【作者】 毕明娟；

【导师】 薛明； 刘同先；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 B型烟粉虱是世界性的入侵害虫,该虫自侵入我国以来,迅速扩展蔓延,已成为多种作物上的害虫的优势种。我们在前期研究中已发现,B型烟粉虱取食诱导的烟草对烟蚜有明显的不利影响,B型烟粉虱在与烟蚜的种间竞争中存在明显的竞争优势。本研究以B型烟粉虱—烟草—烟蚜作为研究体系,研究了B型烟粉虱诱导的植物防御反应对烟蚜和B型烟粉虱生长发育和繁殖的影响；研究了B型烟粉虱取食诱导的烟草防御信号途径,探讨其在对烟蚜防御反应中的作用；通过研究B型烟粉虱和烟蚜取食B型烟粉虱前侵染的烟草植株其生理适应性的差异,明确其生理适应性在B型烟粉虱对烟蚜竞争优势中的作用。本研究对于揭示B型烟粉虱对烟蚜的竞争取代机制,进一步深入探讨外来入侵害虫B型烟粉虱的入侵机制具有重要的意义。本研究主要结果如下：1、B型烟粉虱前侵染的烟草植株对后取食的烟蚜生长发育和繁殖有显著的不利影响,但对后取食的B型烟粉虱却没有明显的不利影响。烟蚜1龄若蚜在B型烟粉虱前侵染的烟草植株系统白脉叶片上取食72h和96h后,存活率仅为35.4%和29.9%,而取食对照叶片的却为94.3%和91.5%,差异极显著；成蚜取食系统白脉叶片12d后,存活率为42.9%,累积单雌产仔量为25.6头／雌,而取食对照叶片的存活率为61.4%,单雌产仔量为32.5头／雌。B型烟粉虱前侵染的烟草植株的局部带虫叶片对烟蚜存活率影响很小,与对照相比略有差异。而B型烟粉虱前侵染的烟草系统白脉叶片和局部带虫叶片对随后取食的B型烟粉虱在存活率、繁殖力和生长发育等方面均没有明显的不利影响。2、B型烟粉虱若虫为害烟草植株后诱导了水杨酸信号途径的强烈表达,对茉莉酸信号途径没有明显的诱导作用。(1)利用生化分析方法,测定了水杨酸和茉莉酸信号途径中关键酶的活性。B型烟粉虱为害烟草植株后水杨酸信号途径关键酶苯丙氨酸解氨酶(PAL)和β-1,3葡聚糖酶活性明显升高。在处理植株的系统白脉叶片中,PAL和β-1,3葡聚糖酶活性分别在B型烟粉虱若虫为害后10和15d升高最明显,分别是对照叶片的1.65倍和2.23倍；在处理植株的带虫叶片中,PAL活性在为害10d时升高最明显,是对照的2.25倍；而β-1,3葡聚糖酶活性与对照相比没有显著变化。茉莉酸合成关键酶脂氧合酶(LOX)活性在诱导的烟草植株的白脉叶片中变化不明显,而在带虫叶片中则明显受到抑制。(2)采用RT-PCR技术测定了水杨酸和茉莉酸信号途径关键基因表达量,结果表明：水杨酸信号途径关键基因PAL、PR1和PR5在B型烟粉虱若虫为害的烟草植株的系统白脉叶片和局部带虫叶片上基因表达量均表现出明显的上调。茉莉酸信号途径基因AOS基因表达量在系统白脉叶片和局部带虫叶片上微弱上调,而LOX和COI基因表达量却没有显著的变化。(3)B型烟粉虱若虫为害烟草后植株内源水杨酸含量较对照显著升高,而茉莉酸含量变化微弱,与对照没有明显差异。在处理植株的系统白脉叶片上,水杨酸含量在B型烟粉虱若虫为害10d时急剧上升,是对照叶片的29.71倍,为害至第15d时较对照也有明显差异,至20d时与对照相似。在处理植株的带虫叶片上,只有在B型烟粉虱若虫为害第10d时,水杨酸含量显著升高,是对照的20.94倍,其余时间段则无与对照没有明显差异。3、外源水杨酸诱导了烟草植株对烟蚜的防御反应,且下部处理叶片的影响均大于上部系统叶片,外源茉莉酸诱导的烟草对烟蚜无不利影响。推测水杨酸防御信号途径在烟草介导的B型烟粉虱对烟蚜的防御反应中起到重要作用。(1)外源水杨酸不同浓度诱导烟草后对烟蚜存活率和发育速率均有显著的不利影响,且1mM>2mM>0.5mM,而对B型烟粉虱若虫的存活率和发育历期几乎没有不良影响。外源茉莉酸诱导烟草,一定程度上加快烟蚜的生长速率,且1mM>2mM>0.5mM；而不同处理浓度对B型烟粉虱若虫发育速率影响不同,0.5mM略为抑制,2mM使发育加快,1mM影响不大。外源茉莉酸诱导烟草植株后对B型烟粉虱和烟蚜存活率均没有显著影响。外源水杨酸和茉莉酸诱导烟草后,下部处理叶片对两种试虫的影响均大于上部系统叶片。(2)分别选择外源水杨酸和茉莉酸对烟蚜影响最为明显的两个浓度,即1mM水杨酸和2mM茉莉酸甲酯诱导烟草进行对烟蚜影响的时间效应实验,结果表明两种外源诱导物喷施后2d对烟蚜略有影响,5d时影响最为明显,处理8d时,影响几乎消除。对烟蚜影响的趋势与浓度实验中趋势相同。4、B型烟粉虱前侵染的烟草植株对随后取食的B型烟粉虱和烟蚜体内解毒代谢酶、保护酶及消化酶活性有不同的影响,说明生理适应性的差异是B型烟粉虱对烟蚜竞争优势的重要原因之一(1)取食前侵染的烟草植株系统白脉叶片后,B型烟粉虱成虫羧酸酯酶活性从取食12h开始显著升高,且一直持续至72h,其中取食12h时差异最明显,是对照的1.71倍。谷胱甘肽S转移酶活性在取食的48和72h时明显升高,取食48h时差异最明显,是对照的2.34倍。烟蚜成蚜羧酸酯酶活性在整个取食时间内均被显著抑制,24h时差异最明显,是对照的0.78倍。谷胱甘肽S转移酶活性在取食的12、48和72h明显降低,在差异最明显的12h时,处理试虫酶活是对照的0.74倍。前侵染的烟草植株白脉叶片对随后取食的两种试虫乙酰胆碱酯酶活性没有显著影响。两种试虫若虫取食系统白脉叶片与成虫的相关酶活规律相似。两种试虫成虫羧酸酯酶和谷胱甘肽-S-转移酶Km和Vmax值亦有不同程度的变化,说明了酶活性的变化不仅限于量变而且可能发生了质变。(2)B型烟粉虱取食其前侵染的烟草植株系统白脉叶片后,其体内超氧化物歧化酶(SOD)和多酚氧化酶(PPO)的活性均显著增加,过氧化物酶(POD)活性在取食12和24h后变化不明显,但在取食6,48和72h后也显著增加,B型烟粉虱体内的这3种酶活性的最高值较对照最高分别增加达1.62,2.71和2.57倍。而烟蚜在多数取食时间段内,体内PPO和SOD的活性仅略有增加,但在取食12h后SOD的活性受到明显的抑制。POD活性除在取食24h后显著高于对照外,在取食其他时间后均受到显著的抑制。(3)B型烟粉虱取食其前侵染的烟草植株系统白脉叶片不同时间后,其体内蛋白酶和淀粉酶活性均显著增加,最大值出现在24和48h,分别是对照的1.54倍和1.33倍；烟蚜取食烟草植株系统白脉叶片6h后,其体内蛋白酶和淀粉酶活性与对照相比没有明显差异,但随着取食时间的延长,这两种酶活性被显著抑制,其最低值分别为对照的0.39和0.72倍。更多还原

【Abstract】 Bemisia tabaci (Gennadius) B biotype spread rapidly when it invades in China. It has been a key pest of various vegetables, cotton and tobacco and so on. We found that B. tabaci was predomination in competing with M. persicae. We chose B. tabaci--tobacco-M. persicae as research system, studied the effect of tobacco’s defense response induced by B. tabaci on development and reproduction of M. persicae and B. tabaci itself. We also investigated defense signial pathway on tobacco induced byB. tabaci to explor the action of it in defensing M. persicae. We explored the difference of physiological adaptability of the two insects to defense response of tobacco induced by B. tabaci. This research had important academic meaning for showing competitive replacement mechanism of B.tabaci to M. persicae, and implementing sustainable control effectively. Major results as follows:1 There was disadvantaged influence of tobacco damaged by B. tabaci on development and fecundity of M. persicae, but no influence on B. tabaci itself. On systemic white-vein leaves, livability of M. persicae nymphae was 35.4% and 29.9% when feeding 72 and 96h, the control was 94.3% and 91.5%. Livability of adult of M. persicae was 42.9% when feeding 12h on systemic white-vein leaves. Fecundity of adult of M. persicae was significantly fall. Accumulative number of eggs per female was 25.6 when feeding 12h on systemic white-vein leaves, control is 32.5. Meanwhile, there was nearly no disadvantaged influence on livability of M. persicae nymphae on local leaves of induced tobacco plants. There was no disadvantaged influence on livability、fecundity and development of B. tabaci itself on systemic and local leaves of induced tobacco plants.2 Nymphae of B. tabaci can induce strong SA signical pathway, but not induce significant JA signical pathway after feeding on tobacco plants.(1) The activity of PAL andβ-1,3-glucanase which are two key enzyme in SA signical pathway were significantly rise. On systemic white-vein leaves, The maximal activity of PAL andβ-1,3-glucanase was as 2.23 and 1.65 times as control on 15d and 10d,respectively. On local leaves,the maximum activity of PAL was as 2.25 times as control on 10d.However the activity ofβ-1,3-glucanase was no significant difference. The activity of LOX which is the key enzyme of JA signial pathway was no significant difference on local leaves,and significantly repressed on systemic white-vwin leaves.(2)The gene expression amout of PAL、PR1 and PR5 in SA signial pathway was significantly rise on systemic and local leaves of induced plants. The gene expression amout of AOS gene in JA signial pathway is a little rise in the two leaves, but the gene expression amout of LOX and COI in JA signial pathway was no significant difference.(3)Content of SA in induced tobacco plants by B. tabaci was significantly rise. On systemic white-vein leaves, content of SA was significantly rise on 10 and 15d, and gradually fall later. Content of SA was as 29.71 times as control plants on 10 days. Meanwhile, on local leaves, content of SA was as 20.94 times as control on 10 days,but no significant difference on later times. Content of SA was nearly no difference in induced plants.3 Application of exogenous SA can induce defence reponses of tobacco plants to M. persicae. It indicated that SA signal pathway had important effect to M. persicae on tobacco plants induced by B. tabaci.(1) Application of exogenous SA on tobacco had disadvantaged influence on livability and development of M. persicae. Degree of the influence on M. persicae of the three concentration was 1mM>2mM>0.5mM. Application of exogenous SA on tobacco plants nearly had no effect on survivol and developmental period of B. tabaci. Application of exogenous JAon tobacco can expedite the development of M. persicae. Degree of the effect on M. persicae of the three concentration was 1mM>2mM>0.5mM. Application of exogenous JA on tobacco had a slight effects on B. tabaci.0.5mM of JA slightly slower the development of B. tabaci nymphae,2mM of JA can expedite the development of it, 1mM of JA had no significant effect on it. Application of exogenous JA on tobacco had no effect on livability of B. tabaci and M. persicae.The systemic leaves had the same trend, but degree of effect was lower than the local leaves.(2) Application of exogenous1mM SA and 2mMeJA on tobacco to research the influence on B. tabaci and M. persicae of time effect. It indicated that the influence was the most significant on 5 days, the influence was slight on 2 days, and was no influence on 8 days.4 There was different influence on activities of detoxification metabolize enzymes、protective enzymes and digest enzymes between B. tabaci and M. persicae. (1) The activity of Carboxylesterase of B. tabaci was significantly increase from 12h to 72h. The maximal activity was as 1.71 times as control on 12h. The activity of Glutathione S-transferase of B. tabaci was significantly increase on 48 and 72 times. The maximal activity was as 2.34 times as control on 48h. The activity of Carboxylesterase of M. persicae was significantly decrease with the time of feeding. The minimal activity was as 0.78 times as control on 24h. The activity of Glutathione S-transferase of M. persicae was significantly decrease on 12、48 and 72h. The minimal activity was as 0.74 times as control on 12h.There was no influence on acetylcholine esterase of the two insects.The nympha of the two insects has the same result.Km and Vmax of the two enzyme of the two insects changed inordinately.It showed that the activities of the two enzymes qualitative changed.(2) The activities of PPO and SOD in B. tabaci significantly increased after feeding on the preinfested tobacco plants for 6,12,24,48 and 72 h, and the POD activity in B. tabaci also significantly increased after feeding on the preinfested tobacco plants for 6,48 and 72 h, respectively.The maximum activities of SOD, PPO and POD in B. tabaci on the preinfested tobacco plants were 1.62,2.71 and 2.57 times higher than those in the control, respectively. In contrast, the activities of PPO and SOD in M. persicae increased a little in most time-span after feeding on the preinfested tobacco plants, while the SOD activity was obviously suppressed after feeding on the preinfested tobacco plants for 12 h. POD activity in M. persicae was significantly inhibited except after feeding on the preinfested tobacco plants for 24 h(3) The activities of protease and amylase in B. tabaci significantly increased in all time-span after feeding on the preinfested tobacco plants, the maximum activities were 1.54 and 1.33 times that in the control, respectively. Activities of protease and amylase in M. persicae did not change after feeding on the preinfested tobacco plants for 6 h(P>0.05); however, the activities were inhibited with the extending of feeding time, the lowest activities in M. persicae feeding on the preinfested tobacco plants was 0.39 and 0.72 times that in the control, respectively.更多还原