| Polycyclic aromatic hydrocarbons(PAHs)are a type of persistent organic pollutants(POPs)that are ubiquitous and persistent in soils.Due to their strong hydrophobicity and absorption by plants,the residual PAHs in farmlands are taken up and accumulated by crops,which eventually endanger human health through their entry into food chains.Furthermore,the current soil survey in China shows that the residual forms of PAH pollution are severe.Therefore,an urgent problem to be solved arises on how to reduce the PAHs pollution of crops growing in these soils.Recently,it has been found that colonizing certain types of functional microbial strains in plant rhizosphere,root surface and body can effectively promote plant growth,reduce the residual concentration of PAHs in the plant,and then reduce the risk of plant contamination.However,few reports have been reported on the mechanisms of PAH reduction in plants regulated by functional strains.It is,therefore,imperative to explore this in planta regulation mechanism of PAHs to make use of this technology to solve the problem of PAH residues in crops.In this study,a phenanthrene-degrading sphingomonad,Sphingobium sp.RS2,isolated from the root surface of Canada horseweed(Conyza Canadensis L.Cronq.),and its gfplabeled strain RS2-gfp(marked by green fluorescent protein,GFP)were used.Phenanthrene was selected as a model PAH pollutant in this study.By exploring the metabolic pathway and degradative genes encoded by strain RS2 for phenanthrene degradation,the strain was colonized on the root surface of water spinach(Ipomoea aquatica Forssk.)in the hydroponic experiment to explore the effects of colonizing the functional isolate RS2 on phenanthrene residues in plants.The mechanisms of reducing phenanthrene pollution by using beneficial bacteria on the root surface were analyzed based on the following four aspects.The number and distribution of functional bacterium RS2 in plants,the distribution and expression of essential genes for phenanthrene degradation,the effect of colonial bacterium RS2 on the structure of bacterial community on the root surface and in vivo,and the activities of PAH-metabolic enzymes in plants,were evaluated.The main results are shown as follows:1.Phenanthrene degradation pathway and colonization-related biological characteristics of strain RS2.The rhizospheric beneficial bacterium Sphingobium sp.RS2 was capable of growing in the environment where phenanthrene was the sole carbon source and energy source.After 72 hours of cultivation with strain RS2,the phenanthrene concentration in culture reduced from 100 mg·L-1 to less than 0.02 mg·L-1,with a degradation rate reaching more than 99%.When using high-performance liquid chromatography-mass spectrometry(HPLC-MS)technology to study the degradation pathway of phenanthrene metabolism,it was found that the intermediate products of phenanthrene metabolism corresponded to the molecular weights of 1-hydroxy-2-naphthalic acid,salicylaldehyde,salicylic acid,and other substances.According to the degradation pathway of phenanthrene metabolism by other bacteria,the strain was predicted to utilize the meta-cleavage pathway for phenanthrene degradation,as evidenced by salicylic acid production.Then,in the study of biological characteristics of RS2,it was found that strain RS2 showed certain chemotaxis to phenanthrene and its intermediate metabolites,as well as some simulated root exudates;it had portrayed biofilm formation in in vitro culture for 120 hours;and through plant growth-promotion experiments,strain RS2 displayed IAA and iron carrier secretion,as well as inorganic and organic phosphorus solubilization.Additionally,similar experiments with strain RS2-gfp showed that the gfplabeled isolate had no significant difference on the degradation and biological characteristics of strain RS2.2.The changes of enzyme activities and gene expressions involved in phenanthrene degradation with induction time in strain RS2.After induction of strain RS2 with different inducers(50 mg·L-1 of phenanthrene,catechol and o-phthalic acid),it was found that the activity of the initial ring hydroxylating dioxygenase(RHD)or catechol-2,3-dioxygenase increased significantly,but the activity of catechol-1,2-dioxygenase did not change much.Then the genome of RS2 was sequenced by whole genome sequencing technology.After homology analysis with the phenanthrenedegrading gene database of bacteria,it was found that there was a unique PAH-degrading gene cluster(composed of three types of gene clusters:nah,bph and xyl genes)of Sphingomonads in strain RS2.Among them,the most critical is the bphA1fA2f gene and the bphA1cA2c gene,which are involved in the coding of the alpha and beta subunits(α and β)of the initial cyclohydroxygenase and salicylic acid hydroxylation dioxygenase,respectively.The bphC gene is responsible for the coding of cyclohydroxygenase(exodiol dioxygenase).Moreover,nine essential phenanthrene-degrading genes(nahD-bphA1cA2cA3-bphC-bphBnahE-bphA1fA2f)were selected and induced by 100 mg·L-1 phenanthrene to study the variation of gene expression by qPCR.It was found that the expression of phenanthrenedegrading genes in RS2 was significantly up-regulated under phenanthrene induction,and the expression of bphA1c gene was the highest,and different genes showed their specificity in different durations.This phenomenon indicated that phenanthrene degradative genes in RS2 are regulated by phenanthrene-induced expression in the environment.3.The colonization of RS2-gfp on root surface could reduce phenanthrene pollution in water spinach.In order to observe the colonization and distribution of strain RS2 on the root surface and in vivo,the strain RS2-gfp labeled with GFP was selected to colonize on the root surface of water spinach,and hydroponic culture experiment was carried out.The effect of strain RS2-gfp colonization on the uptake and accumulation of phenanthrene in water spinach was further studied.The results showed that strain RS2-gfp could colonize well on the root surface of water spinach and the internal root tissues of water spinach,before its distribution to its stem and leaf.Also,the colonization of strain RS2-gfp significantly promoted the biomass of water spinach root and stem and leaf,which reduced phenanthrene accumulation and concentration in the plant bodies,effectively reducing the risk of phenanthrene contamination in water spinach.4.The mechanisms involved in reducing phenanthrene contamination in water spinach by colonization of functional strain RS2-gfp on the root surface.Initially,after plate counting experiments,it was found that the number of culturable bacteria and phenanthrene-degrading strain RS2-gfp on the root surface and in inner part of water spinach increased after RS2-gfp colonization.Secondly,the distribution and expression of bphA1f gene was monitored,with the essential phenanthrene-degrading gene showing that the abundance and expression of this gene in the treatment group colonized with RS2-gfp were significantly higher than those in the control group,and the gene copy and expression s mainly concentrated in the plant root environment.Then,16S rRNA gene amplification analysis was used to study the difference in bacterial community structure in the plant-nutrient fluid system.It was found that the colonization of RS2-gfp in the phenanthrene-contaminated environment could promote the number and synergy of related PAH-degrading bacteria in the rhizosphere environment,such as Burkholderia,Bacillus,Sphingomonas,Massilia among others.The increase in strains promoted the construction of an eco-environmental community system for phenanthrene metabolism.Finally,the changes in the activities of PAH-related enzymes in the plant metabolism were studied.It was found that the colonization of functional strain RS2-gfp could also promote the activities of polyphenol oxidase(PPO)and peroxidase(POD),and accelerate the metabolism of phenanthrene in plants. |
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