【作者】 曹志华；

【导师】 束庆龙； 曹翠萍；

【作者基本信息】 安徽农业大学 ， 森林保护， 2012， 博士

【摘要】 油茶（Camellia oleifera Abel.）是中国南方地区重要的木本油料树种，近年来被安面积种植。油茶炭疽病（Colletotrichum gloeosporioides Penz.）是油茶最主要的病害，发生普遍而严重，一般落果率在30%-50%之间，甚至50%以上。但据长期观察发现：生长在同一油茶林分内，不同油茶植株间抗性差异很安。本论文研究的目的是：1.在室内外病情调查研究的基础上，筛选不同抗病等级的植株作为测试对象；2.在生长季节，对不同抗性植株进行相关生理生化参数的测定，并分析其各物质的含量及变化趋势；3.对不同抗病的油茶品种进行亲缘关系及多样性分析，比较其不同抗性品种在分子水平上的差异；4.采用高分辨液质联用分析法，找出不同抗病品种的差异性物质。具体研究内容如下：1.在植物生长时期，定期对不同抗性品种的果皮内含物进行测定分析，结果表明：果皮中单宁含量呈下降趋势（高抗0.50-0.32，中抗0.46-0.24，高感0.48-0.45），但不同抗性品种之间单宁含量差异不显著（P=0.6597>0.05）。与单宁相反，花青素含量在生长季节则呈现上升趋势，如高抗、中抗、高感品种的果皮花青素含量分别增长了77.3%、69.4%和51.5%，至8月份以后合成速率减慢。不同抗性品种的花青素含量差异达到极显著水平（p=0.0063<0.01），尤其在5月份，高抗品种花青素含量的增长速率高出高感品种12.1%。2.在5-9月间，油茶抗病品种和感病品种间果皮的pH值差异达到显著水平（p=0.0278），尤其在7月份，感病品种pH值均在5以上，但抗病品种的pH值则稳定在4-5之间，抗病品种pH值明显低于感病品种。缓冲液容量随着果实的成熟度增加呈上升的趋势（高抗6.2-7.43，中抗5.33-7.46，高感5.45-7.12）。各油茶品种缓冲容量在6-7月份增长较快，但不同抗病品种间的差异不显著（p=0.7984）。3.三类抗病品种的油茶果实酶活性测定表明：抗病品种PPO、POD和PAL活性随果实成熟度的增加均有升高的趋势。在三种酶中：（1）PPO活性增长的速度较感病品种快，尤其在5月份，抗病品种PPO酶活比感病品种高出18U·g^-1min^-1。感病品种初期也呈上升趋势，但从7月份开始，PPO酶活呈现下降趋势，至9月份下降了14U·g^-1min^-1。不同抗病品种的PPO酶活之间差异极显著（P=0.0001）。（2）POD活性增长速率也是高抗品种快于感病品种，在5月份两者相差达6.2%。中抗、高感品种的酶活在8月份以后均呈下降趋势，酶活分别下降了10、17U·g^-1min^-1，可能由于细胞代谢毒素增加，导致保护酶的功能逐渐下降。各抗性品种之间PPO活性差异极显著（p=0.0001）。（3）在5月份，高抗品种的PAL活性分别比中抗、感病品种提高了17%和30%；7月份，感病品种酶活逐步下降，而高抗和中抗品种仍维持上升趋势。不同油茶品种PAL活性之间的差异达到显著水平（p=0.0004）。4.油茶果实的可溶性糖含量从幼果期到成熟期总体呈递增趋势。在5-9月份，三类不同抗性的油茶品种可溶性糖含量分别增长了60.9%、58.6%和40%，且5月份高抗品种可溶性糖含量比感病品种高45%。各油茶品种之间的可溶性糖含量差异达到显著水平（p=0.0171）。5.不同发病植株的果皮提取物对自由基清除率之间的差异达到显著水平（p=0.0032）。果皮提取液在2.0mg/mL时对自由基清除率均很高，但高抗品种更加明显，加入样品20min后的清除率最低为（89.43±0.34）%，最高达到（91.46±0.32）%，显著高于中抗和感病品种。6.本文共采集30个样株，分别来自于皖南黄山、皖西舒城和皖东凤阳等地选育的抗病性较强的油茶良种以及感病植株（对照）。采用AFLP分子标记方法对其进行亲缘关系及遗传多样性分析，筛选出一对多态性较高的引物进行AFLP分析，产生了147个扩增带，其中，129（87.76%）条是多态性条带，有效等位基因数ne为1.37，Nei’s期望杂合度He为0.23，Shannon多态性信息指数I为0.36。采用UPGMA方法，对30个油茶样株建成的树枝状图，其遗传相似系数介于0.40到0.85之间，平均相似系数为0.63。在62.0%GS处，30个油茶品种被划分为6类。Cluster A只包含感病的安州小青；Cluster C1只包含抗病的安州小红；cluster C2只包含抗病的安州安红，且C1和C2在57%GS处聚类在一起（B1）；Cluster D1只包含感病的舒城品种-重感安青；Cluster D2包含了25个品种，在65%GS处，Cluster D2又可以分为2个类群，即E1：包含14个品种和E2包含11个品种（感病和抗病品种个数分别为5和6）。7.通过高分辨液质联用分析法对高抗的攸县油茶、抗病的安州安红以及感病的安州小青叶片的甲醇提取物进行了成分分析。高抗的攸县油茶叶片中阴离子共有25种化合物，阳离子21种化合物，攸县油茶叶片中化合物安多是酚类、黄酮类、生物碱类、氨基酸以及具有抗菌特性的物质，如（2S,3R）-2-氨基十四碳-3-醇（Xestoaminol C）等；安州安红叶片中阴离子有30种化合物，阴离子以黄酮类及酚类物质为主，也有莽草酸等次生代谢物质；阳离子有30种化合物，以生物碱类、氨基酸、酰胺类物质等为主；安州小青阴离子化合物有18种，有少许的黄酮类和酚类物质，如莽草酸、绿原酸等为主；阳离子有28种化合物，以生物碱类和氨基酸等为主。更多还原

【Abstract】 Camellia oleifera Abel. is an important edible oil tree species from Southern China.Anthracnose, caused by Colletotrichum gloeosporioides （Penz.）, is responsible for morethan50%of C. oleifera production loss, and C. oleifera varieties differ in their resistanceto anthracnose.1. The aim of this study was to Assess resistance mechanisms bymonitoring physiological and biochemical parameters of differentially resistant cultivarsduring the development of C. oleifera;2. Genetic relationship and diversity amongdifferent disease-resistant Camellia oleifera varieties were analyzed to further distinguishbetween varieties at the molecular level;3. The key bioactive materials which wererelavant with resistance were deterimined and studied by HPLC-DAD-MS. The specificresearch was as follows:1. C. oleifera fruit coats were analyzed between May and September for tannins,anthocyanins. Tannin contents generally declined at the developmental stage （highlyresistant0.50-0.32, medium resistant0.46-0.24, highly susceptible0.48-0.45）, whereas nosignificant difference was found in tannin levels between differentially disease-resistantcultivars （P>0.05）. The growth rate of highly resistant varieties was12.1%higher than ofsusceptible ones in May. On the contrary, the anthocyanin contents of fruit coats in highlyresistant, medium resistant and highly susceptible varieties grew by77.3%,69.4%and51.5%, respectively, and the synthesis rate of anthocyanins changed mildly after August.The anthocyanin contents differed significantly among different cultivars （P <0.01）.2. There was no significant difference in pH levels between the disease-resistantvarieties and the susceptible ones at the developmental stage （P <0.05）. In July, fruit coats insusceptible varieties reached a pH value of5, while resistant varieties had a pH of4-5. Thebuffer capacity of different cultivars generally rose with increasing maturation （highlyresistant6.2-7.43, medium resistant5.33-7.46, highly susceptible5.45-7.12）. In addition, thebuffer capacity of different cultivars increased rapidly from June to August. Buffer capacitydid not differ significantly between different cultivars （P>0.05）.3. PPO, POD and PAL activity in the fruit coats of highly resistant, medium resistantand susceptible strains were determined. The results demonstrate that activity of the three enzymes generally rose with increasing maturity.（1） PPO activity in highly resistant plants increased more rapidly during May-Augustand increased slowly after September, especially in May. PPO activity was18U·g^-1min^-1higher than in highly susceptible plants. PPO activity of medium resistant and highlysusceptible cultivars rose slowly in the first three months, and then declined in August andSeptember. PPO activity of highly susceptible varieties decreased by14U·g^-1min^-1from Julyto September. PPO activity differed significantly between disease-resistant and susceptiblevarieties （P <0.01）.（2） POD activity of disease-resistant varieties was higher than of susceptible ones, andthe increase rate of highly resistant varieties was6.2%higher than in highly susceptiblevarieties in May. POD activities of medium resistant and highly susceptible varietiesdeclined after August by10U·g^-1min^-1and17U·g^-1min^-1, respectively. There wassignificant difference in PPO activity between disease-resistant and susceptible varieties （P=0.0001）.（3） In May, PAL activity of high resistant cultivars was higher than that of mediumresistant and high susceptible by17%and30%, respectively. In July, PAL activity ofsusceptible cultivars declined gradually, whereas that of high resistant and mediumresistant cultivars still increased. PAL activity was significantly different betweendisease-resistant and susceptible cultivars （P=0.0004）.4. Soluble sugar contents of fruit coats gradually increased from the fruitlet period tomaturity period. From May to September, the soluble sugar contents in highly resistant,medium resistant and highly susceptible varieties rose by60.9%,58.6%and40%,respectively. Soluble sugar contents of highly resistant varieties were45%higher than ofhighly susceptible ones. Soluble sugar contents differed significantly betweendisease-resistant and susceptible varieties （P <0.05）.5. The free radical scavenging capacities of fruit coat extracts from highly resistant,medium resistant and highly susceptible varieties of C. oleifera were determined. The resultsdemonstrate that fruit coat extracts with a concentration of2.0mg/mL had strong freeradical-scavenging capacity in different incidence cultivars, and that fruit coat extracts from highly resistant varieties had the highest scavenging capacity with a value of91.46%±0.32%.The free radical scavenging capacity of fruit coat extracts differed significantlybetween different cultivars （P <0.05）.6. Colletotrichum gloeosporioides （anthracnose） is the most destructive disease whichcauses severe economic losses in Camellia spp.. A study was conducted to establish ifgenotypes identified as resistant or susceptible to anthracnose in China could bedistinguished using molecular markers. A total of30unrelated C. spp. genotypes wereselected from three ecotype regions （Huangshan, Shucheng and Fengyang） in Anhuiprovince. Resistant selection was based on disease severity in plants following detachedfruit inoculation. One AFLP selective primer combinations were used to genotype theseaccessions, resulting in147amplified bands. Of these,129（87.76%） informativepolymorphic bands were used for genetic diversity analysis. Genetic similarity coefficientsranged from0.40to0.85among the resistant accessions, indicating high genetic diversityamong them. Cluster analysis grouped the30accessions into two major clusters based onpolymorphic bands. This study provides genetic diversity for future breeding of C. spp. foranthracnose resistance.7. Methanol and mixed solvent of choroform and ethyl acetate extracts from leaves ofC.yuhsiensis Hu of high resistant cultivar, Huizhou-dahong of resistant variety andHuizhou-xiaoqing of susceptible variety were analyzed with HPLC-DAD-MS.C.yuhsiensis leaf was rich in25negative compounds and21positive compounds. Thesecompounds mostly were phenolics, flavonoids, alkaloids, amino acid and someantibacterial properties, e.g. the （2S,3R）-2-amino fourteen carbon-3-alcohol（Xestoaminol C）.30negative compounds in Huizhou-dahong leaf were mostly flavonoidsand phenolic substances. In addition, there were secondary metabolites, e.g. shikimic acid;30positive compounds in Huizhou-dahong leaf were mostly alkaloids, amino acids, andamides etc.18negative compounds in Huizhou-xiaoqing leaf were a few flavonoids andphenolics, e.g. shikimic acid, chlorogenic acid;28positive in Huizhou-xiaoqing leaf weremostly alkaloids and amino acid.更多还原