| The rapid spread of antibiotic resistance genes(ARGs)in the environment and their potential threats to human health have attracted considerable attention around the world.The misuse of antibiotics in animal husbandry results in the occurrence of resistant bacteria,ARGs and mobile genetic elements(MGEs)in animal manures.The application of manures to replace chemical fertilizers and improve soil fertility is an important setting for agricultural production,but it also leads to the spread of ARGs harbored by manures in the soil.The Sichuan Basin is a key area for animal husbandry and is also a concentrated distribution area of purple soils.According to the characteristics of the parent rock and the differences in p H during soil development,purple soils are divided into three types,i.e.acidic,neutral and calcareous.Both soil p H and manure application directly or indirectly regulate the accumulation and dissemination of ARGs in soil,but there are few studies on the effects of long-term manure application on the accumulation and migration of ARGs in purple soils with different p H.Hence,this study used four different fertilization treatments(control,chemical fertilizer,manure,manure + chemical fertilizer,and the last three treatments contained equal amounts of nitrogen,phosphorus and potassium nutrients)for purple soils with different p H(i.e.acidic,neutral and calcareous)following a long-term positioning experiment of maize-cabbage rotation.Modern biotechnology was used to study the accumulation and migration characteristics of ARGs in topsoil,soil profile,leachate,and leaf endophyte of cabbage.Our study will provide theoretical and technical supports for the pollution assessment of ARGs in purple soil area and for the scientific application of manure.The main findings are as follows:(1)A total of 408 ARGs of 18 different types were identified in the topsoil of three purple soils with different p H by metagenomic sequencing,in which the abundance of genes conferred to aminoglycoside,β-lactam,glycopeptide,macrolide,multidrug and streptogramin increased in all the manure-amended soils.The effect of manure application on the distribution characteristics of ARGs in acidic and neutral purple soils was stronger than that in calcareous purple soil,and the number of ARGs enriched in acidic and neutral purple soil was 98 and 91,respectively,which was significantly higher than that in calcareous purple soil(8).Structural equation model(SEM)analysis indicated that changes in bacterial abundance were the first important factor driving changes in ARGs,followed by changes in physicochemical properties such as soil p H,and then antibiotics.However,there were differences in the mechanisms regulating the accumulation of ARGs in purple soils with different p H.The effect of MGEs on the relative abundance of ARGs in calcareous purple soil was significantly higher than that in acidic and neutral soils,suggesting that the accumulation and dissemination of ARGs were directly regulated by horizontal gene transfer through the mediation of MGEs.Relative to control treatments,soil p H,bacterial abundance and diversity,and the number and abundance of ARGs in the acidic and neutral purple soils were significantly increased,indicating that the application of manure in the acidic and neutral purple soils,mainly through elevating soil p H,indirectly altered bacterial abundance and facilitated ARGs accumulation and dissemination.(2)The migration results of ARGs in soil profiles showed that a total of 204 ARGs of 9 different types were detected in soil profiles of three purple soils by high throughput quantitative polymerase chain reaction(HT-qPCR),in which aminoglycosides,β-lactams,macrolides,multidrug and tetracycline resistance genes were the predominant ARGs,accounting for 78%-91%.Manure application significantly increased the diversity and abundance of ARGs in the 20-40 cm and 40-60 cm profiles,and the number and abundance of aminoglycosides,β-lactams,macrolides,multidrug-resistant and tetracycline resistance gene ARGs were higher than those in control treatment.The absolute abundance of ARGs increased by 3.26-5.22 times and 10.26-14.02 times in the 20-40 cm and 40-60 cm profiles of acidic purple soil,respectively,3.94-6.23 times and 4.06-4.33 times in neutral purple soil,and 2.04-4.07 times and 2.12-2.45 times in calcareous purple soil.The abundance of ARGs in the 40-60 cm profile of the acidic purple soil with application of manure alone was significantly higher than that in the 20-40 cm profile;however,the abundance of ARGs alone in the 40-60 cm profiles of the neutral and calcareous soil with application of manure was significantly lower than that in the 20-40 cm profile,which indicated that the risk of migration of manure ARGs to deeper soils was the highest in acidic purple soils.Correlation analysis showed that soil physicochemical properties such as p H and bacterial abundance were the main factors affecting the migration of ARGs in the profiles of acidic purple soil.The selection pressure of antibiotics and its synergistic mechanisms with heavy metals can drive the migration of ARGs in profiles of neutral purple soil and the horizontal gene spread of antibiotics and MGEs to promote the migration of ARGs in profiles of calcareous purple soil.(3)HT-qPCR was used to monitor the ARGs in the leachate seepage of 1 m soil layer.A total of 192 ARGs belonging to 9 different types were detected in leachate sampled from the purple soils with different p H,in which aminoglycosides,β-lactams,macrolides,multidrug and tetracycline resistance genes were the dominant ARGs detected in the leachates,accounting for 77%-86%.The absolute abundance and amount of ARGs leached into seepage water were significantly different after manure application in the three purple soils with the following order: acidic purple soil > neutral purple soil >calcareous purple soil.The leachates that might be derived from manure enriched 54-70,44-55 and 39-43 ARGs in acidic,neutral and calcareous purple soil eluents,respectively,indicating that the leaching of ARGs occurred in acidic purple soils and the risk of washing into a water body could be the highest.Variation partitioning analysis showed that the bacteria of Verrucomicrobia and Proteobacteria in acidic soil,those of Proteobacteria,Actinobacteria and Firmicutes in neutral soil,and those of Actinobacteria,Verrucomicrobia and Proteobacteria in calcareous contributed the most to the changes in the abundance of ARGs in the leachates;meanwhile,MGE-mediated horizontal gene transfer also contributed significantly to the changes in the abundance of ARGs in the leachate samples of neutral and calcareous purple soils,while the selection pressure of antibiotics had a greater impact on the spread of ARGs in acidic purple soil.In addition,the content of sand in acidic purple soil affected the leaching of ARGs,while the leaching of ARGs in neutral and calcareous purple soils was affected by the content of silt.(4)A total of 87 ARGs affiliated to 9 different types were detected in leaf endophyte of cabbage by HT-qPCR,in which aminoglycosides,β-lactams,macrolides,multidrug resistance and tetracycline resistance genes were the main ARGs,accounting for 75%-92%.Manure application significantly increased the number and abundance of ARGs in leaf endophyte of cabbage planted in three purple soils with different p H,the abundances of aminoglycosides,β-lactams,macrolides,multidrug and tetracycline resistance genes were also higher.There was no significant difference in the number of ARGs between manure application alone and manure + chemical fertilizer treatment,but the abundance of ARGs in single application manure treatment was significantly higher than that in manure application with chemical fertilizer treatment.The absolute abundance of ARGs in leaf endophyte of cabbage grown in acidic purple soil after manure application was significantly higher than that in neutral and calcareous purple soil.Compared with the control treatment,the absolute abundances of ARGs were increased by 183%-341%,112%-229% and 20%-142%,respectively.SEM analysis showed that antibiotics were the most important factor affecting the changes of ARGs,followed by MGEs and bacterial abundance;however,there were differences in the factors affecting ARGs enrichment in leaf endophyte of cabbage in purple soils with different p H,the bacterial communities and MGEs in cabbage leaves grown in acidic and neutral purple soils contributed greatly to the changes of ARGs,while in the calcareous purple soil,the interaction of bacterial communities,antibiotics and MGEs was the main factor.In conclusion,long-term application of manure facilitated the accumulation of ARGs in the three purple soils and their migration to the environmental medium,in which aminoglycosides,β-lactams,multidrug and tetracycline resistance genes were not only significantly accumulated in the topsoil,but also had the risk of migration to the profiles of soil,leachate,and crop.The accumulation and migration intensities of ARGs in acidic purple soils were higher than those in neutral and calcareous soils.Therefore,ARGs contamination can be reduced by decreasing the use of manure in acidic soil or regulating soil p H;the removal rate of aminoglycosides,β-lactams,multidrug and tetracycline resistance should be improved during composting,so as to reduce the risk of accumulation and control the spread of ARGs from the source. |
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