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Synthetic communities derived from the core endophytic microbiome of hyperaccumulators and their role in cadmium phytoremediation

Microbiome volume 12, Article number: 236 (2024) Cite this article

Abstract

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

Introduction

Soil cadmium (Cd) contamination not only hampers agricultural productivity but also poses significant health risks to humans through the food chain [1]. Existing soil remediation strategies involve physical and chemical methods, which are costly and prone to causing secondary pollution [2]. In contrast, phytoremediation using hyperaccumulator plants offers an in situ, cost-effective, and environmentally friendly alternative for the remediation and resource recovery of heavy metals in farmland [3]. The efficiency of phytoremediation largely depends on the metal accumulation capacity and aboveground biomass of hyperaccumulator plants. Unfortunately, most hyperaccumulator plants have relatively low biomass, presenting a significant bottleneck in the advancement of phytoremediation technology [4].

The plant microbiome extends the functional potential of the host plant and is essential for plant growth, development, and adaptation to environmental stresses [5]. Although microorganisms cannot directly degrade heavy metals in soil, the success of phytoremediation largely depends on the interactions between the plant microbiome and the host plant [6]. Compared to plant-associated microbes in other niches, endophytic bacteria, which colonize the interior of the plant, have a closer symbiotic relationship with the host and pose lower environmental risks, making them advantageous for assisting in heavy metal phytoremediation [7]. Many studies have indicated that the bacterial communities inhabiting hyperaccumulators markedly differ from those in non-hyperaccumulators. For instance, significant differences in the abundance of Bacteroidetes, Firmicutes, and Verrucomicrobia were observed in Cd hyperaccumulator versus non-hyperaccumulator [8]. Additionally, species from the genera Streptomyces, Sphingomonas, and Sphingopyxis were found to be significantly associated with Cd hyperaccumulation in Sedum plants [9]. These endophytic bacteria enhanced heavy metal accumulation in host plants by promoting growth and reducing metal toxicity through siderophore secretion, hormone production, phosphate solubilization, and nitrogen fixation [10]. However, despite the isolation of numerous endophytic bacteria from hyperaccumulators and the identification of mechanisms by which some strains aid in heavy metal phytoremediation, most studies focused on single strains, overlooking their limitations in stability and functionality [11,12,13]. Furthermore, many conclusions are often based on correlations from omics data analyses. Therefore, the synergistic effects of endophytic bacteria in hyperaccumulators on plant heavy metal phytoremediation and their underlying mechanisms warrant further investigation.

The construction of synthetic microbial communities (SynComs) is an effective approach to bridging the gap between single-strain studies and microbiome research [14]. Researchers can construct SynComs using either a bottom-up or top-down approach, based on reductionist or holistic principles. The bottom-up approach constructs a SynCom by starting with known functional strains and progressively adding species based on their interactions, while the top-down approach selectively assembles a subset of a natural microbiome based on its composition and functional responses to environmental conditions [15]. Despite both methods being widely employed, the bottom-up approach relies on extensive trial and error and may overlook key nodes in natural microbial networks [15]. Conversely, the top-down approach depends on prior knowledge of specific genes, pathways, or biological functions, potentially missing interactions and functions within microbial communities that are not well understood or are unknown [16]. To address this, SynComs can be constructed based on the core microbiome. The plant core microbiome consists of microbial assemblages that are stably associated with the plant or its surrounding environment and have significant impacts on plant growth and health [17]. These assemblages represent the minimal subset of microbes that stably and efficiently inherit and maintain community functions [18]. For instance, Luo et al. [19] constructed an artificial microbial consortium based on core taxa from the endophytic microbiome of peanuts, which suppressed root rot disease and increased yield under continuous cropping conditions. Similarly, Zhang et al. [20] developed a SynCom based on core microbiota from maize phloem that demonstrated efficient nitrogen fixation within the plant. Identifying the plant core microbiome in a top-down manner, followed by a bottom-up investigation of the interactions among these community members, can more effectively construct SynComs and better elucidate the processes through which endophytic bacteria synergistically support plant remediation efficiency.

In this study, we investigated (1) the successional changes in the microbiome of Cd hyperaccumulator, (2) whether the endophytic core microbiome, particularly SynComs constructed from these core microbes, can enhance Cd phytoremediation, and (3) the mechanisms by which SynComs affect plant health and Cd accumulation (Fig. 1). We hypothesized that a unique set of core bacteria, crucial for plant Cd hyperaccumulation, existed in the hyperaccumulating ecotype of Sedum but was absent or insignificant in the non-hyperaccumulator. By studying the Cd accumulation characteristics of Sedum plants from different years, regions, and ecotypes, along with their associated endophytic bacterial communities, we identified the core endophytic microbiome associated with plant Cd hyperaccumulation using an integrated network pipeline. Based on the identification results and previously isolated endophytic bacteria from Sedum alfredii, we constructed a preliminary SynCom and investigated the colonization and synergistic effects of its members in sterile S. alfredii seedlings. Guided by these findings, we developed various SynComs and screened for the one with the highest Cd phytoremediation capability through solution culture experiments and genome resequencing. Furthermore, using transcriptomic approaches, we explored its role and underlying mechanisms in regulating the growth and Cd accumulation in two ecotypes of S. alfredii. This study will provide theoretical guidance for identifying core microbiomes associated with specific plant functions and lay a theoretical foundation for the application of SynCom based on endophytic core microbiomes in phytoremediation.

Fig. 1
figure 1

Constructing SynComsbased on endogenous core microbiota for plant Cd remediation. A simplified workflow for identifying core bacterial taxa unique to the hyperaccumulating ecotype (HE), constructing SynComs from core bacteria, and verifying their effects on Cd phytoremediation

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Materials and methods

Sampling data collection

Sedum plants and their corresponding rhizosphere soils were collected in September 2021 from three locations in Zhejiang province, China: Chun'an (CA), Quzhou (QZ), and Jiuxi (JX) (Table S1). The selected sites included two former mining areas (CA, QZ) with severe Cd contamination and one natural site (JX) with mild Cd contamination (Fig. 2a, b). At least five plant and soil samples (ranging from 5 to 10) were collected as biological replicates from each site. Samples were brought back to the laboratory within 24 h. Following the methods described by Bulgarelli et al. [21], plants were separated into shoot and root parts, each divided into two portions for metal analysis and DNA extraction.

Fig. 2
figure 2

Cd-related data and endophytic bacterial diversity and composition of different ecotypes of Sedum collected from various sites and years. a Total soil Cd concentrations. b Available soil Cd concentrations. c Cd concentrations in plant tissues. d Alpha-diversity analysis of endophytic bacterial communities, with box plots representing the quartiles of alpha diversity across different sample groups. Horizontal lines marked with asterisks indicate significant differences between two groups of sample data, with the number of asterisks ranging from 1 to 4, denoting P-values less than 0.05, 0.01, 0.001, and 0.0001, respectively (two-sided Wilcoxon rank-sum test). e Bray–Curtis dissimilarity analysis of endophytic bacterial communities. The endophytic bacterial communities in JX are distinct from those in CA and QZ along two axes (P < 0.001, PERMANOVA by Adonis), with ellipses covering 80% of the sampling data points. f Summarized chord diagrams showing the phylum-level composition of endophytic bacterial communities in both the shoot and root parts of Sedum. The upper half of the chord diagram represents the phylum-level classification of bacterial communities, while the lower half represents the community composition of samples from the three regions in 2019 and 2021. The number of independent samples for each group is as follows: in 2019: JX (n = 12), CA (n = 12), and QZ (n = 12) and in 2021: JX (n = 5), CA (n = 15), and QZ (n = 15). The same applies below

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After surface sterilization, DNA was extracted and purified with Ezup Column Soil DNA Purification Kit (Sangon, China). 16S rRNA genes of V5–V7 hypervariable regions were amplified.

using barcodes and index primers 799f/1193r, and the products were prepared and sequenced on the Illumina NovaSeq 6000 platform (Novogene, China). The amplicon sequence files obtained are available in the NCBI Sequence Read Archive (SRA) database (BioProject ID PRJNA1043636). Additionally, sequencing data of endophytic bacterial communities in Sedum plants collected from the same locations in 2019 were downloaded from the NCBI SRA under BioProject ID PRJNA702986. The methods and platform for DNA extraction and sequencing, as well as the measurements of plant tissue Cd concentration (PCd), soil Cd concentration (SCd), available soil Cd concentration (DTPA-Cd, SaCd), soil pH, and soil organic matter (SOM), were conducted as detailed by Wu et al. [9].

Pre-treatment of plants and soil for experiments

Soil and hyperaccumulator S. alfredii plants were collected from an agricultural field in Quzhou (29.0533°N, 119.0531°E), and non-hyperaccumulator S. alfredii plants were collected from Jiuxi (30.2105°N, 120.1047°E). To minimize the endogenous heavy metal concentrations of plants, they were pre-cultured for three generations under hydroponic conditions. The soil was air-dried for 30 days, sieved through 2 mm, and ground to homogenize. The soil Cd concentration was measured at 1.09 mg/kg (Fig. S11). For sterile soil experiments and DNA extraction, seedlings were surface sterilized using the method described by Chen et al. [22].

Strain origins and trait determination

All 13 candidate strains used to construct the synthetic community in this study were selected from hundreds of endophytic bacteria isolated from hyperaccumulator S. alfredii by Liao et al. [23]. Their IAA production, ACC deaminase activity, and tolerance to Cd were determined according to the methods described by Zhang et al. [24].

Preparation of synthetic community bacterial suspension

Single colonies of the respective candidate bacterial strains were inoculated into LB liquid medium and cultured at 30 °C with shaking at 150 rpm for 72 h. This incubation period was chosen based on preliminary experiments to ensure that the optical density (OD600) of all resuspended bacterial suspensions reached 2.0 or higher. After centrifugation, the supernatant was discarded, and the pellets were washed three times with sterile water. The pellets were then resuspended to form bacterial suspensions, which were diluted to an OD600 of 2.0. The OD600 of each bacterial suspension was adjusted to a proper concentration, ensuring that the final optical density of the mixed SynCom was 10 [7] cells per gram of soil [25].

Microbe transplant experiment

The pot experiment in sterile soil included four treatments: low Cd level (N), high Cd level (H), low Cd level with inoculation (JN), and high Cd level with inoculation (JH), each with four replicates. For soil preparation, 250 g of soil was transferred into tissue culture flasks with vented lids and supplemented with 50 mL of sterile water (Fig. S10). The flasks were autoclaved at 121 °C for 30 min and repeated every 24 h for three times. For the H and JH treatments, the soil Cd concentration was adjusted to approximately 15.0 mg/kg using Cd(NO3)2·4H2O. During seedling transplantation, 5 mL of a suspension containing 13 bacterial strains was applied to the root-soil interface for the JN and JH treatments, while an equal volume of sterile water was added to the N and H treatments [26]. Two surface-sterilized S. alfredi seedlings were planted per flask, and the same batch of surface-sterilized seedlings was used as the initial control group (CK). Except for the CK group, which was sampled immediately, all other treatment groups were cultured for 15 days before sample collection. Finally, all samples underwent metal analysis and DNA extraction. The amplicon sequencing data were submitted to NCBI SRA database under BioProject ID PRJNA1085113.

Hydroponic experiment

Hyperaccumulator S. alfredii seedlings were hydroponically grown in 1.5-L black plastic buckets, four seedlings per bucket, and cultured progressively in 1/8, 1/4, and 1/2 strength nutrient solutions formulated with 2-mM Ca (NO3)2, 0.1-mM KH2PO4, 0.1-mM KCl, 0.5-mM MgSO4, 0.7-mM K2SO4, 10-μM H3BO3, 0.5-μM MnSO4, 5-μM ZnSO4, 0.2-μM CuSO4, 0.01-μM (NH4)6Mo7O24, 100-μM Fe-EDTA, and 100-μM Cd (NO3)2 [27]. Following a 2-week acclimatization period, seedlings were segregated into 12 groups to assess the impact of inoculation with varying SynComs, featuring 6 different SynComs composed of various combinations of five bacterial strains. The treatments were as follows: a control group without any inoculation (CK), single-strain inoculations with SaLS1 from Leifsonia (L), SaNL1 from Novosphingobium (N), SaOA1 from Ochrobactrum (O), SaPI1 from Pseudomonas (P), and SaSP1 from Sphingomonas (S). Additionally, SynComs consisted of four strains, each excluding one specific strain: excluding SaLS1 (SynCom-NL), excluding SaNL1 (SynCom-NN), excluding SaOA1 (SynCom-NO), excluding SaPI1 (SynCom-NP), and excluding SaSP1 (SynCom-NS). The final treatment was a SynCom with all five bacterial strains (SynCom-A).

Pot experiment

After grinding and homogenization, the soil was placed in pots, each holding 2.0 kg of soil. Soil moisture content was maintained at approximately 70% of field capacity. Two ecotypes of S. alfredii were transplanted into separate pots, with three plants per pot. The experiment was conducted using a completely randomized block design, encompassing two ecotypes [hyperaccumulating ecotype (H) and non-hyperaccumulating ecotype (N)] and two treatments [inoculated (I) and uninoculated (U)], resulting in four treatments (HI, HU, NI, NU), each replicated four times. Every 5 days, pots were watered and inoculated. For the inoculated treatments, 5 mL of SynCom-NS bacterial suspension was added near the rhizosphere of each plant. For the uninoculated treatments, an equal volume of sterile water was added as a control. After 40 days of treatment, plants were collected and divided into two parts: one for physiological and biochemical analysis (biomass, Cd content, antioxidant enzyme activities, etc.) and the other for RNA extraction. Details of measuring antioxidant indicators, RNA extraction and sequencing, and qRT-PCR analysis can be found in the Supplementary Methods.

Genome sequencing of strains

Individual colonies of the five target bacterial strains were picked and inoculated into LB liquid medium. The cultures were incubated at 30 °C with shaking at 150 rpm until the logarithmic growth phase. Cells were collected by centrifugation, washed three times with sterile water, and used for DNA extraction and purification. Library preparation followed the standard Illumina TruSeq Nano DNA LT protocol, and sequencing was performed on the Illumina MiSeq platform. The sequencing data were aligned to the reference genome using BWA (version 0.7.17-r1188) and processed for consistency with Picard tools to remove duplicates and ensure paired-end read information integrity. SNP detection and annotation were performed using GATK software. Additionally, CNVs and structural variations (SVs) were detected using CNVnator and BreakDancer, respectively, with the appropriate filters applied to ensure accurate annotation. The genomic sequences were then submitted to NCBI SRA database under BioProject ID PRJNA1085268.

Statistical analysis

The QIIME2 (version 2023.2) pipeline process was taken for the raw sequences: FASTQ files were initially converted individually into QIIME2 artifacts. After removing potential primers with the cutadapt plugin, the sequences underwent quality control, chimera removal, and de-noising using the DADA2 plugin, resulting in the corresponding feature tables and representative sequences [28]. The representative sequences were annotated by a Naive-Bayes classifier, which was pre-trained on Silva 138 99% OTUs full-length sequences [29]. ASVs classified as “mitochondria,” “chloroplast,” “Archaea,” “Eukaryota,” and any unassigned ASVs were excluded. Finally, “phyloseq” package in R software (version 4.1.2) was used to integrate all feature tables, representative sequences, and corresponding classification files [30].

Alpha diversity was assessed through observed richness, Faith’s phylogenetic diversity, Simpson’s diversity, and Pielou’s evenness [31]. Beta diversity was estimated and displayed with Bray–Curtis matrices and principal coordinate analysis (PCoA) [32]. Two methods (ALDEx2 and ZicoSeq) were employed to detect differentially abundant genera between different ecotypes across years and plant compartments [33, 34]. Genera were considered enriched in the hyperaccumulating ecotype only if their abundance differences were significant and consistent across both years. ANCOM-BC2 was used to estimate and correct biases in microbiome count data due to sampling fractions and sequencing efficiency [35]. An innovative network pipeline was employed to identify core taxa in hyperaccumulating Sedum, as detailed in the Supplementary Methods. This process involved three steps: constructing a genus-level sparse network with both linear and nonlinear correlations between taxa using SECOM, detecting critical nodes based on network natural connectivity, and using “NetShift” to identify driver taxa promoting the transition from NHE to HE networks [36,37,38].

All statistical analyses were performed using R (Version 4.1.2). The translocation factor (TF) and bioconcentration factor (BCF) were calculated using the following formulas: TF = metal concentration in shoots/metal concentration in roots and BCF = metal concentration in roots/metal concentration in soil [39]. Significant differences between or among groups were determined using the Wilcoxon rank-sum test and Duncan’s multiple range test (P < 0.05) [40].

Results

Community compositions and Cd concentration in different Sedum ecotypes

Phenotypic data from 2019 and 2021 revealed that soil samples from the CA and QZ sites had significantly higher total Cd and available Cd concentrations compared to those from JX (Fig. 2 a, b). Correspondingly, plant tissues from CA and QZ consistently showed a significant increase in Cd content (Fig. 2c). Although the bacterial alpha diversity did not show significant differences among the three locations, beta-diversity analysis revealed distinct community differences between the samples from JX and those from CA and QZ (Fig. 2d, e, Fig. S1). This significant distinction was evident in both years and was particularly pronounced in root communities. The multivariate analysis of variance (PERMANOVA) underscored significant associations between endophyte community composition and factors such as plant compartment, plant ecotype, sampling year, and location (Table S2).

The endophyte communities were predominantly composed of Proteobacteria (33.52% to 87.09%) and Actinobacteria (3.99 to 53.78%) (Fig. 2f). At the phylum level, JX samples consistently showed the highest proportion of Actinobacteria and the lowest proportion of Proteobacteria across different years and plant compartments. Compared to the JX samples, CA and QZ samples exhibited similar enrichment patterns, suggesting an ecotype-driven rather than a region-specific difference (Figs. S2, S3). Consequently, in subsequent analyses, samples from JX were classified as NHE (non-hyperaccumulating ecotype), while samples from CA and QZ were classified as HE (hyperaccumulating ecotype).

Differential abundance analysis revealed that the genera Leifsonia and Rhodococcus were consistently and significantly enriched in the shoots of HE over 2 years. Additionally, 28 genera, such as Mesorhizobium, Hyphomicrobium, and Rhodoplanes, were significantly enriched in the roots of HE (Fig. S4). These taxa also showed strong correlations with Cd-related indices, including plant Cd (PCd), soil Cd (SCd), and available soil Cd (SaCd) concentrations.

Unique core taxa within hyperaccumulator ecotype

Using “NetShift” and key node detection algorithms in network analysis, we identified a set of core bacteria taxa specifically associated with HE (Fig. 3a). This group primarily consisted of members from the Proteobacteria and Actinobacteria phyla, which were insignificant or absent in the bacterial networks related to NHE (Fig. 3c, e). Among the group, 17 genera, such as Leifsonia, Bacillus, Cupriavidus, Rhodococcus, and Stenotrophomonas, were exclusively associated with the shoot bacterial network in HE; meanwhile, 68 genera, including Rhodoplanes, Hyphomicrobium, Sphingobium, Arthrobacter, and Flavobacterium, were unique in the root endophytic networks of HE; an additional 12 genera, including Sphingomonas, Pseudomonas, Microbacterium, and Bosea, spanned both shoot and root networks. The bias-corrected abundance of these taxa exhibited significant correlations with Cd-related indicators, especially in roots (Fig. 3b, d).

Fig. 3
figure 3

Unique core bacterial genera within the endophytic bacterial communities of hyperaccumulator Sedum. a Phylogenetic tree of core bacterial genera. The coloring of branches and nodes of the phylogenetic tree represents the phyla to which the taxa belong. The color of the genus names indicates the compartment where the taxa are core genera: yellow signifies core genera in both the shoot and root networks of hyperaccumulator Sedum, red indicates core genera only in the shoot network, and blue denotes core genera exclusively in the root network. b Spearman correlation between the bias-adjusted abundance of core genera in shoots and soil Cd (SCd), available soil Cd (SaCd), and plant Cd (PCd) concentrations. c Bias-adjusted abundance of core genera in shoots of different ecotypes of Sedum. d Spearman correlation between the bias-adjusted abundance of core genera in roots and SCd, SaCd, and PCd. e Bias-adjusted abundance of core genera in roots of different ecotypes of Sedum. ANCOM-BC2 was used to correct for the bias in genus abundance

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Selection of SynCom members for enhanced phytoremediation of Cd

Based on the above results, 13 bacterial strains isolated from the hyperaccumulating ecotype of S. alfredii were selected as candidates for constructing the SynComs. These 13 strains belong to 6 different genera. Eight of them, belonging to Pseudomonas, Leifsonia, and Sphingomonas, were identified as core bacteria linked to plant Cd hyperaccumulation. And the other five strains, from Novosphingobium, Ochrobactrum, and Alcaligenes, were chosen for their strong growth-promoting traits and Cd tolerance (Table S4). Then SynCom13, composed of 13 bacterial strains, was inoculated into the roots of hyperaccumulator S. alfredii in sterile soil.

Fifteen days after inoculation, the Cd concentrations in the roots and shoots of inoculated S. alfredii were slightly higher than those uninoculated (Figure S11). Differential abundance analysis showed a significant increase in the relative abundance of six candidate genera in both roots and shoots after inoculation (Fig. 4a). Incorporating plant Cd concentrations into the genus-level endophytic co-occurrence network and extracting Cd-associated sub-networks revealed the correlations of candidate genera within the plant (Fig. 4b). Notably, the abundance of Alcaligenes consistently exhibited a significant negative correlation with Cd concentration in plant tissues. Additionally, Alcaligenes showed a negative correlation with the abundances of other target genera across all treatments. Therefore, Alcaligenes was excluded from further synthetic community studies.

Fig. 4
figure 4

Colonization and interactions of candidate genera within the sterile seedlings and effects of different SynComs on the growth and Cd accumulation of S. alfredii. a Genus-level abundance differences in endophytic bacteria in shoots and roots of sterile S. alfredii seedlings after SynCom13 inoculation. Log fold change and P-values of genera before and after inoculation were calculated using ANCOM-BC2, with the number of asterisks ranging from 1 to 4, denoting P-values less than 0.05, 0.01, 0.001, and 0.0001 (adjusted by Holm-Bonferroni method), respectively. Bolded genera indicate candidate genera used for constructing SynCom. b Genus-level sub-networks of endophytic bacteria associated with plant Cd concentration under different treatments. Correlations were inferred from the bias-adjusted genus abundance table using the Spearman method, selecting only genus significantly associated with plant Cd concentration (P < 0.001) to construct the sub-networks. The statistical test used was two sided. c Images of S. alfredii plants in solution culture experiments under no inoculation (CK), single-strain inoculation (L, P, N, O, S), and SynComs inoculation (A, NL, NP, NN, NO, NS) treatments. d Effects of different inoculation treatments on dry weight and Cd accumulation of S. alfredii in solution culture experiments. Significant differences among treatments were determined using Duncan’s multiple range test

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By comparing the plant growth promotion (PGP) traits of strains within the same genus, we selected the strains SaPI1 (P), SaLS1 (L), SaOA1 (O), SaNL1 (N), and SaSP1 (S) to construct different SynComs. All five strains were capable of producing IAA and exhibited great Cd tolerance. Genome resequencing of the five strains revealed that they share more than 10 common growth-promoting genes, including those involved in phosphate solubilization, nitrogen fixation, IAA production, and siderophore production (Fig. S17). Notably, strain SaSP1 contained the fewest unique growth-promoting genes among the five strains. Moreover, KEGG pathway analysis showed that strain SaSP1 exhibited less potential for metabolic exchange with other strains in essential pathways, such as vitamin and amino acid synthesis (Figs. S18, S19, S20, S21, S22).

Effects of different SynComs on growth and Cd accumulation in S. alfredi

Six different SynComs, composed of four or five selected bacterial strains, were constructed and inoculated into hyperaccumulating S. alfredii under hydroponic conditions. Compared to single-strain inoculations or control treatment, plants inoculated with the SynComs exhibited significantly improved growth (Fig. 4c). Notably, the SynCom-NS treatment group showed a marked increase in plant dry weight and Cd accumulation in both shoots and roots compared to other groups (Fig. 4d). This indicated that SynCom-NS had a significant advantage in promoting plant growth and enhancing Cd uptake.

Effects of SynCom-NS on growth and Cd response in two ecotypes of S. alfredii

In pot experiments, inoculation with SynCom-NS significantly increased leaf area and root length in both ecotypes of S. alfredi (Fig. S23). Following inoculation, the Cd bioconcentration factor (BCF) and translocation factor (TF) significantly increased in both ecotypes (Fig. 5a, b). Additionally, the dry weight and Cd concentration of plant tissues were significantly elevated, particularly in the shoots, where Cd concentrations increased by 87.97% and 58.97%, respectively (Figs. 5c, S24).

Fig. 5
figure 5

Effects of SynCom-NS inoculation on Cd uptake, translocation, oxidative stress indicators, and functional gene expression in two ecotypes of S. alfredii. a Effect of SynCom-NS inoculation on Cd bioconcentration factors in two ecotypes of S. alfredii. b Effect of SynCom-NS inoculation on Cd translocation factors in two ecotypes of S. alfredii. c Effect of SynCom-NS inoculation on Cd concentration in two ecotypes of S. alfredii. d Effect of SynCom-NS inoculation on H2O2 content in leaves of two ecotypes of S. alfredii. e Effect of SynCom-NS inoculation on GSH content in leaves of two ecotypes of S. alfredii. Horizontal lines marked with asterisks indicate significant differences between two groups, with the number of asterisks ranging from one to four, denoting P-values less than 0.05, 0.01, 0.001, 0.0001, respectively (two-sided Wilcoxon rank-sum test). f Heatmap of upregulated genes related to antioxidase, divalent metal transporters, and hormone signaling in two ecotypes of S. alfredii after SynCom-NS inoculation. Upregulated genes (|log2FoldChange|> 1 and P-value < 0.05) were identified using DESeq. Z-score transformation was applied to the abundance of selected genes. g Linear regression of qRT-PCR and RNA-Seq results. The fold change values for the RNA-seq and qRT-PCR of eight genes related to Cd transport are plotted along with the linear fit line. The Pearson’s correlation coefficients (r), coefficients of determination (R [2]), and the P-value (two-sided t-tests) are shown, respectively

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Inoculation with SynCom-NS significantly enhanced the antioxidant defense system in the leaves of hyperaccumulator and non-hyperaccumulator ecotypes of S. alfredii. Hydrogen peroxide (H2O2) levels decreased by 0.57-fold and 0.54-fold, while the reduced glutathione (GSH) content increased by 1.68-fold and 1.41-fold (Fig. 5d, e). Additionally, the activities of various antioxidant enzymes were significantly boosted: ascorbate peroxidase (APX) activity increased by 1.74-fold and 1.55-fold, catalase (CAT) activity by 1.39-fold and 2.28-fold, glutathione reductase (GR) activity by 1.23-fold and 1.79-fold, superoxide dismutase (SOD) activity by 1.48-fold and 1.31-fold, and peroxidase (POD) activity by 1.70-fold and 1.49-fold in the leaves of two ecotypes, respectively (Fig. S26).

Effects of SynCom-NS on transcriptomic regulation in two ecotypes of S. alfredii

Using DESeq, we identified upregulated genes in both ecotypes following inoculation and found significant upregulation of 624 genes related to antioxidant defense, plant hormone signaling, and divalent metal ion transport (Fig. 5f, Supplementary file 2). A total of 76 genes associated with the plant antioxidant system were upregulated, encoding antioxidants such as GR, POD, APX, CAT, and SOD. These genes were broadly upregulated, particularly in the roots of hyperaccumulator S. alfredii. Additionally, 49 genes related to plant hormone signaling were upregulated, involved in pathways of various plant hormones including gibberellin (GA), abscisic acid (ABA), auxin (IAA), cytokinin (TZ), brassinosteroid (BR), salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), with auxin signaling being the most prominently upregulated. Most importantly, 499 upregulated genes associated with divalent metal ion transport were found in two ecotypes, encoding transport proteins for ions such as Ca2+, Cu2+, Mg2+, Zn2+, and Cd2+. This result was consistent with qRT-PCR analysis, confirming that SynCom-NS effectively enhanced the expression of genes related to Cd ion transport proteins in both ecotypes of S. alfredii.

Discussion

Identification of core endophytic microbiome associated with Cd hyperaccumulation

In hyperaccumulator plants, endophytic bacterial communities regulate Cd accumulation through various mechanisms that include promoting plant growth, enhancing Cd transport, and mitigating Cd toxicity [41,42,43]. Our investigation revealed that ecotypic variation in Sedum plants significantly influenced the diversity of their endophytic bacterial communities, independent of geography, year, or plant compartment (Table S2). Core genera within these communities play essential roles in maintaining community stability and functional traits [21, 44].

To conservatively and comprehensively identify unique core bacterial genera within the hyperaccumulators, we designed an innovative integrated network pipeline (Fig. S5). We identified 97 core bacterial genera specifically present in the hyperaccumulator Sedum, which were closely associated with Cd hyperaccumulation (Fig. 3). Among these genera, many are challenging to isolate and culture [45, 46]. However, it is encouraging that several cultivable genera have been repeatedly reported to possess strong potential for aiding phytoremediation. For instance, Pseudomonas, a core genus in the roots and shoots of hyperaccumulator Sedum, has been reported to benefit plants through nitrogen fixation, solubilizing phosphorus and potassium, producing IAA, ACC deaminase activity, and siderophore production [47, 48]. It was found that Pseudomonas fluorescens enhanced photosynthesis, carbon fixation, and Cd accumulation in S. alfredii [49]. Similarly, Leifsonia, a core endophytic genus specifically found in the shoots of hyperaccumulating Sedum, contained genes associated with heavy metal resistance and produced plant hormones such as gibberellins and auxins, promoting shoot growth [50]. Another core genus, Sphingomonas, was identified as enhancing shoot elongation and participating in the transport and detoxification of heavy metals within the plant [51]. And Wang et al. [52] revealed that Sphingomonas spp. SaMR12 alleviated Cd stress in mustard by regulating antioxidant enzyme activities. Although the roles and relevance of these biomarkers, core microbiomes, or potentially beneficial taxa have been proposed, their protective mechanisms and functional roles still require in situ validation through individual and collective culture-based methods.

Functions of core endophytic microbiota in plant growth and Cd uptake

In sterile soil culture experiments, the bias-corrected abundance of six target genera in both the roots and shoots significantly increased after inoculation with SynCom13, indicating successful colonization of the host plant roots and migration to the shoots (Fig. 4a). Network analyses provided evidence of synergistic relationships among inoculated microbes, suggesting that strong interactions within target taxa may ensure the consistent presence and functionality of the core microbiome (Fig. 4b). Although Alcaligenes was considered a plant-beneficial bacterium, it reduces Cd bioavailability through biomineralization by producing large amounts of organic biominerals [53]. In this study, Alcaligenes was negatively correlated with Cd concentration and antagonistic to other target genera in the host plants (Fig. 4b). Conversely, Leifsonia, Novosphingobium, Pseudomonas, Sphingomonas, and Ochrobactrum showed significant positive correlations with each other and with Cd concentrations of plants. These genera likely have synergistic relationships within the plants, enhancing Cd accumulation. Such interactions may provide a selective advantage for co-colonization by community members, stabilizing the core community’s composition and ensuring its persistence in specific habitats [54].

The growth-promoting responses of plants under abiotic and biotic stress induced by single-strain inoculants have been well-documented [55,56,57,58]. However, these strains often failed when applied individually in field conditions due to competition from the native microbiome [59, 60]. Consequently, the “one microbe at a time” approach has evolved into creating synthetic microbial communities. Through hydroponic experiments and the SynCom method, we provided evidence that SynComs constructed from the endophytic core microbiome significantly improve host plant Cd accumulation and growth (Fig. 4c, d). These SynComs included key strains carrying genes essential for host performance, such as those involved in growth-regulating hormone production and nutrient mobilization (Fig. S17). Compared to other SynComs, SynCom-NS demonstrated a significant advantage in promoting plant growth and enhancing Cd hyperaccumulation (Fig. 4d). Metabolic complementarity is crucial for the stability and efficiency of SynComs, as it ensures effective resource and function sharing among strains [61, 62]. Genome resequencing revealed that out of the five candidate strains, strain SaSP1 had the fewest unique growth-promoting genes and limited potential for metabolic exchange (Figs. S18, S19, S20, S21, S22). SynCom-NS improved community performance by excluding strain SaSP1, thereby enhancing metabolic complementarity among the remaining strains.

Mechanisms of SynCom-NS in promoting growth and Cd accumulation in S. alfredii

Compared to non-hyperaccumulating ecotypes, hyperaccumulator S. alfredii can accumulate extremely high concentrations of Cd in shoots without exhibiting toxicity symptoms [63]. This differentiation likely results from a combination of genetic variation and environmental pressures, with the molecular basis of hyperaccumulation involving the high transcriptional expression of genes related to heavy metal uptake, transport, and detoxification [64]. It is well documented that an increase in the number of lateral roots, particularly new lateral roots, contributed to plant Cd uptake from the soil [65, 66]. Following SynCom-NS inoculation, both ecotypes of S. alfredii exhibited a significant increase in the number and length of lateral roots, which enhanced Cd uptake in plants (Fig. S23). Plant hormones are critical in root development by regulating cell division, elongation, and differentiation [67]. Endophytic bacteria promote root development by modulating various plant hormone signaling pathways, such as the interaction between auxin and ethylene/jasmonic acid signaling [68, 69]. Transcriptomic analysis indicated that SynCom-NS upregulated the expression of genes associated with multiple plant hormone signaling pathways, thereby promoting root growth and development, which in turn enhanced Cd uptake and accumulation in the host plants (Fig. 5f).

Efficient metal transport is a key trait that allows hyperaccumulating plants to thrive in contaminated environments while effectively extracting and remediating soil heavy metals [70]. In the pot experiment, SynCom-NS significantly enhanced Cd transport efficiency in two ecotypes of S. alfredii (Fig. 5b, c). Due to the lack of specific Cd transport proteins, the mechanisms for Cd transport in plants are often closely associated with membrane transporters for other metal ions, including heavy metal ATPases, the natural resistance-associated macrophage protein (Nramp) family, and cation diffusion facilitators [71]. Therefore, the increased Cd transport capacity in S. alfredii may be attributed to the upregulation of divalent metal ion transporter proteins within the plant (Fig. 5f, g).

As a toxic heavy metal, Cd induces oxidative stress in plants, leading to the accumulation of reactive oxygen species (ROS), which damage cellular structures and functions [72]. Post-inoculation with SynCom-NS, both ecotypes of S. alfredii showed a significant decrease in H2O2 content and a significant increase in GSH content in their leaves, indicating enhanced antioxidant capacity (Fig. 5d, e). Plants rely on antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPx), and catalase (CAT), as well as nonenzymatic antioxidants like reduced glutathione (GSH) and flavonoids, to scavenge harmful ROS [73, 74]. Genes related to these enzymes were highly upregulated after inoculation (Fig. 5f). The improved antioxidant capacity likely contributes to the stability and robustness of the hyperaccumulator plants under heavy metal stress [75, 76]. Thereby, the upregulation of antioxidant-related genes suggested that SynCom-NS not only supported Cd uptake and accumulation but also mitigated Cd-induced oxidative stress, enhancing overall plant health.

In conclusion, we investigated the variation of endophytic bacterial communities in Sedum across different years, locations, and ecotypes, finding that the ecotype strongly influenced bacterial community assembly. An innovative network pipeline was designed to identify 97 core bacterial taxa specifically present in the hyperaccumulator Sedum and associated with Cd hyperaccumulation. Based on these findings and previously isolated endophytic bacteria from S. alfredii, we constructed different SynComs and identified SynCom-NS as the most effective in enhancing plant phytoremediation. Inoculation with SynCom-NS significantly upregulated genes related to Cd transport, antioxidant enzymes, and plant hormone signaling in both ecotypes of S. alfredii, promoting plant growth and Cd accumulation. This study establishes a theoretical foundation for identifying plant-specific functional core endophytic microbiomes and provides robust evidence for applying synthetic microbial communities derived from these core microbiomes to enhance plant Cd phytoremediation.

Data availability

Sequence data that support the findings of this study have been deposited in the NCBI Sequence Read Archive (SRA) database under BioProject ID PRJNA1043636, PRJNA1085113, and PRJNA1085268.

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (No. 42277002 and No. 42307035) and the Natural Science Foundation of Zhejiang Province, China (No. LZ22D010002).

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Authors and Affiliations

  1. Key Laboratory of Environment Remediation and Ecological Health of Ministry of Education, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China

    Lukuan Huang, Ziyan Fan, Zhipeng Hu, Zhesi Li, Yingyi Fu, Xianyong Lin & Ying Feng

  2. College of Ecology, Taiyuan University of Technology, Taiyuan, 030024, People’s Republic of China

    Qiong Wang

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Contributions

Y. Feng and X. L. designed this study; L. H. and Y. F. collected all the data; Q. W. and Z. L. performed upstream analysis of data; L. H. and Z. H. performed downstream analysis of data; L. H. and Z. F. wrote the paper; L. H. visualized the results.

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Supplementary Information

40168_2024_1959_MOESM1_ESM.xlsx

Supplementary file 1: Supplementary methods: Identification of core genera in HE, network construction, critical nodes detection, core genera comparison, determination of antioxidant indicators, RNA extraction, sequencing and analysis, qRT-PCR analysis. Supplementary Results: differences in endogenous networks among different ecotypes of Sedum, information on sampling sites, repeated measures ANOVA assessing factors influencing diversity differences in Sedum and S. alfredii, topological properties of genus-level co-occurrence networks of endophytic bacteria in Sedum, traits of 13 candidate strains for SynCom construction, genome assembly performance evaluation for 5 candidate strains, list of primers used for qRT-PCR, summary of transcriptome sequencing gene annotation, alpha-diversity of Sedum endophytic bacterial communities in three regions over different years and compartments, differences in phyla of endophytic bacterial communities between Sedum collected from CA, QZ, and JX in 2019 and 2021, enriched genera in the hyperaccumulator Sedum, the network pipeline to identify unique core bacterial genera in the hyperaccumulator Sedum, network robustness tests, changes in genus-level shared bacterial subnetworks in shoots and roots of different ecotypes of Sedum, planting S. alfredii in sterile soil culture system, effects of SynCom13 inoculation on Cd content in soil and S. alfredii, effects of SynCom13 inoculation on alpha diversity of endophytic communities in S. alfredii, Bray–Curtis dissimilarity analysis of endophytic bacterial communities after inoculation of SynCom13, composition histogram of endophytic bacterial communities under each treatment, genus-level co-occurrence network of endophytic bacterial communities in S. alfredii under different treatments, effects of different inoculation treatments on growth and Cd uptake of S. alfredii, KEGG annotated genes in the genomes of the five candidate strains, metabolic pathway diagrams for arginine biosynthesis, histidine metabolism, glycine, serine and threonine metabolism, biotin metabolism, and vitamin B6 metabolism, effects of inoculating SynCom-NS on growth, biomass, Cd accumulation, and peroxidase activity of two ecotypes of S. alfredii, differential gene analysis of two ecotypes after inoculation with SynCom-NS, relative expression levels of 8 Cd transporter genes by qRT-PCR, functional pathway enrichment analysis for GO and KEGG. Supplementary Data: Upregulated genes related to antioxidant defense, divalent metal transport, and plant hormone signaling in each treatment, list of genes related to PGP traits in KEGG database

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Supplementary file 2: Supplementary Figures: Figure S1. Alpha-diversity of Sedum endophytic bacterial communities in three regions under different years and compartments. Figure S2. Differences in phyla of endophytic bacterial communities between Sedum collected from CA and QZ and those from JX in 2019. Figure S3. Differences in phyla of endophytic bacterial communities between Sedum collected from CA and QZ and those from JX in 2021. Figure S4. Persistently and significantly enriched genera in the hyperaccumulator Sedum. Figure S5. The network piepline to identify the unique core bacterial genera in the hyperaccumulator Sedum. Figure S6. Genus-level co-occurrence networks of endophytic bacterial communities in different compartments of two ecotypes of Sedum. Figure S7. Network robustness tests under different node removal orders. Figure S8. Changes in the genus-level shared subnetworks of endophytic bacterial communities in the shoots of different ecotypes of Sedum. Figure 9. Changes in the genus-level shared subnetworks of endophytic bacterial communities in the roots of different ecotypes of Sedum. Figure S10. Planting S. alfredii in sterile soil culture system. Figure S11. Effects of SynCom13 inoculation on Cd content in soil and S. alfredii. Figure S12. Effect of SynCom13 inoculation on alpha diversity of endophytic communitis in S. alfredii. Figure S13. Bray–Curtis dissimilarity analysis of endophytic bacterial communities after inoculation of SynCom13. Figure S14. Composition histogram of endophytic bacterial communities under each treatment. Figure S15. Genus-level co-occurrence network of endophytic bacterial communities in S. alfredii under different treatments. Figure S16. Effects of different inoculation treatments on growth and Cd uptake of S. alfredii. Figure S17. KEGG annotated genes in the genomes of the five candidate strains. Figure S18. Metabolic pathway diagram of 5 candidate strains: arginine biosynthesis. Figure S19. Metabolic pathway diagram of 5 candidate strains: histidine metabolism. Figure S20. Metabolic pathway diagram of 5 candidate strains: glycine, serine and threonine metabolism. Figure S21. Metabolic pathway diagram of 5 candidate strains: biotin metabolism. Figure S22. Metabolic pathway diagram of synthetic community candidate strains: vitamin B6 metabolism. Figure S23. Effects of inoculating SynCom-NS on growth of two ecotypes of S. alfredii. Figure S24. Effects of inoculating SynCom-NS on biomass of two ecotypes of S. alfredii. Figure S25. Effects of inoculating SynCom on Cd content in soil and Cd accumulation in plants. Figure S26. Effect of inoculation of SynCom on peroxidase activity in two ecotypes. Figure S27. Differential gene analysis of two ecotypes after inoculation with SynCom-NS. Figure S28. Relative expression levels of 8 Cd transporter genes by qRT-PCR. Figure S29. Functional pathway enrichment analysis for GO and KEGG. Supplementary Tables: Table S1. Information of sampling sites. Table S2. Repeated measures ANOVA assessing factors influencing diversity differences in Sedum endophytic bacterial communities. Table S3. Topological properties of genus-level co-occurrence networks of endophytic bacteria in different group of Sedum. Table S4. Traits of 13 candidate strains for SynCom construction. Table S5. Repeated measures ANOVA assessing factors influencing diversity differences in endophytic bacterial communities of S. alfredi. Table S6. Genome assembly performance evaluation table for 5 candidate strains. Table S7. List of primers used for qRT-PCR in this study. Table S8. Summary of transcriptome sequencing gene annotation

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Huang, L., Fan, Z., Hu, Z. et al. Synthetic communities derived from the core endophytic microbiome of hyperaccumulators and their role in cadmium phytoremediation. Microbiome 12, 236 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40168-024-01959-x

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Microbiome

ISSN: 2049-2618

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