【作者】 肖明纲；

【导师】 王晓鸣；

【作者基本信息】 中国农业科学院 ， 植物病理， 2004， 硕士

【摘要】 玉米疯顶病是一种毁灭性的玉米病害，由大孢指疫霉(Sclerophthora macrospora (Sacc.)Thirum.,Shaw et Naras)引起，曾在世界多个国家局部地区严重发生。该病于1902年在意大利首次报道，1974年在我国山东省首次发生，近年来其危害日趋严重，已扩展到17省(自治区、直辖市)的48个县市，成为影响很多地区玉米生产的突出问题之一。国内外的有关研究均表明，来自田间发病植株上所结的种子可以传播疯顶病，但从现实的角度考虑种子传播的可能性很小。本研究根据大孢指疫霉的形态特征和细胞壁组成特点，分别从常规染色方法和分子检测技术入手，试图阐明病害大面积突发和种子带菌之间的关系。在此基础上，研究病害发病条件及玉米的抗性，探讨玉米疯顶病的综合治理方法。主要研究结果如下： 1．应用碘—氯化锌染色法、碘液—硫酸染色法、几丁质染色法以及棉蓝乳酚油染色法对不同类群真菌进行了染色。根据病菌细胞壁的显色反应，4种染料都能有效地把细胞壁主要组成成分不同的两类菌区分开，但碘—氯化锌染色法为卵菌细胞壁特异性染料，显色迅速，易于观察，能有效鉴别玉米组织中的卵菌菌丝体。利用碘—氯化锌试剂对不同类型植株来源的种子及病穗苞叶进行特异性染色，结果在种皮、胚、胚乳中都检测到了大孢指疫霉菌丝体的存在，而病穗苞叶中则没有发现菌丝体，表明病菌可以侵染种子而造成种子带菌。 2．通过卵孢子富集、DNA提取方法的筛选，建立了从卵孢子中提取大孢指疫霉DNA的有效方法。利用通用引物(ITS1和ITS4)扩增、克隆和测序了大孢指疫霉ITS区段。以具有较高保守性的ITS区段序列为模板合成了3对特异性引物SM1、SM2、SM3。利用引物SM3对大孢指疫霉进行初步的分子检测，结果表明该引物能特异性地扩增出病原菌DNA而玉米组织则无扩增，说明引物SM3对大孢指疫霉具有专一性。 3．在温室和田间，采取不同湿度处理方式，研究病田土壤、植株病残体和种子在病害传播中的作用和病害诱发条件，但未能充分诱发疯顶病的发生，表明大孢指疫霉卵孢子具有复杂的休眠机制，萌发所需要的条件苛刻。玉米疯顶病的研究仍有许多难点需要攻克。 4．由于田间病害诱发不充分，因此在品种抗性的评价、杀菌剂防治效果试验和土壤湿度控制试验方面未获得预期效果。证明了一些玉米品种和自交系属于感病类型。目前对玉米疯顶病的控制措施主要有以下两点：把采取有效控制田间土壤水分作为预防玉米疯顶病的首选技术措施，同时避免种植已知的感病品种。更多还原

【Abstract】 Crazy top caused by Sclerophthora macrospora, is a destructive maize disease, which is local economic important in many countries in the world. The first report of the disease on maize was published in Italy in 1902. Since it was first found in Shandong Province, China, in 1974, the disease expanded fastly and there were some reports from 17 provinces and 48 counties. In some maize production areas crazy top has becoming a major threat to production, such as in Ningxia and Xinjiang.Maize crazy top is a seed-borne and seed-transmitted disease. Why did the disease expand to some of provinces during a short time although there were few seeds or no seeds harvested from infected plants. Based the morphologic characteristics and cell wall composition of Sclerophthora macrospora, this research tries to make clear of the relationship between mycelia in seeds and the spread of maize crazy top using techniques of hypha special stain method and molecular marker method. Also the conditions of disease development, maize resistance and disease control measureses were studied. The main results are as follows:1. Four methods of mycelia stain were compared, including zinc-chlor-iodide, I2-H2SO4, chitin stain and cotton blue lactopheno, in Pythium inflatum, Phytophthora capsipi, Exserohilum turcicum, Bipolaris maydis and Fusarium moniliforme, which are in different taxonomic groups with celluloses or chitin in the cell wall. Results showed that using cell wall stain methods 5 fungi species can been distinguished in two group: oomycytes and true fungi. Zinc-chlor-iodide is specific to stain the mycelia of Oomycetes quickly and easily. Using the stain method of zinc-chlor-iodide the mycelia fractions in maize seeds and symptomized tissues, collected from infected plants, were detected. The pathogen mycelia fractions were found in the pericarp, endosperm and embryo of maize seeds and were not found in the plant tissues. The fact pointed that Sclerophthora macrospora can infect the seeds and is seed-borne pathogen.2. By the collection of the oospores of Sclerophthora macrospora and comparison of methods of DNA extraction, an effective procedure of DNA extraction from the oospores of Sclerophthora macrospora was made. The internal transcribed spacer (ITS) region was amplified with the general primer (ITS1 and ITS4). The DNA segment amplified was cloned and sequenced. Based on the rRNA-ITS sequence three specific primers were designed and synthesized, including SMI, SM2 and SM3. SM3 primer was specific one for amplifing DNA of Sclerophthora macrospora and no amplification of DNA of maize tissues.3. On glasshouse and in field the effects of the soil collected from crazy top field, infected plant tissues and seeds were evaluated for the disease development under different soil moisture conditions. Only few plants showed typical symptoms. The result showed that it is difficult to induce germination of oospores of Sclerophthora macrospora4. There were no good results on identification of maize resistance, and using fungicides and culture method to control the disease. For reducing the disease severe controlling soil moisture is the effective measure as well as avoiding planting the susceptible varieties.更多还原