【作者】 王忠跃；

【导师】 郭予元；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 葡萄根瘤蚜起源于北美落基山脉东部，是世界范围内葡萄生产中的毁灭性害虫。自葡萄根瘤蚜1867年在法国被发现并迅速传遍欧洲及世界主要葡萄种植区，使葡萄种植受到毁灭性的打击后，科学家们曾经探索和尝试了各种防治手段，包括化学防治、生物防治、农业防治、物理防治、植物检疫等都用于防控葡萄根瘤蚜，但最终证实只有以起源美洲的Vitis属植物为砧木进行嫁接栽培结合植物检疫才是防控葡萄根瘤蚜最为有效的手段，并且自1930年以来广泛的用于葡萄根瘤蚜的防控，而在此之后其它措施的研究也受到削弱。然而嫁接抗虫砧木因为与根瘤蚜的协同进化而产生抗性，1983年第一次监测到葡萄抗根瘤蚜砧木AxR#1抗性失效，并在美国加州造成巨额损失，之后不断有葡萄根瘤蚜遗传多态性及与寄主的协调进化关系或发现抗虫砧木抗性丧失的文献报道。因此，探索其他更加有效的手段防控葡萄根瘤蚜，成为世界上一项重要的技术需求和研究热点，也是我国保障葡萄产业正常发展的重大技术需求。因此，本研究探索了复合种植方法防治葡萄根瘤蚜的可行性，主要研究结果如下：(1)烟草根系提取液对葡萄根瘤蚜生物活性测定结果表明，烟草根系物质及烟碱均对葡萄根瘤蚜具有生物活性，使根瘤蚜死亡率增加、若蚜发育历期延长和成蚜寿命缩短、繁殖率降低。田间试验证实，葡萄园复种烟草能够有效防治葡萄根瘤蚜，葡萄根瘤蚜种群数量下降、新根数量增加、树势得到恢复、葡萄产量和质量得到提高；三年持续种植烟草，使葡萄根瘤蚜种群数量持续降低，树势得到恢复。这一研究结果，证实复种植物之间根系交叉可以把具有杀虫活性的次生代谢物质输送到土壤中的靶标，复合种植植物持续释放次生代谢物质，达到长效控制病虫害种群的目的。本试验证实了复合种植方法防治葡萄根瘤蚜的有效性和可行性，也为寻找葡萄园能够防控根瘤蚜的系列复合种植植物提供思路和依据。(2)按照本研究复合种植植物的筛选要求，通过资料查询和咨询专家，从文献报道中查询到的94种具有杀虫活性物质的中草药种类中，筛选了牛膝、罗勒等18种中草药植物，并进行葡萄园复种种植试验，对出苗率、叶片面积、株高、种子千粒重及发芽率等进行了测定。通过对测定和观测数据进行比较，结果表明：牛膝、紫苑、罗勒、紫苏、薄荷、猫薄荷、决明子等7种中草药的数据与资料上这些植物生长性状的描述一致，能够在葡萄架下正常生长，作为备选植物进行下一步试验。(3)室内生物测定证实了牛膝、紫苑等7种中药的根系中的物质均能降低葡萄根瘤蚜存活率、延长若蚜发育历期和缩短成蚜寿命、减少产卵量等；盆栽试验证实，这7种中草药与葡萄混合种植，也表现出显著影响，表现为新根数量多、种群数量小、新根受害率低，说明中草药的根系中的杀虫物质可以通过根系分泌到达土壤环境，接触根系上的靶标；并且，这种对葡萄根瘤蚜的影响和防治效果，室内试验和盆栽试验一致，防控效果的顺序为：牛膝>紫苑>罗勒>决明子>紫苏>猫薄荷>薄荷。盆栽试验同时证实，葡萄园栽种具有杀虫活性的植物，对防治葡萄根瘤蚜有明显作用；从盆栽试验的两种接种方式结果对比看，葡萄园复种植物的种植时间长短对防控效果有影响，种植时间越长防控效果越好，不但说明了杀虫活性物质可能在土壤中积累，也同时说明在没有被感染的葡萄园种植筛选的防控根瘤蚜复种植物，可能对葡萄根瘤蚜在田间的传播扩散有抑制作用。室内生物测定测试了牛膝、紫苑等7种中草药粗提液对葡萄根瘤蚜一龄若蚜对葡萄根瘤蚜嗅觉行为反应，结果显示各处理之间不存在显著差异，没有明显的吸引或驱避作用。这一结果表明，这些复种植物对葡萄根瘤蚜的影响可能是通过次生代谢物质的触杀或胃毒作用实现的，与挥发性物质的味觉作用无关。(4)对牛膝根系已知的104种化学成分中能够得到的22种进行了对葡萄根瘤蚜活性活性的室内生物测定，证实了β-蜕皮甾酮是对葡萄根瘤蚜的高活性物质。经根系粗提液和纯β-蜕皮甾酮的生物测定结果LC50显示，在同等β-蜕皮甾酮剂量情况下，根系粗提液的活性明显高于纯β-蜕皮甾酮，说明牛膝根系中还有其他对葡萄根瘤蚜有效的活性物质或者是β-蜕皮甾酮与其他化合物有协同增效作用。这一研究结果，不但揭示了牛膝中β-蜕皮甾酮的杀虫活性，也为β-蜕皮甾酮对其他种类害虫的生物活性研究及田间使用等虫害防控技术的开发探讨提供了基础。测定结果显示，葡萄园种植的牛膝根中β-蜕皮甾酮含量与市场上购买的作为中草药的牛膝含量基本一致。(5)携带病毒毒源葡萄混合种植中草药及摩擦接种，试验了5种备选的中草药植物感染、携带和传播10种我国存在的葡萄重要病毒的可能性。RT-PCR检测结果显示，所有试验中草药植株的被测病毒均无目的条带，不携带任何所检验的病毒，证实这5种中草药植物没有携带这10种病毒，也没有通过接种传播到5种中草药植株上；文献报道及网站查询，这五种中草药也没有被葡萄病毒侵染的报道。这些研究结果表明，葡萄园中种植这5种中草药没有携带、感染和传播重要葡萄病毒的风险。(6)使用根际土淋溶液、复合种植等处理方式，对筛选的牛膝、紫苑和荆芥通过葡萄盆栽试验进行了化感作用测定。结果显示：根际土的土壤浸提液对葡萄萌发及葡萄苗生长虽有一定的影响，但差异并不显著；中草药与葡萄混合种植对葡萄枝条萌芽、地上及地下部分生长量也无显著影响。葡萄园复种其他植物，不能对葡萄的种植和生长有负面的化感作用，是选择葡萄园复种植物的前提条件之一。本研究结果说明，葡萄园可以与牛膝、紫苑和荆芥等三种中草药进行单季复合种植；连续复合种植同一种中草药，是否存在对葡萄有负面作用的风险，需要进一步试验测试和验证。更多还原

【Abstract】 Grape phylloxera, Daktulosphaira vitifoliae (Homoptera: Phylloxeridae), is a worldwide pest ofgrapevines. It is native to the northeastern, southwestern, and central United States where it hassuccessfully coevolved with native North American Vitis species. It is a gall-forming pest that attacksthe leaves and roots of grapes. Feeding of Phylloxera does little to affect the health of native grapevinesin eastern North America, but severe viticultural impact of its feeding became evident when it wasimported into France before the1870s. It devastated the European grape, Vitis vinifera L.(Vitaceae:Rhamnales), vineyards first in France, then was spread across the continent, and finally around nearlyevery grape-growing region in the world. It has been the most important pest on grapevines in the worldsince then. Many methods, chemicals, cultural, biological, physical and quarantine, had been trialed,since European vineyards were destroyed by phylloxera since later nineteenth century, even organicfarming was tested recent decade. But only resistant rootstocks, originated from roots of American Vitisspecies and hybrids, adding the quarantines, have been proved to be successful management for the pestand have been used around the world since1930. Researching on other approach of managementstrategies and measures also weakened since then. Though more and more works in the fields ofdetection, quarantine and alternative management has been explored in recent decades, but the moreeffective technologies like resistant rootstocks are far more important by grape industry all over theworld, especially in China because most of vines are planted ungrafted in commercial vineyards.Approaches about multiple cropping, with plants of having insecticidal activity, have been explored tocontrol the pest. The results and conclusions are listed as follow.(1) The impact of aqueous extract of tobacco root and nicotine solution to grape phylloxera wereevaluated by bioassay, and the effects of grape-tobacco intercropping pattern on populations of grapephylloxera and on growth and development of infested vine were studied and evaluated in field. Resultsshowed that aqueous extract of tobacco root and nicotine solution have good biological activity on thepest, its egg hatchability, nymph survival rate, development period, life span and female fecundity weresignificantly affected. In field trial, the populations of grape phylloxera were decreased obviouslycomparing to monoculture pattern and the rate of newly developed root and newly invested roots ofgrape were respectively significantly higher and lower in intercropping patterns than in vinemonoculture. The number of grape phylloxera population on grape roots continued to reduce year byyear and the trees of vine have got renewal gradually, for continuously intercropping with tobacco inthree years. These result confirmed that intercropping grape with tobacco could effectively control grapephylloxera in infested vineyard. The result also indicated that more crops, that could be intercroppedwith gape and have effectiveness to gape phylloxira, should be explored to be an approach ofmanagement measure to control the pest.(2) There are thousands of plants used as Chinese herb medicines, but only94plants of them havebeen evaluated and confirmed containing substances with insecticidal activity, according to reported literatures so far. We selected18kinds of them, according the required condition of intercropping withvine, to test intercropping function in vineyard. The germination rate in soil of vineyard, leaf area, plantheight, seed weight and germination rate of seeds of selected plants were recorded to evaluate if theplants can be growth well with vine. The results showed that achyranthes (Achyranthes bidentata), Aster(Aster tataricus), basil (Ocimum basilicum), purple perilla (Perilla frutescens), Nepeta cataria, mint(Mentha haplocalyx) and Cassia seed (Cassia obtusifolia) have potential to intercrop with grapes invineyard.(3) The impact of aqueous extract of the root of the seven selected plants to grape phylloxera wereevaluated by bioassay, and the effects of intercropping pattern on populations of grape phylloxera andon the rate of newly developed root and newly invested roots of infested vine were studied andevaluated by Potted Plant Test. The results showed that aqueous extract of seven plants have asignificant effect on survival, development and fecundity of grape phylloxera in bioassay, andintercropped with all of seven plants leaded to more new roots, smaller rate of new root damaged andlower population of grape phylloxera. These result confirmed that all of the seven selected plants, thathave insecticidal substances and those activity substances exudated by roots of the plants, could bedelivered to soil and reach and contact the roots and the pest, through roots cross each other. Thebioassay and the Potted Plant Test have the similar results. The achyranthes is the best one, followingare Aster, basil, purple perilla, Cassia seed, Nepeta cataria and mint. The results of Potted Plan Test alsoshowed that the longer the plants intercropped with grape, the better control effects to the pest. Theresults indicated the active substances might be accumulated in the soil and intercropping with thoseplants might have effects to scattering of first instars.Olfactory behavioral responses, of first instars nymphs of grape phylloxera, have tested to aqueousextract of the root of the seven selected plant by bioassay. There is no response of the nymphs to theextracts of the roots. The result indicates the effectiveness of the seven selected plants to grapephylloxera is maybe by the contact or/and stomach toxicity rather than the taste of volatile substances.(4)104chemical compounds of achyranthes root have been analyzed and reported in refereces, butwe got only22from market. We tested the bio-activity of those22chemical compounds to grapephylloxera by bioassay. The results indicated β-ecdysterone is the compound which has insecticidalactivity against grape phylloxera. Aqueous extract of achyranthes root is more effective thenβ-ecdysterone solution at the same dose of β-ecdysterone. This result indicates there is/are maybe otherchemical compounds which have bio-activity or there are chemicals having synergy effects withβ-ecdysterone. We analyzed the contents of achyranthes root from market and the one from tested field.The results indicated they have similar quality.(5) Every plant of5selected plants was inoculated with10important grape viruses by mixedplanting with virus infested grapes and by friction to evaluate if they have opportunity to have/transmitvirus diseases of grapes. RT-PCR test results showed that all of them do not have any of the10important grape viruses. There is no reported reference about the selected plants to be infested orinfection of important grape viruses. The results indicate there is no risk to be infested and transmit important grape viruses, if the5selected plants intercrop with grapes in vineyards.(6) Bioassays of Potted Plant Test, intercropping with grape and leaching solution of rhizospheresoil, had been carried out to evaluate the allelopathic function of the three selected plant to grape. Theresults showed that leaching solution of rhizosphere soil showed some inhibitory effect tobud germination and shoot growth but the difference was not significant, and that intercropping witheach of three did not show inhibitory effect of allelopathic function.The results indicated that grape could be intercropped with achyranthes, aster and basil for at leastone season, but sequential intercropping with one of same crop need more tests to to evaluate theallelopathic function to grape.更多还原