【作者】 高丽娟；

【导师】 张玉星；

【作者基本信息】 河北农业大学 ， 果树学， 2013， 博士

【摘要】 梨是我国的优势树种，也是我国传统的出口创汇果品。梨轮纹病是梨生产上主要病害之一，目前，主要依靠化学杀菌剂防治，但长期使用化学杀菌剂不仅易使病菌产生抗药性，而且存在农药残留等问题，对食品安全、环境保护以及人类健康等造成不利影响。植物诱导抗病性是控制植物病害的重要方法之一，水杨酸是已经被认定的系统获得抗性化学诱导剂。本研究以主栽品种鸭梨（Pyrus bretschneideri‘Yali’）为试材，探讨水杨酸诱导梨抗轮纹病的作用效果，从植物抗氧化酶、病程相关蛋白等方面阐述水杨酸诱导增强梨对轮纹病抗性的生理生化机制。并通过研究水杨酸诱导及轮纹病菌胁迫对梨叶绿素荧光特性、电阻抗参数的影响，试图为利用叶绿素荧光技术、电阻抗技术监测梨轮纹病提供初步的参数。采用实时荧光定量PCR的方法，对水杨酸诱导后梨中NPR1基因转录表达水平进行分析。主要研究结果如下：1.水杨酸处理显著提高鸭梨对梨轮纹病的抗性。用0.2mmol·L-1水杨酸对鸭梨叶片及果实诱导处理，接种轮纹病菌后，叶片的病情指数显著降低，诱抗效果达21.86%；诱导处理后的果实在接种3d时，发病率比对照降低38.64%，接种7d时，果实病斑直径显著低于对照。水杨酸对轮纹病菌毒性测定结果表明0.002~0.2mmol·L-1水杨酸对轮纹病菌的生长无抑制作用，即0.2mmol·L-1水杨酸对轮纹病菌无直接毒性，说明水杨酸诱导后梨对轮纹病抗性的增强是来源于其对植物的诱导抗病性作用。2.外源水杨酸诱导后梨叶片中内源水杨酸总含量升高，其中游离态水杨酸含量降低，结合态水杨酸含量升高，表明水杨酸诱导梨系统获得抗病性的建立与内源水杨酸状态的改变相关。对叶片中超氧化物歧化酶（SOD）、多酚氧化酶（PPO）同工酶谱的分析结果表明，水杨酸诱导处理后SOD、PPO同工酶谱均未出现新的酶带，但酶谱带表达量增强。3.水杨酸诱导处理后梨叶片及果实中苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）、β-1,3-葡聚糖酶（GLU）和几丁质酶（CHI）活性增强，并且降低了膜脂过氧化产物丙二醛（MDA）和超越阴离子（·O2-）的含量，表明水杨酸对其有系统诱导作用，增强梨对轮纹病菌侵染的抵抗能力。4.病菌侵染后叶片的光系统Ⅱ最大原初光能转换效率（Fv/Fm）、实际光化学效率（Y(Ⅱ)）、光化学猝灭系数（qP）、最大表观电子传递效率（ETRmax）减小，说明病菌对光系统Ⅱ反应中心造成破坏。外源水杨酸诱导后梨叶片的非光化学猝灭系数（NPQ）、光系统Ⅱ原初量子效率（Q）和最大表观电子传递效率（ETRmax）提高，光化学猝灭系数无显著变化。诱导叶片接种病菌后，光系统Ⅱ最大原初光能转换效率（Fv/Fm）、光化学猝灭系数（qP）和最大表观电子传递效率（ETRmax）降低幅度减小，表明水杨酸能够缓解病菌对光系统Ⅱ反应中心的伤害，提高光系统Ⅱ反应中心对光能的吸收利用。5.0.2mmol·L-1水杨酸诱导后，接种病菌叶片的高频电阻率、低频电阻率、胞外电阻率、胞内电阻率和弛豫时间分布系数减小；接种病菌果实的低频电阻率、胞外电阻率和弛豫时间增大，高频电阻率、胞内电阻率和弛豫时间分布系数减小。6.克隆鸭梨NPR1基因，研究外源水杨酸对NPR1基因的表达调控。结果表明NPR1基因在梨不同器官中的表达量差异较大，叶片、果肉中NPR1基因的表达量较高，茎中的表达量显著低于其它器官。水杨酸诱导后梨叶片中NPR1表达量有明显升高，果皮中NPR1的表达量在水杨酸处理24h显著升高。梨叶片和果皮中的NPR1基因在水杨酸诱导后表达增强，这将有助于激活下游防卫基因的表达，从而产生系统获得抗性。综上所述，水杨酸通过参与调节梨体内与抗病有关的生理生化过程来增强对轮纹病的抗性，并且能够激活梨防卫基因的表达，产生系统获得抗性。更多还原

【Abstract】 Pear ring rot caused by Physalospora piricola Nose is a major fungal disease of pears inChina, which results in a huge lose annually. Therefore, many methods have been developed inthe past decades, such as planting resistant varieties, strengthening cultivation management andchemicals control. Among these, the applications of fungicides are a traditional approach tocontrol this kind of diseases. Unfortunately, fungicides are becoming less effective because of thedevelopment of fungicide resistance by pathogens. Also, the use of fungicides shows a reducingtrend due to increasing environmental concern about fungicidal residues in fruit. Therefore, thereis need for an effective, environmental-friendly method to control the disease. As a signalmolecule, salicylic acid (SA) could protect different plant species against diseases. To reveal themechanism of resistance of pear against ring rot induced by SA, the influence of SA treatment ondisease severity and plant defense enzymes were studied with Pyrus bretschneideri‘Yali’ andPhysalospora piricola Nose as the materials. In this paper, the physiological and biochemicalcharacteristics of detached pear were measured after application of SA, and the changes ofelectrical impedance spectroscopy (EIS) parameters and chlorophyll fluorescence characteristicswere observed. At the same time, the expression of NPR1gene in pear after SA treatment wastested with Real-Time PCR technique.The main results in this paper were as follows:1. Salicylic acid treatment could significantly improve the resistance of Yali pear toring rot disease. The results showed that the leaves of Yali pear treated with0.2mmol·L-1SA could significantly degrade disease index and the induced resistance reached up to21.86%. While the treatment of0.2mmol·L-1SA could decrease the incidence rate of pearfruit infected with P. piricola. PDA culture results showed that SA at lower concentrationcould not inhibit the growth of P. piricola. The effect of resistance was not by SA killingthe fungi directly, but come from SA activated the systemic acquired resistance of pears.2. The content of total SA was increased in the pear leaves treated with exogenous SA. Itis interesting to note that the content of free states SA was reduced and the content of bindingstates SA was increased. It was suggested that SA induced the expression of systemic acquireresistance by changing of endogenous salicylic acid status.The isozymes analysis resultsshowed that superoxide dismutase (SOD) and polyphenol oxidases (PPO) isozymes had notnew enzymes in pear leaves treated with SA, but the expressions of enzyme were enhanced. 3. In the present study, plant defence responses were induced in pear using markerenzymes such as superoxide dismutase (SOD), peroxidase (POD), phenylalanineammonia-lyase (PAL), polyphenol oxidases (PPO), β-1,3-glucanase (GLU) and chitinase(CHI). In the inoculated leaves and fruits, the SA-treatment enhanced the activities of PAL,PPO, β-1,3-glucanase and chitinase. It was observed that the PR-proteins respondedquickly to pathogen infection and the contents of MDA and·O2-dropped downsimultaneously. These results clearly demonstrated that SA could induce the systemicacquired resistance and strengthen antiviral capability of pear to P. piricola.4. Chlorophyll fluorescence characteristics were observed in pear leaves after treatedwith SA and infected by P. piricola. Under the stress of pathogen, the chlorophyllfluorescence parameter Fv/Fm, Y (II), qP and ETRmaxdecreased. And, the chlorophyllfluorescence parameter NPQ, Q and ETRmaxincreased, whereas qP was no significantchange in leaves teaeted with SA. The decreasing of Fv/Fm, qP and ETRmaxwere inhibitedin induced leaves inoculated with P. piricola. These results suggested that SA couldalleviate the damage of P. piricola on the PSII reaction center and improve absorption andutilization of light energy.5. The effects of SA and pathogen stress on parameters of electrical impedancespectroscopy (EIS) in pear leaves and fruit were investigated. The results showed thatspecific high-frequency resistance, specific low-frequency resistance, specific extracellularresistance, specific intracellular resistance and distribution coefficient of relaxation time inleaves treated with SA and infected by P. piricola decreased. Furthermore, the obtainedresults demonstrated that specific low-frequency resistance, specific extracellularresistance and relaxation time increased, whereas specific high-frequency resistance, specificintracellular resistance and distribution coefficient of relaxation time decreased in fruitstreated with SA and infected by P. piricola.6. According to the eonsensus domain of NPR1gene and by using RT-PCRtechnology, a cDNA fragment of NPR1gene was cloned from Pyrus bretschneideri cv. Yali.Real-time quantitative PCR (qPCR) analysis carried out on mRNAs from leaves and fruitstreated with SA. Results showed that the expression of NPR1gene had significantlydifference in different organs of pear. The expression of NPR1gene in pear could begreatly enhanced by the treatment of0.2mmol·L-1SA.In conclusion, the work presented here showed that SA treatment could significantlyincrease activities of defense-related enzymes and enhance disease resistance in pear. AndSA could induce the systemic acquired resistance and strengthen the expression of defensegene in pear.更多还原