粪肥施用对农田土壤中抗生素耐药基因的影响研究

Due to the abuse of antibiotics in the aquaculture industry, a large number of antibiotics and resistance genes are input into the farmland soil environment. The bacterial resistance in farmland soil is increasingly prominent. The emergence of bacterial resistance in farmland system is closely related to the emission of livestock and poultry farms and/or the mode of fertilization. The antibiotic resistance genes in livestock manure and farmland soil were detected at high frequency. However, it was necessary to clarify the spread of antibiotic resistance genes in farmland soil after manure application. Whether the antibiotic resistance genes could be migrated to soil by fertilizers is not clear. Based on this, this study carried out field experiments, using quantitative PCR, genetic information analysis, high-throughput sequencing technology and mathematical model. Firstly, we clarify the influence of manure application on the survivability and persistence of antibiotic resistant bacteria and the resistance genes in soil during the crop growth and postharvest period and identify the main driving factors affecting the level of antibiotic resistance genes. Meanwhile, horizontal gene transfer was used to elucidate the effect of antibiotic resistant bacteria carried in manure on the transmission of resistant genes in farmland soil, and this study focuses on elucidating the horizontal and vertical migration of antibiotic resistance genes from fertilizer to farmland soil. This study will provide references for improving the non-point source pollution and the spread risk of resistance genes in agricultural system and ensuring crop safety, and establishing a chain of bacterial resistant pollution control from the whole process of fertilizer-soil-crops.

由于抗生素在养殖业的滥用，大量的抗生素和耐药基因输入农田土壤环境。农田土壤中细菌耐药污染日益凸显，农田系统中的细菌耐药的产生与周边畜禽养殖场的排放，施肥模式等密切相关。耐药基因在畜禽粪便、农田土壤已被高频率检出，然而需明确粪肥施用后耐药基因在农田土壤中的变化和传播情况，并且耐药基因是否通过粪肥迁移到农田土壤中尚不清楚，基于此，本研究开展大田试验，采用定量PCR、遗传信息分析、高通量测序、数学模型等技术，首先弄清粪肥施用后在农作物生长期内和收获后农田土壤中耐药菌、耐药基因的适应性、存活能力及持久性的变化情况，识别影响耐药基因变化的主要驱动因子，同时采用基因水平转移的方式阐明粪肥中携带的耐药菌对农田土壤中耐药基因传播的影响，关注耐药基因从粪肥到农田土壤的横纵向迁移过程。该研究为改善农业耐药基因的面源污染及耐药基因传播风险，保证农作物安全，建立肥料-土壤-农作物全过程的耐药污染控制链条提供借鉴。

粪肥施用引起农业系统中致病和细菌耐药的问题已成为一个备受关注的研究热点。在此，探究了施用有机肥、化肥和商品肥等不同肥料后土壤和蔬菜中抗生素抗性基因（ARGs）和病原体的横纵向迁移，从生态群落和分子层面揭示其影响机制。通过对比不同肥料施用前后农田土壤抗生素中耐药基因的丰度变化特征，结果显示有机肥的施用不仅向土壤和农作物中引入了外源 ARGs，更重要的是，促进土壤 ARGs 的增殖，而且在农作物（小白菜）收获十天后，虽然耐药基因有所下降，但仍高于化肥对照组和土壤背景值，并且在小白菜根、茎、叶不同组织中也检测到耐药基因，因此有机肥施用引起农田土壤和农作物耐药增殖的风险仍不能忽视。在剖面土壤中，发现表层和深层土壤中耐药基因丰度没有显著差异，然而耐药宿主菌差异较大，因此猜测可能是由于外源耐药菌在迁移过程与土著菌产生竞争作用，土著菌捕获了外源耐药基因有关，从而导致深层土壤中外源耐药菌无法定殖，而 ARGs 丰度却仍维持与表层土壤相近的水平。该研究摸清了有机肥施用后农田系统中耐药基因的迁移扩散规律，为下一步遏制农田土壤系统中细菌耐药问题提供了理论基础。