长期施用牛粪对褐土抗生素抗性基因多样性的影响
Effects of Long-term Application of Cattle Manure on Antibiotic Resistome in Cinnamon Soil
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摘要: 为探明长期施用牛粪对土壤抗生素抗性基因多样性的影响，以山西寿阳褐土长期定位试验为依托，选择5个施肥处理：CK(不施肥)、N2P1M1(N：120 kg·hm-2；P：37.5 kg·hm-2；牛粪：22 500 kg·hm-2)、N4P2M2(N：240 kg·hm-2；P：75 kg·hm-2；牛粪：45 000 kg·hm-2)、N3P2M3(N：180 kg·hm-2；P：75 kg·hm-2；牛粪：67 500 kg·hm-2)和N0P0M6(牛粪：135 000 kg·hm-2)，采集玉米生长中期耕层(0～20 cm)土样，通过高通量实时荧光定量聚合酶链式反应，对土壤中的抗生素抗性基因(antibiotics resistance genes, ARGs)、可移动遗传元件及相关分类学基因等，共384个基因进行定量检测，分析其多样性及其与土壤理化因子的相关性。结果表明，随着牛粪施用量的增加，ARGs相关基因多样性逐渐增加，5个处理共有的ARGs相关基因亚型有64个，N0P0M6处理特有ARGs相关基因亚型有21个，显著高于其他处理，各处理中含量最高的ARGs相关基因亚型为：AAC(6’)-Im、ANT(4’)-Ia、aadA5、AAC(3)-Via和copA；各处理中含量最高ARGs家族类型为氨基糖苷类、多药类、四环素类、氟喹诺酮类、大环内酯类-林可霉素类-链阳性霉素B类(MLSB)抗生素；不同处理间ARGs相关基因亚型的β-多样性差异显著，冗余分析表明，对ARGs差异影响最大的环境因子是pH、全氮和有机质。综上，畜禽粪肥还田虽然可以提升土壤肥力，促进作物生长，但同时也会造成ARGs的扩散和传播，今后的农业生产中需综合考虑环境生态效益，加大有机肥无害化处理力度，从而控制ARGs的环境风险。Abstract: In order to explore the effects of long-term application of cattle manure on soil antibiotic resistome, five fertilization treatments were selected from the long-term location experiment in the cinnamon soil of Shouyang, Shanxi Province, i.e., CK (No fertilization), N2P1M1 (N: 120 kg·hm-2, P: 37.5 kg·hm-2; Cow dung: 22 500 kg·hm-2), N4P2M2 (N: 240 kg·hm-2; P: 75 kg·hm-2; Cow dung: 45 000 kg·hm-2), N3P2M3 (N: 180 kg·hm-2; P: 75 kg·hm-2; Cow dung: 67 500 kg·hm-2) and N0P0M6 (Cow dung: 135 000 kg·hm-2). Soil samples of surface layer (0～20 cm) at the middle growth stage of maize were collected. A total of 384 antibiotics resistance genes (ARGs), mobile genetic elements and related taxonomic genes in soil were detected by high-throughput quantitative real-time polymerase chain reaction (HT-qPCR), and their diversity and correlation with soil physical and chemical factors were analyzed. The results showed that with the increase of cow manure application amount, the diversity of ARGs related genes increased gradually. There were 64 shared ARGs related gene subtypes in the five treatments, and 21 specific ARGs related gene subtypes in treatment N0P0M6, which was significantly higher than other treatments. The highest abundance of ARGs related gene subtypes in all treatments were AAC(6’)-Im, ANT(4’)-Ia, aadA5, AAC(3)-Via and copA. The highest abundance of ARGs types in all treatments were aminoglycoside, multidrug, tetracycline, fluoroquinolone and macrolide-lincosamide-streptogramin B (MLSB). The β-diversity of ARGs related gene subtypes was significantly different among different treatments. Redundancy analysis showed that pH, total nitrogen and organic matter were the most important environmental factors affecting the difference of ARGs between different treatments. In conclusion, although the returning livestock and poultry manure to the field can improve soil fertility and promote crop growth, it can also cause the dispersal of ARGs in the soil ecosystem. Therefore, in the future of agricultural production, environmental and ecological benefits should be taken into comprehensive consideration to strengthen the harmless treatment of manure, so as to control the environmental risk of ARGs.
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Key words:
- antibiotic resistance genes /
- cinnamon soil /
- ecological risk
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