三七连作障碍根际土壤微生物群落演替研究

Wenshan, the main cultivation region of Panax notoginseng, located in the southeast of Yunnan province, is short of cultivable land resource due to succession cropping obstacle(8-12years) caused by the cultivation of Panax notoginseng. Therefor, elimination of succession cropping obstacle is the key for sustainable development of the Panax notoginseng industry. Studies have revealed that the formation of succession cropping obstacle is involved with the activities of rhizospheric microorganisms of Panax notoginseng during planting season. However, our understanding about this is still limited. In this study, the total RNA and DNA are extracted from the microorganisms in the rhizosphere of Panax notoginseng during planting season and in the soil after planting season. The total and pathogenic microbial communities of the key formation time of succession cropping obstacle will be analyzed by the methods of real-time PCR, sequencing of bacterial 16S rDNA V3 region, fungal 18S rDNA and rRNA transcriptome. Finally, metagenome sequencing will be further used to construct the metabolism network related to the formation of succession cropping obstacle. Systematic studies of the composition of the microbial communities in the rhizosphere of Panax notoginseng and their succession during succession cropping will contribute to reveal the advantages of rhizospheric microorganisms and the succession of pathogenic microorganisms, which will provide scientific and practical bases for the recover of the composition of the microbial communities to inhibit the proliferation of pathogenic microorganisms and eliminate the succession cropping obstacle of Panax notoginseng.

在三七的主产区-云南文山，由于连作障碍（8-12年）的原因已致该地区宜种土地资源极度匮乏，因此如何消除三七的连作障碍成为三七产业可持续发展的关键。已有研究表明，三七连作障碍的形成与其种植期根际微生物的活动密切相关，但我们至今仍缺乏对它们的全面了解，本研究拟以三七栽培期根际及耕后土壤中微生物的总RNA和DNA为材料，通过逆转录实时荧光定量PCR法、细菌16S rDNA V3高变区测序法、真菌18S rDNA测序法、转录组rRNA测序法对三七连作障碍形成关键时间点的致病微生物、总微生物群落进行检测；最后利用宏基因组测序法构建与三七障碍形成可能相关的代谢网络。通过系统研究三七连作根际土壤中微生物群落组成情况，揭示它们的演替规律；明确三七连作中根际微生物中的优势、病原菌的演替规律；为从微生物生态角度恢复原有微生物群落组成，进而抑制和控制病原菌的增殖，为有效消除三七连作障碍提供科学与实践依据。

三七栽培存在着严重的连作障碍问题，一般种植后的土地需要经过8年以上的时间才能再种植，严重制约了我国三七产业的健康发展。本研究采用免培养法，针对三七连作障碍形成过程（三七种植及恢复栽培）根内生、根际土壤及三七采收后土壤微生物群落中各种群如何变化、发展和演替的规律进行了系统研究。在三七的主产区-云南文山选取栽种一年、二年、三年生的三七根、根际土壤与采收后二年与六年的样品分别提取总RNA和总DNA进行分析，结果表明种植与撂荒时间对根际土壤与根内生细菌、真菌多样性的影响十分显著。论文也研究了二年生、三年生根腐病的三七根或根际土壤中细菌与真菌的群落组成，结果表明在DNA水平上，健康植株根际土壤与根内生细菌的多样性均显著高于根腐病植株；而在RNA水平则表现出不同的多样性规律，根腐病植株的细菌多样性最丰富。根腐病的发生影响了根际和根内生细菌与真菌的群落结构和多样性，健康植株的根际与根内真菌多样性均显著高于根腐病样品。土壤环境因子的相关性分析表明土壤总磷，pH 和有机质对三七栽种过程中的细菌群落组成的影响最大，而土壤pH 和有机质对真菌群落组成与多样性的影响最大。宏基因组测序分析表明在发病的三七样品中，其ABC 转运系统(ATP-binding　cassette　transporters)中的支链氨基酸转运蛋白中的底物结合蛋白、通透酶蛋白及ATP 结合蛋白的基因丰度相对较低，这些蛋白是双组分、植物激素转导和磷脂酰肌醇等信号转导系统的重要组成部分。研究结果初步揭示了微生物群落演替在三七连作障碍形成中的规律，为进一步克服三七连作障碍奠定了良好理论基础。截止目前，在本项目的支持下共发表相关论文12篇，其中SCI期刊论文7篇，获授权发明专利5项，9名参与项目的研究生顺利毕业，其中博士5名，硕士4名。