to solve above-mentioned practical problems during timber quarantine,we carried out research as following.First,we investigated the effects of semiochemicals in trapping the bark beetles in import timber and wooden packing material,and some factors that affected the effects in fields and the laboratory.Second,we set the database of morphological characteristics of intercepted and captured bark beetle during quarantine,and the last,we studied the molecular identification of 15 species.The results were showed as below.1.Application of bark beetle semiochemicals in timber quarantine1.1 EAG and walking responses of bark beetle to different semiochemicalsSemiochemicals which can induce the olfactory response and aggregation behavior of bark beetle include host volatile components(kairomones),pheromones secreted by bark beetles itself or synthesized with host compounds by bark beetles,and the compounds released by tissue of decaying wood under the pressure of microorganism after bark beetle colonization.In the experiment,EAG technology was used to study the olfactory response of three typical bark beetle species to each one of the semiochemicals at different concentration,and the walking responses of the three species to each one and the different combinations of the semiochemicals were observed by using Y-tube olfactometer.The research showed that:(1) Dendroctonus micans and Ips nitidus responded to pheromone of low concentration,which demonstrated that the olfactory response threshold of these two species to pheromone was very low,and the response value increased with the concentration.(2) These three species responded to host volatile components of different concentration,but change patterns of EAG response values with the host volatile component concentration.were different among the three species.The EAG response values of D.micans and I.nitidus were maximal when host volatile components concentrations were low or medium,and the EAG values decreased as the concentration increased On the contrary,the EAG response values of X.perforans increased with the concentration increase,and the EAG values were maximal at high concentration of host volatile components.(3) For the two isomeric compounds ofα-pinene,these three tested bark beetle species were sensitive to(1S)-(-)-α-pinene of low concentration(0.0001μL/mL) and R-(+)-α-pinene of high concentration(10μL/mL).(4) The walking responses of the three bark beetle species to different semiochemicals and.their combinations observed by using Y-tube olfactometer showed that ethanol and Monoterpenoids were synergic in attracting bark beetle,and the host volatile components could enhance the responses of bark beetle to pheromone.1.2 Application of attractant in timber quarantine and the influence of environmental factors to trap effectTrapping experiments were carried out on the import timber to evaluate the trapping effect of seven kinds of bark beetle’s attractant.The attractants contain the components of host volatile,bark beetle’s pheromone and the synthetic components of the host pathogen.The result showed:(1) the attractants could trap many species of bark beetle in the main imported timber of China,so far,insects of four families(Scolytidae,Platypodidae, Bostychidae and Cleridae) have been trapped from the imported timber of Southeast Asia, Africa,Oceania and Russia,and there are nine genus in Scolytidae,total 18 species,four species in Platypodidae and one specie in Bostychidae.(2) The attractant of BB06 and BB07,which contain many effective components,have good Trapping effect,and the differences with the Canadian attractant were not significant,but the cost is only 1/5 to 1/8 of Canadian attractant,so they are worth being generalized.(3) Trapping effect was influenced by the weather condition(temperature,wind and rain).The trapping effect under the condition of no wind was much better than that under the condition of wind.The trapping effect under the temperature of 29-36℃was much better than that under the temperature of 23-30℃.Furthermore,the rainfall promoted the trapping effect.by increasing the moisture of timber.(4) In order to get good trapping result,the trap should be established on the top of wood pile during timber quarantine.Establishing time of trap should be 24 hours,to get more accurate result,48 hours is recommended.1.3 Development of controlling released attractants(CRA) and the trial of controlling release functionFour CRA were developed according to different quarantine object.Through trapping trial,it can be concluded that the comparison of effective components releasing speed of four CRA is:porous starch CRA＞sustained release capsule CRA≥aqueous microcapsule CRA＞non-woven fabric CRA.On the early stage,the trapping effect of porous starch CRA is the best.But due to the high speed of effective components releasing,the trapping effect of porous starch CRA decreased conspicuously with the time prolonged,and its function of controlling release is not distinct.This kind of CRA can be used for the quarantine of imported timber with short customs clearance time,such as imported container timber.The persistence of porous starch CRA can be greatly prolonged by adding septa of polyvinyl alcohol(PVA).Many kind of CRA fit for different needs can be developed by adjusting the thickness of PVA septa, which have great prospect of application.Effective components releasing of non-woven fabric CRA is gently and slowly,and its function of controlling release is distinct.The trapping effect of non-woven fabric CRA is good on every stage,and it is very convenient to apply in quarantine.This kind of CRA can be used in quarantining imported timber with long customs clearance time,such as imported ship timber and the monitoring of bark beetle.Effective components releasing of sustained release capsule CRA and aqueous microcapsule CRA are moderate,but the persistence is very short.The persistence of aqueous microcapsule CRA can not be greatly prolonged after adding low temperature binders,and its usage is very complex,so it is not the perfect CRA for application. 2.Preliminary establishment of quarantine bark beetle morphological characteristics databaseOne hundred and thirty two species of quarantine bark beetle were selected from the specimens collected in the plant quarantine laboratory of Jiangsu Entry-Exit Inspection and Quarantine Bureau and the insect specimen’s museum of Institute of Zoology,Chinese Academy of Sciences.The morphological description and photos of these species have been taken.The morphological identification database contains the familiar species intercepted and captured by quarantine department of China,and many species are first described in Chinese.Distinct identification characters are exhibited through accurate morphological description and plenty of photos with primitive colors in the database,which overcome the shortcoming of the previous database.At the same time,the database includes the host and the distribution of related bark beetle species,which provides useful information to quarantine department and insect research.Identification illustrated handbook of bark beetle from imported timber has been published by Shanghai Scientific and Technical Publishers in 2007 according to above-mentioned data.3.Feasibility study on application of DNA bar-coding technology(based on mtDNA CO I gene) in molecular identification of ScolytideaCO I gene of 15 bark beetle species collected from different regions and host is studied to discuss the feasibility of application of DNA bar-coding technology in Scolytidea molecular identification.The result showed that all fragments of the target gene from 15 samples could be amplified with the common primer and the variation was obviously among species.The DNA data analysis showed that the inter-species genetic distance was no more than 0.019,and there were no significant differences among different geography groups or host groups of the same species.While,the intra-species genetic distance was no less than 0.040,and the average intra-species genetic distance was 0.213.The result of neighbor-joining tree clustered basing on the CO I sequence of 15 samples was consistent with that of morphological analysis.The bootstrap values of the original divergence among different geography groups and host groups of the same species amounted to 99%,which illustrated that the neighbor-joining cluster-analysis based on Kimura-2-Parameter could be utilized in identifying the species of Scolytidea.The study demonstrate that using DNA bar-coding technology(based on mtDNA CO I gene) to identify species of Scolytidea is feasible.The research theoretically founded the strategy of establishing DNA bar-coding database of bark beetle from imported timber,so as to improve the level of bark beetle species identification.Generally,to solve the practical problem of bark beetle quarantine in imported timber and wooden packing material,we carried out the research of semiochemicals-trap application,morphological and molecular identification of bark beetle in this project,and the results would contribute to improving the whole level of bark beetle quarantine.更多还原