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Background: Although numerous endophytic bacteria have been isolated and characterized from cadmium (Cd) hyperaccumulators, the contribution and potential application of the core endophytic microbiomes on facilitating phytoremediation were still lack of intensive recognition. Therefore, a 2-year field sampling in different location were firstly conducted to identify the unique core microbiome in Cd hyperaccumulators, among which the representative cultivable bacteria of different genera were then selected to construct synthetic communities (SynComs). Finally, the effects and mechanisms of the optimized SynCom in regulating Cd accumulation in different ecotypes of Sedum alfredii were studied to declare the potential application of the bacterial agents based on core microbiome., Results: Through an innovative network analysis workflow, 97 core bacterial taxa unique to hyperaccumulator Sedum was identified based on a 2-year field 16S rRNA sequencing data. A SynCom comprising 13 selected strains belonging to 6 different genera was then constructed. Under the combined selection pressure of the plant and Cd contamination, Alcaligenes sp. exhibited antagonistic relationships with other genera and plant Cd concentration. Five representative strains of the other five genera were further conducted genome resequencing and developed six SynComs, whose effects on Cd phytoremediation were compared with single strains by hydroponic experiments. The results showed that SynCom-NS comprising four strains (including Leifsonia shinshuensis, Novosphingobium lindaniclasticum, Ochrobactrum anthropi, and Pseudomonas izuensis) had the greatest potential to enhance Cd phytoremediation. After inoculation with SynCom-NS, genes related to Cd transport, antioxidative defense, and phytohormone signaling pathways were significantly upregulated in both ecotypes of S. alfredii, so as to promote plant growth, Cd uptake, and translocation., Conclusion: In this study, we designed an innovative network analysis workflow to identify the core endophytic microbiome in hyperaccumulator. Based on the cultivable core bacteria, an optimized SynCom-NS was constructed and verified to have great potential in enhancing phytoremediation. This work not only provided a framework for identifying core microbiomes associated with specific features but also paved the way for the construction of functional synthetic communities derived from core microbiomes to develop high efficient agricultural agents. Video Abstract., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Biochar and metal-tolerant bacteria have been widely used in the remediation of heavy metal contaminated soil. However, the synergistic effect of biochar-functional microbes on phytoextraction by hyperaccumulators remains unclear. In this study, the heavy metal-tolerant strain Burkholderia contaminans ZCC was selected and loaded on biochar to produce biochar-resistant bacterial material (BM), and the effects of BM on Cd/Zn phytoextraction by Sedum alfredii Hance and rhizospheric microbial community were explored. The results showed that, BM application significantly enhanced the Cd and Zn accumulation of S. alfredii by 230.13% and 381.27%, respectively. Meanwhile, BM alleviated metal toxicity of S. alfredii by reducing oxidative damage and increasing chlorophyll and antioxidant enzyme activity. High-throughput sequencing revealed that BM significantly improved soil bacterial and fungal diversity, and increased the abundance of genera with plant growth promoting and metal solubilizing functions such as Gemmatimonas, Dyella and Pseudarthrobacter. Co-occurrence network analysis showed that BM significantly increased the complexity of the rhizospheric bacterial and fungal network. Structural equation model analysis revealed that soil chemistry property, enzyme activity and microbial diversity contributed directly or indirectly to Cd and Zn extraction by S. alfredii. Overall, our results suggested that biochar- B. contaminans ZCC was able to enhance the growth and Cd/Zn accumulation by S. alfredii. This study enhanced our understanding on the hyperaccumulator-biochar-functional microbe interactions, and provided a feasible strategy for promoting the phytoextraction efficiency of heavy metal contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Soil Cd and Zn contamination has become a serious environmental problem. This work explored the performance of wood vinegar (WV) in enhancing the phytoextraction of Cd/Zn by hyperaccumulator Sedum alfredii Hance. Rhizosphere chemical properties, enzyme activities and bacterial community were analyzed to determine the mechanisms of metal accumulation in this process. Results demonstrated that, after 120 days growth, different times dilution of WV increased the shoot biomass of S. alfredii by 85.2%-148%. In addition, WV application significantly increased soil available Cd and Zn by lowing soil pH, which facilitated plant uptake. The optimal Cd and Zn phytoextraction occurred from the 100 times diluted WV (D100), which increased the Cd and Zn extraction by 188% and 164%, compared to CK. The 100 and 50 times diluted WV significantly increased soil total and available carbon, nitrogen and phosphorus, and enhancing enzyme activities of urease, acid phosphatase, invertase and protease by 10.1-21.4%, 29.1-42.7%,12.2-38.3% and 26.8-85.7%, respectively, compared to CK. High-throughput sequencing revealed that the D 100 significantly increased the bacterial diversity compared to CK. Soil bacterial compositions at phylum, family and genera level were changed by WV addition. Compared to CK, WV application increased the relative abundances of genus with plant growth promotion and metal mobilization function such as, Bacillus, Gemmatimonas, Streptomyces, Sphingomonas and Polycyclovorans, which was positively correlated to biomass, Cd/Zn concentrations and extractions by S. alfredii. Structural equation modeling analysis showed that, soil chemical properties, enzyme activities and bacterial abundance directly or indirectly contributed to the biomass promotion, Cd, and Zn extraction by S. alfredii. To sum up, WV improved phytoextraction efficiency by enhancing plant growth, Cd and Zn extraction and increasing soil nutrients, enzyme activities, and modifying bacterial community., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Combined bioaugmentation inoculants composed of two or more plant growth-promoting bacteria (PGPB) were more effective than single inoculants for plant growth and cadmium (Cd) removal in contaminated soils. However, the principles of consortia construction still need to be discovered. Here, a pot experiment with Cd natural polluted soil was conducted and PGPB consortia with different ecological niches from hyperaccumulator Sedum alfredii Hance were used to compare their effects and mechanisms on plant growth condition, Cd phytoextraction efficiency, soil enzymatic activities, and rhizospheric bacterial community of Brassica juncea L. The results showed that both rhizospheric and endophytic PGPB consortia inoculants promoted plant growth (6.9%-22.1%), facilitated Cd uptake (230.0%-350.0%) of oilseed rape, increased Cd phytoextraction efficiency (343.0%-441.0%), and enhanced soil Cd removal rates (92.0%-144.0%). PGPB consortia inoculants also enhanced soil microbial carbon by 22.2%-50.5%, activated the activities of soil urease and sucrase by 74.7%-158.4% and 8.4%-61.3%, respectively. Simultaneously, PGPB consortia inoculants increased the relative abundance of Flavobacterium, Rhodanobacter, Kosakonia, Pseudomonas and Paraburkholderia at the genus level, which may be beneficial to plant growth promotion and bacterial phytopathogen biocontrol. Although the four PGPB consortia inoculants promoted oilseed growth, amplified Cd phytoextraction, and changed bacterial community structure in rhizosphere soil, their original ecological niches were not a decisive factor for the efficiency of PGPB consortia. therefore, the results enriched the present knowledge regarding the significant roles of PGPB consortia as bioaugmentation agents and preliminarily explored construction principles of effective bioaugmentation inoculants, which will provide insights into the microbial responses to combined inoculation in the Cd-contaminated soils., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pot-culture experiments were conducted to investigate the potential of microorganism-saponin assisted phytoremediation of cadmium (Cd) and benzo(a)pyrene (B[a]P) co-contaminated soil using Cd-hyperaccumulator Sedum alfredii. Results showed that B[a]P-degrading bacterium (Ochrobactrum intermedium B[a]P-16) inoculation significantly increased root (by 22.1-24.1%) and shoot (by 20.5-23.4%) biomass of S. alfredii, whereas the application of saponin had no effect on the growth of S. alfredii. The saponin solution at 2 g L -1 extracted more Cd and B[a]P than water, saponin enhanced Cd and B[a]P bioavailability in soil and thus promoted their uptake and accumulation in S. alfredii. The activity of B[a]P-16, dehydrogenase and polyphenol oxidase in co-contaminated soil was promoted by growing S. alfredii, and the application of B[a]P-16 and saponins caused a significant (P < 0.05) increase in both enzyme activities. The maximum B[a]P removal rate (82.0%) and Cd phytoextraction rate (19.5%) were obtained by co-application of S. alfredii with B[a]P-16 and saponin. The B[a]P-16 and plant promoted biodegradation were the predominant contributors towards removal of B[a]P from soil. A significant (P < 0.05) synergistic effect of B[a]P-16 and saponin on B[a]P and Cd removal efficiency was observed in this study. It is suggested that planting S. alfredii with application of B[a]P-16 and saponin would be an effective method for phytoremediation of soil co-contaminated with Cd and PAHs., (Copyright © 2019. Published by Elsevier Ltd.)

Plant growth promoting bacteria (PGPB) have been reported to have the ability to promote plant growth, development and increase heavy metals (HMs) uptake. Therefore, PGPB inoculation as soil remediation agents into plants with larger biomass and potential of phytoextraction is of great importance to increase bioremediation efficiency. In this study, 12 PGPB strains isolated from a cadmium (Cd)/zinc hyperaccumulator Sedum alfredii Hance were inoculated into non-host plant Brassica juncea and their effects on plant growth and Cd uptake were determined. The results showed that inoculation of most PGPB strains promoted plant growth, boosted root development and improved chlorophyll content in the absence of Cd. Inoculation of PGPB strains promoted plant growth up to 111% in shoot and 358% in root when treated with 2 μM Cd. In addition, PGPB inoculation not only ameliorated plant root morphology including the total root length (RL), total surface area (SA), total root volume (RV) and number of root tips (RT), but also facilitated Cd uptake up to 126%. Furthermore, inoculation of PGPB strains promoted plant Cd accumulation up to 261% in shoot and up to 8.93-fold increase in root. Among all the 12 PGPB strains, Burkholdria SaMR10 and Sphingomonas SaMR12 were identified as the promising microbes for improving phytoremediation efficiency of Cd contaminated soils. These results not only provided useful findings for further investigation of interacting mechanisms between different bacterial strains and plants, but also facilitated the development of microbe-assisted phytoremediation application for HM contaminated soil., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Endophyte-assisted phytoremediation has gained increasing attention. However, the interacting mechanisms of endophytes and metal hyperaccumulators are still not clear. An endophytic bacterium Pseudomonas fluorescens Sasm05 inoculation promoted Sedum alfredii Hance rooting and root development, in which the specific root length (SRL) and average number of root tips (ART) increased to 2.09- and 3.35-fold, respectively. Sasm05 inoculation promoted plant growth, increased the chlorophyll content, and elevated Zn uptake of plant at excess Zn supply. At 200 μM Zn treatment level, Sasm05 inoculation increased plant biomass and the chlorophyll content by more than 40%, and root Zn content by 40%. Furthermore, Sasm05 inoculation upregulated the expression of the Zn transporter SaIRT1 to 3.43-fold in the roots, while another transporter SaNramp1 expression was increased to 38.66-fold in the roots and 7.53-fold in the shoots. Time course study showed the best effects of Sasm05 on plant biomass and the chlorophyll content were detected at 30 d, while for Zn content at 3 d. These results firstly provided molecular evidences of endophytic bacteria in facilitating host plant Zn uptake, which will absolutely benefit the understanding of interacting mechanisms between hyperaccumulators and their endophytes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interactions between roots and microbes affect plant's resistance to abiotic stress. However, the structural and functional variation of root-associated microbiomes and their effects on metal accumulation in hyperaccumulators remain poorly understood. Here, we characterize the root-associated microbiota of a hyperaccumulating (HP) and a non-hyperaccumulating (NHP) genotype of Sedum alfredii by 16S ribosomal RNA gene profiling. We show that distinct microbiomes are observed in four spatially separable compartments: the bulk soil, rhizosphere, rhizoplane, and endosphere. Both the rhizosphere and rhizoplane were preferentially colonized by Proteobacteria, and the endosphere by Actinobacteria. The rhizosphere and endophytic microbiomes were dominated by the family of Sphingomonadaceae and Streptomycetaceae, respectively, which benefited for their survival and adaptation. The bacterial α-diversity decreases along the spatial gradient from the rhizosphere to the endosphere. Soil type and compartment were strongest determinants of root-associated community variation, and host genotype explained a small, but significant amount of variation. The enrichment of Bacteroidetes and depletion of Firmicutes and Planctomycetes in the HP endosphere compared with that of the NHP genotype may affect metal hyperaccumulation. Program PICRUSt predicted moderate functional differences in bacterial consortia across rhizocompartments and soil types. The functional categories involved in membrane transporters (specifically ATP-binding cassette transporters) and energy metabolism were overrepresented in endosphere of HP in comparison with NHP genotypes. Taken together, our study reveals substantial variation in structure and function of microbiomes colonizing different compartments, with the endophytic microbiota potentially playing an important role in heavy metal hyperaccumulation.

A hydroponic experiment was conducted to verify the effects of inoculation with endophytic bacteria Sphingomonas SaMR12 on root growth, cadmium (Cd) uptake, reactive oxygen species (ROS), antioxidases, glutathione (GSH) and the related gene expression of Sedum alfredii Hance under different levels of Cd such as 0, 10, 25, 100 and 400 μM. The results showed that inoculation of SaMR12 improved Cd accumulation and upregulated glutathione synthase (GS) expression, but slightly reduced malondialdehyde (MDA) concentration and alleviated Cd-induced damage in roots. However it didn't alter the activities of antioxidant enzymes. When Cd concentration exceeded 25 μM, SaMR12 increased the concentration of GSH and the expression level of GSH1. At high Cd treatment levels (100 and 400 μM), SaMR12 significantly reduced H2O2 concentration and enhanced expression level of 1-Cys peroxiredoxin PER1 and ATPS genes. These results indicate that although SaMR12 has no significant effects on antioxidases activities, it reduces H2O2 concentration by enhancing GSH concentration and relevant genes expression, and subsequently improves Cd tolerance and accumulation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Inoculating endophytic bacteria was proven as a promising way to enhance phytoremediation. By a hydroponic experiment, the role of this study was to clarify the effects of inoculating endophytic bacterium Sphingomonas SaMR12 on phytoremediation, with special emphasis on changes of cadmium uptake, plant growth, root morphology, and organic acids secretion at different cadmium treated levels (0, 5, 50, and 100 μM). The results showed that SaMR12 inoculation improved the accumulation of cadmium as well as plant biomass, length of roots, number of root tips, and root surface area. Root secretion of oxalic, citric, and succinic acids was also increased after inoculated, which may alleviate the cadmium toxicity to plant or inhibit the rising trend of oxidative stress of plant. The major finding of this work suggested that in the root, SaMR12 improves cadmium bioavailability and absorption facility by increasing root-soil contact area and root organic acid secretion; and in the shoot, SaMR12 increases cadmium tolerance by alleviating oxidative stress of plant, so as to enhance the capability of cadmium extraction by plant., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A greenhouse pot experiment was conducted to compare the phytoextraction efficiencies of Cd by hyper-accumulating Alfred stonecrop (Sedum alfredii Hance) and fast-growing perennial ryegrass (Lolium perenne L.) from a Cd-contaminated (1.6 mg kg(-1)) acidic soil, and their responses to the inoculations of two arbuscular mycorrhizal (AM) fungal strains, Glomus caledonium 90036 (Gc) and Glomus mosseae M47V (Gm). Ryegrass and stonecrop were harvested after growing for 9 and 27 wk, respectively. Without AM fungal inoculation, the weekly Cd extraction by stonecrop (8.0 μg pot(-1)) was 4.3 times higher than that by ryegrass (1.5 μg pot(-1)). Both Gc and Gm significantly increased (P < 0.05) root mycorrhizal colonization rates, soil acid phosphatase activities, and available P concentrations, and thereby plant P absorptions (except for Gm-inoculated ryegrass), shoot biomasses, and Cd absorptions (except for Gm-inoculated stonecrop), while only Gc-inoculated stonecrop significantly accelerated (P < 0.05) the phytoextraction efficiency of Cd by 78%. In addition, both Gc and Gm significantly decreased (P < 0.05) phytoavailable Cd concentrations by 21-38% via elevating soil pH. The results suggested the potential application of hyper-accumulating Alfred stonecrop associated with AM fungi (notably Gc) for both extraction and stabilization of Cd in the in situ treatment of Cd-contaminated acidic soil., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.

This study was the first attempt to examine the possible role of the naturally occurring rhizospheric bacteria in heavy metal removal by Sedum alfredii Hance, a terrestrial Zn/Cd hyperaccumuluator, from Zn, Cd, Cu and Pb contaminated water using antibiotic ampicillin. Moreover, the toxicity symptom in plants under heavy metal stress expressed as total chlorophyll, chlorophyll a and b content, growth inhibition, root length, and N, P contents were studied, and the possible relationship among them were also discussed. These results indicate that rhizospheric bacteria may play an important role in the uptake of N and P by S. alfredii, and consequently result in the increase of Chlorophyll content in the leaves and plant biomass due to improved photosynthesis. At the same time, root length significantly decreased under the treatment with ampicillin, which suggested that rhizospheric bacteria appeared to protect the roots against heavy metal toxicity. The Pb, Zn, Cu and Cd concentrations in the roots, stems and leaves of S. alfredii were much higher than those exposed to ampicillin. Accordingly, metal concentrations in the contaminated water without ampicillin treatment were lower than those treated with ampicillin. These results suggest that the rhizospheric bacteria may be useful in plant tolerance to heavy metal toxicity, and also accelerate the metal removal from contaminated water.

Although Pteris vittata L. and Sedum alfredii Hance have been identified as an As hyperaccumulator and a Zn/Cd hyperaccumulator, respectively, for a few years, variations in metal accumulation among populations and their arbuscular mycorrhizal (AM) status have not been fully explored. Six populations of P. vittata and four populations of S. alfredii from southeast China were investigated. Up to 1,373 As, 680 Pb, 376 Zn, 4.8 Cd, 169 Cu mg kg(-1) in fronds of P. vittata and 358 As, 2,290 Pb, 23,403 Zn, 708 Cd, 342 Cu mg kg(-1 )in shoots of S. alfredii were detected. Constitutive properties of As and Zn hyperaccumulation in metallicolous populations of P. vittata and S. alfredii, respectively, were confirmed. However, Cd hyperaccumulation in S. alfredii varied among populations. The two hyperaccumulators varied in efficiency in taking up other heavy metals. Different metal tolerance strategies adopted by the two hyperaccumulators varied among plant species and metal species. Low to moderate levels of AM colonization in P. vittata (4.2-12.8%) and S. alfredii (8.5-45.8%) were observed at uncontaminated and metal-contaminated sites. The relationship between metal concentrations and AM colonization in the two hyperacumulators was also examined. The abundance of AM fungal spores ranged from 16 to 190 spores per 25 g soil. Glomus microaggregatum, Glomus mosseae, Glomus brohultii and Glomus geosporum were the most common species associated with both P. vittata and S. alfredii. To our knowledge, this is the first report of AM fungal status in rhizosphere of P. vittata and S. alfredii.

To investigate the effects of bacteria (Burkholderia cepacia) on metal uptake by the hyperaccumulating plant, Sedum alfredii, a hydroponic experiment with different concentrations of Cd and Zn was conducted. It was found that inoculation of bacteria on S. alfredii significantly enhanced plant growth (up to 110% with Zn treatment), P (up to 56.1% with Cd treatment), and metal uptake (up to 243% and 96.3% with Cd and Zn treatment, respectively) in shoots, tolerance index (up to 134% with Zn added treatment), and better translocation of metals (up to 296% and 135% with Cd and Zn treatment, respectively) from root to shoot. In the ampicillin added treatment with metal addition, stimulation of organic acid production (up to an increase of 133% of tartaric acid with Cd treatment) by roots of S. alfredii was observed. The secretion of organic acids appears to be a functional metal resistance mechanism that chelates the metal ions extracellularly, reducing their uptake and subsequent impacts on root physiological processes.