Effect of cadmium stress on the bacterial community in the rhizosphere of mulberry (Morus alba L.)

Abstract

Mulberry has a good tolerance to cadmium (Cd) and is considered a candidate plant for phytoremediation. The rhizosphere microbial community plays an important role in phytoremediation. Nevertheless, little information on the rhizosphere microbial community mechanisms in mulberry during the phytoremediation of Cd-contaminated soil is available. In this study, the remediation efficiency of mulberry in pots subjected to three simulated Cd pollution levels and their rhizosphere bacterial communities during the remediation process were analyzed. “Yuesang 11” was used as the test mulberry variety, and three simulated Cd pollution levels were set by adding three concentrations of Cd (Cd5, 5 mg kg⁻¹; Cd3, 3 mg kg⁻¹; Cd2, 2 mg kg⁻¹). The results showed that the elimination rates of Cd in the rhizosphere soil were 81.7%, 85.3%, and 57.9% under the stress of the Cd2, Cd3, and Cd5 conditions, respectively. Meanwhile, 3,082,583 high-quality sequence reads and 976 operational taxonomic units were successfully obtained from the mulberry rhizosphere soil by high-throughput absolute quantification sequencing and further assigned to 11 bacterial phyla and 26 families. Of these, decreased abundances of 19 bacteria at the family level and increased abundances of seven bacteria under Cd stress were revealed by comparative analysis. Based on the alpha diversity indices (Chaol, Shannon and Simpson) and principal component analysis, the rhizosphere bacterial diversity of the Cd5 condition was significantly decreased, but that of the Cd2 and Cd3 conditions was not different from that of soil without Cd (CK). Likewise, redundancy analysis showed that the abundances of Acidobacteria Gp2, Acidobacteria Gp13, and Sphingobacteria were significantly positively associated with the elimination rates of Cd. This study suggested that the mulberry rhizosphere contains a relatively stable bacterial community consisting of diverse Cd-resistant bacteria, providing a scientific basis for remediating heavy-metal polluted soils using mulberry.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01090-3.

Introduction

Cadmium (Cd), a heavy metal belonging to group 12 (IIB) [1], is extremely toxic and was first listed among the 12 chemicals that pose global hazards by the United Nations Environment Program in 1984 [2]. A report in China Youth Daily published on 1st February 2012 stated that nearly 20% of arable land was contaminated with heavy metals and that Cd pollution was one of the most serious soil heavy metal pollutants in China [3]. Numerous studies have demonstrated that Cd in the soil not only is toxic to plants but also causes serious health issues for animals and humans by entering the food chain [4]. It is therefore urgent to find ways to effectively remediate Cd-contaminated soils [5].

Phytoremediation is a green technology with good public reception and is a preferable option to clean up heavy metal-contaminated sites [6, 7]. In phytoremediation, most removal of heavy metals occurs because of the activity of microbes in the rhizosphere [8, 9]. Rhizosphere microorganisms have evolved many strategies to counter heavy metal stress, including the active efflux of metals, metal ion sequestration, Cd accumulation, and enzymatic detoxification [10–12]. Therefore, various rhizosphere microbes support different functions, indicating the significance of functional diversity within microbial communities in plant and heavy metal-contaminated soil systems [13, 14]. Therefore, the characterization of rhizosphere microbial diversity and its community is crucial to further understanding microbe-heavy metal interactions in phytoremediation.

Mulberry (Morus alba L.) is a perennial woody plant characterized by large biomass, well-developed roots, and high resistance to drought, salts, and heavy metals [15]. Numerous studies in recent years have focused on the accumulation, mobility, and environmental safety of Cd in the soil-mulberry-silkworm system, indicating that mulberry has an excellent tolerance and enrichment capacity for Cd [16–21]. Although the micro-environmental characteristics of Cd-polluted soil after 3 years of remediation by mulberry using microbiological isolation culture methods have been reported [22], there is still limited information about the interaction of Cd and rhizosphere microbiota in phytoremediation by mulberry because more than 99% of soil bacteria are non-culturable [23]. In this study, the characterization of mulberry rhizosphere bacterial communities using high-throughput sequencing technology and the efficiency of Cd remediation under different simulated Cd pollution conditions were analyzed to further understand the phytoremediation mechanisms of mulberry

Materials and methods

Plant cultivar and soil preparation

This study was conducted using pots. The Yuesang 11 mulberry variety is a Cd-tolerant ecological cultivar originating from Guangdong Province and was used as test material for this study. The test soil was collected from suburban farmland in Nanchang Province, China (116°014′N, 28°375′E). Its physical and chemical properties were as follows: pH 5.02 (soil to water ratio 1:2.5); total nitrogen (TN), 0.85 g kg⁻¹; soil organic matter (SOM), 2.96 g kg⁻¹; total phosphorus (TP), 0.32 g kg⁻¹; total potassium (TK), 68.57 g kg⁻¹; and an undetectable level of total Cd, meeting the third level of soil environmental quality in accordance with GB15618-1995 (Soil Environmental Quality Standards, GB15618-1995, China) [23].

Trial design and index determination

Trial design

Three Cd-contaminated soil levels were prepared by adding soil with 5 mg, 3 mg, or 2 mg of Cd per kg after treatment with CdCl₂·2H₂O solution [24] to simulate three realistic Cd pollution levels: severe (Cd5), moderate (Cd3), and light (Cd2). Soil without Cd (CK) was used as a blank control. The concentration of CdCl₂·2H₂O in the stock solution was 1 g L⁻¹. The treated soil was first stabilized for 30 days and then added to one pot; also, the 10g soil per each treatment was collected to analyze the initial concentration of Cd. Finally, one mulberry seedling with consistent growth was planted. All mulberry plants were grown in an illumination incubator at 25±2°C under a 16-h photo-period with a light intensity of 3,600 l×. The soil moisture content was maintained at approximately 60%. The experiment design was a randomized block and each treatment was replicated three times, and each time with 3 pots, for a total of 9 pots per treatment.

Soil sample collection

The rhizosphere soil was collected after 90 days of growth; that mulberry plant was at the vigorous growth period named the eight-leaf stage [25]. Samples were collected as follows: we removed loosely adherent soil from the rhizosphere soil by shaking gently, leaving only a proportion of tightly adherent soil as rhizosphere soil, and three plants per treatment were selected. Approximately 10 g of rhizosphere soil per independent replication for each treatment was mixed, homogenized, and then divided into two parts [26]. One was used to analysis of the final concentration of Cd and its solubility after being air-dried, ground, and sieved at 0.25 mm. The other was transferred to 15-mL sterile centrifuge tubes, stored in a liquid nitrogen tank, and transported to the laboratory for determination of the bacterial community structure.

Analysis of Cd solubility in rhizosphere soils

Cd solubility was analyzed using Tessier extraction [27]. The extracted Cd fractions, including the exchangeable (EXC), carbonate-bound (CAB), Fe-Mn oxide (FMO), organic matter (SOM), and residue (RES) fractions, were extracted and then analyzed by inductively coupled plasma–mass spectrometry (ICP–MS, Thermo 6300) [28]. The elimination rate of Cd was calculated using the following formula: R = (C_i − C_f)/C_i, in this formula, R represented the elimination rate of Cd, C_i represented the initial concentration of Cd, and C_f represented the final concentration of Cd.

DNA extraction and absolute quantification 16S-seq

The DNA extraction of the rhizosphere soil (0.5g) was performed using the MP soil reagent kit (CAT NO. 116560) according to the manufacturer’s instructions. The integrity of the extracted DNA was assessed through agarose gel electrophoresis, and the concentration and purity of extracted DNA were tested using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., USA) and a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA ), for assessing the sequence in the library that was between 120 and 200 bp without nonspecific amplification. Profiling was set to 20 ng μL⁻¹, and total content was required to be ≥ 500 ng. To obtain an accurate and reliable absolute abundance of soil bacteria [29], the absolute quantification 16S-seq by MiSeq [30] was conducted in Genesky Biotechnologies Inc., Shanghai, China. The spike-in sequences with identical conserved regions to natural 16S rRNA genes and variable regions replaced by random sequences with approximately 40% GC content were artificially synthesized. Furthermore, nine different spike-in sequences with known gradient copy numbers were added to the sampled DNA pools, functioning as internal standard and allowing the absolution quantification across samples. Thus, the V3-V4 hypervariable regions of microbial 16S rRNA gene and spike-in sequences were amplified with a forward primer (Illumina adapter sequence 1 + CCTACGGGNGGCWGCAG) and reverse primer (Illumina adapter sequence 2 + GACTACHVGGGTATCTAATCC) [31–33].

PCR amplification was achieved on an ABI 2720 thermal cycler (Thermo Fisher Scientific, MA, USA) with a TopTaq DNA polymerase kit (Transgen, Beijing, China). After library quantification, pooling, and quality check, all samples were sequenced using Illumina NovaSeq 6000 (Illumina, USA) sequencing (2×250 bp). Each biological replicate was replicated three times. The detected numbers of spike-in reads can be used for read amount normalization of different samples DNA and further for absolute quantification of microbial taxa.

Illumina MiSeq sequence processing and analysis

The raw read sequences were processed in Quantitative Insights Into Microbial Ecology 2 (QIIME 2™, version 2019.10) [34]. The adaptor and primer sequences were trimmed using the cutadapt plugin. The DADA2 plugin was used for quality control and to identify amplicon sequence variants (ASVs) [35]. The bacteria taxonomic assignments of ASV-representative sequences were performed with a confidence threshold of 0.8 by a pre-trained Naive Bayes classifier in RDP (version 11.5) [36]. Thus, the raw sequencing data which was the bacterial 16S rRNA was deposited in Genome Sequence Archive (GSA) in China National Center for Bioinformation (CNCB) with the accession number CRA009661.

Data analysis

The alpha-diversity (Chaol index, Simpson index, Shannon index) of bacteria was calculated using QIIME2 [34]. Microbiota dissimilarity among the four treatments (Cd2, Cd3, Cd4, and CK) was depicted by principal coordinate analysis (PCoA). Associations between rhizosphere bacteria and environmental parameters (the concentration of different Cd chemical forms and elimination rate of Cd) were performed using the Spearman correlation test. The statistical significance of data was determined using a one-way ANOVA analysis followed by the Duncan’s test (SPSS 12.0, SPSS Inc., USA). The visualization of data and analysis results was all accomplished by R software (v 4.0.5).

Results and analysis

Composition and forms of Cd in mulberry rhizosphere soil subjected to Cd stress

As can be seen in Table 1, there was considerable interspecific variation in the composition and forms of Cd in mulberry rhizosphere soil subjected to Cd stress. Specificity, the total concentrations of Cd in the rhizosphere soil subjected to various treatments differed (0.367 mg/kg for Cd2, 0.440 mg/kg for Cd3, 2.107 mg/kg for Cd5) (Table 1). The speciation of Cd in the rhizosphere soil under different Cd stresses consisted of five forms, including EXC-Cd, FMO-Cd, RES-Cd, CAB-Cd, and SOM-Cd. Among them, the highest relative Cd content was found for EXC-Cd (33.52% for Cd2, 34.78% for Cd3, 44.76% for Cd5), followed by FMO-Cd (28.07% for Cd2, 34.09% for Cd3, 34.15% for Cd5), and the lowest content was found for RES-Cd (3.54% for Cd2, 2.95% for Cd3, 1.9% for Cd5). Additionally, the elimination rate of Cd was highest for the Cd3 condition, 85.3%, followed by the Cd2 condition, 81.7%, and the lowest rate was observed for the Cd5 condition, 57.9%.

Table 1.

Concentration and forms of Cd in mulberry rhizosphere soil subjected to different Cd stress

Treatment	EXC-Cd		CAB-Cd		FMO-Cd		SOM-Cd		RES-Cd		Total-Cd (mg kg−1)	Elimination rate of Cd
	Concentration (mg kg−1)	Relative content rate(%)	Concentration(mg kg−1)	Relative content rate (%)	Concentration(mg kg−1)	Relative content rate (%)	Concentration(mg Kg−1)	Relative content rate (%)	Concentration(mg kg−1)	Relative content rate (%)
Cd2	0.123±0.001b	33.52±2.126b	0.110±0.001b	29.97±1.854a	0.103±0.001b	28.07±3.407ab	0.017±0.001b	4.63±0.016a	0.013±0.001a	3.54±0.016a	0.367±0.016b	0.817±0.026ab
Cd3	0.153±0.001b	34.78±2.846b	0.110±0.001b	25.00±1.864a	0.150±0.001b	34.09±2.564a	0.013±0.001b	2.95±0.018bc	0.013±0.001a	2.95±0.016ab	0.440±0.018b	0.853±0.024a
Cd5	0.943±0.002a	44.76±3.715a	0.350±0.002a	16.61±1.281b	0.707±0.003a	34.15±2.491a	0.067±0.001a	3.18±0.014b	0.040±0.002b	1.90±0.008b	2.107±0.013a	0.579±0.019b
CK	ND		ND		ND		ND		ND		ND	ND

EXC-Cd, exchangeable cadmium; CAB-Cd, carbonate-bound cadmium; FMO-Cd, Fe-Mn-oxide-associated cadmium; SOM-Cd, stands for organic matter-bound cadmium; RES-Cd, residual cadmium; relative content rate = (the concentration of a certain form Cd/the concentration of total Cd)×100%; ND indicates the Cd was not detected; the value represents mean ± standard deviations, and columns with different small letters indicate a significant difference (P=0.05)

Rhizosphere bacterial community in mulberry subjected to different Cd stress levels

From all rhizosphere soil samples, sequencing analysis yielded a total of 3,082,583 high-quality sequence reads with an average length of approximately 400 bp of effective sequence, 976 OTUs, and a 97% sequence identity threshold. The obtained soil OTUs were assigned to 12 bacterial phyla and 27 classes (Fig. 1, Table 2). However, the rhizosphere bacteria responded differently to Cd stress. The relative abundance of most rhizosphere bacteria at the family level after Cd stress treatment was significantly lower than that in the control (CK), including Acidobacteria Gp1, Gammaproteobacteria, Betaproteobacteria, and Gemmatimonadetes. Nevertheless, seven classes, including Acidobacteria Gp2, Acidobacteria Gp13, Alphaproteobacteria, Sphingobacteriia, Chloroflexia, Anaerolineae, and Fimbriimonadia, exhibited an increased relative abundance in the Cd stress condition compared to the CK condition, and a negative correlation with the concentrations was observed in the Cd stress condition.

Fig. 1.

Comparisons of the absolute abundance of rhizosphere bacterial (at the family level) of mulberry subjected to different Cd stress

Table 2.

The absolute abundance of the rhizosphere bacteria in mulberry subjected to different Cd stress

Taxon		Treatment
phylum	family	Cd2	Cd3	Cd5	CK
Acidobacteria	Acidobacteria Gp2	2409a	2253ab	1953b	1054c
	Acidobacteria Gp1	1097b	1008b	1443a	1443a
	Acidobacteria Gp13	523a	533a	379b	282c
	Acidobacteria Gp6	1b	0b	4b	17a
	Acidobacteria Gp3	5b	3b	4b	27a
	Acidobacteria Gp7	2b	0b	3b	13a
Proteobacteria	Gammaproteobacteria	622b	608b	679ab	725a
	Alphaproteobacteria	117b	161b	150b	436a
	Betaproteobacteria	38b	47b	59b	109a
	Deltaproteobacteria	5b	10b	17b	30a
Bacteroidetes	Sphingobacteriia	93a	91a	22b	22b
	Cytophagia	3a	1b	1b	1b
Actinobacteria	Actinobacteria	73a	43b	21c	15cd
Chloroflexi	Ktedonobacteria	229c	250c	322b	430a
	Thermomicrobia	4b	3b	4b	46a
	Unassigned	2b	3b	2b	28a
	Chloroflexia	0b	0b	0b	65a
	Anaerolineae	1b	0b	1b	13a
Gemmatimonadetes	Gemmatimonadetes	96bc	163b	217b	735a
Armatimonadetes	Fimbriimonadia	0b	2b	1b	14a
	Armatimonadia	2b	3b	4b	34a
Verrucomicrobia	Opitutae	0b	4b	5b	64a
	Subdivision3	2b	3b	4a	3b
Planctomycetes	Planctomycetia	2b	6b	4b	28a
Firmicutes	Bacilli	0b	1b	0b	36a
Chloroplast	Chloroplast	1b	0b	1b	21a
Others	No rank	90b	181a	5c	22b

Different lowercase letters in the same line indicate a significant difference (P < 0.05) between different treatments

Alpha diversity of the rhizosphere bacterial community in mulberry subjected to different Cd stress levels

The alpha diversities of rhizosphere soil bacteria in each treatment were analyzed based on the Chao1 and Simpson and Shannon diversity indices and are shown in Table 3. Compared with the control values, the Shannon diversity index and the Chaol index were decreased, while the Simpson diversity index was increased in Cd-stressed soil. Interestingly, for the Shannon, Chaol, and Simpson indices, there was a significant difference only between the Cd5 treatment and the control (p=0.023, 0.047, 0.042).

Table 3.

Alpha diversity of rhizosphere bacteria community in mulberry subjected to different Cd stress

Treatment	Chaol	Simpson	Shannon
Cd2	250.01±21.63ab	0.0549±0.0017b	3.19±0.19a
Cd3	256.36±19.27ab	0.0533±0.0018b	3.27±0.27a
Cd5	225.20±18.46b	0.0573±0.0013a	3.02±0.24b
CK	260.62±19.34a	0.0531±0.0021b	3.33±0.25a

Columns with different small letters indicate significant differences (P=0.05)

Changes in the rhizosphere bacterial community in mulberry under different Cd stress levels

To further understand the rhizosphere bacterial community response to Cd stress, changes in the mulberry rhizosphere bacterial community under Cd stress were detected using PCoA. The PCoA biplot with scores and loading in Fig. 2 displays the variance between the samples. The first principal component (PC1) accounts for 63.49% of the variance between samples, and the second principal component (PC2) accounts for 13.36% of the variance between samples. The bacterial communities in the Cd2 and Cd3 conditions were the most similar but were significantly different from those in the Cd5 and CK conditions, indicating that low-concentration Cd stress promoted the richness of rhizosphere soil bacteria, while it inhibited the richness and diversity.

Fig. 2.

PCoA analysis of the mulberry rhizosphere bacterial community under different Cd stress. This PCoA plot was generated using the matrix of unweighted Unifrac distances. A point represents a sample in the plot. Axis 1 represents 63.49% of the variation in the bacteria community genetic distance and axis 2 represents 13.36% of the variation in the bacteria community genetic distance among samples

Correlation between rhizosphere bacteria and the forms and elimination rate of Cd

To further reveal the intrinsic connection of the rhizosphere soil bacteria and Cd forms as well as the elimination rate of Cd, the relative abundance of 26 classes of bacteria in the mulberry rhizosphere, Cd occurrence concentration, and the elimination rate of Cd were examined using the Spearman correlation analysis. The correlation coefficients (Table 4), indicated by the heatmap shown in Fig. 3, showed that the Cd concentration (TOTAL-Cd) was significantly negatively correlated with the relative abundance of Alphaproteobacteria, Fimbriimonadia, and Chloroflexia (p<0.05), with correlation coefficients of −0.676, −0.595, and −0.646, respectively. Acidobacteria Gp3 was significantly negatively correlated with EXC-Cd (p<0.05), with a correlation coefficient of −0.579. Anaerolineae was significantly negatively correlated with EXC-Cd, CAB-Cd, and RES-Cd (p<0.05), with correlation coefficients of −0.636, −0.603, and −0.656, respectively. Acidobacteria Gp2, Acidobacteria Gp13, and Sphingobacteriia were significantly positively correlated with the Cd elimination rate in soil, with correlation coefficients of 0.774, 0.589, and 0.746, respectively. However, Acidobacteria Gp1, Ktedonobacteria, Gemmatimonadetes, Deltaproteobacteria, Acidobacteria Gp6, and Anaerolineae were significantly negatively correlated with the Cd elimination rate in soil (p<0.05), with correlation coefficients of −0.634, −0.704, −0.676, −0.606, −0.749, and −0.669, respectively.

Table 4.

Correlation between rhizosphere bacteria and the forms and elimination rate of Cd

Rhizosphere bacteria	Factor	Correlation coefficients	P value
Alphaproteobacteria	TOTAL-Cd	−0.676	0.015*
Fimbriimonadia	TOTAL-Cd	−0.595	0.041*
Chloroflexia	TOTAL-Cd	−0.645	0.023*
Acidobacteria_Gp3	EXC-Cd	−0.578	0.048*
Anaerolineae	EXC-Cd	−0.636	0.026*
Anaerolineae	CAB-Cd	−0.602	0.037*
Anaerolineae	RES-Cd	−0.655	0.020*
Acidobacteria_Gp2	Elimination rate of Cd	0.774	0.003**
Acidobacteria_Gp1	Elimination rate of Cd	−0.633	0.026*
Acidobacteria_Gp13	Elimination rate of Cd	0.589	0.043*
Ktedonobacteria	Elimination rate of Cd	−0.704	0.010*
Gemmatimonadetes	Elimination rate of Cd	−0.676	0.015*
Sphingobacteriia	Elimination rate of Cd	0.746	0.005**
Deltaproteobacteria	Elimination rate of Cd	−0.606	0.036*
Acidobacteria_Gp6	Elimination rate of Cd	−0.749	0.004**
Anaerolineae	Elimination rate of Cd	−0.669	0.017*

Asterisk (*) and (**) indicates significant difference between rhizosphere bacteria and the forms and elimination rate of Cd at P<0.05 and P<0.01

Fig. 3.

Heatmap of correlation between rhizosphere bacteria and the forms and elimination rate of Cd. The values in the scale bar mean the correlation coefficient between individual bacteria at the level of class and the forms and elimination rate of Cd, and the asterisks indicate statistically significant differences according to Kruskal-Wallis tests (*P < 0.05 and **P < 0.01) in the plot

Discussion

In our study, A great difference in the elimination rate of Cd in mulberry rhizosphere soil was observed, with Cd elimination rates of 85.3% in the Cd3, 81.7% in the Cd2, and 57.9% in the Cd5 conditions, demonstrating that the Cd elimination capacity of mulberry rhizosphere soil initially increased and then decreased with increasing Cd stress level, which might be related to the strong accumulating and translocating heavy metals capacity of mulberry [21, 37–39]. Additionally, a similar phenomenon in which low concentrations of heavy metals may promote the accumulation capacity of heavy metals and higher concentrations inhibit accumulation capacity has also been observed in other hyperaccumulator plants, such as Alfalfa [40] and Amaranthus caudatus [41].

Generally, when faced with heavy metal stress, the rhizosphere soil microbial system responds rapidly and sensitively, leading to rapid iteration and complex shifts in bacterial community structures [42–44]. Studies have found that the abundance of some low-resistance microorganisms, such as Proteobacteria and Bacillus, is reduced by heavy metal toxicity [45, 46], but the abundance of metal-resistant bacteria, such as Bacteroidetes and Acidobacteria, is increased [47, 48]. Similar results were found in our study. Most bacteria in mulberry rhizosphere soil displayed a decreased abundance, while seven classes, Acidobacteria Gp2, Acidobacteria Gp13, Alphaproteobacteria, Sphingobacteriia, Chloroflexia, Anaerolineae, and Fimbriimonadia, showed an increased abundance.

Several studies have shown that chronic exposure to heavy metals reduces the biomass of microorganisms and their activity as well as the diversity of their communities [49, 50]. Normally, microbial community diversity is positively correlated with heavy metal pollutant concentrations [42]. However, as shown in Table 4, the Shannon, Chaol, and Simpson indices, as comprehensive indicators of species richness and evenness [51], showed a significant difference under Cd stress at concentrations less than 3 mg kg⁻¹, while these indices significantly decreased under Cd stress at 5 mg kg⁻¹ compared with the control condition. In addition, PCoA (Fig. 2) showed that the bacterial community structures of mulberry rhizosphere soil in the Cd2 and Cd3 conditions were similar and were significantly different from those in the Cd5 and CK conditions. This result indicated that the mulberry rhizosphere bacterial diversity could tolerate elevated Cd concentrations, except for excessively high Cd concentrations. A similar finding was reported by Wang et al. [52], in which low mercury concentration had accelerated the growth of pakchoi and increased the abundance of nitrogen-fixing microorganism and the richness of the community, whereas the medium and high mercury concentration had represented inhibiting effect.

Furthermore, in this study, the correlation heatmap showed that Acidobacteria Gp2, Acidobacteria Gp13, and Sphingobacteriia were significantly positively correlated with the rate of Cd decline in soil, indicating that these bacteria have the competitive advantages of material circulation and soil energy transformation, thus becoming the dominant bacteria participating in heavy metal accumulation and compartmentalization.

Bacteria with similar functions included Actinobacteria, Firmicutes, Actinomycetes, Acidobacteria, and Proteobacteria [42, 53]. Additionally, Alphaproteobacteria, Fimbriimonadia, and Chloroflexia were significantly negatively correlated with the Cd concentration (TOTAL-Cd), Acidobacteria Gp3 was significantly negatively correlated with EXC-Cd, and Anaerolineae was significantly negatively correlated with EXC-Cd, CAB-Cd, and RES-Cd. These results indicate that the functional bacteria targeting the detoxification and tolerance of Cd pollution in the mulberry rhizosphere soil represent adaptive differentiation, which in turn affected the rhizoremediation rate and performance [54, 55], consistent with previous reports [56–58]. The results also further confirm that rhizosphere microorganisms contribute to metal speciation in soil, which may be a key factor for effective phytoremediation [59]. Therefore, a new method for bioremediation using the synergistic relationships between plants and microbes in the rhizosphere has been created and applied in the remediation of heavy metal contamination in soil, which will be the main focus of further study.

Author contributions

Experimental design and writing—original draft preparation, Guiping Hu; the determination of Cd solubility, Hongmei Cao; writing—review and editing, Chuan Ye; sequencing data analysis, Feng Wang. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Youth Foundation of Jiangxi Province (20202BABL215022), the High-level and high-skilled leading personnel training project of Jiangxi Province (2022) and the Modern Agricultural Industry Technology System of Jiangxi Province (JARS-23-3).