【作者】 菅文磊；

【导师】 卢钢；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 硕士

【摘要】 芦笋（Asparagus officinalis L.）别名石刁柏，属百合科天门冬属，以嫩茎为食的雌雄异株多年生植物，是出口创汇的一种高档蔬菜。芦笋茎枯病是一种世界性分布的毁灭性病害，在中国、日本、美国、巴西、法国等20个国家发生危害，一般发病率50％～100％，病茎枯死率10％～30％，重者全田毁灭，每年造成巨大的经济损失。我国芦笋产区常常发生因本病的严重为害而造成基地成片翻耕改种或边种边毁的现象。本研究以芦笋品种美国加州UC308 F₁为材料，利用苗期接种天门冬拟茎点霉，研究营养液加硅和喷施甲壳素处理对芦笋幼苗主要抗氧化系统及抗病相关代谢酶活性的影响，及对茎枯病抗性的诱导，旨在阐明硅与甲壳素在抵御、缓解芦笋茎枯病病害中的调节机制，为芦笋茎枯病的防治提供技术依据；另外本研究还比较了不同芦笋品种愈伤再生能力，研究了烷化剂甲基磺酸乙酯（EMS）处理对芦笋愈伤组织分化的影响，为芦笋抗病突变体的筛选提供研究基础。主要结果如下：1．芦笋茎枯病菌——天门冬拟茎点霉[Phomopsis aspasagi（Sacc）Bubak]在含不同浓度NaCl、Na₂SiO₃溶液的PDA培养基中培养，结果发现NaCl处理对病菌的生长没有抑制作用；而不同浓度的Na₂SiO₃均能明显抑制病菌的生长。虽然低浓度（3 mmol／L）的Na₂SiO₃对病菌生长的抑制作用与对照相比没有显著差异，但Na₂SiO₃浓度大于6 mmol／L时，对病菌生长的抑制作用显著地高于对照，且随Na₂SiO₃浓度升高对病菌的生长抑制越明显。2．接种天门冬拟茎点霉后，施2.0 mmol／L硅处理提高了芦笋体内过氧化氢酶（CAT）、愈创木酚过氧化物（G-POD）、超氧化物歧化酶（SOD）、苯丙氨酸解氮酶（PAL）、多酚氧化酶（PPO）、几丁质酶、β-1，3-葡聚糖酶活性，极显著的降低了芦笋茎枯病发病率和发病指数，相对防治效果达39.33％，提高了芦笋对天门冬拟茎点霉的抗病能力。3．接种天门冬拟茎点霉后，在不同时期喷施甲壳素都能使芦笋体内过氧化氢酶（CAT）、愈创木酚过氧化物酶（G-POD）、超氧化物歧化酶（SOD）、多酚氧化酶（PPO）活性明显提高，但不同时期喷施对各种酶的活性影响不同，接种后马上喷施影响最显著；在接种前2 d、接种当天、接种后2 d喷施甲壳素都能显著降低了芦笋经枯病的发病率和发病指数，相对防治效果分别为39.33％、45.45％和23.68％，喷施甲壳素提高了芦笋对茎枯病的抵抗能力。4．在相同培养条件下芦笋不同品种的脱分化率及愈伤生长速度不同，品种‘硕丰’脱分化率最高。0.7％EMS对愈伤组织的致死率及褐化率显著高于0.5％EMS。相同浓度的EMS处理对不同材料的影响有显著差异，对品种‘冠军’影响极显著。培养的愈伤组织在含有激动素1 mg／ml KT的培养基上分化率最高，达61.8％；‘冠军’的分化率显著高于其它两个品种。该体系可用于芦笋抗性突变体的筛选。更多还原

【Abstract】 Asparagus （Asparagus officinalis L.） is a dioecious, herbaceous perennial species in the family Liliaceae （lily） and it is also an important vegetable for exported purpose in China. The phomopsis stem blight caused by phomopsis aspasagi（Sacc） Bubak is a destructive disease of asparagus in many parts of the world such as China, Japan, America, Brazil, France. The incidence of the disease is 50% 100%, and the rate of stem blight is 10% 30%, all of asparagus die away when the disease is very serious. Asparagus commercial fields are often made a clean sweep because of the phomopsis stem blight in china.The aims in the present study are to demonstrate the effects of Si and chitin on the growth of disease-resistance and to investigate the possible mechanism by which Si and chitin increases the tolerance to this disease in Asparagus officinalis seedlings. On the other hand, the effect of Ethyl methyl sulfonate （EMS） treatment on the proliferation and regeneration of in vitro callus from Asparagus officinalis seedling was studied using three varieties, ’UC800’, ’Guanjun’ and ’Shuofeng’, which provided the basis of screening the mutant with biotic and abiotic resistance in asparagus breeding program. The main results are presented as following:1. The PDA medium with NaCl and Na₂SiO₃ at different level was used to culture Phomopsis aspasagi（Sacc） Bubak in the experiment. The growth of mycelium wasn’t inhibited on medium containing NaCl compared with control, but that was obviously inhibited on the medium with Na₂SiO₃ when its concentration was above 6 mmol/L. Morever, the inhibition was more significant with the increase of the Na₂SiO₃ concentration in the medium.2. Hydroponic culture experiment were conducted with asparagus cultivars to investigate the effects of Si and inocultation with phomopsis aspasagi （Sacc） Bubak. The results showed that the inoculation of phomopsis aspasagi（Sacc） Bubak enhanced the activity of catalase （CAT）, guaiacol peroxidase （G-POD）, superoxide dismutase （SOD）, phenylalanine ammonia-lyase （PAL）, polyphenoloxidase （PPO）. chitinase andβ-1.3-glucanase activity, while Si supply increased the activity of CAT, SOD, G-POD, PAL, PPO, chitinase andβ-1.3-glucanase in vivo of inoculation plants. The disease index was significantly decreased by Si-supply after inoculation, and the relative against efficiency was 39.33%. Theaddition of Si improved the ability of resistance to phomopsis stem blight.3. To investigate the effects of chitin on induction of the tolerance of phomopsis stem blight in asparagus, different asparagus cultivars were used and inocultated with phomopsis aspasagi （Sacc） Bubak in seedlings. The results showed that the inoculation of phomopsis aspasagi（Sacc） Bubak enhanced the activity of catalase （CAT）, guaiacol peroxidase （G-POD）, superoxide dismutase （SOD）, polyphenoloxidase （PPO） activity, while chitin supplement increased the activity of CAT, SOD, G-POD and PPO in vivo of inoculation plants. The effect of the time supplying chitin on activity of antioxidant systema enzyme was significantly different and the change was the most obvious when supplied the chitin in 2 days after inoculation. The disease index significantly reduced with chitin-supply, and the relative against efficiency was respective 39.33%, 45.45% and 23.68%when supplied the chitin 2 days before inoculation, the day of inoculation and 2 days after inoculation. So we concluded that chitin addition improved the ability of resistance to phomopsis stem blight.4. The proliferation rate and growth rate of the callus was found to be significantly different among different genotypes, and the proliferation rate of the variety ’Shuofeng’ was the highest. The callus was obviously inhibited on the medium containing 0.5% EMS, and more significantly on the higher concentration of 0.7% EMS. The effect was depended on the genotypes, and the callus from ’Guangjun’ was most susceptible to EMS treatment among three test varieties. The regeneration rate of the in vitro callus was significantly affected by the combinations of different plant growth regulators in the media and the highest rate with 61.8% was found in percent of bulblet when cultured in the MS media containing with 1 mg·L^-1 KT. When compared with different genotypes, the variety ’Guanjun’ was most easy for induction the stem. The combination of in vitro callus culture and EMS treatment will provide a potential tool for screening the mutant with biotic and abiotic resistance in asparagus breeding program.更多还原