【作者】 张玉丰；

【导师】 刘华山；

【作者基本信息】 河南农业大学 ， 作物栽培学与耕作学， 2006， 硕士

【摘要】 本研究通过盆栽试验，针对烟草普通花叶病毒（TMV）的不同寄主（烤烟K326和心叶烟），采用人工对健康烟株接种TMV诱导其发病，然后用不同种类的植物提取液，采用不同方法对烟株进行处理，测定烟株叶片各种生理生化指标变化（叶片抗氧化酶活性如POD、SOD、CAT、PPO等；C、N代谢、MDA含量等）、叶片光合特性、叶绿素荧光动力学参数、烟株根系活力、病程相关蛋白（PR）的产生、烟株的体外防治效果以及烤后烟叶的品质等，研究了不同植物提取液的不同处理方法防治烟草TMV的防治效果及作用机理。试验结果如下：1、植物提取液具有较好的清除超氧阴离子自由基和羟基自由基的效果，不同清除体系的效果明显不同，其中以纯提取液清除超氧阴离子自由基和羟基自由基的效果最好。处理感染TMV烟株后的清除效果明显弱于纯植物提取液、健康叶和病叶体系。不同提取液清除超氧阴离子自由基的能力为：制剂＞商陆＞落葵＞毛叶子花：清除羟基自由基的能力为：制剂＞落葵＞毛叶子花＞商陆。2、感染TMV烟株叶片经提取液处理后，均不同程度的提高了叶片抗氧化酶活性，降低了膜脂过氧化程度，叶片总酚含量明显增多。与健康株相比，提取液处理后的烟株叶片生理生化指标均达到或接近健康株，甚至超过健康株，进而促进了烟株生长、增强了烟株抗病力。其中以先喷施提取液后接毒液的烟株抗TMV效果好。各种提取液的效果为：制剂＞商陆＞落葵＞毛叶子花。3、经提取液处理的烟株叶片的光合速率、蒸腾速率、气孔导度、胞间CO₂浓度均非常接近健康烟株。采用先喷施提取液再接毒液的处理方法，在处理后第12d达到最大值，用落葵、毛叶子花、商陆和制剂处理的烟株净光合速率分别相当于健康烟株的95.07％、92.22％、94.90％和98.33％；叶绿素含量明显增多；叶片PsⅡ潜在的最大光化学活性Fv／Fm比值均有所增加，从而增强了叶片光能转化，促进了叶片光合作用。其效果为：制剂＞落葵＞商陆＞毛叶子花。4、提取液处理明显提高了烟株根系活力，增强了叶片硝酸还原酶和转化酶活性。其中先喷施制剂处理效果最为明显，硝酸还原酶和转化酶活性分别达到了健康烟株的107％和104％。5、电泳实验结果表明，经提取液处理的烟株叶片，产生新的病程相关蛋白量有差异。除了一些共有的蛋白相继产生和消失外，还产生一些各自特有的新蛋白，其中在处理后的第3d，制剂就诱导产生一条分子量为15.9KDa的特有蛋白，又分别在处理后的第6d、18d和24d，诱导出三条分子量分别为49.1KDa、54.1KDa和16.9KDa的新病程相关蛋白；落葵在处理后的第9d诱导出所特有的27.5 KDa和26.9 KDa新蛋白；毛叶子花分别在处理后的第6d、12d、15d、21d和24d，诱导出分子量分别为54.2KDa、67.6KDa、84.5KDa、88.3KDa和69.4KDa的特有新蛋白；商陆则在处理后的第15d诱导出19.5 KDa的特有蛋白。随着病程的延长，新产生的条带明显减少，诱导抗性减弱；制剂和毛叶子花防治TMV效果持续时间长可能与这些新蛋白的产生有关。6、提取液在防治普通烟草花叶病和抑制TMV增殖方面均具有较好的作用，且预防效果明显好于治疗效果。无论是先喷施提取液后接病毒还是先接种病毒后喷施提取液，其中制剂的防治效果明显好于单一植物提取液处理，其防效分别达到82.3％和77.3％；抑制TMV增殖效果分别达到了58.03％和49.29％。各种提取液综合效果为：制剂＞落葵＞商陆＞毛叶子花。7、烟叶化学成分检测结果表明，制剂处理能协调烟叶的化学成分，改善烟叶品质，烟叶可用性明显提高。其中先喷施提取液再接毒液的烟叶中，钾、烟碱、总氮、总糖和还原糖含量分别相当于健康烟株的103.4％、125.2％、136.01％、103.6％、102.2％和97.2％，比毒液株的烟叶分别提高了21.1％、4.28％、5.18％、12.9％、25.4％和18.8％，其中还原糖／总糖、总糖／烟碱、总氮／烟碱的比值分别为0.938、7.50和0.985，均处于比较适宜的范围。作为天然植物提取液，对烟草花叶病毒的防治具有明显的效果。其作用机理主要表现在钝化病毒、诱导植物产生病程相关蛋白、提高植物自身的代谢能力等方面；这种提取液对动植物无毒、安全无公害、环保，应用前景可观。更多还原

【Abstract】 Pots experiments were conducted to study the effects of extracts from different plant （Basella rural L.shorted for L,Bougainvillea spectabilis Willd shorted for M and Phytolacca americana shorted for S） and inhibitor on tobacco mosaic virus and their mechanism to control disease,by inoculating TMV on healthy tobacco,which in 2004 ～2005 in Henan Agricultural University. The host plant were Nicotiana tobacum cv K326 and N.glutinosa L. The following results were obtained through analysizing or measuring the changes of physiologic and biochemical indexes（including the activity of protectingenzyme such as superoxide dismutase（SOD）, peroxidase（POD）, catalase（CAT） and Polyp -henol oxidase（PPO）、 C、 N metabolism and lipid peroxidation levels ）、 photosynthetic character、 fluorescent induction kinetics parameters、 vitality of root system、 inhibitory effect and the quantity of flue-cured tobacco leaf..1. The extracts of plant and inhibitor could scavenge the superoxide radical and hydroxyl radical significantly. there is different effect in different scavenging system, the pure extracts has the best scavenge effect on superoxide radical and hydroxyl radical than any other scavenging system, the effect of after being treated to the tobacco was poor to the pure extract system、 healthy leaf system and diseased leaf system obviously, the integrated effect on scavenging superoxide radical all of the agents were that: Z>S>L> M; and scavenging hydroxyl radical were: Z>L>M>S. enzyme such as superoxide dismutase（SOD）, peroxidase（POD）, catalase（CAT） and Polyp -henol oxidase（PPO）、C、N metabolism and lipid peroxidation levels)、photosynthetic character、fluorescent induction kinetics parameters、vitality of root system、inhibitory effect and the quantity of flue-cured tobacco leaf..1. The extracts of plant and inhibitor could scavenge the superoxide radical and hydroxyl radical significantly, there is different effect in different scavenging system, the pure extracts has the best scavenge effect on superoxide radical and hydroxyl radical than any other scavenging system, the effect of after being treated to the tobacco was poor to the pure extract system, healthy leaf system and diseased leaf system obviously. the integrated effect on scavenging superoxide radical all of the agents were that: Z＞S＞L＞M; and scavenging hydroxyl radical were: Z＞L＞M＞S.2. After being treated by different extracts and inhibitor,the protecting enzymes activities in leaves infected with TMV were all increased in some content, lipid peroxidation levels were decreased and the content of total phenolics compounds was increased obviously; compared with the healthy plant, the physiologic and biochemical indexes in leaves induced by extracts and inhibitor were nearly or reached to it; Finally, which promote the plant growth, and strength the anti-mosaic activity, as far as measure is concerned, using extracts and inhibitor before inoculation has better effect, the integrated effect all of the agents were that: Z＞S＞L＞M.3. After treated by the extracts and inhibitor,the net photosynthetic rate（Pn）、transpiration rate （Tr）、stomatal conductance（Gs） and intercellular CO₂ concentration（Ci） in leaf were nearly or reached to healthy plant greatly, as far as the measure using extracts and inhibitor before inoculation is concerned, after treatment for 12d, the net photosynthetic rate in leaf treated by L、M、S and Z, which were 95.07％、92.22％、94.90％and 98.33％compared to healthy plant respectively; the content of chlorophyll has been increased significantly; the biggest efficiency of photochemistry of PSII（Fv/Fo） and potential activation of PSII（Fv/Fm） were all increased, and strengthened conversion of light energy, promoted the photosynthetic character. the integrated effect all of the agents were that: Z＞L＞S＞M.4. The extracts and inhibitor could increase the vitality of root system obviously; and strengthen the activity of NR and IvR. The effect of Z treatment was best during all of the treatment, and the activity of NR and IvR were reached to 107％and 104％compared to the healthy plant respectively.5. The result based on routine PAGE showed that, There ere new Pathogenesis-related proteins（PR） in the leaves treated with extracts and inhibitor. On the third day after treatment, there is a special PR about 15.9 KDa induced with inhibitor; and there are three pieces of PR about 49.1KDa、54.1 KDa and 16.9 KDa induced on sixth day、twelfth day and twenty-forth day after treatment, respectively; There are special PR about 27.5KDa and 26.9 KDa induced with Basella rural L. on third day after treatment.; and there are five special pieces of PR about 54.2KDa, 67.6 KDa, 84.5KDa, 88.3 KDa and 69.4KDa induced with Bougainvillea spectabilis Willd on sixth day、twelfth day and、fifteenth day and twenty-forth day respectively; and about Phytolacca Americana The special PR about 19.5 KDa was induced on fifteenth day after treatment.6. The extracts and inhibitor have significant effect in controlling Tobacco mosaic disease and inhibitory multiplication of TMV. And the defend effect is excelled to cured effect. the inhibitor has better effect than any single extract in in controlling Tobacco mosaic disease and inhibitory multiplication of TMV either before or after inoculation,the inhibitory effect was 82.3％and 77.3％respectively, and the inhibitory multiplication effect was 58.03％and 49.29％respectively, the integrated effect all of the agents were that: Z＞L＞S＞M.7. The chemistry ingredient in tobacco leaf showed that, the quality of leaf disposed by inhibitor was more harmonized and content was in reason, the usability of leaf is increased obviously.as far as the measure using extracts and inhibitor further inoculation is concerned, the content of K₂O、nicotine、total nitrogen、total sugar and deoxidize sugar were equivalent 103.4％、125.2％、136.01％、103.6％、102.2％and 97.2％to healthy plant respectively, and improved 21.1％、4.28％、5.18％、12.9％、25.4％and 18.8％to diseased plant respectively.As natured extracts and inhibitor, The effect on controlling tobacco mosaic disease including many aspects. Be the same as other vegetable inhibitor, the mechanism of controlling TMV manifest following aspects mainly: inactive virus, induce producing anti-disease activity、improve the resist-contradictorily of plant itself, and so on. The extracts and inhibitor are of the superiority to propagation that has innocuity and security, and has a promising application prospect in production.更多还原