甘蔗鞭黑粉菌侵染甘蔗的早期可视化观察

Visualization of the early infection stage of Sporisorium scitamineum in sugarcane cultivars

摘要:

[目的] 甘蔗鞭黑粉菌（Sporisorium scitamineum）引起的甘蔗黑穗病是我国甘蔗生产重要的病害。示踪甘蔗鞭黑粉菌侵染甘蔗的过程将有助于揭示其致病性和甘蔗抗黑穗病机制，为抗病品种的选育以及黑穗病的防治奠定基础。[方法] 利用农杆菌介导的遗传转化技术对甘蔗鞭黑粉菌进行黄色荧光标记，对转化子进行配合及致病力检测，将标记菌株接种甘蔗感病品种ROC22及抗病品种中蔗1号、中蔗6号和中蔗9号并进行早期可视化观察。[结果] 组成型表达的eYFP不影响标记菌株的配合及致病能力，而且黄色荧光性状能通过冬孢子稳定遗传。激光共聚焦显微观察表明，注射接种病原菌第5天，在感病品种ROC22的生长点已可见荧光菌丝及少量聚集状菌丝体，在抗病品种中蔗1号、中蔗6号和中蔗9号中可见少量单一丝状菌丝，无聚集状菌丝体。接种后35 d，在ROC22中可见大量聚集状菌丝体，但在中蔗品种中的聚集状菌丝体明显较少，而以中蔗1号最少。[结论] 成功构建了甘蔗鞭黑粉菌侵染甘蔗的荧光示踪系统，并发现中蔗系列品种存在抑制甘蔗鞭黑粉菌菌丝体在细胞间扩展的机制。

Abstract:

[Objective] Sporisorium scitamineum isa pathogenic fungus that causes sugarcane smut, the most important disease of sugarcane in China. Tracing the infection progression of the disease will help to reveal the mechanism of resistance to smut and lay a foundation for the selection of resistant cultivars and the control of smut. [Methods] We labeled the basidiospores of the pathogen with a gene that encodes the enhanced yellow fluorescent protein (eYFP) by Agrobacterium tumefaciens-mediated transformation (ATMT). We tested the mating and pathogenicity of the transformants. To visualize the early stage of infection, we injected the eYFP-tagged strains into smut susceptible sugarcane cultivar ROC22 and smut resistant cultivar Zhongzhe 1, Zhongzhe 6, Zhongzhe 9. [Results] The eYFP-tagged strains were indistinguishable to the wild-type strain in mating and pathogenicity, and the fluorescence was able to inherit into the teliospores. Laser confocal microscopic examination showed that at 5 days post inoculation, a few aggregated as well as separated mycelia were observed in the susceptible sugarcane cultivar ROC22, but only a small amount of single mycelium was found at the same time in the resistant Zhongzhe cultivars. Much more aggregated mycelia were found at 35 days post infection in ROC22 than in Zhongzhe cultivars, with the lowest number in Zhongzhe 1. [Conclusion] A fluorescent tracer system for sugarcane infection by S. scitamineum was successfully constructed, and there may be a mechanism to constrain the mycelial growth of S. scitamineum in Zhongzhe cultivars.