【作者】 殷丽华；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 果树学， 2013， 博士

【摘要】 苹果褐斑病是造成我国苹果早期落叶的主要病害之一，目前对该病害的抗病机制尚不明确。本试验从苹果属资源对苹果褐斑病的抗性评价入手，利用组织细胞学、生理生化及分子生物学等多种技术和方法，研究了苹果叶片在接种褐斑病菌后，叶片对病菌入侵和扩展的响应情况，以及寄主中活性氧迸发、抗氧化系统、抗病相关蛋白、抗病信号通路和酚类物质代谢等方面的变化情况，探索了苹果属资源对苹果褐斑病的抗性差异及其机制，并发现外源褪黑素处理可显著提高感病材料对苹果褐斑病的抗性，为生产上防控苹果褐斑病提供依据。取得的主要结果如下：1.建立了人工接种苹果褐斑病菌的抗性评价方法，并利用该方法对国内外主栽的28个苹果品种和39个野生资源进行了抗苹果褐斑病评价。共鉴定获得5个抗病品种（‘皮诺娃’、‘蜜脆’、‘首红’、‘粉红女士’和‘秦冠’）和8个抗病的野生材料（武山变叶海棠、新疆野苹果、三叶海棠（西南农大）、滇池海棠、海棠花、野苹果9号、中熟红果子和腾冲三叶海棠）。该研究结果为抗病品种选育提供了种质基础。2.利用电镜技术研究了苹果褐斑病菌在不同抗性材料叶片表面和叶组织内的发育情况。扫描电镜观察发现，接种3d后（3dpi），在感病材料‘长富2号’和冬红果的叶片表面，病菌分生孢子萌发率较高，分别为60%和79%，萌发的芽管可以直接侵入也可通过形成附着胞后再侵入寄主细胞；而在抗病材料‘秦冠’和新疆野苹果叶片表面，病菌孢子的萌发率分别为36%和32%，且孢子萌发后出现干瘪现象，很难进一步侵入寄主细胞。细胞学观察表明，接种7d后，抗病品种可以形成胞壁沉积物阻止菌丝体进一步扩展和吸器的形成。以上结果初步证实了苹果属资源对褐斑病菌的抗性表现为抑制分生孢子萌发和抗扩展两个方面。3.抗病材料新疆野苹果在接种苹果褐斑病菌2d后，其叶片中H₂O₂迅速积累，而感病材料冬红果中H₂O₂的含量在第4d、6d和10d时均显著低于对照，同时，本研究还发现在抗病材料与苹果褐斑病菌互作前期，超氧化物歧化酶（SOD）活性上升，抗坏血酸过氧化物酶（APX）的活性下降，从而使得H₂O₂得到了有效的积累，为氧迸发提供了有利条件，而在感病材料与苹果褐斑病菌互作的前期，APX和过氧化氢酶（CAT）的活性显著上升，这与感病材料中H₂O₂含量变化相一致，说明氧迸发在苹果属资源抗褐斑病过程中可能起重要作用。4.在接种苹果褐斑病菌后，抗病材料新疆野苹果和感病材料冬红果中几丁质酶活性均有所上升，但在抗病材料中的上升时间（6dpi）明显早于感病材料（20dpi），且抗病材料的几丁质酶活性明显高于感病材料。对β-1,3-葡聚糖酶的测定结果表明，抗病材料中β-1,3-葡聚糖酶活性远高于感病材料。同时，利用qRT-PCR对这两个基因在接种后不同时间的表达情况进行了研究，结果也进一步证实了几丁质酶和β-1,3-葡聚糖酶在苹果抵御褐斑病菌的侵染过程中起重要作用。5.采用高效液相色谱法测定了抗病材料新疆野苹果和感病材料冬红果接种苹果褐斑病菌后，叶片中18种主要的酚含量变化情况，在抗病材料中，其没食子酸含量在接种后第4d迅速上升，显著高于未接种对照，之后一直保持较高水平，而在感病材料中，没食子酸含量在接种后4d开始下降，在接种后6d达到最低点，之后一直保持较低水平；苹果褐斑病菌的入侵引起表儿茶素含量的显著升高，且在抗病材料中的上升程度要高于感病材料，说明表儿茶素可能与苹果抗褐斑病密切相关；对香豆酸的含量在抗病和感病材料中均表现出一定的下降，说明没食子酸、表儿茶素和对香豆酸与苹果对褐斑病的抗性密切相关。另外，对苯丙氨酸解氨酶（PAL）的活性和其编码的基因在不同抗性材料中转录水平的研究发现，抗病材料中PAL的活性和其编码基因的表达均表现出较高水平的上调，表明PAL与苹果对褐斑病的抗性相关。6.利用qRT-PCR对SA，JA和ET信号途径的关键基因在不同抗性材料中的表达情况进行研究，结果表明在抗病材料新疆野苹果中，SA途径响应基因PR1和PR5受苹果褐斑病菌诱导后表达上调；JA途径关键基因COI1和PLD也呈现上调表达，而乙烯途径响应基因ERF3没有显著变化；在感病材料冬红果中，其叶片PR1、COI1和PLD等基因均没有显著变化，ERF3则在接种后显著上调。外源施加SA和MeJA均提高了冬红果对苹果褐斑病的抗性，而施加1-氨基环丙烷羧酸（ACC）未能诱导冬红果产生抗病性。以上结果表明，苹果资源对褐斑病的抗性可能与SA、JA和ET信号途径相互协调作用相关。7.根施0.1mM褪黑素预处理3d显著提高了感病材料冬红果对苹果褐斑病的抗性。褪黑素处理可以使植物体内的活性氧维持在一个稳定水平，而且显著提高了过氧化物酶（POD）、PAL、几丁质酶和β-1,3-葡聚糖酶的活性。本研究首次证实了褪黑素可以诱导植物产生抗病性。外源施加褪黑素对苹果褐斑病的防治具有重要的应用价值和现实意义，并且为进一步深入研究褪黑素与植物抗病关系提供了理论参考。更多还原

【Abstract】 Marssonina apple blotch, caused by Diplocarpon mali Y. Harada&Sawanura （anamorph:Marssonina coronariae （Ellis&J. J. Davis） J. J. Davis）, is one of the most severe diseases ofapple （Malus×domestica）. This disease leads to premature defoliation in the main regions ofapple production, thereby weakening tree vigor and affecting the size, color, quality, andquantity of the fruit. However, the knowledge of mechanism on marssonina apple blotch ispoorly documented that has become an impediment to effectively control the disease in theorchard. In this study, we previously evaluated the resistance of28cultivars and39Malusspecies or biotypes after inoculating leaves with conidial suspensions of D. mali in vitro andvivo. Using M. sieversii and M. prunifolia cv. Donghongguo, we investigated the infectionprocess of D. mali on apple leaves, and monitored the levels of hydrogen peroxide, activitiesof antioxidant and pathogenesis-related enzymes, expression of marker genes for salicylicacid （SA）, jasmonic acid （JA）, and ethylene （ET） in pathways for defense regulation, and theconcentrations of18types of phenolic comounds during incompatible and compatibleinteractions. We also examined whether abiotic stress, plant hormones, and melatonin couldimprove resistance to Marssonina apple blotch. Our goal was to offer useful information fordeveloping and optimizing the disease management. The main results are as follows:1. Using artificial inoculations, the resistance of28cultivars and39Malus species orbiotypes to Marssonina apple blotch were evaluated. Of the28apple cultivars tested here, five（‘Pinova’,‘Honeycrisp’,‘Pink Lady’,‘Qinguan’, and ‘Redchief Delicious’） proved to beresistance genotypes and a low incidence of infection. The39species and biotypes showedsignificant differences in their degree of resistance to D. mali. Eight accessions wereclassified as resistant resources, including M. toringoides （Rehd.） Hughes cv.Wushanbianyehaitang, M. sieversii Ledeb. cv. Xinjiangyepingguo, M. sieboldii （Reg.） Rehd.cv. Sanyehaitang （xinannongda）, M. yunnanensis Schneid. cv. Dianchihaitang, M. spectabilisBorkh. cv. Haitanghua, M. sieversii （Ledeb.） Roemer cv. Yepingguo9, M. sieversii （Ledeb.）Roemer cv. Zhongshuhongguozi, and M. sieboldii （Reg.） Rehd. cv. Tengchongsanyehaitang.Those that proved most resistant are potential resources in programs for resitance breedingand disease management.2. The germination and growth of D. mali on leaves of resistance and susceptible Malusplants was disclosed by fluorecence and electromicroscopy.‘Naganofji No.2’ and M. prunifolia cv. Donghongguo, which were susceptible to Marssonina apple blotch, with highergermination of condia on their leaves surface. The pathogen penetrated the cuticle either bygerm tube or by formation of appressoria. Compared with the susceptible plants, the resistantplants ‘Qinguan’ and M. sieversii significantly suppress condia germinated. On their leavessurface, largely condia were wizened. Seven days post inoculation （dpi）, abundant hyphaewas observed in leaves tissue of ‘Naganofji No.2’, but fewer in ‘Qinguan’. These resultsdemonstrate that the disease resistance to Marssonina apple blotch of host plants responsed asinvading-resistance and conidia germinating inhibition.3. A ROS burst occurred in the infected plants of disease-resistance M. sieversii but notin those of the susceptible M. prunifolia. Compared with the uninfected M. sieversii control,infected plants of that species had a significant H₂O₂burst at2dpi, and then maintained thathigh level throughout the plant-pathogen interaction. Infected plants of M. prunifolia hadsignificantly lower H₂O₂concentrations at4,6, and10dpi when compared with the controlfor that species. This meant that ROS burst has a key role in the resistance response by M.sieversii. We observed a large increase in SOD activity and suppression of APX activity in M.sieversii plants at the early stage of infection, suggesting that this species produced andaccumulated much more H₂O₂. However, in M. prunifolia, the significant rise in APX andCAT activities during that early stage may have explained why those plants had no ROS burst.4. Compared with the uninfected M. sieversii plants, chitinase activity in the infectedplants increased rapidly after6dpi. For M. prunifolia, activity did not differ significantlybetween infected and uninfected plants, except at Day20, meaning that chitinase respondedearlier in M. sieversii. Relative expression followed a similar trend, with up-regulation ofchitinase transcripts occurring8d earlier in M. sieversii. Furthermore, β-1,3-glucanaseactivities and transcription levels were higher in M. sieversii than in M. prunifolia under eitherinoculated or uninoculated conditions. This demonstrated that chitinase and β-1,3-glucanasewere more effective in limiting pathogen growth and invasion in M. sieversii.5. Using M. sieversii and M. prunifolia cv. Donghongguo, we monitored theconcentrations of18types of phenolic compounds, transcripts for key enzymes in thephenylpropanoid pathway, and activities by phenylalanine ammonia lyase （PAL）, polyphenoloxidases （PPO）, and peroxidases （POD） when plants interacted with D. mali. We found gallicacid, epicatechin, and ρ-coumaric acid showed sensitivity to inoculation with D. mali. Levelsof gallic acid followed opposite trends in M. sieversii and M. prunifolia after inoculation.Compared with the uninfected M. sieversii control, infected plants of that species had asignificantly high concentration of gallic acid at4dpi, which was then maintained throughoutthe remainder of that interaction period. Infected plants of M. prunifolia had significantly lower gallic acid concentrations after4dpi than did the control for that species. In bothspecies, infection with D. mali enhanced their concentrations of epicatechin and reduced theirlevels of ρ-coumaric acid. PAL activity in M. sieversii plants was dramatically elevated after10dpi, peaking to3.14-fold higher than the control at20dpi. For M. prunifolia, PAL activitygradually increased before being decreased in infected plants.6. Up-regulation of the SA and JA pathway, along with suppression of the ET pathway,proved to be essential to resistance by M. sieversii plants. PR1and PR5were strongly inducedby D. mali only in M. sieversii, indicating that effective manipulation of the SA pathway wasessential for conferring resistance by that species. The transcription of COI1and PLD wasinduced in the incompatible interaction of M. sieversii-D. mali but not in the susceptible M.prunifolia plants, meaning that this JA pathway was also essential to the resistance by M.sieversii. However, we also found that JA-signaling acted antagonistically on SA-dependentdefenses such that, during the later stages of infection, expression of the JA markers COI1andPLD was increased while that of PR1and PR5in the SA pathway was decreased. ERF3wasinduced by D. mali in the susceptible genotype M. prunifolia but not in the resistant M.sieversii. We verified this theory by pre-treating those plants with exogenous ACC, and foundthat such an application made them even more susceptible to D. mali. By contrast, exogenousSA and JA improved the resistance of M. prunifolia plants, which was consistent with ourconclusion that the up-regulation of the SA and JA pathway, along with suppression of the ETpathway enable plants of M. sieversii to be resistant to D. mali.7. Pre-treatment with exogenous melatonin improved resistance to D. mali by susceptiblegenotype M. prunifolia. Pre-treatment enabled plants to maintain intracellular H₂O₂concentrations at steady-state levels and enhance the activities of plant defense-relatedenzymes, possibly improving disease resistance. Because melatonin is safe and beneficial toanimals and humans, exogenous pre-treatment might represent a promising cultivationstrategy to protect plants against this pathogen infection.更多还原