西红花内生真菌对宿主种球退化、耐盐性与药材品质的影响及栽培应用

The dry stigma of Crocus sativus L. is a kind of unique Chinese medicinal. The bottle-neck of its industrialization are the degeneration and low propagation coefficient of seed corms caused by pathogens. So enhancing the pathogen resistance and reproductive capacity is an important way of increasing both the quantity and quality of C. sativus. Previous studies have found that the population distribution of endophytic fungi in C. sativus is specific. Some isolates can promote the growth of hostplant under drying stress and accumulate the active components in suspension cells of C. sativus. In this project, we plan: (1) to study the endophytes population structure of C. sativus through the application of molecular ecology technology and screen out the target bacteria which is closely related to the quality of C. sativus; (2) to build a hairy root culture system of C. sativus, study the impact of fungi on the growth and secondary metabolism of their host plant, analyze the role of active strains on the accumulation laws of metabolic intermediates of host plant and gene expression pattern of the key enzyme by using metabonomics and RT-PCR technique respectively; (3) to explore the possible mechanisms for the plant growth and drought resistance promoted by targeting bacteria through the application of the model plant Arabidopsis thaliana; (4) to investigate the effects of the target bacteria on the quality of the cultivated C. sativus in the artificial climate chamber and the experimental field. This project would enrich the research of endophyte fungi resources and functions in extreme environment of our country, for the sake of the improvement of the quality of Chinese medicinal materials by applying endophytic fungi, thus exploring a new way to realize the large-scale cultivation of Arabidopsis thaliana.

西红花Crocus sativus 的干燥柱头为传统名贵药材。制约西红花产业发展的瓶颈为种球退化和繁殖系数。提高其抗病性和繁殖能力是提升药材产量和品质的重要途径；前期研究发现，西红花内生真菌的分布规律存在特异性，且某些菌株能促进盐胁迫下宿主西红花的生长、拮抗病原菌以及促进西红花药效成分积累。本项目计划：（1）应用分子生态学技术研究西红花内生真菌的种群结构，筛选促生长、增抗性的目标菌株；（2）构建西红花组培与真菌共培养体系，考察目标菌对其生长和次生代谢的影响；应用代谢组学和RT-PCR分析目标菌影响宿主次生代谢及关键酶基因的表达规律；（3）应用模式植物拟南芥探讨目标菌促进植物生长、抗性的可能机制；（4）在人工气候室和试验田内考察目标菌对西红花药材品质的影响。期望通过本研究丰富西红花内生菌资源与功能，为应用内生真菌提高中药材品质，形成西红花优质种球的繁育体系探索一条新的途径。

西红花Crocus sativus L.的干燥柱头为传统名贵药材，制约西红花产业发展的瓶颈为种球退化和繁殖系数。为此，我们首先完成了西红花种质资源收集、生长性状、药材品质与抗病性评价，建立了西红花内生真菌与根际微生物菌种资源库，阐明了内生真菌菌群结构与药材品质的关系，发现当粘帚霉属Clonostachys sp.、木霉属Trichoderma sp.真菌为高丰度优势菌群时，西红花中西红花苷含量较高，根际土真菌Tetrapisispora sp.，细菌Methylosulfonomonas sp.、Sharpea sp.高丰度存在时，西红花种球具有较强抗病性；其次，筛选得到一株蓝状菌属内生真菌CS04（Talaromyces sp.）对盐胁迫环境下西红花无菌苗具有较好的促生长作用，并发现PPF（糖蛋白）部位为CS04促生作用的主要活性因子，并发现CS04-PPF主要通过上调西红花光合作用途径关键基因NAD-ME，GLK2，PHYB，PPDK，PPT2与RBCS的表达，提高光合作用速率，从而促进西红花生长；最后，以内生真菌CS04为主要原料菌，研制了西红花CS04生物菌肥，应用于大田西红花栽培，经CS04生物菌肥处理的西红花植株，其柱头产量相比对照提高14.70%、球茎产量提高29.08%，西红花总苷含量提高31.80%。课题的顺利实施丰富了西红花内生真菌资源与功能，为中药材生物菌肥应用推广及西红花产业发展提供了重要参考依据。