桉树枝瘿姬小蜂侵害机理及寄主桉树化学防御研究
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摘要
桉树枝瘿姬小蜂Leptocybe invasa Fisher et La Salle是危害桉属Eucalyptus植物枝叶的重要危险性害虫,该虫自2007年入侵我国以来,已对我国桉树资源造成严重的危害。本文通过对该虫在福建发生危害特点、生物学和生态学特性的调查研究基础上,从该虫产卵趋性、寄主选择行为和寄主的适合度,以及不同桉树品系(种)叶片生理生化指标与抗虫性关系等方面进行深入分析,综合评价了不同桉树品系(种)的抗虫性。以巨桉E. grandis为研究对象,深入探讨桉树枝瘿姬小蜂产卵危害、机械损伤和喷施外源茉莉酸甲酯(MeJA)等诱导因子处理后,其叶片中化学物质的时序变化。从营养物质、次生物质、防御酶系、蛋白酶抑制剂和植物挥发物等角度探讨了不同诱导因子对桉树化学防御反应机理,以及诱导防御对桉树枝瘿姬小蜂生长发育和种群趋势的影响。研究揭示了该虫侵害、选择寄主的行为机理和桉树诱导化学防御反应策略的互作关系,从抗性选育和提高寄主防御能力方面,为该虫的防治提供新的思路和技术途径。通过试验研究,得到如下结果:
     1、调查了桉树枝瘿姬小蜂在福建省危害现状及不同桉树品系(种)受害情况。结果表明:(1)福建省桉树主栽品系(种)大部分都可遭受桉树枝瘿姬小蜂危害,并可在尾赤桉DH201-2、巨桉QG3、邓恩桉、尾叶桉U6、巨尾桉GL9、大叶桉和窿缘桉等7种桉树品系(种)上形成虫瘿,正常生长发育,并完成其生活史。(2)研究发现不同桉树品系(种)受害程度和对桉树枝瘿姬小蜂的适合程度是不同的。桉树枝瘿姬小蜂对不同桉树寄主的选择频次率、产卵量和幼虫数量均有差异。(3)以不同桉树品系(种)为食的桉树枝瘿姬小蜂,除雌性比差别不大外,其余如出蜂数、雌成虫体重、怀卵量、寿命等生长发育和繁殖力指标都有极显著的差异。(4)对寄主进行适合度分析,并用聚类分析法将桉树枝瘿姬小蜂的寄主划分为适宜寄主、较适宜寄主、较不适宜寄主、不适宜寄主4类。在适宜寄主上寄生取食,每雌怀卵量都达70粒以上,存在种群暴发的生物学潜能。在生产上可使用寄主适合度指标做为抗虫品系筛选的综合性评价指标。
     2、研究了桉树枝瘿姬小蜂雌成虫对不同桉树品系(种)气味及颜色的行为趋性,以及对各桉树品系(种)产卵选择情况。结果表明:(1)桉树枝瘿姬小蜂对13种桉树品系(种)挥发性气味的趋性存在明显的差异,其中对尾赤桉DH201-2、尾巨桉DH32-28和巨桉QG3等10种品系(种)有不同程度的正趋性,尤其是对尾赤桉DH201-2的趋性最强,而对韦赤桉GL3和大花序桉则表现出负趋性或不选择性;(2)不同色卡对桉树枝瘿姬小蜂的引诱效果差异明显,其中纯白色(RAL9010)和信号黄(RAL1003)引诱效果较好,选择系数分别为0.29和0.28;(3)不同桉树品系(种)上的桉树枝瘿姬小蜂产卵刻痕数明显不同,其中尾赤桉DH201-2上的产卵刻痕数明显大于其它品系(种)。
     3、调查了13种福建主栽的桉树品系(种)幼苗受害情况,按制定的抗性分级标准,并用灰色关联分析法,综合评价了桉树品系(种)的水分、营养物质及次生物质含量与受害程度和抗性值关联度情况。结果表明:(1)13个品系(种)中,尾巨桉DH32-28、尾巨桉DH32-26、韦赤桉GL3、尾细桉M1、巨桉JG1、大花序桉和柳桉等7个品系为抗性品系(种),其余的品系(种)具有不同程度的感虫性。研究发现同一桉树品种不同无性系的抗性也存在显著区别。(2)不同感虫品系(种)的样枝着生的虫瘿数和每瘿孕虫数差异极显著。(3)不同桉树品系(种)嫩枝嫩叶的含水率差异极显著,感虫品系(种)显著低于抗虫品系(种);不同桉树品系(种)可溶性糖、多糖和蛋白质等营养物质的含量随着抗性的增强而减少;而黄酮、单宁和总酚等次生代谢物质含量随着抗性的增强而增加。(4)灰色关联分析表明,桉树嫩枝、嫩叶中的营养物质,特别是多糖的含量对桉树枝瘿姬小蜂生长发育影响和抗性影响较大。
     4、研究了桉树枝瘿姬小蜂危害及不同诱导方式对桉树叶片化学物质和挥发物影响。结果表明:(1)桉树枝瘿姬小蜂产卵危害、机械损伤和外源MeJA处理都会诱导桉树叶中可溶性糖、多糖和蛋白质等营养物质的含量发生变化,与对照相比有明显的下降趋势,但MeJA处理引起桉树防御反应的变化幅度更大。(2)3种诱导处理都能明显影响桉树叶黄酮、单宁和总酚等次生代谢物质的含量,与对照相比有明显的上升趋势,并在时序上各种次生物质含量呈现不同的变化规律。(3)桉树叶PAL、SOD、POD和PPO等4种保护酶活性变化与不同诱导处理有一定的相关性,表明了不同的诱导方式对桉树叶中保护酶活力的作用不同,总体上虫害和MeJA处理诱导的酶活力较机械损伤更为明显。(4)不同诱导处理均可激发桉树叶中TI和CI2种蛋白酶抑制剂活性发生变化,且不同处理在时序上有不同的反应表现。与机械损伤处理相比,桉树受MeJA诱导处理后,桉树叶的TI和CI活性增幅更显著,且更迅速,与虫害诱导的变化情况相近。(5)采用动态顶空吸附法和气相色谱-质谱法分析表明,不同处理的桉树植株释放出的挥发物的种类和相对含量存在显著差异。不同诱导处理后,大部分萜烯类化合物在诱导后1-3d大量释放,5-7d后释放量逐渐减少。
     5、对不同诱导处理后,桉树叶片生理生化指标的变化,以及对桉树枝瘿姬小蜂的生长发育、繁殖力和种群趋势等生物学指标的影响进行研究。结果表明:(1)用诱导处理后的植株接入桉树枝瘿姬小蜂,发现其子代存活率降低、雌虫体重下降、怀卵量减少、寿命缩短和种群趋势指数下降。(2)用模糊相似优先比法分析综合比较评价了桉树枝瘿姬小蜂产卵危害诱导与其它诱导处理的相似程度,结果表明,喷施1.0mmol·L~(-1)MeJA诱导能够产生与桉树枝瘿姬小蜂产卵危害较为相似的结果,证明了喷施外源MeJA在诱导抗性中有积极的作用,可作为害虫综合治理的一种有效辅助手段。
The Eucalyptus gall wasp, Leptocybe invasa, is one of the most devastating pests, whichcauses severe injury to young foliage of Eucalyptus spp. Since its first recorded occurrence inChina in2007, the pest had caused serious damage to the eucalypt plantation resource in thiscountry. Based on investigations and studies of the biological and ecological characteristics of thepest in Fujian, this paper provides detailed analysis regarding host preference, oviposition taxis,resistance among eucalypt taxa and the relationship between some physio-biochemical indices inleaves of different species and plant resistance. Further research into chemical defense of E.grandis by different induced treatments, which included by gall wasp oviposition, physicaldamage and exogenous methyl jasmonate (MeJA), was carried out. Result analysed includedtemporal changes of chemical substances in leaves. From the aspects of nutrients, secondarymetabolites, defense enzymes, protease inhibitors and plant volatiles, we investigated themechanisms involved in chemical defenses of E. grandis and the effects of these changes on thegrowth, development and population trend of L. invasa. This research revealed interactionsbetween the pest infestation and chemical defense of Eucalyptus species, and provided newinsights for technical approaches to control the pest by breeding insect-resistant species andimproving host defense capability. After three years of research, the major results obtained are asfollows:
     1. The harm status and levels of damage to different varieties caused by L. invasa weresurveyed in Fujian. Results showed:(1) Most of the infested eucalypt varieties can attacked by L.invasa in Fujian; when the larvae feed on E. urophylla×E. camaldulensis clone DH201-2, E.gradis clone QG3, E. dunnii, E. urophylla clone U6, E. grandis×E. urophylla clone GL9, E.robusta and E. exserta respectively, they can form galls, grow normally and complete their lifecycle.(2) There were differences among varieties for the degree of damage and host fitness of L.invasa, which resulted in changes to frequency through selection, and the fecundity and number oflarvae on differ among different Eucalyptus varieties.(3) L. invasa larvae fed by differentEucalyptus varieties, aside from the female ratio, differed significantly in other attributesincluding emergence rate, female weight, fecundity and longevity of female.(4) According to thefitness index (FI), the host plants of L. invasa were divided into4categories by cluster analysis:the most suitable, suitable, secondary and unsuitable. Fed on suitable host, fecundity reaches morethan70eggs per female which is adequate to lead to potential population explosion of the pest.We can use host fitness index as a comprehensive evaluation index for the screening of resistant clones in practice.
     2. The effects of smell and color of different Eucalyptus varieties on host preference by L.invasa were studied in laboratory. In addition, selection behavior of oviposition of the pest wasstudied in the field. The results revealed that:(1) There were significant differences amongeucalypt species for female adult preference by smell–out of10Eucalyptus varieties femaleadults showed the strongest preference for E. urophylla×E. camaldulensis clone DH201-2.However, the pest had negative taxis or no selection for either E. wetarensis×E.camaldulensisclone GL3or E. cloeziana.(2) Color tests showed that there were obvious differences amongcolor cards for their pest-attracting effect, pure white cards (RAL9010) and signal yellow cards(RAL1003) had an obvious attraction effect, with the selection coefficients accounting for0.29and0.28respectively.(3) The analyzed results showed that the number of oviposition scars weresignificantly different among Eucalyptus varieties. It is obvious that the oviposition scars on E.urophylla×E. camaldulensis clone DH201-2were more than other varieties.
     3. In order to investigate relationships between L. invasa and different Eucalyptus varietiesand to examine the change process of the host stress response after being damaged, we screened13infested eucalypt clones and species in Fujian and graded these for resistance, then measuredthe contents of water, nutrients and secondary substances in young foliage of these differentvarieties. Data obtained from this work was analyzed by the grey relational analysis. The resultsshowed that:(1)13varieties showed resistance, including E. urophylla×E. grandis clonesDH32-28and, DH32-26, E. grandis×E. urophylla clone GL3, E. urophylla×E. tereticornisclone M1, E. grandis clone JG1, E. cloeziana and E. saligna. All other varieties screened haddifferent degrees of susceptibility. The research also showed that the resistance of different clonesfrom same species differed significantly.(2) The number of galls per sample branchlet and thenumber of wasp larvae per gall differed greatly among varieties.(3) The water content in youngfoliage of different varieties differed greatly, with susceptible varieties having much lower foliagewater contents than did resistant varieties. As the level of resistance increased, the contents ofsoluble sugars, polysaccharides and proteins in young foliage decreased, whilst the content offlavonoids, tannins and total phenols increased.(4) Grey relational analysis between contents ofnutrients and secondary substances in young foliage of different varieties and damage degrees bythe gall wasp, showed that the content of nutrients, especially the content of polysaccharide, hadgreater impact on the growth of the wasp and varieties resistance than did other attributesmeasured.
     4. Chemical analysis of some chemical substances in leave and volatiles emitted from E.grandis after inductions by gall wasp oviposition, physical damage and exogenous MeJA was studied. The results showed that:(1) Induction treatments can all induce changes in the nutrientcontent of Eucalyptus leaves, including contents of soluble sugars, polysaccharides and proteins.Compared with the control group, the content of these compounds tended to decline. But, theintensity of defense reaction caused by MeJA treatment was higher.(2) In regard to three methodsof induction, compared with the control group, the content of secondary metabolites, such asflavones, tannins and total phenols, all had obvious tendencies to increase, and the contents of allkinds of secondary metabolites showed different results over time.(3) Variation in the activitylevels of PAL、SOD、POD and PPO enzymes in Eucalyptus leaves showed some correlations withdifferent induction treatments, indicating that different methods of induction had differing effectson the activity of protective enzymes. Generally speaking, enzymatic activity induced by insectattack and MeJA treatment was more obvious than physical damage.(4) Different inductiontreatments can all stimulate change to the activity of TI and CI in eucalyptus leaves, moreover,different treatments had different results over time. Induced by MeJA, compared with physicaldamage, increased the amplitude of TI and CI activities in Eucalyptus leaves–the amplitude washigher and peaked quicker–which was similar to the change caused by insect attack.(5) VOCsof E. grandis were studied by the methods of headspace enrichment and GC-MS in this paper. Theresults showed that the composition and quantity of the VOCs were significantly different amongthe four treatments. After different inductions, terpene volatiles emitted largely in1-3days, butthe volatile emissions decreased gradually after5-7days.
     5. Through different induction treatments, changes on physiological and biochemical indicesof Eucalyptus leaves were studied, along with the effects on indices of the growth, development,reproductive activity and population trends of L. invasa. The results showed that:(1) Plants withinduction treatments that were inoculated with L. invasa, revealed that the survival rate ofoffsprings of L. invasa decreased, average weight of females declined, fecundity reduced, averagelife span shortened, and the index of population trend decreased.(2) Vague comparative methodon similarity evolution between oviposition-induction and other induction treatments, showed thatspraying1.0mmol·L~(-1)MeJA can make the similar result as oviposition, demonstrated sprayingMeJA plays an active role in induced resistance, which can be used as the supplementary methodfor integrated pest management.
引文
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