Response Mechanisms And Assembly Processes Of Rhizosphere Microbes To Typical Emerging Contaminants In Black Soil

The black soil region in Northeast China is an important commodity grain base.In recent years,research on the protection and utilization of black soil has focused on improving soil fertility and crop yield.However,insufficient attention has been given to the health of black soil and its impact on root-associated microbial communities,particularly the migration and transformation of new pollutants in crops under human activities such as continuous cropping and excessive fertilizer application.Emerging pollutants are toxic and harmful chemicals that are discharged into black soil,possessing characteristics such as biological toxicity,environmental persistence,and bioaccumulation.They pose significant risks to agro-ecosystems and human health,but have not yet been effectively regulated or are inadequately managed.Emerging pollutants are relatively "new" compared to traditional regulated pollutants and are not currently under effective regulation.Large amounts of microplastics(MPs)and antibiotic resistance genes(ARGs)have been found in black soil farmland.Therefore,this study focuses on these two typical emerging pollutants.These human activities can lead to selective enrichment of antibiotic resistance genes(ARGs)and the continuous accumulation of microplastics(MPs)from field mulching and other sources.This study focused on typical black soil in Northeast China and used sampling,sequencing,and analysis of root-associated and non-root-associated microbial communities of typical crops,along with field and greenhouse experiments involving different fertilization types,tillage methods,and cropping systems.We elucidated the impact of MPs and ARGs on the structure and function of root-associated microbial communities of typical crops(rice and maize),identified the driving mechanisms of microbial communities in response to new pollutant stress in black soil,clarified the interaction mechanisms between new pollutants and heavy metals,revealed the community assembly and regulation mechanisms of soil microorganisms under new pollutant stress,and isolated and screened root-associated microbial strains for antibiotic resistance phenotype monitoring and analysis.The main research findings are as follows:(1)In this study,a rice pot experiment was conducted to investigate the effects of two biodegradable microplastics(PLA and PPC)of the same particle size on the composition of the rice rhizosphere bacterial community using absolute quantitative amplicon sequencing technology.It was found that the available cadmium content in the soil treated with PLA and cadmium was significantly higher than that in the other treatment groups.Meanwhile,the cadmium content in the plant tissue(including aboveground and underground parts)treated with PLA and cadmium was significantly higher than that in the PPC and cadmium-treated groups.Absolute quantification revealed that the absolute contents of Cyanobacteria and Firmicutes in the rhizosphere were significantly higher in the CDPPC group than in the CDPLA group.PCo A results showed significant differences in the rhizosphere bacterial community composition between the two microplastic treatments,and random forest analysis identified Burkholderiales and Xanthomonadales as biomarkers for microplastic and cadmium co-exposure.Procrustes analysis showed a significant correlation between rich and rare bacterial groups in all samples.In addition,skewed Mantel tests showed that soil p H was the main environmental factor influencing the rare bacterial community in the CDPPC group.Based on the NST value of the Bray-Curtis matrix,except for the PPC group,the rich bacterial groups were mainly controlled by random processes.Deterministic processes dominated the rare bacterial groups,except in the PPC group.It is noteworthy that different bacterial taxa exhibited opposite trends for PPC and PLA.If the rich bacterial groups are mediated by deterministic processes,the rare bacterial groups must be accompanied by random processes and the relative contribution of the random processes decreases with the addition of coexisting pollutants.SEM analysis results showed that the enriched groups in the CDPPC treatment had a negative regulatory effect on the cadmium content in rice,while the rare species in the CDPPC population positively regulated rice biomass,p H,and soil enzyme activity.(2)Based on a long-term field positioning experiment of fertilization and straw return,using metagenomic sequencing technology,we found that the diversity of antibiotic resistance genes(ARGs)in both rhizosphere and non-rhizosphere soils treated with chemical fertilizers was significantly higher than that in the unfertilized group,especially in non-rhizosphere soils,where multiple resistance genes were the most abundant type of ARG.The results of mobile genetic elements(MGEs)were similar to ARGs,with their diversity and abundance significantly higher in both non-rhizosphere and rhizosphere samples treated with fertilizer.Procrustes analysis confirmed a significant correlation between ARG abundance and bacterial community composition,especially in rhizosphere soil.Fertilizer application decreased microbial diversity,emphasizing the importance of microbial diversity in stabilizing microbial communities and serving as a protective barrier against negative stress.In addition,the microbial community after straw addition did not show statistical changes.According to Bray-Curtis distance and Permanova analysis,long-term fertilizer use led to selective enrichment of ARGs.Using machine learning methods,we identified31 microbial genera associated with ARGs and MGEs.Combining Source Tracker algorithm and bipartite network analysis,we found that Pseudomonas,Streptomyces,and Bacillus were the main genera responsible for the spread and horizontal transfer of ARGs.After large-scale isolation of corresponding strains from the maize rhizosphere,we conducted culture,antibiotic resistance phenotype,and species identification tests.Three genera showed strong antibiotic resistance characteristics,with Pseudomonas demonstrating particularly outstanding resistance to a range of Response Mechanisms and Assembly Processes of Black Soil Rhizosphere Microbes to Typical Emerging commonly used antibiotic classes.All relevant strains showed widespread resistance to semi-synthetic or synthetic antibiotics such as sulfamethoxazole,ampicillin,and ciprofloxacin,as well as high levels of resistance to other tested antibiotics.Macro-genome binning analysis of Pseudomonas indicated the extensive presence of multiple ARGs and MGEs in its contigs,particularly with int I1 mediating the horizontal transfer of multidrugs resistance genes.(3)Based on the metagenomic analysis of antibiotic resistance genes(ARGs),neutral theory,null model,and ecological niche theory models were constructed to investigate the response of microbial communities and ARGs to human agricultural practices.The study found that the tolerance of microbial communities and ARGs varied,resulting in inconsistent community assembly processes.Environmental filtering and niche partitioning caused changes in microbial community structure,while random events such as diffusion and stochastic drift played an important role in shaping the ARGs structure.Compared to rhizosphere soil,non-rhizosphere soil exhibited relatively higher spatial heterogeneity in the ARGs community assembly process,making it a more stable habitat.The antibiotic resistance genome in the rhizosphere soil was relatively random,indicating that rhizosphere ARGs were relatively less influenced by abiotic factors,and cross-boundary interactions between microbial communities would become more important.Deterministic and random factors interacted simultaneously to affect the assembly of microbial communities.Therefore,even if the assembly process is completely random,a smaller ecological niche species pool surviving under long-term fertilizer addition conditions could lead to higher similarity between ARGs communities and ecological niche assembly structure.The study shows that the impacts of MPs pollution and ARGs presence on agricultural root-associated microbial communities are complex.Degradable MPs pollution can lead to reduced microbial diversity,changes in community structure,and significant differences in ecological construction processes.In addition,the presence of PLA may accelerate the spread of antibiotic resistance genes.The presence of ARGs may lead to a decrease in antibiotic sensitivity of root-associated microbial isolates,resulting in the emergence of antibiotic resistance phenotypes.Furthermore,ARGs may also cause changes in root-associated microbial community structure and a reduction in ecosystem functions.In summary,the impacts of MPs pollution and ARGs presence on agricultural root-associated microbial communities differ significantly in ecological functional composition.To reduce long-term ecological impacts,measures need to be taken to reduce MPs pollution and to use fertilizers rationally to decrease the spread of ARGs and increase antibiotic resistance in microbial strains.