【作者】 张莹；

【导师】 杨国庆； 陆宴辉；

【作者基本信息】 扬州大学 ， 农业昆虫与害虫防治， 2025， 博士

【摘要】 昆虫依赖其嗅觉系统感知环境中的化学信号,而植物挥发物在调节昆虫行为方面发挥着多重生态功能。植物挥发物种类繁多,其中有的能够驱避害虫,从而保护寄主植物;有的则能干扰害虫的寄主定位,或被天敌用作寻找猎物的化学信号。利用植物源挥发物开发驱避剂,为实现害虫的绿色防控开辟了新的技术途径。植物精油(EOs)作为天然挥发性化合物的浓缩形式,在干扰害虫嗅觉行为与实现综合防控方面展现出广阔应用前景。棉蚜Aphis gossypii是全球重要的农作物害虫,其化学防治因抗药性加剧而面临严峻挑战,亟需开发环境友好型的替代防治策略。尽管已有研究表明部分植物精油对棉蚜具有驱避作用,但其行为效应及嗅觉分子机制尚缺乏系统解析为此,本文在温室条件下系统评估了8种植物精油(万寿菊、圣罗勒、甜罗勒、热带罗勒、丁香罗勒、胡椒薄荷、亚洲薄荷和薰衣草)对棉蚜行为选择和种群动态的调控效果,筛选出驱避活性显著的精油,并深入探讨其对棉蚜选择、取食、繁殖及翅型分化的影响。通过电生理测定与活性成分筛选,结合RNA干扰技术,进一步解析了关键气味结合蛋白在驱避作用中的分子功能,为构建以植物源挥发物为基础的绿色控蚜策略提供理论依据与技术支撑。主要研究结果如下:(1)植物精油对棉蚜具有显著的驱避和抑制种群增长的效应。精油处理组使无翅棉蚜从中央植物(0 cm)向周围扩散,驱避指数(Dispersion Index,DI)分析表明,万寿菊、圣罗勒、甜罗勒、热带罗勒、胡椒薄荷、亚洲薄荷和薰衣草7种植物精油处理在8 cm和12 cm两个距离下均有显著的驱避效果。万寿菊、胡椒薄荷和亚洲薄荷精油在8 cm处的驱避效果最佳(DI>0.5)。罩笼试验结果表明,万寿菊、甜罗勒、热带罗勒、胡椒薄荷、亚洲薄荷和薰衣草精油显著抑制了有翅棉蚜的选择和产蚜量,圣罗勒精油显著降低了有翅棉蚜的产蚜量。在无翅棉蚜的种群增长试验中,植物精油处理组均抑制了棉蚜的种群增长。8种植物精油处理下的棉蚜种群增长率均显著低于对照组。其中,胡椒薄荷、亚洲薄荷和薰衣草精油组的处理组的棉蚜种群增长率最低,出现负增长。(2)植物精油显著干扰了棉蚜的选择、取食、产蚜行为,并促进了其翅型分化。在无翅棉蚜定居试验中,万寿菊、圣罗勒、丁香罗勒、甜罗勒、热带罗勒、胡椒薄荷、亚洲薄荷和薰衣草精油均表现出对无翅棉蚜显著的驱避作用(P<0.05)。Y管嗅觉仪试验显示,胡椒薄荷、甜罗勒、热带罗勒和薰衣草精油显著降低了有翅棉蚜的寄主选择率(P<0.01),万寿菊和亚洲薄荷精油同样表现出显著的驱避活性(P<0.05)。取食行为分析表明,所有植物精油均抑制了有翅棉蚜对棉花的取食并延长了非取食的时间。其中,8种植物精油均显著降低了棉蚜分泌水溶性唾液及被动取食韧皮部汁液(E1+E2波)在总取食时间中所占的比例,胡椒薄荷和热带罗勒精油显著延长了非刺探波(Np)的时间,薰衣草和亚洲薄荷精油显著增加了C波和Pd波时间。产蚜行为测定结果显示,8种植物精油处理后会减少有翅棉蚜总产蚜量并显著缩短其产蚜天数,而对单日产蚜数无影响,其中万寿菊、热带罗勒、胡椒薄荷、亚洲薄荷和薰衣草和精油的效果最好;万寿菊、甜罗勒、胡椒薄荷、亚洲薄荷和薰衣草精油处理后显著减少无翅棉蚜总产蚜量,热带罗勒、甜罗勒、亚洲薄荷和薰衣草精油处理后产蚜期显著缩短。此外,翅型分化试验表明,万寿菊、甜罗勒、热带罗勒、胡椒薄荷、亚洲薄荷和薰衣草精油显著促进了无翅棉蚜向有翅蚜的分化,而圣罗勒和丁香罗勒精油则未对该行为表现出显著影响。(3)鉴定并验证了植物精油中共有7种活性物质对棉蚜具有显著的驱避作用。通过气相色谱-触角电位联用仪(GC-EAD)和气相色谱-质谱联用(GC-MS)分析,共鉴定出8种植物精油中11种引起棉蚜触角反应的化合物。具体包括:万寿菊精油中的1,2,6-二甲基辛-7-烯-4-酮;胡椒薄荷精油中的薄荷酮、异薄荷酮、新薄荷醇和薄荷醇;亚洲薄荷精油中的薄荷酮和薄荷醇;薰衣草精油中的芳樟醇和乙酸芳樟酯;甜罗勒精油中的芳樟醇;热带罗勒精油中的芳樟醇和蒿脑;圣罗勒精油中的反式石竹烯、丁香酚和甲基丁香酚;以及丁香罗勒精油中的芳樟醇、反式石竹烯和丁香酚。EAG剂量反应试验表明,有7种化合物(薄荷酮,异薄荷酮,薄荷醇,新薄荷醇,芳樟醇,乙酸芳樟酯,蒿脑)表现出显著的剂量依赖性效应。室内行为选择试验进一步证实了这7种化合物对棉蚜具有显著的驱避作用(P<0.01)。田间释放挥发物试验表明,这7种化合物对棉蚜的种群增长具有显著抑制作用。(4)AgosOBP3和AgosOBP7是能够识别植物精油活性物质的关键气味结合蛋白。克隆并重组表达了9个棉蚜气味结合蛋白(AgosOBP2-10),并通过荧光竞争结合试验测定其与7种植物精油活性挥发物的结合能力。结果表明,AgosOBP3能够与异薄荷酮、芳樟醇和乙酸芳樟酯结合,AgosOBP4能够结合芳樟醇和薄荷醇,AgosOBP7能够结合薄荷醇、蒿脑、乙酸芳樟酯、新薄荷醇和芳樟醇。结合指数分析显示,AgosOBP3和AgosOBP7对多种挥发物具有较强的结合能力。进一步利用RNAi技术探讨AgosOBP3和AgosOBP7在植物精油活性化合物识别过程中的功能。在干扰处理后24h、48 h和72 h,目的基因的表达水平均显著低于对照组(未注射ds RNA),其中48 h时干扰效率最高。EAG试验结果显示,相较于未干扰组,干扰处理组显著降低了有翅棉蚜对芳樟醇、异薄荷酮和乙酸芳樟酯的电生理反应(P<0.05),而对蒿脑、新薄荷醇和薄荷醇的反应则无显著变化(P>0.05)。Y型嗅觉仪试验进一步表明,干扰处理后有翅棉蚜对异薄荷酮、芳樟醇、乙酸芳樟酯和新薄荷醇的趋避行为消失,但蒿脑和薄荷醇处理组仍然对有翅棉蚜具有显著的驱避作用,说明AgosOBP3和AgosOBP7是棉蚜能够识别植物精油活性物质的关键基因。综上所述,万寿菊、甜罗勒、热带罗勒、胡椒薄荷、亚洲薄荷及薰衣草精油对棉蚜的行为表现出显著的调控作用,能够干扰其寄主选择、取食行为和繁殖活动,并促进翅型分化。通过电生理试验分析,鉴定出植物精油中对棉蚜具有驱避作用的关键活性成分,并阐明了相关气味结合蛋白在精油驱避效应中的功能,为植物精油及其活性成分在棉蚜绿色防控中的应用提供了重要的理论依据。更多还原

【Abstract】 Insects rely on their olfactory systems to detect chemical signals in the environment,and plant volatiles play a crucial ecological role in modulating insect behavior.Plant volatiles comprise a diverse array of chemical compounds,some of which can repel herbivorous insects and thereby protect host plants,while others may interfere with host location or serve as kairomones for natural enemies to locate prey.The development of repellents derived from plant volatiles offers a promising approach for achieving environmentally friendly pest management.As concentrated forms of natural volatiles,plant essential oils(EOs)have demonstrated broad application potential in disrupting insect olfactory behavior and enhancing integrated pest control.Aphis gossypii,a globally significant pest of various crops,has become increasingly difficult to manage due to the rapid development of resistance to chemical insecticides,highlighting the urgent need for alternative,eco-friendly control strategies.Although some plant essential oils have been reported to repel A.gossypii,their behavioral effects and underlying olfactory mechanisms remain insufficiently understood.To address this,the present study systematically evaluated the behavioral responses and population dynamics of A.gossypii under greenhouse conditions in response to eight plant EOs(Tagetes erecta,Ocimum sanctum,Ocimum basilicum,Ocimum basilicum var.basilicum,Ocimum gratissimum,Mentha piperita,Mentha arvensis and Lavandula angustifolia).The study aimed to identify EOs with significant repellent activity and to investigate their effects on aphid host selection,feeding behavior,reproduction,and wing morph differentiation.Through electrophysiological recordings and active compound screening,combined with RNA interference(RNAi)techniques,the molecular functions of key odorant-binding proteins involved in the repellent effects were further elucidated.These findings provide theoretical support and technical guidance for developing green aphid control strategies based on plant-derived volatiles.The main research findings are summarized as follows:(1)Plant EOs exhibited significant repellent effects on A.gossypii and effectively suppressed its population growth.In treated groups,apterous A.gossypii dispersed from the central plant(0 cm)to surrounding plants,and analysis of the dispersion index(DI)indicated that the essential oils of T.erecta,O.sanctum,O.basilicum,O.basilicum var.basilicum,M.piperita,M.arvensis,and L.angustifolia exhibited significant repellent effects at distances of8 cm and 12 cm.Among them,T.erecta,M.piperita,and M.arvensis showed the strongest repellent effects at 8 cm(DI>0.5).The cage experiment demonstrated that T.erecta,O.basilicum,O.basilicum var.basilicum,M.piperita,and M.arvensis,and L.angustifolia significantly inhibited the host selection and reproduction of alate A.gossypii,while O.sanctum significantly reduced the number of aphids produced.In population growth experiments,all essential oil treatments suppressed aphid population growth,with growth rates lower than those of the control group.M.piperita,M.arvensis,and L.angustifolia showed the strongest inhibition,even leading to negative growth rates.(2)Plant EOs significantly disrupted the host selection,feeding,and reproductive behaviors of Aphis gossypii,and promoted its wing morph differentiation.In the aphid settling experiment,essential oils from T.erecta,O.sanctum,O.basilicum var.basilicum,O.basilicum,M.piperita,M.arvensis,and L.angustifolia significantly repelled apterous A.gossypii(P<0.05).Y-tube olfactometer assays revealed that M.piperita,O.basilicum,O.basilicum var.basilicum,and L.angustifolia significantly reduced host selection by alate A.gossypii(P<0.01),while T.erecta and M.arvensis also had significant repellent effects(P<0.05).Feeding behavior analysis showed that all essential oils inhibited phloem sap ingestion(E2 wave)and extended non-probing(Np)duration.Additionally,all eight essential oils significantly reduced the proportion of time spent secreting aqueous saliva and passively feeding on phloem sap(E1+E2 wave).M.piperita and O.basilicum var.basilicum significantly prolonged Np duration,while L.angustifolia and M.arvensis increased C-wave and Pd-wave durations.Reproductive behavior assays demonstrated that all essential oils reduced total offspring production and significantly shortened the reproductive period of alate A.gossypii,with T.erecta,O.basilicum var.basilicum,M.piperita,M.arvensis,and L.angustifolia exhibiting the strongest effects.Additionally,wing polyphenism experiments indicated that T.erecta,O.basilicum,O.basilicum var.basilicum,M.piperita,M.arvensis,and L.angustifolia significantly promoted the transition from apterous to alate A.gossypii,whereas O.sanctum and O.gratissimum showed no significant impact.(3)Seven active compounds commonly present in the plant essential oils were identified and validated to have significant repellent effects on A.gossypii.GC-EAD and GC-MS analyses identified 11 antennally active compounds across the eight EOs.These included menthone,isomenthone,neomenthol,and menthol from M.piperita;menthone and menthol from M.arvensis;linalool and linalyl acetate from L.angustifolia;linalool from O.basilicum;linalool and estragole from O.basilicum var.basilicum;andβ-caryophyllene,eugenol,and methyl eugenol from O.sanctum.linalool,β-caryophyllene,and eugenol from O.gratissimum and 1,2,6-trimethyloct-7-en-4-one from T.erecta.EAG dose-response experiments demonstrated that seven compounds(menthone,isomenthone,menthol,neomenthol,linalool,linalyl acetate,and estragole)exhibited significant dose-dependent effects.Behavioral assays confirmed their strong repellent activity against A.gossypii(P<0.01).Field-release experiments further validated their significant and consistent suppression of aphid population growth under natural conditions.(4)AgosOBP3 and AgosOBP7 were identified as key odorant-binding proteins responsible for recognizing active compounds in plant EOs.Nine A.gossypii odorant-binding proteins(AgosOBP2-10)were cloned and expressed,and fluorescence competitive binding assays evaluated their binding affinity to the seven active volatiles.AgosOBP3 bound to isomenthone,linalool,and linalyl acetate;AgosOBP4 bound to linalool and menthol;and AgosOBP7 bound to menthol,estragole,linalyl acetate,neomenthol,and linalool.EAG results showed that,compared to the non-interference group,the interference treatments significantly reduced the antennal responses of alate cotton aphids to linalool,isomenthone,and linalyl acetate(P<0.05),while responses to estragole,neomenthol,and menthol remained unchanged(P>0.05).Y-tube olfactometer assays further demonstrated that,following gene interference,the behavioral responses of alate A.gossypii to isomenthone,linalool,linalyl acetate,and neomenthol were abolished,whereas estragole and menthol continued to exhibit significant repellent effects.These findings indicate that AgosOBP3 and AgosOBP7 are key genes enabling A.gossypii to recognize active components in plant EOs.In summary,plant EOs from T.erecta,O.basilicum,O.basilicum var.basilicum,M.piperita,M.arvensis,and L.angustifolia exhibited significant behavioral regulatory effects on A.gossypii,interfering with host selection,feeding behavior,and reproduction,while also promoting wing differentiation.Through electrophysiological analysis,key active components in plant essential oils with repellent effects against A.gossypii were identified,and the functional roles of related odorant-binding proteins in mediating the repellent activity were elucidated.These findings provide important theoretical support for the application of plant EOs and their active constituents in the environmentally friendly control of A.gossypii.更多还原