Bacterial Community Structure,degrading Genes And Ecological Functions In Soil-root Surface-plant System Under Different Levels Of PAH Pollution

Polycyclic aromatic hydrocarbons（PAHs）are a group of persistent organic pollutants（POPs）that have a potential "teratogenic,carcinogenic,mutagenic" effect and are difficult to degrade.PAHs can be absorbed and accumulated by plants,which will cause great threat to human health through the food chain.The combination of plant and microbe is often used for remediation of PAH-contaminated soils and the reduction of PAH pollution risk of crops in polluted areas.Rhizosphere microdomain is the most active area of microorganism in soil-plant system,which is important for the removal of organic pollutants.In recent years,it has been found that the soil-plant system under PAH contamination contains a large number of PAH-degrading microorganisms,which carry a considerable number of genes responsible for PAH degradation.Although the existence of the PAH-degrading gene has been confirmed in soils and plants,its abundance and distribution in rhizosphere and plants is unclear.Moreover,there are few reports on whether degradation of PAHs interacts with the processes of nitrification,denitrification and methane oxidation,and how the relevant genes are distributed under the stress of PAHs.The soil,collected from PAH-contaminated areas,was artificially enhanced with phenanthrene and pyrene,and then set up as different pollution levels.Alfalfa（Medicago sativa L.）was used as the target plant to conduct greenhouse potted experiments in the root box which divided the soil into rhizosphere soil,near rhizosphere soil,far from rhizosphere soil and control soil.Sphingobium sp.RS2,a PAHs degrading strain isolated in our lab previously,was used as the test strain.It has been proved that RS2 can mineralize the phenanthrene The distribution of key genes responsible for PAH degradation in soil-root surface-plant system under different levels of PAH pollution were studied by HPLC and real-time quantitative PCR.By high throughput sequencing technology,the composition and diversity of endophytic bacteria in the root and shoot of alfalfa,root surface bacteria and soil bacteria communities were analyzed,and the response characteristics of bacterial communities to PAH pollution were revealed.Moreover,the distribution of functional genes related to carbon cycle and nitrogen cycle in soil-root surface-plant system under different levels of PAH pollution were also studied.This study provides relevant technical support and theoretical basis for the prevention and control of PAH pollution by the combination of plant and microorganism.The main research results are as follows:1.The distribution of PAH degrading genes in soil-root surface-alfalfa system under different PAH pollution levels.The growth of alfalfa and PAHs residual concentration in soil-root surface-plant system under different PAH pollution levels were detected.High levels of PAH contamination could inhibit the growth of alfalfa,while the inoculation of RS2 with alfalfa could promote the plant growth.The total PAH contents in rhizosphere soils were significantly lower than that in non-rhizosphere soils.Furthermore,there are a number of PAH-degrading genes such as nidA,nahAc and phe in soil-root surface-plant system.The copy numbers of genes responsible for PAH degradation increased with the increase of soil PAH content.Furthermore,the abundances of these genes on root surfaces were the highest,followed by those in soils and the abundances of these genes in shoots and roots were the least.In the same sampling area,the copy number of phe gene was significantly higher than that of nahAc or nidA.Under different levels of contamination,the residue content of phenanthrene or pyrene was positively correlated with the richness of nahAc gene.2.The characteristics and diversity of bacterial community structure in soil-root surface-alfalfa system under different PAH pollution levels.The high-throughput sequencing analysis of the V4-V5 region of 16S rRNA gene was carried out to investigate the bacterial communities of alfalfa planting soil,root surface,root and shoot tissues of alfalfa.The results showed that the order of diversity of bacterial community structure was soil>root surface>root>shoot,which indicated that the diversity of bacterial community structure in soil was much higher than that in plant.Secondly,many bacterial groups in the plant also exist in the soil,which means that some bacteria in the soil can enter the plant after multiple choices of rhizosphere,root surface and root internal environment.Next,there are obvious differences in the composition of bacterial community structure among different sampling sites.The dominant genus of each site are different.Bacillus occupies an absolute dominant position on the root surface,and Ensifer has an obvious advantage in the root.In addition,RS2 can promote the relative abundance and interactions of PAH degrading bacteria（such as Sphingomonas,Paenibacillus,Nocardioids）in the root surface.3.The abundance and distribution of genes related to carbon cycle and nitrogen cycle in soil-root surface alfalfa-system under different PAH pollution levels.In the soil-root surface-plant system,PAH pollution has a certain impact on the abundance and distribution of functional genes related to carbon and nitrogen cycles.In general,the abundances of these genes on root surfaces were the highest,followed by those in soils and the abundances of these genes in shoots and roots were the least.With the increase of phenanthrene and pyrene content,all kinds of functional genes are affected to varying degrees.With the increase of pollution level,the abundance of amoA and narG increased and the abundance of nirK,cbbL,mcrA,and pmoA decreased.According to the correlation analysis of high-throughput sequencing results in Chapter 3,nitrifying bacteria,denitrifying bacteria,ammonia-oxidizing bacteria,methane-oxidizing bacteria and other related bacteria were detected in soil-root surface-plant samples.Due to the different effects of PAH contamination on related bacteria,the correlation between different the richness of functional bacteria and genes is also different.The richness of Nitrospira was positively correlated with the copy number of nirK gene.The richness of Rhodobacter was negatively correlated with the copy number of amoA,and the richness of Bradyrhizobium was negatively correlated with the copy number of narG.Furthermore,the richness of Methylobacter and Methylophilus was positively correlated with the copy number of cbbL,mrcA and pmoA gene,respectively.