Effect Of Robinia Pseudoacacia-Rhizobia Symbiosis On Microbiome In The Phytoremediation Of Heavy Metal Contaminated Soil In Mine Area

Currently,the enhancement of phytoremediation technology by inoculated microorganism is one of the most important way for repairing heavy metal contaminated soils.In this study,based on the survey of heavy metal contaminated soil in mining area,we analyzed the influence of heavy metal on the microbial diversity and community;and isolated some strains possessing heavy metal tolerance and promoting-plant growth traits from the root nodule of legume?Robinia pseudoacacia?.Then,we choose woody legume plants?R.pseudoacacia?inoculated with the selected rhizobia to repair heavy metal contaminated soil and explore the effect of the symbiotic system between legume and rhizobia on microbial diversity and communities in the rhizosphere and plant.Furthermore,we also studied the influence of this symbiotic system on the microbial function in the rhizosphere.The major results were in following.1.Mine drainage from Pb-Zn smelter leads to the variation of microbial diversity and microbial community in riparian soils along a settling pond used for mine drainage treatment.The heavy metal was the main factor to affect microbial diversity and community in riparian soils and more microorganisms related to sulphur-and carbon-cycles may exist in soils heavily contaminated by mine drainage.We used an Illumina Mi Seq platform to explore the soil microbial composition and diversity.Non-metric multidimensional scaling analysis showed that the microbial communities differed significantly among the four sampling zones?influent,upstream,downstream and effluent?,but not seasonally.Constrained analysis of principal coordinates indicated heavy metals?zinc,lead and copper?,total sulphur,pH and available potassium significantly influenced the microbial community compositions.Heavy metals were the key determinants separating the influent zone from the other three zones.Lower diversity indices were observed in the influent zone.However,more potential indicator species,related to sulphur and organic matter metabolism were found there,such as the sulphur-oxidizing genera Acidiferrobacter,Thermithiobacillus,Limnobacter,Thioprofundum and Thiovirga,and the sulphur-reducing genera Desulfotomaculum and Desulfobulbus;the organic matter degrading genera,Porphyrobacter and Paucimonas,were also identified.2.Eighty-two endophytic bacteria were isolated from the root nodules of R.pseudoacacia grown in Pb-Zn mine area.Based on the 16S rRNA genes analysis,those isolates were classified into 21 genera,14 families,7 orders,5 classes and 3 phyla.Mesorhizbium,Pseudomonas and Rhizobium were the three main genera and accounted for45.1%,12.2%and 7.3%of the total isolates.The tolerance to heavy metals Cd²⁺,Zn²⁺,Pb²⁺and Cu²⁺and plant growth promoting?PGP?features of indole-3-acetic acid?IAA?production,ACC deaminase activity,siderophore production and phosphate solubilization were conducted.Most isolates showed MIC?mmol/L?less than 1.0 for Cd²⁺?78.0%?,Zn²⁺?51.2%?,Pb²⁺?29.3%?and Cu²⁺?95.1%?.The resistance of Herbaspirillum chlorophenolicum HZ10 for Cu²⁺was 1.4 mmol/L;the resistance of Mesorhizobium amorphae HZ64 for Pb²⁺was 2.2mmol/L;and the resistance of Enterobacter mori HZ21 for Zn²⁺and Cd²⁺were 40.0 and 18.0mmol/L respectively.IAA production was identified in 50 isolates ranged from 0.13 to 7.12?g/ml.ACC deaminase activity was detected in 43 isolates ranged from 0.19 to 385.61?M?-KB mg of protein^-1 h^-1.Sixty-three isolates belonging to 17 genera produced siderophore and the isolates of Variovorax and Pseudomonas showed the greatest halos.Only three isolates?HZ4,HZ35 and HZ59?belonging to the species Pseudomonas chlororaphis solubilized tricalcium phosphate in vitro.Three isolates?Mesorhizobium loti HZ76,Ensifer adhaerens HZ14 and Agrobacterium radiobacter HZ6?as the inoculant associated with R.pseudoacacia,which presented both higher resistance to multiple heavy metals and better PGP traits?positively correlated with all the considered factors?could be a preferable system for phytoremediation.3.To assess the influence of R.pseudoacacia assocation of rhizobia on bacterial diversity and community structure in heavy metal-contaminated soils,contaminated soils were collected from two sites surrounding a Pb-Zn smelter in Mianxian County,Shaanxi Province,China,in June 2014.This area has been contaminated for almost 10 years by dust?lightly contaminated?and mine drainage?heavily contaminated?.The experiment design involved two levels of heavy-metal contamination?lightly and heavily?×two levels of planting?unplanted and planted with R.pseudoacacia?×four levels of rhizobial inoculation?uninoculated[WJ],and inoculated separately with PGP traits strain M.loti HZ76[J76],E.adhaerens HZ14[J14],or A.radiobacter HZ6[J6]?.In this study,we used 16S rRNA gene V4-V5 sequencing and metagenome sequencing to analyze the bacterial communities and functions in the rhizosphere of R.pseudoacacia with or without rhizobial inoculation compared with bulk soils.Soil bacterial communities varied significantly depending on the level of soil contamination,and planting also had some influence.Although inoculation of M.loti HZ76?a natural microsymbiont of R.pseudoacacia?was a relatively minor factor,it did influence the soil bacterial community.Under the selective pressure,plant growth promotion-related biomarkers in the rhizosphere,especially those associated with Mesorhizobium,Variovorax,Streptomyces,and Rhodococcus genera,increased following inoculation.Genes encoding ATP-binding cassette transporters were up-regulated in the rhizosphere after inoculation compared with genes related to sulfur/nitrogen metabolism.These results provide insight into soil bacterial communities and their functions in the R.pseudoacacia rhizosphere in response to rhizobial inoculation and heavy metal contamination.This knowledge may prove useful for improving phytoremediation of metal-contaminated soils.4.The bacterial communities associated with plants could help the plants in adaptation and remediation of the HM contaminants.To understand the interactions among the bacterial communities,the plants,and the contaminants,we used an Illumina Mi Seq platform to explore the bacterial composition in the roots and nodules of R.pseudoacacia grown in HM contaminated soils,in comparison with those in rhizosphere and bulk soils.As results,the bacterial communities were clearly distinct based on sampling compartments?rhizoshpere,root and nodule?and the compartment was the largest source of variation in the bacterial community,but the contamination level was the minor source of variation.In the HM contaminated conditions,rhizocomparments is the main factor to influence the bacterial communities in all studied samples;while the contamination levels showed greater effects on soil and rhizosphere microbiotas,but not on the root and nodule endosphere microbiotas.Bacterial colonization of R.pseudoacacia roots and nodules is a niched-process and plants select for certain bacterial consortia to fill the root and nodule niches.Furthermore,root and nodules formed two endospheric niches that selected the bacterial microbiota in a parallel based upon their functions:N fixation in nodules,and more metabolic functions in the root compartment.This study provides the knowledge of plant-associated bacterial communities in the contamination environments.