Farmland ecosystems are essential sources and sinks of antibiotic resistance genes (ARGs), and the application of livestock manure is a major contributor to ARGs in soil. The massive application of livestock manure to vegetable fields has intensified the pollution caused by ARGs in soil. Raw consumption of edible vegetables is one of the most direct ways to introduce ARGs from the soil–plant system to humans, which poses a potential threat to human health. However, few studies have investigated the effects of different fertilizer types on ARGs and bacterial communities in vegetable fields. In this study, 21 soil samples (0–20 cm) were collected from vegetable fields in Hebei Province using different fertilizer types (fresh fowl manure, fresh sheep manure, fresh cattle manure, commercial organic fertilizer, and chemical fertilizer). The distributions and characteristics of ARGs and bacterial communities in vegetable fields were investigated using real-time quantitative polymerase chain reaction (PCR) and high-throughput sequencing techniques. Eight tetracycline resistance genes (tetA, tetC, tetG, tetL, tetO, tetM, tetW, and tetQ), two sulfonamide resistance genes (sul1 and sul2), and one intI1 gene were detected in all vegetable fields. The absolute abundance of sulfonamide resistance genes (9.96×109 copies·g−1 in dry soil) was significantly higher than that of tetracycline resistance genes (1.07×109 copies·g−1 in dry soil). The application of livestock manure and chemical fertilizer both significantly increased the abundance of ARGs in vegetable fields. The highest abundance of ARGs (6.34×109 copies∙g−1 in dry soil) was found in vegetable fields with higher chemical fertilizer amendment, while the lowest abundance of ARGs (3.09×108 copies∙g−1 in dry soil) was found in vegetable soil with commercial organic fertilizer. In addition, the Shannon and Chao1 indices, representing the α diversity of the soil bacterial community, were significantly higher in soil fertilized with livestock manure compared to high-chemical fertilizer application but not in low-chemical fertilization soil, indicating that livestock manure application significantly increased the abundance and diversity of the soil bacterial community. Pearson’s correlation analysis showed that soil bacterial community structure was an important factor influencing the distribution of ARGs. Proteobacteriota, Bacteroidota, Actinobacteriota, and Firmicutes were the dominant potential hosts of ARGs and were significantly correlated with sulfonamide and tetracycline resistance genes (P<0.05). The distribution of ARGs was also affected by soil organic matter and total nitrogen content. The intI1 gene had significant and positive correlations with the sul2, tetG, tetQ, and tetW genes, suggesting its crucial role in ARGs dissemination. In the present study, the use of higher concentrations of chemical fertilizers led to a significantly increased abundance of ARGs in the soil of vegetable fields, whereas the application of commercial organic fertilizers had the least effect on ARGs abundance. This study serves as a guide for evaluating the status of ARGs pollution in vegetable fields with different fertilizer types.

Long-term excessive application of nitrogen fertilizer under diverse land uses has caused serious environmental problems on the North China Plain. Current studies focus on denitrifiers and bacterial communities in topsoil under diverse land-use types; however, few studies have studied denitrifiers and microbial communities in subsoils. The variations in soil bacterial communities and denitrifiers with soil profiles (0–300 cm) under crop, apple orchard, and vegetable fields were investigated through high-throughput sequencing and quantitative PCR technologies. NO3−-N largely accumulated in the deeper soil layers (100–300 cm) in the apple orchard, resulting in a higher risk of NO3−-N leaching. The soil bacterial community structure at the 0–100 cm soil depth had a marketable difference from that at 100–300 cm under these three land-use types, and the C:N ratio was the main driving factor for their vertical distribution. The bacterial α-diversity decreased with soil depth; the crop field had the highest α-diversity across all horizons except 100–200 cm, and total carbon was the most important factor driving α-diversity. In addition, the absolute abundance of the nirK, nirS, and nosZ genes decreased with soil depth and varied with land-use type, which was deeply affected by multiple soil properties, such as soil organic matter and total nitrogen. Our findings highlighted that potentially important and unique functions remain to be revealed in subsoils, which may provide new insights into mitigating nitrate leaching in various land-use types.

本文通过对养殖场猪粪和鸡粪堆放地0～100 cm土壤样品的采集和分析,研究了长期堆放畜禽粪便对土壤中抗生素抗性基因(简称"抗性基因")和细菌群落结构垂直分布的影响.定量PCR结果表明,与对照土壤相比,猪粪和鸡粪堆放增加了0～100 cm土壤中四环素类抗性基因(tetC、tetG、tetL、tetW)、磺胺类抗性基因(sulI、sulII)以及整合酶基因(intI1)的检出率和检出丰度,说明粪肥堆放造成堆放地土壤中抗性基因污染.聚类分析结果表明,抗性基因和intI1基因的丰度随土壤深度呈递减趋势,且主要集中在0～30 cm土层,表明堆放地土壤中抗性基因存在向下层土壤迁移的风险.相关性分析表明,intI1基因分别与抗性基因呈显著正相关,说明intI1基因可能在抗性基因传播中起着重要作用.同时高通量测序结果表明,与对照土壤相比,猪粪和鸡粪堆放显著降低了0～10 cm和10～30 cm土层细菌群落结构的多样性,0～30 cm土层中,猪粪和鸡粪堆放地土壤中细菌群落结构与对照土壤的差异要高于深层土壤.此外,方差分解分析结果表明,土壤化学性质和细菌群落结构均影响了土壤中抗性基因的垂直分布,且细菌群落结构的变化是其主要的影响因素.本研究可为控制畜禽粪便堆放地土壤中抗性基因污染提供科学依据.

Long-term fertilization is known to impact the biodiversity and community structures of soil organisms, which are responsible for multiple soil ecosystem functions (multifunctionality). However the relationship between the alterations of soil organisms and ecosystem multifunctionality remains unclear, especially in the case of long-term fertilization. To explore the contribution of soil organismal biodiversity and community structures to ecosystem multifunctionality, we took soil samples from a nearly 25-year field fertilization experiment. Organic matter significantly improved the soil ecosystem multifunctionality. Ecosystem multifunctionality was found to be closely linked to the biodiversity and communities of soil organisms within the major ecological clustering of soil organisms (Module 1) according to the trophic co-occurrence network, rather than the entire community of soil organisms. This indicated that ecological clusters of soil organisms within the network were critical in maintaining soil ecosystem multifunctionality. The application of organic fertilization could enrich specialized soil organisms and increase interactions of soil organisms in the ecological cluster. As a result, our findings emphasize the role of ecological clusters in the soil organismal co-occurrence network in controlling soil multifunctionality after long-term fertilization, presenting a novel perspective on the link between soil biodiversity and ecosystem multifunctionality.

Manure, which contains large amounts of antibiotics and antibiotic resistance genes (ARGs), is widely used in agricultural soils and may lead to the evolution and dispersal of ARGs in the soil environment. In the present study, soils that received manure or chemical fertilizers for 15 years were sampled on the North China Plain (NCP), which is one of the primary areas of intensive agriculture in China. High-throughput quantitative PCR and sequencing technologies were employed to assess the effects of long-term manure or chemical fertilizer application on the distribution of ARGs and microbial communities. A total of 114 unique ARGs were successfully amplified from all soil samples. Manure application markedly increased the relative abundance and detectable numbers of ARGs, with up to 0.23 copies/16S rRNA gene and 81 unique ARGs. The increased abundance of ARGs in manure-fertilized soil was mainly due to the manure increasing the abundance of indigenous soil ARGs. In contrast, chemical fertilizers only moderately affected the diversity of ARGs and had no significant effect on the relative abundance of the total ARGs. In addition, manure application increased the abundance of mobile genetic elements (MGEs), which were significantly and positively correlated with most types of ARGs, indicating that horizontal gene transfer via MGEs may play an important role in the spread of ARGs. Furthermore, the application of manure and chemical fertilizers significantly affected microbial community structure, and variation partitioning analysis showed that microbial community shifts represented the major driver shaping the antibiotic resistome. Taken together, our results provide insight into the long-term effects of manure and chemical fertilization on the dissemination of ARGs in intensive agricultural ecosystems.

Impacts of manure application on the soil fungal community in agricultural systems have been extensively explored. However, the contribution of manure-sourced exogenous species in shaping soil fungal diversity and community assemblage are still open questions. In this study, fungal communities in soils that received manure with or without chemical fertilizers, as well as a no fertilizer control, were characterized using high throughput sequencing. We found that diverse taxa in manure, including some potential pathogens, were transmitted into soil through manure application, accounting for 9.80–10.94% of soil fungal richness, but only for 1.10–2.04% in the relative abundance of soil fungal community. Manure application impacted soil fungal community assemblage mainly through alterations of soil characteristics, especially soil carbon pool. Nutrients from manure showed great selection on fungal taxa, thus strengthening the effect of niche filtering on fungal community. As a consequence, manure application resulted in lower fungal richness than chemical fertilizers through enhancing species extinction. These findings suggested that the transmission of manure-sourced exogenous species is a key source of fungal richness but contributed little to soil fungal community assemblage. Manure incorporation structured soil fungal diversity and community assemblage primarily through changing niche breadth and the types of substrates available in the soil.