土存镰刀菌病毒对太子参连作的致害机制研究

In modern agriculture of traditional Chinese medicine，the consecutive monoculture of Chinese medicine in the same field results in the outbreak of virus disease, and its mechanism of causing disaster is still unknown. In this study, the Radix pseudostellariae variety (Zheshen 2), which has been mostly planted on a large-scale in the geo-authentic production zones will be used as the test plant material. And we construct the system of virus-free tissue culture of R. pseudostellariae. First, RNA deep sequencing and qRT-PCR will be used to analyse virus communities and their distribution characteristics in the plant of R. pseudostellariae, and further study will be used to clarify the effects of R. pseudostellariae virus on the plant functional traits. Second, RNA deep sequencing, mycelium tip isolation and PCR amplification will be used to screen the pathogenic Fusarium carrying the virus of R. pseudostellariae and virus-free fungi. Moreover, study will also conducted on the effect of mycovirus on the physiological characteristics of Fusarium. Third, the transcriptome, widely-targeted metabolomics and electron microscope will be used to analyse the difference of gene expression and metabolomics of virus-free tissue culture of R. pseudostellariae under infection of the above virus Fusarium and virus-free Fusarium, and the potential disaster mechanism of mycovirus infection to R. pseudostellariae will be elucidated. Finally, the interactions between the mycovirus and Fusarium mediated by root exudates will be analysed. The study will expound the mechanism of disaster caused by soil born mycovirus under the continuously monocultured R. pseudstellariae regime, and provides the theoretical basis for the effective prevention and control of the outbreak of virus diseases such as medicinal plants.

连作导致药用植物病毒病爆发是现代药用植物栽培普遍存在的问题，其发生过程与机制仍是空白，亟待人们予以揭示。本研究以道地“柘参2号”为材料，构建太子参脱毒苗繁育体系，首先采用小RNA深度测序和qRT-PCR分析连作太子参不同生长时期病毒种类和含量变化，太子参病毒对植株功能性状的影响；采用小RNA测序、菌丝尖端分离法和PCR验证等筛选鉴定携带太子参主要病毒的致病镰刀菌及其脱毒真菌，分析病毒对真菌生理特性的影响；利用转录组学、广泛靶向代谢组学和电镜观察分析上述病毒镰刀菌及其脱毒真菌侵染太子参的基因和代谢表达差异，阐明真菌病毒侵染对太子参的致害机制；分析太子参关键根系分泌物介导的病毒—真菌间互作关系，阐明连作栽培太子参病毒病增加、土存真菌病毒介入药用植物连作根际演变的生态学过程及其致害机制，以期为探索药用植物病毒病的生态控制提供理论依据。

连作导致药用植物土传病害和病毒病爆发是现代药用植物栽培普遍存在的问题，而病毒-微生物-药用植物之间的互作关系在连作病害发生过程中的作用仍是空白。本项目采用小RNA深度测序、qRT-PCR、转录组学和代谢组学等研究方法，系统探究了连作促进太子参土壤病毒传播、致病真菌病毒介入药用植物连作根际演变的生态学过程。主要研究结果如下：1. 病毒侵染会改变太子参植株功能性状，造成太子参生物量的锐减。2. 连作会显著增加植株中芜菁花叶病毒、蚕豆萎焉病毒2号和黄瓜花叶病毒丰度，加剧了太子参病毒病的发生和发展。3. 宏转录组学分析表明连作改变根际土壤RNA病毒群落结构和组成，造成黄瓜花叶病毒、蚕豆萎焉病毒2号、马铃薯病毒M型、芜菁花叶病毒等植物病毒在土壤中逐年累积。4. 采用小RNA深度测序等技术筛选携带病毒的致病尖孢镰刀菌N1-YS及其脱毒菌YT-N1，二者均能造成太子参死亡，其中脱毒菌菌丝生长速率、产3A-DON和15A-DON真菌毒素含量要显著高于带毒菌株，而带毒菌产孢子能力显著高于脱毒菌。比较转录组学分析表明真菌病毒通过调控菌的剪接体、氨基糖和核苷酸糖代谢途径、磷酸戊糖途径等代谢途径，从而导致其生理特性发生变化。5. 分别采用N1-YS和YT-N1侵染脱毒太子参组培苗，结果表明携带病毒真菌会促使太子参MAPK信号通路-植物、甘氨酸、丝氨酸和苏氨酸代谢、糖酵解/糖异生、植物-病原体相互作用通路中基因上调表达；携带病毒真菌也会促使太子参半乳糖代谢、ABC转运体、甘油脂代谢、抗坏血酸和醛酸代谢中代谢物下调表达，进而破坏了太子参生理代谢。6. 太子参根系分泌酚酸和有机酸能显著促进脱毒YT-N1菌的菌丝生长、产毒素能力，促进带毒N1-YS菌产孢子能力。7. 生物炭和根际促生菌能有效减少太子参根际土壤、植物中尖孢镰刀菌等致病菌丰度，改善太子参根际微环境，进而减缓太子参连作障碍的发生。综上所述，太子参连作会造成土壤和植株中病毒累积，也促进了带毒致病尖孢镰刀菌的增殖，进而造成太子参代谢紊乱，加重了太子参病害发生。本项目的研究成果为药用植物栽培中土传病害的致病过程以及生态防控提供了新机制和新策略。