Rhizosphere Microbial Diversity Of Cynodon Dactylon And Their Combined Remediation In Open-pit Lead-zinc Mining Area In Yunnan Province

Mountain-like low-grade slag heaps are usally abandoned and piled up randomly in the mining area,and the heavy metal contaminants are continuedly dispersed and pollutes to the neighboring ecosystems via the aeolian and hydric erosion,which thus become the main sources of heavy metal pollants.So,vegetated phytocaps can effectively limit the diffusion of heavy metals and promote the ecological restoration of mining areas.However,due to the poor physiochemical characteristics of mine slag soils,such as high and steep terrain,low soil water holding capacity,inadequate nutrition,etc.,there are a shortage of plant species used for the restoration of steep slopes of wasteland in the mining area.Previous field surveys found that Cynodon dactylon naturally grows on the steep slope of a heavy metal mine abandoned dump in Jianshui,Yunnan Province,exhibited an excellent mine-tailing adaptation characteristic.While,the rhizosphere microbial diversity of C.dactylon and their combined remediation in open-pit lead-zinc mining area were still obscure.In this paper,the accumulation characteristics of heavy metals and phytoremediation potentials of C.dactylon,and its rhizosphere microbial diversity and their combined remediation benefits were comparatively studied,and the main results and conclusions are as follows:（1）The soil of the slag of the Jianshui open-pit lead-zinc mine in Yunnan province is seriously polluted by heavy metals.The concentration of Cd,Zn,and Pb are 4.34±1.03,1836.83±265.47,4105.66±507.48 mg kg^-1 respectively.The naturally growing C.dactylon settled on it to form a single excellent community,indicating that it has good adaptability to abandoned slag heap.Under laboratory conditions,the responses of two origins of C.dactylon to heavy metal stress under single cadmium stress and complex heavy metal（Pb,Zn,Cd）stress were comparatively studied.The experimental study found that both mining and non-mining areas of the two origins of C.dactylon grew well and there was no significant difference in biomass under high concentrations of compound heavy metal stress or single Cd stress.It shows that C.dactylon has good characteristics of heavy metal resistance.（2）In the study of the response of C.dactylon to heavy metal stress,it was also found that the two origins of C.dactylon had strong heavy metal accumulation ability,but there was no significant difference.The concentration of Pb,Zn,and Cd in C.dactylon rhizosphere can reach 1399.30±83.724219.39±1129.05134.74±37.82 mg kg^-1 by cultured in the soil of compound tailings area.Under the condition of single Cd pollutionhydroponic culture,the maximum Cd concentration in the rhizosphere was 5031.41±682.79 mg kg^-1.The heavy metal migration ability of C.dactylon is weak.The heavy metal transfer coefficients of C.dactylon under compound heavy metal polluted soil culture and single Cd hydroponic culture are less than 1.The previous study also found that there was no significant difference in the concentration of extractable heavy metals in the rhizosphere soil of C.dactylon grown naturally on the waste slag heap and the neighboring bare soil,compared with bare soil,the concentration of extractable Cd and Pb in rhizosphere soil decreased by 4.19%and 0.01%respectively.This shows that C.dactylon has a strong phytostabilization effect on heavy metals,and there is no risk of heavy metal weathering.（3）The physical and chemical properties and soil enzyme activities of mine area were improved significantly by C.dactylon.Compared with bare soil,the C.dactylon rhizosphere soil organic matter,total nitrogen,hydrolyzable nitrogen,and available potassium increased significantly.In particular,the contents of organic matter and available potassium of rhizosphere soil are 2.07 and 2.70 times higher than bare soil respectively.Acid phosphatase（ACP）,alkaline phosphatase（AKP）,sucrase（SC）,andβ-Glucosidase（β-GC）,which are 2.68 times,37.71 times,18.29 times,and 2.70 times of bare soil respectively.（4）Through high-throughput sequencing and comparing the changes of microbial diversity in C.dactylon root,rhizosphere soil,and bare soil,it was found that the colonization of C.dactylon can promote the restoration of soil microbial diversity and function.Combined with microbial diversity analysis and q-PCR experiment,it can be found that the diversity,abundance,and biomass of bacteria in soil are significantly improved after C.dactylon colonization.Through functional prediction analysis,it was found that heterotrophic and urea degradation-related bacteria were significantly enriched in rhizosphere soil,and pathological fungi were concentrated in bare soil.The colonization of C.dactylon also changed the interaction of microorganisms,made the interaction network simpler,and the positive correlation ratio between bacteria and fungi increased;The key species changed from Dokmaia of Ascomycetes to Acremonium.Through the correlation analysis of environmental factors,it was found that bacteria had a very significant positive correlation with soil enzyme activities.In soil physical and chemical properties,AK is the main influencing factor of bacterial and fungal communities.AK is significantly correlated with 42%of bacteria and 34%of fungaldominant genera（the top 50 of abundance）.（5）It was found that the roots of C.dactylon from two origins were inoculated with dark septal endophytic fungi（DSE）in Cd contaminated soil,DSE can promote the phytostabilization of C.dactylon under Cd stress.After inoculation with DSE,the concentration of Cd in the underground part of C.dactylon from non-mining origins was as high as 1433.72±55.25 mg kg^-1,which was 8.84 times higher than that without inoculation.DSE also significantly inhibited the transfer of Cd to the aboveground part of C.dactylon.The translocation factor of C.dactylon from non-mining origins inoculated with DSE was significantly reduced by 88.82%.In conclusion,C.dactylon is would be ideal for optimizing revegetation for numerous barren rare earth mine tailing soils,due to its strong adapatibility and the phytostailization potentials of metal comtaminants.The revegetation of C.dactylon in the mining area can phytostabilize heavy metals in the rhizosphere and prevent the dispersal of heavy metal contaminants from the mine tailing soils to the surrounding environments.At the same time,C.dactylon also improves soil physical and chemical properties and soil enzyme activities,restores soil microorganisms,and promotes bacterial abundance,diversity,and biomass,which contribute to the acceleration of the restoration of the soil ecosystem and the potential secondary succession of wasteland in the mining area.Vise versa,the microbial restoration,e.g.inoculation of DSE,can enhance the accumulation of Cd in the roots of C.dactylon,and it can be used as the preferred strain for microbial C.dactylon combined remediation in heavy metal mining areas.