Research On The Contamination And Regulation Of Antibiotic Resistance Genes In Arable Black Soils Of Northeast China

Black soil areas are an important food production base in northeast China,which also plays a critical role in maintaining national food and ecological security.Recently,soil degradation becomes more and more serious due to the excessive reclamation and extensive management measures in the black soil region of northeast China.Applying animal manure can effectively boost soil fertility,improve soil physicochemical processes and alleviate soil degradation process.However,antibiotic resistance genes(ARGs)and antibiotic resistant pathogens are also introduced,which pose a potential threat to human and animal health.Therefore,clarifying the effects of different fertilization systems on ARGs in black soil of northeast China and revealing the spread of ARGs in arable black soils with different soil organic matter(SOM)content as well as effect of antibiotics on the dissemination of ARGs were explored by combining field long-term localization test with microcosms experiment.In addition,the effects of lime addition on the dissemination of ARGs were analyzed by pot experiment.The results are showed as follows:(1)The distribution of ARGs in arable black soil was tested by using highthroughput quantitative polymerase chain reaction(HT-q PCR)with soil samples collected from four fixed long-term fertilization experimental stations and two open farmland regions.The results showed that the application of animal manure significantly increased the number and abundance of ARGs and mobile genetic elements(MGEs)in soils,mainly conferred resistance to multidrug resistance.The composition of ARGs is not affected by geographical distribution,and the difference of antibiotic resistome is mainly related to fertilization systems.A significant positive correlation between the relative abundance of ARGs and MGEs including transposon and integron genes was observed.In addition,aad A1-01 and mex F,conferring resistance to aminoglycoside and multidrug,respectively,as predictive genes could monitor ARGs contamination.(2)Using HT-q PCR and Illumina Mi Seq techniques,the temporary changes of ARGs in black soils with different organic matter content were studied by a microcosm experiment.The results showed that the number of ARGs in soils with low SOM content was significantly higher than that in soils with high SOM content,and the absolute of ARGs in the low OM soils significantly increased with the extension of incubation time.The application of animal manure can increase the abundance of ARGs and MGEs,but it had litter effect on the ? diversity of bacterial communities in low OM soils.The SOM content was negatively correlated with the relative abundance of ARGs,as well as 23 ARGs from animal manure.The structural equation model(SEM)analysis showed that the decrease of SOM content can increase soil bacterial diversity and reduce soil bacterial abundance,and boost the abundance of MGEs,which ultimately promotes the spread of ARGs in soils.(3)The antibiotics can increase the selection pressure of soils and increase the number of resistant bacteria.We investigated the effects of veterinary tetracycline and sulfonamide antibiotics on the ARGs and bacterial communities in soils by a microcosm experiment.The results showed that the number of culturable bacteria was the largest in high OM soils,and the antibiotics could reduce the number of culturable bacteria in soils.With the increase of antibiotic concentration,the number of culturable resistant bacteria decreased gradually.The decreased number of culturable bacterial was positively correlated with SOM content.The absolute abundances of ARGs(tet Q,tet W,sul1 and sul2)in low OM soils were generally higher than that in high OM soils.With the extension of incubation time,the abundance of ARGs in low OM soils increased gradually,but the bacterial abundance had little changes.The variation of gene abundance was related to soil,incubation time and antibiotic concentration,among which soil was the dominant factor.Antibiotic addition altered soil bacterial community composition and structure,as well as microbial interactions,but has little effect on OTU abundance and Shannon diversity index.In addition,resistance genes,integrons and bacterial diversity were negatively correlated with SOM content.(4)Lime is a common conditioner for acid soils,which can improve soil fertility and increase crop production.However,studies about the effects of lime on the dissemination and migration of ARGs in soils are rarely reported.Hence,we found that the application of lime significantly reduced the number and abundance of ARGs in the bulk soils,rhizosphere soils and leaf endophyte by the indoor pot experiment with lettuce.The structure of ARGs and bacterial communities were significantly different among bulk soils,rhizosphere soils and leaf endophyte as well as the lime treatments of the same sample.Lime application changed bacterial community composition and increased bacterial community diversity,the number of manure-derived ARGs and OTU were gradually decreased from soil to lettuce leaves.SEM analysis showed that lime application could directly affect ARGs abundance and indirectly affect ARGs abundance by affecting MGEs abundance.Bacterial diversity has a strong negative effect on ARGs,but bacterial abundance has no effect on ARGs and MGEs.