Effect of Long-term Biogas Slurry Fertilization on Distribution of Tetracycline and Sulfonamide Resistance Genes in Soil
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摘要:
目的 探讨长期施用猪粪沼液对土壤环境中抗生素抗性基因（Antibiotic resistance genes, ARGs）分布的影响及其季节变化。 方法 分别在春、夏、秋、冬4个季节采集养猪场周边长期用沼液作为肥料的土壤，同时采集当地未施用过沼液的土壤作为对照，采用荧光定量PCR方法，对土壤中四环素类和磺胺类ARGs进行检测和定量分析。 结果 在施用沼液的土壤中能检测到11～13种ARGs，沼液的长期施用提高了土壤中ARGs的多样性，显著增加了土壤中tetG、sul1和sul2等基因的相对丰度，使四环素类和磺胺类ARGs在土壤中的存在水平升高。上述2类ARGs在土壤中的分布具有明显的季节性，均在春季具有更高的相对丰度。在长期施用沼液的土壤中存在大量的tetG、tetZ、tetM、tetO、tetW、sul1和sul2基因，这些基因在不同季节里都是优势ARGs。 结论 沼液农用会增加土壤中ARGs的存在水平，因此需要加强优化畜禽废弃物发酵工艺，以减少ARGs在沼液肥中的残留。 Abstract:Objective Effect of long-term application of pig manure biogas slurry on the abundance and seasonal variations of antibiotic resistance genes (ARGs) presented in the soil was studied. Method Soil specimens at the fields using biogas slurry of pig manure for fertilization for many years were collected in four seasons to compare with those at a nearby location without such practice. The presence and abundance of tetracycline and sulfonamide ARGs in the soils were determined by quantitative PCR. Result There were 11 to 13 classes of ARGs detected in the biogas slurry fertilized soils. The ARG diversity was significantly greater in them than control with a relative abundance of tetG, sul1 and sul2 genes. The increased levels of tetracycline and sulfonamide ARGs also showed significant seasonal variations with the highest relative abundance in spring. Long application of biogas slurry on land resulted in abundant tetG, tetZ, tetM, tetO, tetW, sul1, and sul2 genes, which were the dominant ARGs year-round, in the soil. Conclusion Long-term biogas slurry application could elevate the ARG level in the soil. Therefore, it was highly recommended the waste material utilization be optimized by a fermentation process prior to fertilization for antibiotic contamination mitigation on farmland. -
Key words:
- soil /
- biogas slurry /
- resistance gene /
- Real time PCR /
- seasonal characteristics
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图 1 不同季节土壤中四环素类ARGs相对丰度总和
注：sum tet，四环素类ARGs总和；BS+，长期施用沼液土壤；BS−，未施用沼液土壤；SP、SU、AU和WI分别代表春季、夏季、秋季和冬季；条形图上不同字母表示处理间存在显著性差异（P＜0.05），下同。
Figure 1. Sum of relative abundance of tetracycline ARGs in soil specimens collected in different seasons
Note: sum tet: sum of tetracycline ARGs; BS+: soil with long-term biogas slurry fertilization; BS−: soil with no biogas slurry application; SP, SU, AU and WI: spring, summer, autumn, and winter, respectively; data with different letters on bars mean significant difference among treatments (P＜0.05). Same for below.
图 2 不同季节土壤中四环素类ARGs的相对丰度
Figure 2. Relative abundance of tetracycline ARGs in soil specimens collected in different seasons
图 3 长期施用沼液的土壤中四环素类ARGs的相对丰度
注：图中tetS和tetT的数值部分有缺失是因为未检出。
Figure 3. Relative abundance of tetracycline ARGs in soils with long-term application of biogas slurry
Note: Absence of numeric data on tetS and tetT genes indicates non-detection.
图 4 不同季节土壤中磺胺类ARGs相对丰度总和
注：sum sul，磺胺类ARGs总和。
Figure 4. Sum of relative abundance of sulfonamide ARGs in soil specimens collected in different seasons
Note: sum sul: sum of sulfonamide ARGs.
图 5 不同季节土壤中磺胺类ARGs的相对丰度
Figure 5. Relative abundance of sulfonamide ARGs in soil specimens collected in different seasons
表 1 不同季节土壤中检测到的抗性基因（n=3）
Table 1. Resistance genes detected in the soils in different seasons
抗性基因
ARGs长期施用沼液土壤
Soil with long-term of
biogas fertilization未施用沼液土壤
Soil with no biogas
fertilization春季
Spring夏季
Summer秋季
Autumn冬季
Winter春季
Spring夏季
Summer秋季
Autumn冬季
WintertetA − − − − − − − − tetB + − − − + + − + tetC − − − − − − − − tetG + + + + + + + + tetH + − − + − − − − tetK − − − − − − − − tetM + + + + − − − − tetO + + + + − − − − tetQ + + + + − − − − tetS − − − + − − − − tetT + + + − − − − − tetW + + + + − − − − tetY + + + + + + + + tetZ + + + + + + + + sul1 + + + + + + + + sul2 + + + + + + + + sul3 + + + + + + + + 注：+，阳性；−，阴性。Note: +, positive; −, negative. 
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