百合抗尖孢镰刀菌细胞突变系抗病机制的研究

Lily blight disease caused by the soil-borne pathogen Fusarium oxysporum f. sp. lilii is one of the most serious diseases in lilies. One way of controlling the disease would be the breeding and screening for resistant cultivars. It is a new method of obtaining disease-resistance material that lily clones screen by toxin of Fusarium oxysporum f.sp. lilii. We have achieved 2 mutant clones of oriental lily cultivars "Casa Blanca" and "Acapulco" by screening clones adding toxin to medium. But it is not clear to disease-resistant mechanism of mutant clones. So this study will explore disease-resistance mechanism of oriental lily cultivar "Casa Blanca" mutant clones from cytology, chromosome and protein. First, the pathogenic processes of the basal stem and tissue bulb in the oriental lily "Casa Blanca" mutant clones and normal clones were studied cell structurally following inoculation with Fusarium oxysporum f.sp. lilii and treating with toxin. Second, the peroxydase, phenylalnine ammonialyase, superoxide dismutase activity of bulblets were determined by inoculation with Fusarium oxysporum f.sp. lilii and treating with toxin. Third, we focus on proteomics as a tool for the discovery of differentially expressed proteins closely related to the disease resistance. Total proteins were extracted from bulblets sampled at 24, 48 hour after inoculation, and separated by two dimensional electrophoresis. This study will find effect of cell structure oriental lily "Casa Blanca" mutant clones to Fusarium oxysporum f.sp. lilii and toxin. The relationship between resistance to Fusarium and peroxydase, phenylalnine ammonialyase, superoxide dismutase contents in lily "Casa Blanca" mutant clones were investigated. This study will achieve pathogenesis-related(PR) proteins and reveal the resistant mechanism. These results will provide theory basis of lily disease-resistance.

百合茎腐病是百合种球和鲜切花生产中的重要土传病害，由百合尖孢镰刀菌百合专化型（Fusarium oxysporum f.sp. lilii）引起，防治困难。利用百合尖孢镰刀菌分泌的毒素对百合细胞无性系进行筛选是一条获得抗病种质的新途径。本研究利用已经获得的东方百合品种卡萨布兰卡（Casa Blanca）抗镰刀菌毒素细胞突变植株研究其抗病机制，以正常生长的组培苗为对照，应用光学显微镜、荧光显微镜及透射电镜观察经百合尖孢镰刀菌及其毒素处理后的细胞突变植株及正常植株的茎基部和鳞茎；并利用分光光度计分析细胞突变植株经百合尖孢镰刀菌及其毒素侵染后体内POD、PAL、SOD等防御酶变化趋势；同时利用蛋白双向凝胶电泳技术，对百合尖孢镰刀菌及其毒素诱导后特定时间段的细胞突变植株材料进行差异蛋白分析。揭示抗病突变株在细胞学水平、生化水平及分子水平上的抗病机理，为百合抗病育种提供理论依据。

百合茎腐病是百合种球和鲜切花生产中的重要土传病害，由百合尖孢镰刀菌百合专化型（Fusarium oxysporum f.sp. lilii）引起，防治困难。利用百合尖孢镰刀菌分泌的毒素对百合细胞无性系进行筛选是一条获得抗病种质的新途径。本研究利用已经获得的东方百合品种卡萨布兰卡（Casa Blanca）抗镰刀菌毒素细胞突变植株研究其抗病机制，以正常生长的东方百合品种卡萨布兰卡（Casa Blanca）组培苗为对照，应用光学显微镜、荧光显微镜及透射电镜观察经百合尖孢镰刀菌处理后的细胞突变植株及正常植株的根部；并利用分光光度计分析细胞突变植株经百合尖孢镰刀菌侵染后体内POD、PAL、SOD等防御酶变化趋势；同时利用蛋白双向凝胶电泳技术，对百合尖孢镰刀菌诱导后特定时间段的细胞突变植株材料进行差异蛋白分析。.本项目基本明确了百合尖孢镰刀菌对植株细胞结构的影响。感病东方百合品种卡萨布兰卡（‘Casa Blanca’）在经过尖孢镰刀菌毒素筛选后，其细胞结构发生了很大的变化。在接种前，抗病无性系细胞排列比感病无性系更紧密，木质部细胞间隙和直径更小，细胞壁加厚，这些特征可能是感病无性系在经历长期的毒素选择过程中形成的抗病性的变化，这种变化变成了抗病无性系固有的组织学特征，从而改进了细胞机械抗性，对抗病性起到十分重要的作用。在接种之后，从细胞和组织的超微结构来看，抗病无性系比感病无性系抗扩展能力更好。项目揭示了抗百合尖孢镰刀菌细胞突变植株的抗性与防御酶活性的关系。卡萨布兰卡（‘Casa Blanca’）突变植株和对照株的POD、PPO、PAL的活性均呈现先升高后降低的变化趋势。而β-1，3-葡聚糖酶则呈现逐渐升高的变化趋势。由此说明突变植株与防御酶的活性紧密相关。项目还获得了与抗百合尖孢镰刀菌相关的蛋白，主要是参与防御反应的病程相关蛋白即推定的抗病蛋白RPS2、推定的晚疫病同族蛋白R1C-3、过氧化物酶Q、抗坏血酸过氧化物酶和NBS-LRR类似蛋白等5个蛋白。. 本项目揭示了抗病突变株在细胞学水平、生化水平及分子水平上的抗病机理，为百合抗病育种提供了理论依据。