| Heavy metal contaminations occur in soil due to mining, anthropogenic activities and soil amendment using industrial, agricultural and municipal wastes. Phytoremediation is a novel, low-cost, sustainable technique for remediation of contaminated soil in situ. Metal hyperaccumulating plants are used for remediation of heavy metal-polluted soils, sediments, and water resources. The efficiency of phytoremediation, particularly, for large scale metal phytoextraction, is often limited by the slow growth and low aboveground biomass of the metal hyperaccumulators and the metal bioavailability in soil. However, most of hyperaccumulators grow slowly and produce shallow root systems and low shoot biomass, limiting their application for heavy metal phytoextraction. Therefore, new techniques that significantly increase metal bioavailability and accumulation and plant biomass would enhance the efficacy of phytoextraction by hyperaccumulators for future phytoremediation application.Sedum alfredii Hance is a vegetatively propagated perennial of the Crassulaceae family and was first found in an old Pb/Zn mined area in Quzhou, Zhejiang Province, southeast of China. It is a relatively fast-growing, high-biomass Zn/Cd co-hyperaccumulator and Pb accumulator. S. alfredii has now been used as a model plant to study metal hyperaccumulation and develop new techniques to enhance phytoextraction. We aim to use indigenous plant growth-promoting microbes to enhance S. alfredii phytoextraction of heavy metals.1. Eighty-five metal-resistant bacterial strains were isolated from rhizosphere soils and Sedum alfredii plants grown in the old Pb/Zn mining area using mineral-minimal media containing1-aminocyclopropane-l-carboxylic acid (ACC) as the sole nitrogen source and high levels of Zn, Cd and/or Pb. Analyses of bacterial16S ribosomal RNA genes (16S rDNA) revealed that the85strains were affiliated to20genera of Proteobacteria (55%), Actinobacteria (27%), Firmicutes (11%) and Bacteroidetes (7%) and that45strains (53%) might represent15-17novel species of eight genera. ACC deaminase structure genes (acdS) were amplified from28strains; ACC deaminase activities were detected from21of them in free-living states. The phylogenies of the acdS sequences from the other seven strains that did not show ACC deaminase activities were incongruent with those of their16S rDNA; these acdS genes may have evolved through horizontal transfer. All the85strains showed differential resistance to high levels of Zn, Cd and/or Pb. The percentages of the obtained bacterial strains with relatively higher metal-resistance were positively correlated to the metal concentrations of the rhizosphere soil, root and shoot tissues. This indicated the nature selection of the high level metals in the soils and plants on the bacterial metal-resistance and adaption. Most of the bacterial strains could produce indole acetic acids and siderophores, or solubilize mineral phosphate, and thus had potentials to promote plant growth and increase metal-solubility from soils.2. Four native endophytic bacteria, Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12and Variovorax sp. SaNR1were used to investigate their endophytic nature and root colonization patterns and effects on phytoextraction of Zn and Cd. Laser scanning confocal microscopy revealed that gfp-tagged SaZR4, SaMR12, and SaNR1cells formed biofilms on roots and that SaZR4and SaMR12cells could invade root tissues. SaMR10showed the lowest total population associated with S. alfredii and little effect on plant growth and phytoextraction. SaZR4significantly promoted Zn-extraction but not Cd-extraction. SaMR12and SaNR1significantly promoted plant growth in substrates supplemented with Zn or Cd and phytoextraction of Zn and Cd. Together, this study have shown that the four native endophytic bacteria differently colonize the host plants and modulate metal uptake and growth of host plant, and that SaMR12and SaNR1strains are promising assistants of S. alfredii plants for phytoremediation of Zn/Cd-contaminated soil.3. Soil experiments showed that endophytic bacteria SaZR4, SaMR12and SaNR1could increase the shoot biomass of S. alfredii in the two harvest and root biomass in the second harvest in original Pb/Zn mined soil and a multi-metal contaminated paddy soil. The phytoextraction of the four heavy metals was significantly enhanced in the two soils. Phytoextraction of Zn was approximately2-fold after inoculation with SaZR4. This study showed that continual inoculation of endophytic bacteria in the perennial Zn/Cd hyperaccumulator S. alfredii is applicable.4. A heavy metal-resistant fungus belonged to the Fusarium oxysporum complex was isolated from the Zn/Cd co-hyperaccumulator Sedum alfredii Hance grown in a Pb/Zn mined area. This Fusarium fungus was not pathogenic to plants but promoted host growth. Hydroponic experiments showed that500μM Zn or50μM Cd combined with the fungus increased root length, branches, and surface areas, enhanced nutrient uptake and chlorophyll synthesis, leading to more vigorous hyperaccumulators with greater root systems. Soil experiments showed that the fungus increased root and shoot biomass and S. alfredii-mediated heavy metal availabilities, uptake, translocation or concentrations, and thus increased phytoextraction of Zn (144and44%), Cd (139and55%), Pb (84and85%) and Cu (63and77%) from the original Pb/Zn mined soil and a multi-metal contaminated paddy soil. Together, the nonpathogenic Fusarium fungus was able to increase S. alfredii root systems and function, metal availability and accumulation, plant biomass, and thus phytoextraction efficiency. This is the first report that showed an indigenous culturable Fusarium fungus other than mycorrhizal fungi to enhance phytoextraction by hyperaccumulators. This report showed a new avenue of microorganism-assisted phytoextraction for hyperaccumulators that are mainly non-mycorrhized Brassicaceae plants. Moreover, this report showed a new function and application potential for the worldwide distributed soil and plant-associated nonpathogenic Fusarium oxysproum fungi.5. In hydroponic experiments, HE plants exhibited stronger tolerance and higher translocation of Zn and Cd compared with NHE. The endophytic fungus Piriformospora indica could colonize in the roots of two ecotypes and promote the root development, nitrogen and phosphorus uptake. P. indica enhanced the heavy metals tolerance of the two ecotypes. The shoots of HE accumulated1.17-1.75fold amount of Zn or1.83-2.21amount of Cd compared to the uninoculation. While for NHE, even in the treatment of200μM Zn or10μM Cd, the plant exhibited heavy metal stress and the growth was inhibited. P. indica decreased Zn or Cd uptake in the heavy metal stress conditions in the NHE. However, under the higher Zn (400μM) or Cd (20μM) treatments for NHE, both noninfested and infested plants exhibited a severe biomass reduction. The results showed that the endophytic fungus P. indica has a potential application in the phytoextraction of heavy metals by the Zn/Cd hyperaccumulator Sedum alfredii Hance. In addition, results from NHE indicated that P. indica may be used for phytostabilization of heavy metals by agricultural crops. |
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