铁皮石斛核心共生微生物类群确定及其对驯化栽培宿主抗病性与药效的影响

The prevalence of type II diabetes is growing at a distressing rate globally and type II diabetes is becoming one of the most serious disease that threaten human’s lives. Dendrobium officinale, which has the effects of "tonifying stomach and promoting fluid" and “nourishing yin and clearing heat”, has been used to treat diabetes in China for hundreds of years. However, wild Dendrobium officinale has very low natural fertility and growth rate, while artificial cultivation of Dendrobium officinale is facing variety degeneration and constant fungous and bacterial infections. Microbiome is an indispensible factor for the growth and development of Dendrobium officinale. But the relationship between core microbiome and disease resistance or drug potency of Dendrobium officinale remains unknown. The current research will employ high-throughput sequencing technique to explore core microbiome of Dendrobium officinale, and employ infinite dilution technique combined with CARD-FISH to culture its core bacteria and fungi. The research will also screen the bacteria or fungi that could promote growth, increase disease resistance and drug potency of Dendrobium officinale by inoculating them into seedlings that cultured by Dendrobium officinale tissue. The result of present study is benefit for protecting the wild resource of Dendrobium officinale, and guiding to resolve the problems of domestication of other traditional Chinese medicine plant. Meanwhile, it is help for revealing driving factor of construction of orchid-microbiome and interaction of microbiome and host plant physiologically.

Ⅱ型糖尿病发病率在发展中国家呈现流行势态，已成为继心血管病和肿瘤之后严重威胁人们健康的非传染性疾病。铁皮石斛具有益胃生津、滋阴清热的功效，自古就是中医治疗糖尿病的必用中药。野生铁皮石斛的自然繁殖能力低、生长缓慢，被列为国家重点二级保护植物，禁止采摘。人工栽培铁皮石斛面临品种退化与微生物病害防治难题。石斛微生物是其生长发育不可或缺的重要生物因子，但由于研究技术限制，目前对石斛核心微生物及其对石斛药效与抗病性的影响未见研究。本项目拟用高通量测序技术分析石斛核心微生物类群，并在类群信息指导下，采用CARD-FISH结合无限稀释技术培养石斛核心细菌与真菌，进一步通过接种石斛组培苗筛选促进生长、提高抗病能力与提高石斛药效的菌株。研究结果不仅可以保护濒危野生铁皮石斛资源，也可指导解决其他中药植物驯化面临的问题；同时在生理学上可以揭示兰科植物微生物组形成的驱动因素以及宿主与微生物的互作机制。

石斛微生物是其生长中必不可少的生物因子，尤其是植物长期选择和适应的、稳定的核心微生物群，目前对石斛核心微生物及其对石斛生长与抗病性的影响未见研究。本项目采用高通量测序技术等方法分析铁皮石斛共生微生物时空动态变化，发现了生长期、生长方式、组织部位对内生菌类群结构形成的驱动影响，确定了Burkholderiaceae、 Enterobacteriaceae、Bacillaceae 、Paenibacillaceae是石斛中丰度较高的核心细菌类群，Aspergillaceae、Nectriaceae、Cladosporiac、Rhynchogastremataceae 、Cortinariaceae是石斛中丰度较高的核心真菌类群，从核心微生物的角度揭示宿主植物的生理需求及植物与微生物互作机制。在此基础上确定分离得到核心类群菌株，进行活性筛选后获得一批具有生防和促生活性的菌株，可以作为开发微生物菌剂应用于人工仿生环境铁皮石斛种植的良好备选。最后将活性核心类群菌株回接组培苗验证核心微生物对宿主植物组培苗生长具有一定有益的作用。研究结果有利于高效栽培铁皮石斛满足市场需求，从而保护野生铁皮石斛种质资源，对于其他中药植物的驯化与栽培也有重要参考意义。