Rhizosphere Microbiota In The Control Of Lead Extraction By Trifolium Repens L. During Soil Remediation

In recent years,the problem of soil heavy metal pollution in China has become increasingly prominent and has attracted great attention.In situ extraction with Trifolium repens L.has great potential in the remediation of heavy metal-contaminated soil.However,the rhizosphere regulation mechanism of Trifolium repens L.and the interaction mode of root microorganisms are still unclear.The intrinsic link between the biological process of lead in the rhizosphere microenvironment of Trifolium repens L.was also unclarified.Investigating the lead tolerance mechanism of Trifolium repens L.,as well as its rhizosphere regulation mechanism,will provide novel insights for revealing the key mechanism of plant accumulation of heavy metals and promoting the practical application of phytoremediation with heavy metal pollution.The results of the study are as follows:（1）A root box experiment was conducted using Trifolium repens L.under four concentrations(CK,137 mg·kg^-1,521 mg·kg^-1,and 812 mg·kg^-1)lead stress.The results showed that low-concentration lead stress promoted the root length,plant height,dry weight,and the chlorophyll a contents of Trifolium repens L.,but had no effect on the phenotypes and malondialdehyde contents.Medium and high concentrations of lead stress inhibited the growth of Trifolium repens L.with the increase of lead stress concentrations,and the underground parts were more effected than those in the overground parts.However,with the increase of planting time,this inhibited effect was alleviated.The lead contents in roots,stems,and leaves of Trifolium repens L.were significantly positively correlated with lead pollution concentrations（P<0.05）.Under the same lead concentration stress,the sequence of lead accumulation in Trifolium repens L.were roots,stems,and leaves.At the end,under the low-concentration lead exposure,the lead enrichment coefficients and migration coefficients reached the maximum value.（2）The effective lead contents in the soil increased with increasing lead stress concentrations,and the change trends were the same in the treatment groups and the control group,showing a trend of decreasing first and then increasing.Compared with the unplanted soil,the ratio of reduced lead and acid-soluble lead in the non-rhizosphere and rhizosphere soils of Trifolium repens L.gradually increased,and the proportion of residual lead and oxidized lead in the soil gradually decreased.The results showed that the lead in the soil was transformed from the residue and oxidation state to the reduced state and acid dissolved state through the remediation.Compared with non-rhizosphere soil,the p H values of rhizosphere soil decreased by 0.39,0.40,0.24 and 0.14,respectively,after 90 d of phytoremediation.With increasing plant growth time,the soil organic matter content first decreased and then increased.Under different concentrations of lead stress,microbial biomass carbon contents in rhizosphere soil increased by 7.73%12.79%16.46%and 15.26%,respectively,compared with non-rhizosphere soil.ANOVA showed that the growth of Trifolium repens L.and soil lead stress concentrations had significant effects on soil microbial biomass carbon contents.The activities of urease,sucrase and acid phosphatase in the soil decreased with increasinglead concentrations in the soil and showed an increasing trend after phytoremediation.The growth of Trifolium repens L.and lead stress concentrations in the soil had significant effects on the activities of the three soil enzymes（P<0.001）.In addition,the interaction between the growth of Trifolium repens L.and lead stress concentrations in soil had significant effects on soil urease and acid phosphatase activities（P<0.01）.（3）The characteristic of bacterial community structure and composition of root associated microbiome of Trifolium repens L.were explored by 16S r RNA gene amplicon sequencing technology.Theβ-diversity analysis indicated that the bacterial community structure and composition were significantly different in soil samples and plant samples,and the bacterial community structure was also significantly different in different soil sampling sites.The relative abundance of Proteobacteria,Actinobacteria,Bacteroidetes and Patescibacteria in rhizosphere soil was higher than that in unplanted soil.The results showed that the roots growth of Trifolium repens L.and the lead in soil jointly shaped a special rhizosphere bacterial community.The rhizosphere microbial interaction network analysis showed that the correlation between the physicochemical properties of the unplanted soil and the microorganisms was strongest,indicating that the unplanted soil was more significantly affected by lead ions in the soil.The correlation between the rhizosphere bacterial community and soil environmental factors was conducted by RDA,indicating that the rhizosphere microorganisms of Trifolium repens L.were induced by factors such as the effective lead concentration in the soil and soil p H to produce specific assemblies and shape the specific rhizosphere.soil microenvironment.PICRUSt functional prediction analysis showed that soil lead stress enhanced the bacterial functions related to amino acid metabolism in the rhizosphere soil.