根肿病菌生理小种的鉴别寄主及分子鉴定研究

Clubroot is a serious disease in cruciferous crops，forming a strong threat to the production of cruciferous vegetables and oil rape in China. There are differentiations in pathogenicity in Plasmodiophora brassicae. In China, the composition and distribution of physiological races in P. brassicae have not been clear. These situations restrict the effective control of the disease. Two identification systems with different differential hosts for identification of the physiological races of P. brassicae are applied at home and abroad, including Williams and ECD. But the two systems have their own limitations. They are more suitable in Europe than in Asia. Additionally, the hosts in the two systems are well adapted to the European conditions. In the southern China they grow slowly and are difficult to bear seed due to their vernalization and daylength reactions. The aim of the present study is to construct a suitable host system and a molecular identification system for the identification of physiological races in P. brassicae, using the innovative single spore isolates and in vivo subculture propagation technology. The inoculation methods, inoculant concentration and grading standards will be studied, and technical regulations will be formulated for the identification of P. brassicae physiological races, in order to study the composition, the distribution and the prevailing physiological races of P. brassicae in China. The results from this study will provide a theoretical basis for breeding and rational use of resistant varieties against clubroot. It has important practical significance in guidance of the integrated prevention of clubroot.

根肿病是我国十字花科作物上的严重病害，对我国十字花科蔬菜和油料作物油菜的生产构成严重威胁。根肿菌具致病性分化，我国根肿菌生理小种组成及分布尚不明确，这一现状制约了根肿病有效控制。目前国内外应用的根肿菌生理小种鉴别寄主体系有Williams和ECD，但两套鉴别体系也有其自身的局限性，它们较适于欧洲根肿菌生理小种的鉴定，而对亚洲根肿菌生理小种鉴别力较差。此外，由于两个鉴别系统的寄主属欧洲品种，在我国南方因它们在春化和光照反应上的特点，生长缓慢，且难以结实。本研究以构建适于我国的根肿菌生理小种鉴别寄主体系为目标，采用改良的单孢分离、寄主继代扩繁技术，通过研究接种方法、浓度和分级标准，制订出我国的根肿菌生理小种鉴别寄主体系的鉴定技术规程，探明我国根肿病菌生理小种组成、分布和优势小种。本研究成果对我国根肿病的抗病育种和抗病品种的合理利用提供理论依据，对指导根肿病的综合防治具有实践意义。

根肿病是十字花科作物上的重要病害。芸薹根肿菌具有生理小种的分化。目前，国内外通用的生理小种鉴别系统主要有Williams（1965）和ECD（1996）两个鉴别系统。这两个鉴别系统的不足是：在非欧洲国家种植生长势弱；对非欧洲地区根肿菌鉴别力较差；鉴定结果不太稳定。本研究以构建中国根肿菌生理小种鉴别系统和分子鉴定为内容和目标，对我国乃至全球根肿菌生理小种的鉴定及在此基础上的抗病育种和品种的科学利用均具有重要意义。通过两年的研究，取得了以下重要成果：.1.中国根肿菌生理小种鉴别系统的构建. ⑴ 白与黑鉴别系统 十字花科作物既有质量性状遗传，又有数量性状遗传。白与黑鉴别系统区别于Williams和ECD（1996）鉴别系统以数量遗传为基础，以病情指数（DI）“量”的差异划分抗、感，创新以质量性状遗传为基础，即以不发病与发病“质”的差异划分抗、感（白与黑）。该鉴别系统的特点为：① 鉴别寄主数适中，由4个品种构成，理论上能鉴定出16个生理小种；② 4个鉴别寄主均来自国内，适应性好。③ 鉴别力强，不仅能区别国内不同地区来源的菌株，还能进一步区分Williams4号小种。④ 小种命名易理解，与国际上通用的Williams生理小种命名一致。 . ⑵ 阈值鉴别系统 该系统由3个鉴别寄主构成，以病情指数“量”的差异划分抗、感。将DI＜30的为抗病，DI≧30的为感病。其主要特点同Williams鉴别系统。.2. 建立根肿菌生理小种鉴定方法.建立了有系统、规范和适用，又与创新构建的白与黑和阈值鉴别系统相配套使用特点的生理小种鉴定方法。.3.单孢系的建立. 建立了从单孢的分离、接种和培养及继代扩繁的寄主、播种、接种、人工气候箱中培养全过程的方法，并申报了国家发明专利。.4. 根肿菌遗传多样性分析、小种特异性分子标记.应用ISSR分子标记，从100对引物中筛选出827、890两对扩增条带清晰、多态性明显、重复性好的引物，对国内10省的34个根肿菌样进行ISSR-PCR扩增、聚类分析。827引物能将34个菌株分为13类群。这为进一步设计特异性分子标记，分子鉴定生理小种奠定了基础。