| Phytoremediation is an emerging in-situ remediation technology due to its friendly-environment, less cost and convenient operation for remediation of heavy metal-contaminated soils. A large number of microorganisms which could colonize plant tissue interiors or rhizosphere soils, can affect growth and heavy metal accumulation of plants and metal formation in heavy metal-polluted environments by many approaches, and then improve phytoremediation efficiency. Research on endophytes and rhizobacteria diversity of hyperaccumulator or metal-tolerant plants and their interaction with plants will further understand microbial resource and biodiversity, clarify the relationship between diversity and heavy metal accumulation of plants, and can obtain and applicate plant growth promoting bacteria, offer the theoretical and experimental basis for endophytic bacteria-assisted phytoremediation of Cu-contaminated soil.Total DNA were extracted from plant roots of Elsholtzia splendens and Commelina communis growing on different contaminated sites, partial16S rDNA were amplified with the primers799F and1492R, and then16S rDNA clone libraries were constructed. Phylogenetic analysis based on16S rDNA sequences showed that endophytic bacteria of Elsholtzia splendens roots belonged to two major groups:a-, β,γ-Proteobacteria and Bacteroidetes. Clones belonging to γ-Proteobacteria accounted for73.9%and79.0%in the high-and low-contaminated clone libraries respectively, and were predominant in clone libraries of Elsholtzia splendens roots; Endophytic bacteria of Commelina communis roots belonged to three major groups:a-,β, γ-Proteobacteria, Firmicutes and Actinobacteria. Clones belonging to y-Proteobacteria accounted for41.0%,77.1%and89.9%in the high-, low-and non-contaminated clone libraries respectively, and were predominant in clone libraries of Commelina communis roots. In addition, clones belonging to Firmicutes accounted for4.8%,22.9%and10.1%in the high-, low-and non-contaminated clone libraries of Commelina communis roots respectively.38.6%of clones affiliated with Actinobacteria were only found in the high-contaminated clone library of Commelina communis roots. An increasing abundance of gram-positive bacteria (Firmicutes and Actinobacteria) was found in Commelina communis roots, and root bacterial communities of the two plant species exhibited a decrease in y-Proteobacteria with increasing copper contamination.Bacterial community and diversity of rhizosphere soils were studied from Elsholtzia splendens and Commelina communis growing on copper mine wasteland by16S rDNA clone libraries, the results showed that rhizobacterial communities from the two plant species were affiliated with ten major groups. Proteobacteria were the most abundant group in the clone libraries. Nitrospira, Chloroflexi and Actinobacteria were only found in the rhizosphere soil of Elsholtzia splendens, and Cyanobacteria were only found in Commelina communis. Study on bacterial community of rhizosphere soils from the two plant species in different contaminated sites by DGGE found that rhizosphere bacteria from Elsholtzia splendens belonged to four major groups:a-, β-, γ-Proteobacteria, Bacteroidetes, Chloroflexi and TM7; Rhizosphere bacteria from Commelina communis belonged to four major groups:Acidobacteria, Bacteroidetes, a-, y-Proteobacteria and Firmicutes. Community composition of rhizosphere bacteria may be related to plant species, metal concentration and soil conditions. As copper contamination decreased, an increasing abundance of y-Proteobacteria was found in Elsholtzia splendens rhizosphere, rhizosphere communities of Commelina communis exhibited a decrease in Firmicutes, but an increase in Acidobacteria, and a-, γ-Proteobacteria appeared in low-, non-contaminated sites.Sixty two strains that were resistant to Cu (64mg.L-1) and19strains that can grow on ACC as the sole N source were screened out from plant tissue interiors and rhizosphere soils of copper-tolerant plant species Elsholtzia splendens and Commelina communis by conventional plate culture technique.16S rDNA of the tested strains were amplified by PCR with the universal bacterial primers, and were digested with the restriction endonucleases Msp I and Hae III respectively.16S rDNA sequences phylogenetic analysis of representative strains with diverse ARDRA patterns indicated that the Cu-resistant-endophytic bacteria and rhizobacteria showed abundant diversity. γ-Proteobacteria (mainly Acinetobacter, Enterobacter and Pantoea) was predominant in Cu-resistant bactaria isolates from Elsholtzia splendens; Firmicutes (mainly Bacillus) was predominant in Cu-resistant bactaria isolates from Commelina communis.Three strains with plant growth promotioning characteristics, heavy metal resistance and copper solubilization were screened out based on plant growth promotion and improvement of copper uptake of the plant and identified as Burkholderia sp.GL12, Bacillus megaterium JL35, Sphingomonas sp.YM22. Strains GL12, JL35and YM22could produce ACC deaminase, arginine decarboxylase, IAA, siderophore, and be resistant to metals; In the Cu2(OH)2CO3solubilization experiment, inoculation with the strains was found to increase the Cu2+concentration compared to the controls.The effects of Strains GL12, JL35and YM22on plant growth and Cu uptake were studied by pot experiment. The results showed that the inoculation with tested endophytic bacteria GL12, JL35and YM22could promote rape and maize growth in Cu-contaminated soils, and increase dry weights by11-56%in shoots and by48-125%in roots compared to the controls. The Cu uptakes of inoculated rape and maize plants were increased from31%to48%and from42%to91%compared to the uninoculated control, respectively.Strains GL12, JL35and YM22could colonize in rhizosphere and root interiors of the rape and maize, and strain JL35could colonize in leaves of rape and maize. The water-extracted Cu concentration of rhizosphere soils of the inoculated rape and maize plants was increased from16%to113%and from63%to94%compared to the controls, respectively. Moreover, the inoculation with strain GL12was found to significantly increase NH4OAc-extracted Cu concentration by51%for rape and by14%for maize, compared to the controls, respectively. The inoculation with the strains can alleviate peroxidation level under copper stress by the improvement of SOD activity and ASA concentration in maize roots. Furthermore, the research on rhizosphere bacterial and root endophytic bacterial community from maize by DGGE showed that there were some influences of the inoculation with strains on rhizosphere bacterial and root endophytic bacterial community compared to the uninoculated control. |
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