植物微生物群抗生素抗性水平与抗性基因的消长机制

Manure has become a reservoir of antibiotics and antibiotic resistance genes (ARGs), and its application to agricultural soils greatly stimulates the antibiotic resistance bacteria and ARGs in soils, posing a huge threat to the environment and human health. Most studies about ARGs in plant microorganism are focused on single compartment. Exploring the co-occurrence and fate of ARGs and heavy metal resistance genes (MRGs) in soil-plant-microbial system under the cross-stress of heavy metals and antibiotics are still major challenges. Based on two green manure-rice cropping rotation systems, vetch (Vicia sativa)-rice and ryegrass (Lolium perenne)-rice, the distribution and dynamics of ARGs in plant microbiota including rhizosphere, rhizoplane, leaf and endosphere bacteria following pig manure application will be studied. Roman and stable isotope probing (2H-SIP) will be used to characterize phenotypic changes, physiological states and functions of cells of plant microbiota in situ under antibiotic and heavy metal stress. Combining Raman spectroscopy, metagenomic sequencing and functional gene array, the co-occurrence patterns and underlying mechanisms will be studied from single cell to community. Root-inhabiting bacterial microbiota will be cultured. Then several functional strains will be sorted and assembled to reveal the process of horizontal gene transfer of ARGs and the relationship with mobile genetic elements such as plasmids, integrons and transposable elements. Heavy metal (Cu, Zn)-mediated enhancing effect on antibiotic resistance in rhizosphere will be identified. Finally, this study will further explore the main factors that influence the fate and dissemination of ARGs in the soil-plant-microbial systems. Our study can provide theoretical basis for the control of dissemination of ARGs in agroecosystems.

畜禽粪便是抗生素和抗性基因的储存库，通过施肥等方式会刺激土壤中耐药微生物和抗性基因（ARGs）的富集与传播，对生态环境和人类健康造成极大的威胁。目前对植物微生物ARGs的研究多集中在局部组织和单因素，缺乏重金属和抗生素交叉污染下土壤-植物系统中ARGs与重金属抗性基因的赋存与转归及协同作用机制。本项目拟通过对绿肥-水稻轮作农田生态系统的研究，揭示不同绿肥（禾本科及豆科）及水稻植物微生物群（根际、根表、茎叶及内生微生物）的抗生素抗性水平及ARGs动态分布特征；利用单细胞拉曼同位素标记和单细胞弹射技术，结合宏基因组测序，从单细胞水平到群落水平，研究植物不同生长期功能微生物群落演替及ARGs在植物不同组织间的消长及传输机制；阐明抗生素与重金属交叉胁迫下ARGs和重金属抗性基因的共现关系和主控因素，识别控制ARGs水平转移的主要因子，为消减与控制土壤中有机肥源ARGs提供理论依据。

研究畜禽粪便-土壤-植物系统中抗生素抗性基因（ARGs）的来源、分布和扩散机制，揭示微生物组抗性水平的主要影响因素及ARGs 在环境中的消长机制，对于阻断有机肥源抗性基因的传播、减少耐药菌和ARGs 在环境中的污染问题有着重要意义。本项目通过对绿肥-水稻轮作农田生态系统的研究，揭示了生境决定了土壤-植物系统中细菌、抗性基因和可移动遗传元件之间的关系，基于细菌16s rRNA测序和RGs（包括ARGs、MRGs和MDRs）和可移动遗传元件（MGEs）的高通量定量PCR，发现虽然细菌的α-多样性和RGs和MGEs的丰度在根际中并不是最高的，但是细菌与根际中的RGs和MGEs的关联最强，而不是土壤和叶际中的；根际细菌之间的网络相互作用比土壤和叶际细菌之间的网络相互作用更强，根际是土壤-植物系统中ARG交换的热点；阐明抗生素与重金属交叉胁迫下ARGs和重金属抗性基因（MRGs）的共现关系和主控因素，明确了抗生素抗性基因与重金属抗性基因的共存性是促进稻田土壤抗生素抗性传播的重要机制；农业施用粪肥增加了农业生态系统中多药耐药性抗性基因（MDR）和MRGs的丰度和共存，主要是由于直接引入了抗性基因，抗生素和金属的共选择压力增加，以及刺激了在粪肥施用下获得和转移抗性基因的宿主；这种共存会进一步增加土壤抗生素抗性，对农业生态系统功能和人类健康构成威胁；进一步识别了控制ARGs扩散的主要因子，阐明了跨气候带稻田土壤微生物抗砷、镉功能基因的分布模式及控制重金属含量对减少ARGs传播潜在重要性，为消减与控制土壤中有机肥源ARGs提供理论依据。