疯草内生真菌分生孢子水平传播机理研究

Locoweed is the main toxic plants on grassland. Livestock would be poisoned or killed. The annual loss caused by locoweed was more than 2 billion RMB in China. Sainsonine is the main toxic substance, and their endophyte was the the key reason of the toxicity of locoweed. Up to data, they were isolated from twenty locoweed species or varieties, and assigned as a same fungal species (Undifilum oxytropis), which 8 locoweed species from China. The strategies for pest management are mainly based on their occurrence dynamics. However, locoweed and locoweed endophyte can not control effectively since the dynamics of endophyte has not been studied intensively. Transmission is the most difficult in the dynamics. Some studies reported locoweed endophyte was transmitted by seeds only. Nevertheless, it can not explain why they exist in different locoweed all of world, and they were diversity in ecological, morphological, physiological and genetical aspects. The applicant of this project found that locoweed endophyte can transmit horizontally. Based on the finding, a hypothesis is presented: the diversity of locoweed endophyte possibly has some association with horizontal transmission of endophyte. This project is going to study transmission mechanism of locoweed endophyte from 8 locoweed from China, including the sproulation capacity, infection ability, and host range, to study the effect of horizontal transmission on the diversity, with the methods of field investigation, artifical inoculation, isolation, PCR detection, hybrid between isolates, gene sequencing. The objective of this project is to determine the geographical area, conditions, dynamics, and to provide information to manage locoweed endophyte.

疯草是天然草地主要毒草，家畜误食导致中毒或死亡，我国因之每年损失二十多亿。疯草的主要毒性为苦马豆素，而疯草内生真菌是此类疯草毒性的主要原因，已发现于国外20种和我国8种疯草，均属于同一种真菌，存在丰富的形态、生理、生态和遗传多样性。然而，由于对其发生规律缺乏深入研究，目前尚无法防除。发生规律中最大的难点为传播环节，已报道其仅通过种子垂直传播，但该观点与疯草内生真菌已分布于不同地区不同疯草体内的现实存在矛盾。本项目申请人近期研究发现其既可在植株上产孢，又可发生侵染，故存在水平传播，并提出假设：其多样性形成的原因可能与水平传播有关。本项目拟采用田间调查、室内接种、分离培养、菌株杂交、PCR检测、基因测序等方法，研究我国8种疯草中内生真菌的产孢能力、侵染能力、寄主范围等水平传播机理，以及水平传播对其种内多样性的影响，旨在阐明疯草内生真菌在我国的水平传播地区、条件和规律，为有效防治提供理论依据。

本项目分离得到了采集自西藏、新疆、内蒙、甘肃、青海等5省区甘肃棘豆、黄花棘豆、小花棘豆、镰形棘豆、毛瓣棘豆、变异黄芪、茎直黄芪的内生真菌，测定了ITS、gpd、钙调素蛋白编码基因、肌动蛋白编码基因、几丁质合成酶编码基因等序列，通过构建系统发育树分析了其亲缘关系，显微观察了植株表面的疯草内生真菌的分生孢子，利用离体叶片接种了分生孢子，比较了不同疯草菌株的互作，分析了疯草内生真菌在植株体内的分布规律。首次发现镰形棘豆的内生真菌，确定我国的疯草内生真菌和与美国的蓝伯棘豆和绢毛棘豆内生真菌均属于棘豆链格孢(Alternaria oxytropis)，可与已报道的其他疯草内生真菌(3个种：A. cinereum、A. bornmuelleri和A. fulvum，均分布于美国)明显分开，但分属于不同亚群，我国的疯草内生真菌可分为3个类群：毛瓣棘豆的内生真菌、甘肃棘豆-黄花棘豆的内生真菌和变异黄芪-茎直黄芪-小花棘豆-镰荚棘豆的内生真菌）；发现疯草内生真菌在植株体内分布不均匀，在同一植株上大部分枝条及其种子带菌的情况下也有少量枝条及其种子不带菌，在所有供试疯草叶片表面均检出内生真菌的分生孢子，且分生孢子可侵染叶片，最终证明了其可通过分生孢子水平传播。本研究颠覆了长期认为疯草内生真菌仅通过种子垂直传播的认识，在体内分布的不均匀特性的发现有力证明了植株之间苦马豆素含量差异较大的原因等悬念，为将来根据其水平传播的途径设计防治措施，继而减低疯草的毒性提供了理论依据。