菊花连作土壤微生物区系变化及致病机制研究

Chrysanthemum is one of the ten Chinese traditional flowers and the four biggest cut flowers in the world, which has high ornamental and economic value. In the production process, the continuous cropping obstacle causes significant economic losses to the farmers. Until now, there is less reports detailed the mechanism of this problem. Our previously study indicated that the worsen of the rhizosphere microbial communities and developed of the pathogen were the main reasons for this problem. The proposal intends to use the 454 Pyrosequencing technology analysis of the specific soil microbial and pathogen's community and diversiry, supplemented by the Real-Time PCR to determinate of the B/F ratio to analyize the relationship between the chrysanthemum continuous cropping obstacle and the soil microbial community; furthermore, high-throughput RNA sequencing technology will be used to study the response mechanisms of chrysanthemum when pathogen infected, and key gene cloning and functional analysis. The results of this project will provide important theoretical basis to clarify the continuous cropping soil microflora changes and pathogenic mechanism, and will give deep insight in plants and soil microbial groups of interactions,which will help to solve the chrysanthemum scientific, ecological continuous cropping obstacles and provide theoretical foundation.The subject have important theoretical and application value.

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值极高。在生产过程中，连作障碍现象发生普遍，给生产企业造成了极大的经济损失。而针对菊花连作障碍的具体成因目前鲜有报道。我们通过前期研究，认为菊花产生连作障碍的主要原因为根际微生物区系的恶化，病原菌的增加造成的土传病害。本项目拟利用454焦磷酸高通量测序技术连续跟踪分析土壤微生物区系及病原微生物具体类群的变化，辅以Real-Time PCR技术测定细菌、真菌及B/F值 (细菌总量/真菌总量)，系统地分析菊花连作对根际和土体土壤微生物区系的影响；随后采用转录组高通量测序技术研究菊花连作后病原菌侵染的致病机制，对相关关键基因进行克隆与功能分析。通过系统地研究菊花连作土壤微生物区系变化及致病机制，将为理清植物与土壤微生物类群的互作关系，为科学、生态地解决菊花连作障碍提供理论基础。本课题既具有较高的理论深度，又有很强的应用价值。

本研究利用454焦磷酸高通量测序技术连续2年跟踪分析了菊花枯萎病田间发病率，土壤微生物区系及病原微生物数量的变化。结果表明：连作5年后，菊花田间发病率为45.1%，菊花外观品质指标与连作年限呈显著负相关。连作对菊花‘神马’根际真菌和细菌影响显著，而对土体土壤微生物数量没有显著影响。连作5年后，菊花‘神马’根际土壤中尖孢镰刀菌数量最高，为连作1年根际土壤的4.55倍；细菌/真菌比连作1年土壤降低了44%。DGGE和454焦磷酸测序结果显示，连作5年后，菊花‘神马’根际和土体土壤中稻瘟病病原菌Magnaporthe grisea、纹枯病菌Rhizoctonia solani、镰刀菌Fusarium sp.、尖孢镰刀菌F. oxysporum和茄病镰刀菌F. solani等显著增加，菊花连作土壤中子囊菌（Ascomycete）所占比例最大（67.9%），担子菌（Basidiomycetes）次之（21.4%），细菌群类大多为土壤常见细菌。采用转录组高通量测序技术研究了菊花连作后病原菌侵染的致病机制，并且成功的筛选出了受尖孢镰刀菌诱导表达的3个CmWRKY候选基因CmWRKY1、6和8，对CmWRKY1进行了克隆与功能分析。初步明确了CmWRKY 1基因参与植物-病原物互作机制。培养研究生3名，发表论文6篇(SCI 4篇)。