| Heavy metal contamination in soils is one of the world's major environmental problems,posing significant risks to human health as well as to ecosystems.Phytoremediation,with its in situ,low-cost and environmental friendly nature,is being developed as a novel and highly promising technology for the remediation of heavy metal-contaminated soils.However,this plant-based technique is often inefficient due to the phytotoxicity and low metal bioavailability in soil,which hinder its large-scale application.Therefore,how to improve the efficiency of phytoremediation has become the research hostpot at home and abroad.Bioaugmentation-assisted phytoremediation is one of a promising method for enhancing the efficiency of phytoremediation.Against this background,the selection of functional microbes and their role in assistance of phytoremediation of heavy metal-contaminated soils were studied in this thesis.In this thesis' Cd-hyperaccumulator Solanum nigrum L.and its bacterial endophytes were selected as the material and objects for the study of "plant-microbe"combined remediation.First of all,we analysed the diversity and structure of endophytic bacterial community in S.nigrum and its dynamic changes under heavy metal-contaminated environment,thereby getting the characteristic of bacterial endophytic bacterial community and the interactions among endophytes-heavy metal-plants.On this basis,we isolated and selected the potential functional bacterial endophytes for bioaugmentation-assisted phytoremediation of heavy metal-contaminated soils,to build the new type and efficient "S.nigrum-functional endophytes”and discussed the mechanism involved in the interaction between host plants and inocula in detail.The details are summarized as follows:(1)Investigations on the diversity of endophytic bacterial populations associated with Cd-hyperaccumulator plant S.nigrum growing in a metalliferous soil,thereby achieving the information of endophytic bacterial community in S.nigrum.It was found that the diversity of endophytic bacterial populations was abundant in S.nigrum after analyzed by 16S rDNA cloning and sequencing.Phylogenetic analysis reveled that the majority of clones were affiliated with Proteobacteria(61.93%),which included a(41.12%),?(6.09%)and ?(14.72%)subclasses.Other clones belonged to Bacteroidetes(12.18%),Firmicutes(2.03%),Actinobacteria(18.78%)and uncultured bacteria(5.08%).The dominant genera were Sphingomonas and Pseudomonas,which accounted for 24.37%and 12.18%of total clones respectively.In addition,different bacterial communities were found in different plant tissues which indicated that endophytes were plant tissues-sensitive.(2)A comparative analysis of endophytic bacterial communities associated with two hyperaccumulators(Solanum nigrum L.and Phytolacca acinosa Roxb.)growing in metalliferous soils.The denaturing gradient gel electrophoresis analysis(DGGE)showed that the endophytic bacterial community structures were affected by both the level of heavy-metals pollution and the plant species.Heavy-metals in contaminated soil determined,to a large extent,the composition of the different endophytic bacterial communities in S.nigrum growing across soil series.Detailed analysis of endophytic bacterial populations by cloning of 16S rRNA genes amplified from the stems of the two plants at same site revealed a different composition.The most abundant genus in S.nigrum was Sphingomonas(23.35%)and Pseudomonas(12.18%),while Pseudomonas(21.40%)and Agrobacterium(12.44%)prevailed in P.acinosa.These results suggest that both heavy-metals pollution and plant species contribute to the shaping of the dynamic endophytic bacterial communities associated with hyperaccumulators.(3)Analysis and characterization of cultivable heavy metal-resistant bacterial endophytes isolated from S.nigrum and their potential use for phytoremediation.Thirty Cd-tolerant bacterial endophytes were isolated from roots,stems,and leaves of S.nigrum,phylogenetic analysis based on 16S rDNA sequences showed that these isolates belonged to Bacillus,Arthrobacter,Flavobacterium,Chryseobacterium,Serratia,Microbacterium,Pseudomonas,Sphingomonas,etc.A majority of isolates(70%)had the capacity to produce indole-3-acetic acid(IAA),and about 50%and 56%of the isolates had the ability to produce siderophores and 1-aminocyclo-propane-1-carboxylic acid(ACC)deaminase,and few isolates has the ability of nitrogen fixation and phosphorus solubilization(only 10 and 8 strains,respectively).To investigate the actual role of the bacterial isolates in plant growth and health,re-infection experiments was conducted in laboratory condition.No clear connection was found between traits in vitro and promoting activities of seedling growth.However,the data show that in most cases there is significant growth promotion when both IAA and ACC deaminase are present.(4)Four heavy metal-resistance and plant growth-promoting endophytes(PGPE)were used for assisted phytoextraction of Cd-polluted soils by S.nigrum.The performances of bioaugmentation-assisted phytoextraction were assessed and mechanisms of S.nigrum-PGPE interactions were discussed.Results showed that the inoculation with PGPE stimulated the growth of S.nigrum.At the low Cd-contaminated(12.1 mg/kg)soil,the greatest effect was found for Enterobacter sp.LSE04,of which the plants shoot length,fresh weight and dry weight enhanced by 13.7,28.2 and 41.4%,respectively.At the moderate Cd-contaminated(63.7 mg/kg)soil,Acinetobacter sp.LSE06 showed the most excellent growth effect,which the increase were up to 10.9%for shoot length,15.7%for fresh weight and 23.1%for dry weight;for the high Cd-contaminated(116.5 mg/kg)soil,Serratia nematodiphila LRE07 was the best and the plants shoot length,fresh weight and dry weight gone up by 18.9,23.1 and 19.8%respectively.Inoculation of PGPE also influenced the accumulation of Cd in the root,stem and leaf tissue of S.nigrum.For example,in moderate Cd-contaminated(63.7 mg/kg)soil,the largest effect on Cd uptake was found for LRE07,which enhance the concentration of Cd in root,stem and leaf by 21.3%,15.6%and 11.9%,respectively,compared with the control.As the biomass increased,the total Cd accumulation in S.nigrum was significantly increased.For instance,in moderate Cd-contaminated(63.7 mg/kg)soil,the total Cd in root and aerial part of S.nigrum with LRE07 inoculation were 1.69-fold and 1.37-fold compared with the non-inoculated control.(5)A pot study was conducted to evaluate the effect of functional bacterial endophyte Pseudomonas sp.Lk9 which can produce biosurfactants,siderophores and organic acids on the growth and metal uptake of Cd-hyperaccumulator S.nigrum growing in Cd/Zn/Cu multi-metal-contaminated soil.The performances of phytoremediation by "S.nigrum-Lk9" partnerships were assessed and the mechanisms of plant-endophyte were discussed.The results revealed that Lk9 inoculation could improve soil Fe and P mineral nutrition supplies,enhance soil heavy metal availability,and affect the secretion of plant-mediate low-molecular-weight organic acids,significantly increasing S.nigrum shoot dry biomass by 14%and the total of Cd by 46.6%,Zn by 16.4%and Cu by 16.0%accumulated in aerial parts,compared to those of non-inoculated control.The assessment of phytoextraction showed that Lk9 inoculation elevated the bioaccumulation factor of Cd(28.9%)and phytoextraction rates of all metals(17.4%,48.6%and 104.6%for Cd,Zn and Cu,respectively),while the translocation factors had negligible difference between Lk9 inoculation(3.30,0.50 and 0.40 for Cd,Zn and Cu,respectively)and non-inoculated control(2.95,0.53 and 0.42 for Cd,Zn and Cu,respectively).It was also found that the symbiotic association between S.nigrum and Lk9 significantly increased the soil microbial biomass C by 39.2%and acid phosphatase activity by 28.6%compared to those in S.nigrum without Lk9.These have great significance for the restore soil health and functioning. |
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