稻曲病菌菌核分化形成相关基因的筛选与功能研究

As extension of high yield rice cultivars with large and compact panicles and long growth periods in large scale in the past decades, the occurrance of rice false smut has become more and more frequent and extended to south China. Now it has become one of the most important rice diseases in the major rice-growing regions of China. It has been certified that the pathogen sclerotia were found to be common and abundant in the late-ripening rice both in north and south China in recent years, and they can over-winter successfully and germinate to produce great amount ascospores as the primary infection inocula in the coming year, covering the main growth periods of rice. This implys that control the sclerotia formation and their germination in the coming year will be the critical targets in the disease management. We found the low temperature during rice filling stage is one of the important factors to induce the fungus producing sclerotia, and treatment with low temperature at 15ºC for 3 nights are enough to induce the sclerotia differentiation at the early developmental stage of rice false smut balls in laboratory. In this project the sclerotia differentiation and formation related genes will be selected primarily based on the transcriptomic analysis of various sclerotium-containing balls at different developmental stages by means of our sclerotia-induced protocol. The gene knockout and complementation experiments will be carried out to confirm their functions in the sclerotia differentiation and formation. Finally molecularly cytological methods will be employed to reveal the mechanism of these genes in sclerotia formation. The identification and biofunctions of sclerotia induction and formation related genes will favor to make novel strategy on the disease control.

随着高产、密穗、长生育期水稻品种的大范围推广，稻曲病的发生呈现不断加重和向南方蔓延的趋势，已成为我国水稻最为重要的病害之一。近年来最新的研究表明，菌核可在我国各主要稻区的晚熟水稻上大量产生，并能够在田间越冬和作为来年初侵染源。因此菌核及其控制技术的研究成为稻曲病防控的焦点问题。本课题组研究表明，水稻灌浆期初期低温是诱导稻曲病菌菌核分化的主要环境因子，连续3个夜间低温(15ºC)就足以满足诱导菌核的分化形成；并在此基础上结合光照条件建立了高效、重复性良好的稻曲病菌室内菌核诱导方法。本项目拟借助低温菌核诱导技术，对菌核分化与形成不同阶段的稻曲球进行转录组分析，初筛出与菌核分化形成相关的重要基因，然后采用基因敲除与补回试验分析和验证这些基因与菌核分化形成的相关性，并进一步利用分子细胞学技术对其作用机制做进一步分析。该项目的目标是揭示稻曲病菌核分化的分子机制，为开发新的防控策略提供科学依据。

稻曲病是目前我国最为严重的水稻真菌病害。本项目研究发现，成熟的稻曲病菌菌核可安全越冬，并经过4-5个月的休眠后陆续开始萌发，产生子囊孢子和进一步产生分生孢子，并持续2-4个月产生子实体的能力，因此菌核成为稻曲病菌的主要初侵染源。稻曲病菌的厚垣孢子虽然数量庞大，但多种模拟试验中未发现能够越冬后萌发并产生分生孢子。因此，菌核是稻曲病菌的主要越冬形式和来年病害发生的主要初侵染源，也是稻曲病防控预警的前提和基础。稻曲球发育中早期的相对低温是菌核形成的重要条件，本项目成功建立了菌核诱导率为67%的稻曲球接种试验，并在此基础上进行了菌核形成过程的转录组学分析，结果发现低温处理可诱导793 个基因显著差异表达，涉及多种代谢途径。此外本项目还对稻曲病菌营养生长中的感光基因进行了转录组水平的分析。两个转录组对比后筛选出了40多个候选的菌核诱导相关基因。首先从中筛选了14个本底表达水平较低、诱导上调幅度较大的基因进行功能研究，其中evA基因敲除后，菌株无法完成侵染，而对照的菌核率高达66.7%。基因回补试验后，菌株恢复原有的致病性和致病力，且菌核率达到了29.6%。说明该基因对菌核形成有重要作用。对另外13个候选基因的功能研究尚在进行中。此外，针对稻曲病菌菌核形成时间周期过长的问题，通过对83个菌株的筛选，获得了13个可低温诱导产生拟菌核的菌株，不同菌株的菌核诱导时间为50-80天。田间试验表明其在正常双季晚稻上菌核形成率达到了54.3-79.5%。这些菌株可为室内菌核诱导试验提供了快速的替代方法。针对菌核形成过程的研究表明，菌核约在接种后15-20天即稻曲球破膜后5-10天开始形成，并在可见隐含菌核后7-10天成熟。