| In the present study,two ecotypes（hyperaccumulation and non-hyperaccumulation）of Sedum alfredii were planted in different Cd concentrations(0,10,50,100,500 and1000 mg·kg-1),Repectively,We aimed to investigate the changes of root microenvironment under a Cd concentration gradient and their responses to the assemblage of root microbiomes of two ecotypes of Sedum alfredii.Furthermore,we investigated the response of environmental factors to microbial structure and function genes.The main results are as follows:（1）The test conditions of HPLC for simultaneous determination of eight low molecular weight organic acids（LMWOA）（including oxalic acid,tartaric acid,malic acid,malonic acid,acetic acid,citric acid,fumaric acid and propionic acid）were optimized.The optimized conditions were as follows:column temperature 30℃,mobile phase flow rate 0.5ml·min-1,phosphate buffer solution concentration 0.01mol·L-1,pH=2.6,and UV detection external wavelength was 210 nm,the mobile phase ratio was 1:99,the retention time was 20 min,and the injection volume was 10 μL.Such that,the recoveries of eight organic acids can reached to 93.19%-103.6%,and the relative standard deviations were at 0.17%-1.7%.（2）Veen analysis demonstrated that over 90%of the microorganisms presentted in the root and bulk soil of two ecotypes of Sedum alfredii.The statistical analysis revealed that the compositioned of these microorganisms were of significant differences between two habits,among which Alphaprotobacteria,Gammaproteobacteria and Actinobacteria were enriched in the root environment,while Deltaproteobacteria,Acidobacteria and Chloroflexi enriched in the bulk soil.Furthermore,we also identified that the microbial functions were significantly different between root and bulk soil,in which signaling pathway and degradation were significantly enriched in root environment,while transportation system and biosynthesis were significantly enriched in bulk soil.（3）Our results showed that the physiological indexes（including fresh weight,dry weight,and root length）of the two plants had different responses under Cd treatment.Specifically,the growth of hyperaccumulative Sedum alfredii was not affected with elevated Cd concentrations,whereas the growth of non-hyperaccumulative Sedum alfredii was inhibited or even died.Furthermore,we demonstrated that the environmental parameters of the two plants also had different responses under various Cd treatments.For example,the soil pH of root soil of hyperaccumulators decreased rapidly to about 5.8,while that of non-hyperaccumulators decreased slowly to about 6.5;In hyperaccumulator root soils,oxalic acid increased with the increase of Cd treatment;citric acid,tartaric acid and malonic acid increased first and then decreased.In non-hyperaccumulator root soils,tartaric acid was not secreted by high concentration of Cd,and the contents of the other three organic acids increased first and then decreased.（4）The root soil and bulk soil microbial composition of two ecotypes of the Sedum alfredii were basically same at the phylum level,but significant differences in genus level.Among them,Arthrobacteria,Nocardia and Bosea were significantly enriched in hyperaccumulator rhizosphere soil;The root soil of non-hyperaccumulator was significantly enriched with lysobacter,Microvirga and Tumebacillus.According to metagenomic function analysis,plant root are mainly involved nutrient cycling microorganisms,such as carbon cycling（C fixation,C degradation,TCA cycling）,nitrogen cycling（nitrification,denitrification,nitrogen transfer）,P cycling（phosphate dissolution and mineralization,phosphorus transfer and regulation）,and s cycling（sulfur degradation,sulfur oxidation,and sulfur metabolism）.（5）The CCA analysis showed that pH and tartaric acid were the important environmental factors regulating the overall microbial community structure of hyperaccumulation plant root soils,while citric acid had the greatest impact on the microbial composition of non-hyperaccumulation plant root soils.It was found that oxalic acid,tartaric acid and pH had the greatest effect on the root microorganisms of two ecotypes of Sedum alfredii.In hyperaccumulator plants,Paraburkholderi,Ptenobacteria and Pyntrophaceticu were significantly correlated with these mentioned factors,while in non-hyperaccumulator plants,Pseudolabrys,Cuprophytic bacteria and Klebsiella were closely correlated with oxalic acid,tartaric acid and pH.（6）We also identified that the environmental factors have an important impact on microbial functions.For example,oxalic acid was positively correlated with C,N,P and S cycling related genes,while tartaric acid,citric acid,malonic acid and pH were negatively correlated with C,N,P and S cycling related genes.In addition,TOC and TN were positively correlated with C and N cycling related genes,but negatively correlated with P and S cycling related genes.In this study,we have revealed the differences of the assembly patterns of two ecotypes of Sedum alfredii and their effects on plant growth.Furthermore,we have identified the regulatory factors of root microbiomes of two ecotypes of Sedum alfredii under Cd stress.Also,the relationship between these regulatory factors and the utilization of nutrient elements in plants under stress environment was explained.This study may provide new insight better understanding of plant growth in metal stress environment,and provide basis information for practical ecological restoration engineering. |
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