Conjugative Transfer And Its Mechanism Of Antibiotic Resistance Genes Mediated By Plasmid RP4 Under Agricultural Fungicide Stress

The substantial and frequent applications of fungicides and organic fertilizers in greenhouse agricultural production have led to an increasingly serious pollution problem of both fungicide residues and antibiotic resistance genes(ARGs)in soils.Previous studies have found that exposure to disinfectants,o-xylene,heavy metals and other environmental pollutants could promote the horizontal gene transfer of ARGs,but the dissemination characteristics and regulatory mechanisms of ARGs under the stress of agricultural fungicides are still unclear.In this thesis,four commonly used fungicides(triadimefon,chlorothalonil,azoxystrobin,and carbendazim)were used as target compounds to investigate the effects of fungicides on soil ARGs and their potential molecular mechanisms using metagenomic sequencing technique based on the indoor simulated soil ecosystem.The intragenera(donor: E.coli MG1655;recipient: E.coli HB101)and intergenera(donor: E.coli MG1655;recipient: P.putida KT2440)conjugative transfer systems of antibiotic resistant plasmid RP4 were established to explore the conjugative transfer frequency under fungicide stress.Meanwhile,the mechanism was elucidated at the cellular and molecular levels using transmission electron microscopy,flow cytometry,RT-q PCR,and RNA-seq.The abundance and diversity of ARGs in the greenhouse soils were significantly higher than those in the mountain soils.After four fungicides treatment for 60 d,the total abundance of ARGs in soils was 1.02-to 1.98-fold higher(greenhouse soils)and 1.12-to2.16-fold higher(mountain soils)than the corresponding controls,among which the abundance of sulfonamide and tetracycline ARGs in the greenhouse soils and the abundance of multidrug ARGs in the mountain soils increased significantly.The maximum increment was observed in the carbendazim-treated soils,followed by azoxystrobin and chlorothalonil,while triadimefon had less effect on the abundance of soil ARGs.Meanwhile,fungicide treatment increased the abundance of plasmid and integron genes in the soil.There was a significant positive correlation between the abundance of ARGs and the abundance of integron genes in the fungicide-treated greenhouse soils,but there was no correlation in the mountain soils.Metagenomic assembly also confirmed the co-occurrence patterns of ARGs(e.g.,sul2 and sul1 genes)and mobile genetic elements(e.g.,transposase and integrase genes)in the greenhouse soils.These results suggested that the increasing abundance of ARGs in the fungicidetreated greenhouse soils was mainly associated with the horizontal gene transfer mediated by mobile genetic elements.The intragenera conjugative transfer frequency of plasmid RP4 gradually increased with the increasing treatment concentrations(0-10 ?g/m L)under chlorothalonil,azoxystrobin,and carbendazim exposure,and the corresponding fold changes were 2.62,2.38,and 3.08 times higher than the control at 10 ?g/m L.Triadimefon had no significant effect on the conjugative transfer.In the intergenera system,the conjugative transfer frequency of plasmid RP4 also increased with the increasing treatment concentrations of fungicides(? 1 ?g/m L),and it decreased when the concentration reached 10 ?g/m L.The maximal intergenera conjugative transfer frequency of plasmid RP4 was achieved at 1?g/m L fungicides exposure,which was 1.38-fold(triadimefon),3.52-fold(chlorothalonil),4.40-fold(azoxystrobin),and 2.77-fold(carbendazim)of the control,respectively.The promoting effect of chlorothalonil,azoxystrobin,and carbendazim on the conjugative transfer might be related to the expression of conjugation-related regulatory genes on the plasmid RP4,and this effect was also varied with intra-and intergenera conjugative transfer systems.These three fungicides could promote the conjugative transfer of plasmid RP4 by suppressing the expression of the regulatory genes kor A and kor B and increasing the expression of the conjugative transfer system genes trb Bp,tra F,trf Ap and tra J.Meanwhile,azoxystrobin and carbendazim could increase the expression of plasmid vertical transfer inhibitor gene kil A to promote the conjugative transfer of RP4 plasmid in intragenera conjugation.RNA-seq results suggested that fungicide could increase the expression level of multiple genes on the tra and trb operons.Triadimefon and chlorothalonil also decrease the expression level of entry exclusion gene trb K.Fungicides exposure increased the intracellular ROS production in the donor and recipient bacteria.When exposed to chlorothalonil and azoxystrobin(10 ?g/m L),the intracellular ROS production in the recipient P.putida KT2440 was 2.33-and 1.50-fold of that in the control,respectively.RT-q PCR and RNA-seq results showed that chlorothalonil,azoxystrobin,and carbendazim increased the expression of ROS,and SOS response genes.Meanwhile,fungicides also increased the damage to the cell membrane of donor/recipient bacteria and enhanced their cell membrane permeability.The cell membrane permeability of the recipient P.putida KT2440 was prone to be affected by the high concentration of chlorothalonil,azoxystrobin,and carbendazim.RT-q PCR and RNA-seq results also showed that chlorothalonil,azoxystrobin,and carbendazim increased the expression of outer membrane protein genes.These results indicated that fungicide stress could enhance the oxidative stress response,SOS respsonse,and cell membrane permeability to promote the conjugative transfer of plasmid RP4.In addition,this research also found that the four fungicides increased the expression of pili protein-related genes in donor E.coli MG1655 and recipient P.putida KT2440.Chlorothalonil,azoxystrobin and carbendazim could induce up-regulation of flagellar assembly function genes in the recipient,which may also play a role in the conjugative transfer of plasmid RP4 caused by fungicides.This study reveals the shift and molecular mechanism of ARGs in the fungicidescontaminated soils,and elucidates the conjugative transfer characteristics and its mechanisms of plasmid-mediated ARGs under fungicide stress.These results provide scientific guidance for the comprehensive evaluation of the ecological risk of fungicide pollution and the reasonable use of fungicides,and also provide a theoretical basis for revealing the spread mechanism of ARGs in the environment polluted by fungicides.