Research On Colonization Characteristics Of Enterobacter And Its Phytoremediation Mechanism Of Cadmium Contaminated Soil

In the face of increasingly serious soil pollution by heavy metals,plant-microbe combined remediation technology has become a research focus in the remediation of heavy metal contaminated soil due to its advantages of green,convenience and low cost.In order to enrich the plant growth-promoting bacteria and strengthen the mechanism of hyperaccumulator phytoremediation of cadmium(Cd)contaminated soil,Enterobacter sp.FM-1,a heavy metal-tolerant microorganism screened by our laboratory in the early stage was used as the research object in this study and Enterobacter sp.FM-1 was labeled with green fluorescent protein(GFP)labeling technology.The genetic stability,fluorescence detection,growth curve and Cd tolerance characteristics of the labeled strains were analyzed,the differences in physiological characteristics between the wild strains and the labeled strains were compared.Wild strains and labeled strains were inoculated into heavy metal contaminated soil and plants,the colonization number and distribution of Enterobacter sp.FM-1 in rhizosphere soil and plants were studied,the effects of the growth-promoting properties of Enterobacter sp.FM-1 on plant biomass,indole acetic acid(IAA)content,phosphorus content,antioxidant enzyme system and Cd absorption were discussed.Draws the following conclusions:1.The GFP-labeled Enterobacter sp.FM-1 that could grow on kanamycin plates containing50?g/ml were selected.After PCR amplification of the labeled strains,a clear positive band can be observed at 726 bp(target gene)of the gel electrophoresis imaging system.It shows that the GFP gene has been transferred into Enterobacter sp.FM-1.The green fluorescence can be observed through the fluorescence microscope,indicating that the GFP gene has been successfully expressed in Enterobacter sp.FM-1 cells.In addition,the labeled strain could still grow on the resistant plate and the plasmid stability reached more than 95%after 50 times consecutive passages,indicating that its genetic stability was good.Not only that,there was no significant difference between the labeled strains and the wild strains in growth and tolerance to Cd²⁺.The results indicated that Enterobacter sp.FM-1-GFP was a strain with good tolerance to heavy metals and good fluorescence characteristics.2.By the pot experiments,the fluorescent labeled Enterobacter sp.FM-1-GFP were inoculated into two soils in the recovery area and the downstream area of the mining area.The strains could be stably colonized in the soil for about 14 days,but the number of colonies in the two soils showed a downward trend with the extension of treatment time.Observed by fluorescence microscope,green fluorescence could be successfully detected in a variety of plant tissues such as the break of roots,main lateral root junctions,vascular tissue of roots,root apical meristems and inner-outer root epidermis of hyperaccumulation plants Bidens pilosa L.,Centella asiatica L.and Solanum nigrum L.,which indicated that the strains could colonize in the rhizosphere of three hyperaccumulators and the amount of colonization was about 10⁵-10⁶CFU/g.3.With medium-high inoculation concentrations,the Enterobacter significantly increased the biomass of plants,the content of IAA,iron,phosphorus in roots,stems,leaves and the absorption of Cd by plants.Compared with the control,the plant height and weight of Bidens pilosa L.,Centella asiatica L.and Solanum nigrum L.were increased by 50.73-85.19%and161.49-269.58%,64.75-134.91%and 190.44-327.30%,72.67-158.04%and 197.94-487.68%,respectively.In addition,the content of IAA,iron and phosphorus in the roots,stems and leaves were significantly increased compared to the control under the treatment with Enterobacter,among which the IAA increased by 24.17-337.59%,the content of iron and phosphorus increased by 145.47-195.65%and 32.72-100.30%,respectively.Moreover,the accumulation of heavy metals in the roots,stems and leaves of plants also increased with the increase of inoculation concentration.In the downstream area,the concentration of Cd in the leaves of Bidens pilosa L.reached 111.67 mg/kg under the treatment of 1.1×10⁹ CFU/ml,which was 69.71%higher than CK.The Cd content in the stems and leaves of Centella asiatica L.were all greater than 100 mg/kg under the treatment of adding Enterobacter.The Cd content in the roots,stems and leaves of Solanum nigrum L.also reached the maximum value under high concentration of Enterobacter inoculation,which were 1.54,1.87 and 2.82 times of the control,respectively.4.Enterobacter sp.FM-1 has great potential effect on vegetables alleviating oxidative stress under heavy metal stress.The bacteria can not only secrete IAA and phosphorus-soluble substances to promote plant growth,but also increase the content of chlorophyll and carotenoids by activating plant pigment synthase,thereby enhancing photosynthesis to promote vegetables growth.Among them,the chlorophyll content increased by 48.03-155.09%and the carotenoids increased by 48.31-124.35%under the medium-high inoculation concentration.In addition,Enterobacter sp.FM-1 also participated in the regulation of plant antioxidant mechanism,promoting the synthesis of peroxidase(POD)and catalase(CAT),accelerating the elimination of reactive oxygen species(ROS)such as superoxide(·O₂^-)by superoxide dismutase(SOD)and activated the antioxidant enzyme system of vegetables.At the same time,the inoculation of Enterobacter sp.FM-1 helped to convert a large amount of glutathione(GSH)into phytochelatins(PCs),accelerating the chelation and removal of heavy metals by acid-soluble thiol(-SH)and alleviated the toxic effects of heavy metals caused by Cd stress.