Studies On The Leucaena Leucocephala-Rhizobia Symbioses In The Bioremediation Of Heavy-metal Polluted Soils

Mineral resources are fundamental to the development of society.Long term over-exploitation of mineral resources has caused soil pollutions in the mining area and depleted mines are a great waste of land resources.The fact that heavy metals are difficult to degrade and are bioaccumulative means they can possibly be remediated using plants.Legumes-rhizobacteria symbiotic systems have a unique combination of biological remediation advantages,and thus have been deemed as a prospective application for the remediation of heavy metal contaminated sites.This research delved deep into Leucaena leucocephala-rhizobia associations in the remediation of vanadium titanium magnetite tailings soil.Root nodules of L.leucocephala were collected,from which rhizobia were isolated.Phylogenetic and systematic analyses of the genes were performed and assays of rhizobial growth promoting properties were carried out.Strains with outstanding ability of tolerance to metals in contaminated soils were screened and analysed.Pot-culture methods were utilized to evaluate leucaena-rhizobia symbiosis systems in the bioremediation of metal tainted soils.The main results are as follows:(1)A total of 21 strains were isolated from nodules of L.leucocephala in V-Ti magnetite tailings.Analysis of 16S rDNA similarity and phylogenetic results showed that 11 strains were identified as belonging to the genus of Sinorhizobium,10 strains belonged to fast-growing Rhizobium;MLS A of housekeeping gene showed that YH7,YH8,YH9,YH10,YH11,YH12,YH13,YH14,YH15 and YH16 were most similar to Sinorhizobium morelense,which were clustered into group ?;YH2,YH3,YH17,YH18,YH19,YH20,YH21 were found to bear closest relations to Rhizobium alkalisoli,Rhizobium galegae,and Rhizobium vignae,which were clustered into group ?;YH4,YH5,and YH6 shared the same similarities to Rhizobium radiobacter and Rhizobium pusense in group ?;YH1,which had the highest similarity to Sinorhizobium saheli,was clustered into group ?.(2)Sixteen strains of rhizobia had their nodD gene amplified as the results of the analyses of symbiotic nodulation and N-fixation genes(nodD and nifH),clustering them into 3 main sub-branches of group ?,? and ? in the phylogenetic tree,which were found to be similar to Sinorhzobium sp.HAMBI1489.It might be due to the fact that these strains shared similar nodD genes to that of Sinorhizobium but did not exhibit any genetic similarity to that of Rhizobium.Eight rhizobial strains which could be amplified for gene nifH,were chiefly clustered into group ? and ?.Therefore,symbiotic nodulation and N-fixation genes were different among different rhizobia.It is supposed that some of these genes may have mutated or been lost and that may be one of the reasons for rhizobia having host specificity.(3)Fourteen strains of rhizobia could secrete IAA in PGP activity tests,with the concentrations ranging from 6.87?g/mL to 166.77?g/mL in the broth;Twelve strains were competent siderophore producers,with siderophore volumes ranging from 4.06?g/mL to 61.55?g/mL in liquid media;Nine strains dissolved phosphorus,with soluble phosphorus contents ranging from 6.87?g/mL to 122.01?g/mL.As a whole,YH1 and YH2 achieved the best overall results in PGP tests.Pot-culture experiments using mine tailings showed that both YH1 and YH2 could establish symbiotic relations with Ducocephala,which indicated that they had strong stress tolerance.Plants inoculated with YH1 and YH2 demonstrated significantly higher(p<0.05)plant height,root length,biomass and nitrogen contents than the controls,while nitrogen contents were increased by 12.1%and 10.2%respectively.(4)L.leucocephala plants were found to be able to accumulate multiple metals including Fe,Ti,V,Mn,Cd,Cu and Ni.Strain YH1 and YH2 not only showed considerably good PGP activities,they could also change the metal enrichment coeficients of the plants.Compared with the controls,Fe and Mn enrichment rates were increased by 25.7%and 47.6%in plants inoculated with YH2,however the accumulation rates of Ti,V,Cd,Cu and Ni were reduced while strain YH1 helped to alleviate the uptake of Fe,Ti,V,Mn,Cd,Cu and Ni by the plants.As a result,strain YH1 exerted better metal concentraion effects on L.leucocephala than strain YH2,while both of them could lower metal transfer abilities of L.leucocephala.(5)Strain YH1 and YH2 both showed strong tolerance to Cd and Mn.The maximum tolerance capacities to Mn of YH1 and YH2 were respectively 1500?g/mL and 1000?g/mL,and to Cd were 250?g/mL and 400?gg/mL.Therefore,Cd and Mn contaminated soils were chosen for the study of leucaena-rhizobia bioremediation.In Mn contaminated soil,YH1 and YH2 have both increased Mn enrichment coefficient of the plants.In soils spiked with 5 mg/kg Mn,the enrichment coefficients increased by 84.1%and 65.1%respectively and in the soils with 50 mg/kg Mn,119.8%and 24.2%respectively.However,in soils tainted with 5 mg/kg Cd,YH1 and YH2 reduced the enrichment coefficients of the plants for Cd,while in the soils supplemented with 50 mg/kg Cd,the enrichment coefficients of L.leucocephala were increased by 278.3%and 180.3%.To sum up,the leucaena-rhizobia(YH1/YH2)symbiotic systems established in this research,which could effectively remediate metal pollutions both in V-Ti tailings and soils with Cd or Mn pollutants,have provided the theory basis for the bioremediation of soils with heavy metal pollutants.