Effects Of Application Of Cow Manure/Compost On The Transmission Of Antibiotic Resistance Genes In Farmland Soil And Lettuce

Antibiotic resistance genes(ARGs),which are different from traditional chemical pollutants,have become a new type of environmental pollutants due to their characteristics of persistence,diffusion and explosiveness.They were considered as one of the major challenges to human health in the report of the World Health Organization in 2014.With the rapid development of global intensive economy,ARGs are widely distributed in the world due to human activities,and can be detected in soil,atmosphere,water and sediment.In agricultural production activities,people often use animal manure as organic fertilizer to increase the nutrient substrate content of the soil.However,the manure usually contains a large number of antibiotic residues,ARGs,antibiotic resistant bacteria(ARB)and human pathogenic bacteria(HPB),which increases the pollution level and risk of ARGs in the soil.At present,the risk of manure application on ARGs transmission in farmland soil and vegetables is still unclear.Therefore,we conducted field experiments to reveal the effect of manure application on ARGs and their migration in soil and lettuce.In this study,three treatments included unfertilized treatment(CK),application of cow manure treatment(CM)and application of cow manure compost treatment(CMC)were set up in the farmland.Lettuce was planted in each treatment,and rhizosphere soil and lettuce samples were collected at harvest time,respectively.DNA was extracted from original soil,cow manure,cow manure compost,rhizosphere soil and different parts of lettuce(i.e.root endophyte,leaf endophyte and phyllosphere).By using high-throughput quantitative PCR,amplicon sequencing and constructing the HPB database,we studied the abundance and diversity of ARGs and HPB in different samples and the structure and composition of bacterial communities.The main results of this paper are as follows:(1)A total of 211 ARGs were detected in all samples through high-throughput quantitative PCR,.The detected ARGs belonged to three main resistance mechanisms:antibiotic deactivation,cellular protection and efflux pump.The genes conferring resistance to aminoglycosides,beta-lactamase,MLSB,multidrug and tetracycline were the most frequently detected ARGs in all samples.Application of cow manure and compost significantly increased the diversity of ARGs in rhizosphere soil.The application of cow manure/compost did not significantly increase the absolute abundance of ARGs in rhizosphere soil,root endophyte,leaf endophyte and phyllosphere of lettuce.The abundance and diversity of mobile genetic elements(MGEs)in rhizosphere soil and lettuce tissue were not obviously altered by cow manure and compost.(2)The non-metric multidimensional scalings(NMDS)analysis revealed that there were significant differences in the patterns of ARGs in rhizosphere soil,root endophyte,leaf endophyte and phyllosphere of lettuce(Adonis test,P<0.001,R~2=0.67).Cluster analysis showed that the profiles of ARGs were clearly separated among different sample types rather than different treatments.Bipartite network analysis showed that the application of cow manure/compost could introduce new ARGs into soil and lettuce,and more diverse transposon genes(tnp A-05 and Tp614)were introduced into the soil by cow manure compost than cow manure.It indicated that application of cow manure and compost might introduce new ARGs and MGEs into the soil and lettuce,which had the potential risk of ARGs transmission.(3)High-throughput sequencing was used to analyze the characteristics of bacterial community structure in different samples,and the results showed that the application of manure and compost significantly increased the diversity of bacterial community in rhizosphere soil and root endophyte of lettuce.A total of 35 HPB species were identified based on BLASTN sequence alignment,and Proteobacteria and Firmicutes were the most frequently detected HPB in all samples.Compost treatment significantly increased the diversity of HPB in leaf endophyte of lettuce and the relative abundance of Enterobacter cloacae isolate Nr.3 in the phyllosphere of lettuce.(4)Ordinary least squares(OLS)regression analysis showed that the transposon gene was more closely related to ARGs than the integrase gene,indicating that application of manure could promote the risk of horizontal gene transfer of ARGs.The results of Procrustes test and Mantel test showed that bacterial community structure was also the important factor affecting the spread of ARGs.The network co-occurrence analysis between ARGs and HPB indicated that the 11 pathogenic bacteria containing Mycobacteria might be the potential hosts of ARGs.