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Article

Comprehensive Analysis of Microbiomes and Metabolomics Reveals the Mechanism of Adaptation to Cadmium Stress in Rhizosphere Soil of Rhododendron decorum subsp. Diaprepes

by
Ming Tang
1,2,3,†,
Lanlan Chen
1,2,3,†,
Li Wang
1,2,3,†,
Yin Yi
1,2,3,
Jianfeng Wang
4,5,6,7,8,
Chao Wang
4,5,6,7,8,
Xianlei Chen
1,2,3,
Jie Liu
1,2,3,
Yongsong Yang
9,
Kamran Malik
4,5,6,7,8 and
Jiyi Gong
1,2,3,*
1
Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Karst Mountainous Areas of Southwestern China, Guizhou Normal University, Guiyang 550025, China
2
College of Life Sciences, Guizhou Normal University, Guiyang 550025, China
3
Engineering Research Center of Carbon Neutrality in Karst Areas, Ministry of Education, Guizhou Normal University, Guiyang 550025, China
4
State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, Lanzhou University, Lanzhou 730000, China
5
Center for Grassland Microbiome, Lanzhou University, Lanzhou 730000, China
6
Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou University, Lanzhou 730000, China
7
Engineering Research Center of Grassland Industry, Ministry of Education, Lanzhou University, Lanzhou 730000, China
8
College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China
9
Guizhou Baili Dujuan Management Area Forestry Bureau, Bijie 551614, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(8), 884; https://doi.org/10.3390/horticulturae10080884
Submission received: 12 June 2024 / Revised: 18 August 2024 / Accepted: 19 August 2024 / Published: 21 August 2024
(This article belongs to the Special Issue Physiological and Molecular Biology of Ornamental Plants—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
The toxicity of cadmium (Cd) not only affects the growth and development of plants but also has an impact on human health. In this study, high-throughput sequencing and LC-MS were conducted to analyze the effect of CdCl2 treatment on the microbial community and soil metabolomics of rhizosphere soil in Rhododendron decorum subsp. diaprepes. The results showed that CdCl2 treatment reduced the quality of the rhizosphere soil by significantly decreasing the soil organic carbon (SOC) content, urease, and invertase activities, increasing the percentage of the exchangeable Cd fraction. CdCl2 treatment did not significantly change the Chao1 and Shannon indices of bacterial and fungal communities in the rhizosphere soil. R. decorum was more likely to recruit Cd-resistant bacteria (e.g., Proteobacteria, Chloroflexi) and increase the abundance of Cd-resistant fungi (e.g., Basidiomycota, Rozellomycota). Moreover, CdCl2 treatment decreased the content of secondary metabolites associated with plants’ resistance to Cd. Rhizosphere soil urease, invertase activities, alkaline phosphatase (ALP), SOC, total potassium (TK), Cd, and nitrate nitrogen (NN) were the main drivers of the composition of rhizosphere bacterial and fungal communities. CdCl2 treatment weakened the relationships among bacterial/fungi, differential metabolites, and physicochemical properties in rhizosphere soil.

1. Introduction

Soil contamination caused by cadmium (Cd) is a serious problem worldwide, representing a broadly distributed contaminant in the soil ecosystem [1]. It not only causes nutritional imbalances and deficiencies in plants growing in contaminated soil but also passes through those same plants into the food chain, posing a threat to environmental and food security [2,3].
Microorganisms are essential for the soil ecosystem to maintain biodiversity, control nutrient cycling, and regulate soil fertility [4]. Cd has been reported to inhibit soil microbial diversity and plant growth even at low doses [5,6,7]. According to Jin et al. (2023), the composition of the rhizosphere’s microbial community was significantly negatively affected by Cd [8]. Since the adaptation of microbes to the soil environment influences the accumulation of metals in plant species in contaminated environments, the characteristics of the microbial community structure have been used as markers of soil quality [9,10]. Rhizosphere microorganisms show different responses to Cd-contaminated soil. For example, Actinobacteria tolerate high Cd concentrations found in heavy-metal-contaminated soil [11,12]. In addition, rhizosphere microorganisms stimulate plant growth and enhance resistance to heavy metals through releasing amino acids and plant hormones [13]. In heavy-metal-contaminated soils, the metabolic capacity of microbial communities has been constantly altered to adapt to different environments [14]. Rhizosphere soil metabolites reflect changes in the soil microbial community, as changes experienced at the organism and enzyme levels will manifest as a modified metabolite profile. In addition, under stress, plants play a driving role in regulating the distribution of micro-organisms to ensure metabolite activity [15]. Therefore, rhizosphere soil metabolite communication is a key mechanism of plant–microbe interactions [16].
Rhododendron decorum subsp. diaprepes belongs to the Ericaceae and is a variety of Rhododendron decorum. R. decorum has certain ornamental and ecological restoration value. R. decorum is mainly distributed in Yunnan, Guizhou, and Sichuan, China, and usually grows in shrubland or forests at an altitude of 1000–4000 m. More and more researchers have been paying attention to the growth of rhododendrons under alkaline soil conditions, and they select alkali-resistant species to adapt to the growth of an alkaline soil environment. Research has reported that R. decorum can survive in heavy-metal-contaminated soil [17,18]. Furthermore, a study showed that Cd stress altered the composition of endophytic microbial communities in R. decorum leaves [19]. However, there is limited information on rhizosphere soil microbial communities, metabolites, and physicochemical properties, as well as responses to Cd stress in R. decorum.
Therefore, this study aimed to (1) explore the effects of different CdCl2 treatments on rhizosphere soil physicochemical properties in alkaline soil, (2) evaluate the effects of different CdCl2 treatments on the composition of rhizosphere bacterial and fungal communities in alkaline soil and rhizosphere soil metabolomic characteristics in alkaline soil, (3) determine the key factors affecting the changes in the rhizosphere microbial communities under CdCl2 treatment, and (4) investigate the relationships among the rhizosphere bacterial/fungal community, differential metabolites, and physicochemical properties of rhizosphere soil under CdCl2 treatment.

2. Materials and Methods

2.1. Experiments on Plant Growth and Cd Addition

The seeds of Rhododendron decorum subsp. diaprepes were provided by associate professor Jie Liu from the Key Laboratory of Plant Physiology and Development Regulation, School of Life Sciences, Guizhou Normal University. Seeds with full and evenly sized particles were selected and disinfected by soaking in a 10% NaClO solution for 10 min, followed by rinsing 5 times with sterile water. Then, the seeds were disinfected again with 75% ethanol for 30 s and rinsed another 5 times with sterile water. Later, the seeds were soaked in 600 mg/L gibberellin (GA3) for 24 h and evenly dispersed in a 90 mm culture dish containing sterile water filter paper with sufficient absorption. The seeds were allowed to germinate in an incubator at a constant temperature of 22 ± 2 °C, with 50% relative humidity, a light cycle of 16 h/8 h (light/dark), and light intensity of 2500 lx. After growing 3 leaves, the seedlings were transplanted into pinnacle peat soil/perlite = (3:1 ratio) and allowed to grow for one and a half years in a culture chamber under the same growth parameters as mentioned above.
The 1.5-year-old robust seedlings with consistent growth status were selected and transplanted to flower pots (top diameter: 92 mm, bottom diameter: 81 mm, height: 80 mm), with 50 g of soil for Cd stress experiments (the soil was obtained from Fengxiang Mountain of Guizhou Normal University, pH 8.05 ± 0.04). There were five biological replicates for each treatment. For CdCl2 treatment, 1.5-year-old robust seedlings were treated with 50 mL of distilled water and CdCl2 once every five days. The soil samples were air-dried and sieved to a 2 mm size. The three treatments were as follows: (1) CK (soil + R. decorum); (2) 2.5 mM CdCl2 (1690 mg kg−1 Cd2+ contaminated soil + R. decorum); and (3) 5 mM CdCl2 (3370 mg kg−1 Cd2+ contaminated soil + R. decorum). After 6 Cd treatments, the treatments were stopped, and the seedlings were allowed to grow for another two weeks. After the experiment, the loose soil on the surface of the roots was gently removed, and the soil firmly attached to the roots was collected as rhizosphere soil. After gently shaking away the loosely attached soil, the soil attached to the root system (0–0.5 cm from the root) was carefully brushed to remove the mixed roots in the rhizosphere soil. The rhizosphere soil samples were collected and divided into two parts. A portion of the rhizosphere soil was stored at −80 °C to analyze soil enzyme activities. Another portion was air-dried to measure nutrient contents, pH, and chemical forms of Cd. The separation for roots and rhizosphere soil was carried out as described in our previously published paper [20]. Briefly, the excised roots were placed in a 50 mL sterile centrifuge tube, and 35 mL of sterile phosphate buffer (PBS: 3 mM NaH2PO4, 7 mM Na2HPO4, 130 mM NaCl, pH 7.4) was added. Then, the sterile centrifuge tubes were shaken for 3 min and centrifuged at 3000× g for 5 min to obtain the rhizosphere soil. After the supernatant was discarded, the rhizosphere soil was placed in a new 2 mL sterile centrifuge tube and stored at −80 °C until we extracted DNA.

2.2. Analysis of Rhizosphere Soil’s Physical Physicochemical Properties

The rhizosphere soil’s available phosphorus (AP) was extracted with 0.5 M NaHCO3 and measured using the molybdenum blue method at 710 nm. The rhizosphere soil’s total phosphorus (TP) was extracted HCl and then with 0.5 M NaHCO3. After mixing, the extracts were mineralized with a triple acid mixture of HNO3-HClO4-H2SO4 (10:1:4, v/v) and determined at 660 nm using a spectrophotometer (made by Shimadzu Co., Ltd., Kyoto, Japan). The rhizosphere soil’s total nitrogen (TN) was determined using the Kjeldahl method. Soil ammonium nitrogen (AN) and nitrate nitrogen (NN) were extracted with 1 M KCl and measured by a continuous-flow analyzer (Skalar san++, Skalar, Breda, The Netherlands). Soil organic carbon (SOC) was determined by the dichromate oxidation method. Available potassium (AK) was extracted with 1 M NH4OAc and determined by inductively coupled plasma mass spectrometry (ICP-MS) (SHIMADZU, Kyoto, Japan, 2030). The rhizosphere soil’s total potassium (TK) was digested with HF-HClO4, and TK in the digest was determined by ICP-MS. A pH meter (Mettler Toledo Delta 320, Greifensee, Switzerland) was used to measure the rhizosphere soil’s pH (soil/distilled water = 1:2.5). A 5 g rhizosphere soil was used to analyze the activity of invertase, and alkaline phosphatase was measured using 3,5-dinitrosalicylic acid colorimetry and disodium phenyl phosphate was measured using the colorimetric method [21]. The urease activity was measured with 5 g of rhizosphere soil, 1 mL of toluene, 10 mL of 10% urea solution, and 20 mL of citrate buffer (pH 6.7) added to a 50 mL volumetric flask, which was then incubated at 37 °C for 24 h. The reaction mixture was filtered, and 1 mL of the filtered solution was reacted with 3 mL of 0.9% NaClO solution and 4 mL of sodium phenol solution. After 20 min, the mixture was measured with an ultraviolet spectrophotometer (made by Shimadzu Co., Ltd.) at 578 nm. Catalase activity was determined with 2 g of rhizosphere soil, 40 mL of water, and 5 mL of 0.3% H2O2, which were thoroughly mixed for 20 min at 150 rpm. The mixture was added to 5 mL of 1.5 mol/L H2SO4 and then filtered. Finally, the filtrate was titrated with 0.1 mol/L KMnO4. The activity of catalase was analyzed in soil according to the method of Hackenberger et al. (2018) [22]. Total Cd was extracted with a triple acid mixture of HF-HNO3:HClO4 (5:5:1, v/v) and determined by ICP-MS. The Cd fractions were determined according to the method of He et al. (2018) [23]. Five fractions were determined as follows: extractable with 1 M MgCl2 (pH 7) (exchangeable, Ex-Cd), extractable with 1 M NaOAc (pH 5) (carbonate-bound, CB-Cd), extractable with 0.04 M NH2OH·HCl in 25% (v/v%) CH3COOH solution (Fe-Mn oxide-bound, OX-Cd), extractable with 0.02 M HNO3 in 30% H2O2 (organic matter-bound, OM-Cd), and HNO3-HF-HClO4 digested (residual, Res-Cd). The extracted Cd solutions were filtered into 25 mL tubes, diluted with distilled water to volume, and stored at 4 °C. The extracted Cd concentrations were determined by ICP-MS. The validation of ICP-MS measurement results was carried out using working calibration solutions of all investigated Cd ions. These solutions were prepared using appropriate stepwise dilutions of 100 mg L−1 certified standard stock solutions. The ICP-MS instrument (SHIMADZU, Kyoto, Japan, 2030) was regulated to measure the samples and the correlation coefficient; the RSE and BEC were 1.00, 3.03% and 0.003, respectively. The recovery percentages between the four steps used to obtain Cd fractions and total Cd (BCR/total) are shown in Table S1. Blanks and standard soil material (GBW07564) from the Chinese Academy of Geological Science were used for quality control.

2.3. DNA Extraction, Amplicon Sequencing, and Bioinformatics Analysis

The total genomic DNA was extracted from 350 mg of rhizosphere soil using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China), according to the manufacturer’s instructions. A NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA) was used to measure the concentration and purity of the extracted DNA, and electrophoresis on a 1.8% agarose gel was used to evaluate the quality and amount of the material. The V3V4 region of the 16S rRNA and the ITS1 region of the rRNA gene was used with 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers and ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) primers, respectively. The PCR was carried out in a reaction volume of 10 μL: 4 ng of DNA template, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 5 μL of KOD FX Neo Buffer, 2 μL of dNTP (2 mM each), and 0.2 μL of KOD FX Neo. The remaining volume was adjusted to 20 μL with ddH2O. The amplified products were quantified using the Qsep-400 (BiOptic, Inc., New Taipei City, Taiwan) and purified with the Omega DNA purification kit (Omega Inc., Norcross, GA, USA). Utilizing an Illumina novaseq6000 (Beijing Biomarker Technologies Co., Ltd., Beijing, China), the amplicon library underwent paired-end sequencing (2 × 250). The raw data processing followed the method described by Jin et al. [8].

2.4. Rhizosphere Soil Metabolomics and Data Analysis

A 50 mg sample was added to 1000 μL of extraction solution containing the inner target. The extraction solution was prepared with methanol, acetonitrile, and water at a ratio of 2:2:1 and an internal standard concentration of 20 mg/L. The solution was thoroughly mixed for 30 s by swirling and vortexing, followed by the addition of steel balls, a 45 Hz grinding machine for 10 min, and ultrasonication for 10 min (ice-water bath). After being stored at −20 °C for an hour, the sample was centrifuged for 15 min at 12,000 rpm and 4 °C. Then, 500 μL of supernatant was carefully removed, transferred to an EP tube, and dried in a vacuum concentrator. The dried metabolites were then redissolved by adding 160 μL of extraction solution (acetonitrile-to-water volume ratio = 1:1), vortexing for 30 s, and ice-water bath sonication for 10 min. The samples were centrifuged at 12,000 rpm for 15 min at 4 °C. Then, we carefully removed 120 μL of supernatant into a 2 mL injection vial; 10 μL of each sample was taken and mixed into the QC sample for automated testing. The chromatographic column used for LC-MS analysis was the Acquire UPLC HSS T3 (1.8 μm, 2.1 × 100 mm), (mobile phase A: 0.1% aqueous formic acid solution; mobile phase B: 0.1% formic acid acetonitrile). ESI ion source parameters and raw data were obtained using the method of Jin et al. [8]. Metabolite data analysis was performed on the BMKCloud platform (www.biocloud.net).

2.5. Statistical Analysis

Statistical analyses of the rhizosphere soil’s physicochemical properties were performed using the SPSS software (v27.0.1). The significant differences in the rhizosphere soil’s physicochemical properties were determined using Duncan’s test in a one-way analysis of variance (ANOVA). Statistical significance was defined as the 95% confidence level, and the data results are expressed as the mean ± standard deviation (mean ± SD) and were plotted using GraphPad Prism software version 8 (GraphPad Software Inc.; San Diego, CA, USA). The numbers of up- and down-regulated differential metabolites in the rhizosphere soil were analyzed by Origin (2021). Based on the Bray–Curtis dissimilarity, PCoA analysis was performed using the R “vegan” package (v3.1.1). The Chao1 and Shannon indices, the relative abundance of rhizosphere bacterial and fungal communities at the phylum and genus levels, and a random forest classification were analyzed using R (v3.1.1). We applied linear discriminant analysis (LDA) of effect size (LEfSe) to identify the differential abundance of rhizosphere bacteria and fungi, with a threshold set to LDA ≥ 3.0, p < 0.05. Principal component (PCA) analysis and heat map analysis of rhizosphere soil metabolites were carried out in R (v3.1.1). The rhizosphere soil’s differential metabolite abundance score map clustering and supervised orthogonal partial least squares analysis (OPLS-DA) were performed using the BMKCloud platform (www.biocloud.net). The co-occurrence network analysis was conducted using the “igraph” package in R and illustrated using Gephi software (v0.9.2). Redundancy analysis (RDA) was conducted using R (v3.1.1) to determine the impact of environmental factors on the composition of rhizosphere bacterial and fungal communities.

3. Results

3.1. The Physicochemical Properties of Rhizosphere Soil at Different CdCl2 Concentrations

Compared with CK, 2.5 and 5 mM CdCl2 had significant toxic effects on R. decorum and inhibited the growth of R. decorum (Figure 1). After one month of treatment with different concentrations of CdCl2 (CK, 2.5 mM, and 5 mM), there was a noticeable difference in the physicochemical properties of the rhizosphere soil in R. decorum. The concentration of CdCl2 had a significant impact on the total Cd content of the rhizosphere soil. The maximum value was reached under 5 mM treatment (Figure 2A). The Cd content of the different chemical forms was the highest in EX-Cd (12.8–39.5%), followed by CB-Cd (25.4–36.6%), OX-Cd (29.6–30.4%), OM-Cd (2.4–16.0%), and Res-Cd (0.5–4.3%) (Figure 2G). The results indicated that the chemical forms of EX-Cd and CB-Cd were dominant in the rhizosphere soil. Under the CK, 2.5 mM and 5 mM CdCl2 treatments, EX-Cd occupied 12.8%, 39.8%, and 39.5%; CB-Cd occupied 36.6%, 25.4%, and 29.6%; OX-Cd occupied 30.4%, 31.5%, and 29.6%; OM-Cd occupied 16.0%, 2.8%, and 2.4%; and Res-Cd occupied 4.3%, 0.5%, and 0.5%, respectively.
Compared with the CK, the addition of CdCl2 did not significantly alter the catalase activity and the contents of TN, AN, NN, TP, and AK in the rhizosphere soil of R. decorum, but the TK content was significantly altered by CdCl2 treatment (Figure 3). Compared with CK, the CdCl2 treatments significantly decreased the SOC content and the urease and invertase activities in rhizosphere soil (Figure 3). The AP content only significantly increased under the 2.5 mM treatment compared to CK (Figure 3E). Compared with CK, the 2.5 mM CdCl2 treatment did not significantly change the ALP activity and pH, but they were decreased by the 5 mM CdCl2 treatment (Figure 3J).

3.2. Effects of CdCl2 on Diversity and Composition of Rhizosphere Soil Bacterial and Fungal Communities

Our results suggested that the Chao1 and Shannon indices of bacterial and fungal communities in the rhizosphere soil showed no significant difference among all groups (Figure 4A,B,D,E). Furthermore, principal coordinates analysis (PCoA) revealed that the rhizosphere’s bacterial and fungal community composition showed differences at different soil Cd content. Moreover, the first and second principal coordinates accounted for 48.9% and 53.9% of the total variation in the rhizosphere bacterial and fungal communities under different treatments, respectively (Figure 4C,F).
The most abundant phyla in the bacterial community of the rhizosphere soil, across all three treatments, were Proteobacteria, Acidobacteriota, and Actinobacteriota, which accounted for over 70% of the total sequences (Figure 5A). We observed that the increase in CdCl2 concentration could significantly affect the relative abundance of Proteobacteria (45.4%, 45.9%, 45.5%), Acidobacteriota (12.1%, 11.8%, 13.0%), Myxococcota (9.5%, 9.9%, 8.8%), Bacteroidota (7.9%, 9.4%, 7.7%), and Chloroflexi (3.8%, 5.3%, 4.0%) in rhizosphere soil (Figure 5A). The relative abundance of the genera SWB02, Haliangium, Bauldia, and Acidibacter was the highest under the 2.5 mM CdCl2 treatment (Figure 5B). The relative abundance of genera Pseudolabrys and Streptomyces was the highest at 5 mM CdCl2 treatment (Figure 5B). As shown by the cladogram, the bacterial genera Terrimonas, Actinospica, Dokdonella, Pseudolabrys, and Streptomyces were significantly more abundant in rhizosphere soil with CdCl2 treatment (Figure S1A). In addition, the random forest analysis showed that the bacteria Chloroflexi, Bacteroidota, and Chytridiomycota were the key phyla in the CK vs 2.5 mM group, whereas Firmicutes, Fibrobacterota, and Chytridiomycota were the key phyla in the CK vs. 5 mM group (Figure S2).
The most abundant phyla in the fungal community of rhizosphere soil, across all three treatments were Basidiomycota, Ascomycota, unclassified_Fungi, and Chytridiomycota, which accounted for over 95% of the total sequences (Figure 5C). CdCl2 treatment increased the relative abundance of Basidiomycota and Rozellomycota, while it decreased the relative abundance of Ascomycota, Chytridiomycota, Mortierellomycota, and Glomeromycota in rhizosphere soil. Compared with CK, the 2.5 mM CdCl2 treatment decreased the relative abundance of Clitopilus, Byssochlamys, and Serendipita in rhizosphere soil, but the 5 mM CdCl2 treatment increased the relative abundance of Clitopilus (Figure 5D). As shown by the cladogram, the fungal genera unclassified_Agaricomycetes, Simplicillium, Leptodontidium, Phialemonium, and Penicillium were significantly enriched in rhizosphere soil under Cd stress (Figure S1B).
For RDA, the rhizosphere soil’s physicochemical characteristics and the enzyme activities were selected. Our results showed that the three rhizosphere soil samples could be distinguished from one another (Figure 6), implying that the CdCl2 treatment altered the rhizosphere soil’s environment. At the phylum level, the first and second coordinate axes of the RDA explained 35.8% and 22.2% of the total variance in the rhizosphere bacterial and fungal communities, respectively. Rhizosphere soil urease, invertase, and ALP enzymes, along with SOC, TK, Cd, and NN were the main factors in the composition of the rhizosphere bacterial and fungal communities. Most of the phyla, including Chytridiomycota, Verrucomicrobiota, Ascomycota, and Actinobacteriota, had positive correlations with invertase, urease, and catalase enzymes, as well as SOC and pH, while they had negative correlations with Cd, NN, and AP. Myxococcota, Chloroflexi, and Bacteroidota were positively correlated with ALP, TP, AK, TK, AN, and NN, while Myxococcota were negatively correlated with NN and Cd. Basidiomycota, Proteobacteria, and Rozellomycota were positively correlated with NN, Cd, AP, and AN, while they were negatively correlated with pH, SOC, catalase, invertase, and urease.

3.3. Measurement of Metabolites in Rhizosphere Soil Treated with CdCl2

To determine the response of rhizosphere soil’s metabolic profiles to the different CdCl2 treatments, we used LC-MS to identify the main metabolites in rhizosphere soil. PCA demonstrated that the different CdCl2 treatment concentrations significantly altered the rhizosphere soil’s metabolic profiles in R. decorum, where PC1 and PC2 explained 49.6% and 17.9% of the total variance, respectively (Figure 7A). We also used the supervised discriminant analysis OPLS-DA model to evaluate the effect of CdCl2 stress on rhizosphere soil metabolic profiles, and the results showed a significant separation between CK and 2.5 mM (R2Y = 1, Q2Y = 1.0) (Figure S3A), as well as between CK and 5 mM (R2Y = 1, Q2Y = 1.0) (Figure S3B). Compared with CK, the 2.5 mM CdCl2 up-regulated 418 differential metabolites (DMs) and down-regulated 699 DMs, while the 5 mM CdCl2 up-regulated 441 DMs and down-regulated 791 DMs (Figure 7B and Figure S5A,B).
Compared with CK, the DMs up-regulated by the 2.5 mM CdCl2 treatment were mainly enriched in the “Aminobenzoate degradation pathway”, while down-regulated DMs were mainly enriched in the “Cutin, suberine”, “wax biosynthesis” and “Tryptophan metabolism pathway” (Figure S3B). Compared with CK, the DMs up-regulated by the 5 mM CdCl2 treatment were mainly enriched in the “Aminobenzoate degradation pathway”, while down-regulated DMs were mainly enriched in the “Purine metabolism” and “Tryptophan metabolism” pathways (Figure S3D). Compared with CK, the 2.5 mM CdCl2 treatment increased the content of taurine, but 2.5 mM and 5 mM CdCl2 treatment decreased the contents of hydroxyspirilloxanthin, 8 (R)-HPETE, trioxilin A3, and farnesylcysteine in rhizosphere soil (Figure S4).

3.4. Correlations among Microbial Communities, Physicochemical Properties, and Metabolites in Rhizosphere Soil

In the rhizosphere’s microbial networks, angelicin, dopaxanthin, epsilon-Caprolactam, dihydroxyflavone, nicotinamide D-ribonucleotide, and L-1-pyrroline-3-hydroxy-5-carboxylate were identified as key differential metabolites that were highly connected to other nodes in CK vs. 2.5 mM CdCl2 treatment (Figure 8A,C), while scolymoside, farnesylcysteine, triethanolamine, L-threonine emerged in CK vs. 5 mM CdCl2 (Figure 8B,D). Moreover, invertase, urease, and ALP activity, along with CB-Cd, AP, OX-Cd, TK, OM-Cd, Res-Cd, pH, EX-Cd, and SOC were the key rhizosphere soil properties to connect other nodes in CK vs. 2.5 mM CdCl2 treatment (Figure 8A,C), while urease and invertase activity, EX-Cd, pH, CB-Cd, OM-Cd, Cd, and TK were identified in CK vs. 5 mM CdCl2 treatment (Figure 8B,D). Additionally, bacterial species hubs affiliated with Actinobacteriota, Chloroflexi, and Bacteroidota were identified in CK vs. 2.5 mM CdCl2 treatment (Figure 8A), while bacterial hubs at the phylum level affiliated with Firmicutes, Actinobacteriota, and Planctomycetota were identified in CK vs. 5 mM CdCl2 treatment (Figure 8B). Meanwhile, the fungal phylum Chytridiomycota was identified as the keystone taxon in samples for CK vs. 2.5 mM CdCl2 treatment and CK vs. 5 mM CdCl2 treatment (Figure 8C,D). The fungal phylum Basidiomycota was the keystone taxon for CK vs. 5 mM CdCl2 treatment (Figure 8D).
As shown in Table 1, in the rhizosphere bacterial and fungal networks, CdCl2 treatments decreased the numbers of total edges, negative edges, connectance, average degree, and diameter, indicating that CdCl2 treatments decreased the complexity of the networks.

4. Discussion

Microorganisms are recognized as perturbation sensors, being much more sensitive to environmental stress than macro-organisms [24,25]. They make crucial contributions to environmental adaptation, but there has been little research on the response of the rhizosphere environment and metabolites to Cd stress, especially the bacterial and fungal communities associated with rhizosphere soil. In this study, the prime objective was to explore the effects of Cd stress on the composition of rhizosphere microbial communities and rhizosphere soil metabolites in R. decorum.
Cd leads to a change in rhizosphere soil’s plant environment, which in turn affects plant growth. Boampong reported that Cd treatment (1000 mg kg−1) decreased the dry shoot weight by 39%, 45.8%, and 49.3% in Acacia saligna, Eucalyptus rostrate, and Conocarpus erectu, respectively [26]. A study reported that a 500 µM Cd nutrient solution clearly inhibited the physiological and biochemical process in K. paniculata, caused membrane lipid peroxidation and severe membrane damages, and increased MDA and H2O2 contents [27]. Previous studies have reported that CdCl2 treatment resulted in significant enrichment of Cd2+ in the soil [8,28]. In this study, 2.5 and 5 mM CdCl2 treatments had significant toxic effects on R. decorum and inhibited the growth of R. decorum (Figure 1), suggesting that CdCl2 treatment might inhibit the physiological, biochemical, and molecular process in R. decorum. In addition, the biological toxicity of heavy metals in soil is not only determined by their total content but is also closely related to their chemical forms [29]. Soil pH significantly affects the availability of heavy metals in soil [30]. Under alkaline conditions, Cd is present as CdHCO3+ or CdCO3, which is conducive to the adsorption of heavy metal ions in soil and reduces the availability of Cd in soil [31]. Jin et al. (2023) reported that CdCl2 decreased the pH in A. inebrians rhizosphere soil [8], which was consistent with our results. The decrease in pH results in more H+ to exchange with the Cd absorbed by soil colloids, increasing the Cd2+ content in the root–soil system, thereby increasing Cd’s mobility [32]. A decrease in soil pH will increase the transformation of Cd from a stable form (e.g., carbonates, Fe, and Mn oxide-bound forms) to an effective bioavailable (e.g., exchangeable form) form [33]. In addition, the possible risks of Cd transfer from rhizosphere soil to plants can be divided into three categories: high-risk, including EX-Cd (exchangeable fraction) and CB-Cd (carbonate-bound fraction); medium-risk, including OX-Cd (Fe/Mn oxides-bound fraction) and OM-Cd (organics fraction); and low-risk, including Res-Cd (residual fraction) [34]. In this study, CdCl2 increased the percentage of the exchangeable Cd fraction but decreased the percentages of the organic matter-bound and residual fractions in the rhizosphere soil, indicating that CdCl2 increased the effective bioavailability in R. decorum rhizosphere soil. These results are consistent with the findings of Dong et al. (2016) on rice under Cd stress [35]. In addition, the recovery percentages of Cd were 94% and 105% under 2.5 and 5 mM CdCl2 treatments, respectively. The results are in accordance with the specification, and the test results are reliable. However, in the CK treatment, the recovery percentage of Cd was 197%, which might be due to contamination of the container used during the operation, and the error could be reduced by increasing the number of replicates of experiments.
Soil enzyme activities are very sensitive to heavy metal pollution and are involved in functions related to soil C, N, and P cycling [36]. In rhizosphere soil, CdCl2 treatment significantly decreased the SOC content, urease, and invertase activity. SOC plays an important role in providing carbon sources, promoting soil fertility and contributing to microorganism energy [37]. Therefore, the reduction in SOC affected soil quality. A study reported that Cd pollution reduces the distribution of carbon in roots and leads to a decrease in root exudation, resulting in a decrease in soil organic carbon content [38]. Moreover, the increases in Basidiomycota abundance under Cd stress were associated with increased lignin degradation, releasing SOC [39]. Xiao et al. (2023) reported that SOC loss is caused by increasing the abundance of Basidiomycota that promotes carbon degradation [40], which is consistent with our findings. Soil invertase and urease are key enzymes in the soil–plant C and N cycles [41,42]. Previous studies have shown that heavy metals decrease the activities and community composition of soil microorganisms, thereby affecting the synthesis of soil enzymes when the soil is contaminated by heavy metals [43]. In addition, the decrease in enzyme activity may be due to the interaction of heavy metals with enzyme–substrate complexes, denaturing the enzyme proteins or interacting with the protein active moiety [44].
As a hazardous heavy metals, Cd seriously jeopardizes the community and activity of soil microorganisms [45]. In addition, the effects of different Cd fractions on microorganisms vary significantly [46]. The variety and quantity of sensitive bacteria are adversely affected in heavy-metal-contaminated soils, whereas bacteria that are resistant to the metals are able to adapt and proliferate, increasing their abundance [14,47,48]. Microbial diversity is a crucial indicator for assessing the functioning of ecosystem [49]. In this study, Cd pollution had a negative impact on fungal and bacterial diversity (according to the Shannon and Chao1 indices) (Figure 4). The PCoA results indicated that CdCl2 changed the composition of rhizosphere microbial communities (Figure 4). Microorganisms possess robust metabolic and physiological adaptations to changing environmental conditions, enabling microbial communities to withstand adverse environments [50]. Studies have shown that the structure of the microbial community is extremely sensitive to changes in the toxicity of heavy metals in the soil [16,51]. In this study, CdCl2 increased the relative abundance of Proteobacteria and Chloroflexi, which might be a way for bacteria to resist Cd in R. decorum rhizosphere soil. Hou et al. (2018) reported that Proteobacteria were significantly enriched in Sedum alfredii rhizosphere soil under high Cd stress [52]. It has been reported that Proteobacteria are the dominant bacterial phylum in areas with severe heavy metal contamination [53,54,55]. Proteobacteria are important for the carbon and nitrogen cycles [55]. The present findings are consistent with those of Zhao et al. (2019), who discovered that the relative abundance of Proteobacteria in the microbial communities of soil with long-term heavy metal pollution, was inversely linked with soil AK [14]. Previous studies have demonstrated that Chloroflexi is the dominant phylum in heavy-metal-contaminated soils. This is due to the fact that bacteria belonging to this phylum are tolerant and resistant to heavy metal stress [56,57,58]. Meanwhile, members of Chloroflexi are oligotrophic bacteria that prefer to live in nutrient-poor soil habitats [59,60,61,62]. Therefore, Chloroflexi could survive and thrive better than other bacteria in heavy-metal-contaminated areas characterized by low soil nutrient levels [63]. For fungal communities, with the increase in CdCl2 concentration, the relative abundance of Basidiomycota and Rozellomycota increased in the rhizosphere soil. Basidiomycota play a key role in preventing the migration of Cd from the soil to the plants [64]. Basidiomycota are filamentous fungi that have been used to remove heavy metals [65]. In addition, Rozellomycota exhibits a high degree of tolerance to heavy metals [66]. Rozellomycota is able to directly obtain nutrients from the organic matter in the environment through phagocytosis [67]. Rhizosphere soil urease, invertase activities, ALP, SOC, TK, Cd, and NN were the main drivers of the composition of bacterial and fungal communities. In addition, the nutrients (TN, AN, NN, TP, AP, and TK) had a positive correlation with Myxococcota and Bacteroidota and Chloroflexi but had a negative correlation with Acidobacteriota and Actinobacteriota. This may be attributed to the observation that rhizosphere soil nutrients could influence microbial abundance [68]. The report suggests that competition among microorganisms for nutrients may result in a decreased abundance of some microorganisms [69].
Plant and rhizosphere soil microbes respond to environmental stress by regulating their metabolic processes [43]. In this study, Cd exposure reduced the content of secondary metabolites (SMs) in rhizosphere soil, such as hydroxyspirilloxanthin, 8(R)-HPETE, farnesylcysteine, trioxilin A3, and lividamine. The accumulation of SMs is thought to be how plants adapt and protect against environmental stress. The accumulation of SMs is considered to be a way for plants to adapt and protect themselves against heavy metal stress [70]. One study has shown a reduction in SMs with increasing stress time or intensity in rhizosphere soil [71]. Under this situation, plants expend all of their energy to sustain life instead of synthesizing SMs [72]. Additionally, rhizosphere metabolism may shape specific rhizosphere microbial communities through changes in the rhizosphere soil’s physicochemical properties [73,74,75]. In addition to changes in rhizosphere soil’s microbial communities, physicochemical properties, and metabolites, R. decorum-mediated co-occurrence networks among these variables were clearly influenced by Cd stress. In this study, CdCl2 treatment decreased the number of total edges, negative edges, connectance, average degree, and diameter of bacterial and fungal co-occurrence networks, indicating that Cd decreased the complexity of the co-occurrence networks (Figure 8). It has been reported that rhizosphere microbial communities with simple networks exhibit less resistance to abiotic stress than those with complex ones [76,77]. Reduced network complexity leads to reduced ecological community stability and impaired ecosystem functioning [78,79]. Different microbial taxa may complement one another through metabolites under Cd stress, thereby improving their tolerance to adverse environmental conditions. Farnesylcysteine is one of the key rhizosphere soil metabolites nodes in bacterial and fungal networks belonging to SMs. Plants exposed to Cd stress exhibit higher levels of SMs, which help them against Cd stress [43]. As the key bacterial node, Actinobacteriota is beneficial to plant growth [80]. Meanwhile, the fungal phylum Chytridiomycota contributes to nutrient cycling and the flow of energy [81].

5. Conclusions

In this study, we found that CdCl2 treatment significantly increased the total Cd content and the percentage of exchangeable Cd fraction in rhizosphere soil, while it decreased the SOC content and the urease and invertase activities, reducing the quality of the rhizosphere soil. CdCl2 treatment did not significantly change the Chao1 and Shannon indices of the rhizosphere bacterial and fungal communities. R. decorum was more likely to recruit beneficial bacteria (Proteobacteria and Chloroflexi) to resist Cd stress and increased the relative abundance of Cd-resistant fungi (Basidiomycota and Rozellomycota) in rhizosphere soil. Rhizosphere soil urease and invertase activities, ALP, SOC, TK, Cd, and NN, were the main drivers of the composition of the bacterial and fungal communities. The random forest analysis showed that the bacterial Firmicutes and the fungal Chytridiomycota contributed the most. Moreover, CdCl2 treatment decreased the contents of SMs in rhizosphere soil, such as hydroxyspirilloxanthin, farnesylcysteinen, and lividamine. Furthermore, CdCl2 treatment decreased the complexity of the co-occurrence network of rhizosphere soil’s differential metabolites, physicochemical properties, and bacteria/fungi. This study could enhance the understanding of the rhizosphere’s microbiological mechanisms of R. decorum’s adaptation to Cd stress.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae10080884/s1, Figure S1: Analysis of LDA effect size (LEfSe) of soil bacteria in three treatment groups; LDA > 3.0, and the classification label is genus. Branch diagram of (A) bacterial and (B) fungal species evolution under different CdCl2 concentrations, and histogram of LDA value distribution for different species. Figure S2: A random forest was used to analyze characteristic species with significant effects on phylum level bacteria in (A) CK vs. 2.5 mM and (B) CK vs. 5 mM, and fungi in (C) CK vs 2.5 mM and (D) CK vs. 5 mM. Figure S3: OPLS-DA score map showing the separation of metabolites in rhizosphere soil treated with Cd in comparison groups of (A) CK vs. 2.5 mM, and (C) CK vs 5 mM. A differential metabolite abundance score map revealed the differential metabolite pathways in the (B) CK vs. 2.5 mM and (D) CK vs. 5 mM CdCl2 comparative treatment groups. Figure S4. Effects of CdCl2 on rhizosphere soil metabolites: The different rhizosphere soil metabolites (top 20) were identified (A) between CK and the 2.5 mM treatment and (B) between CK and the 5 mM treatment. Figure S5: The volcano map shows differences in metabolites for (A) CK vs. 2.5 mM and (B) CK vs. 5 mM. All identified metabolite data were plotted in a log2 (FC) versus-log10 (p-value) relationship. Table S1: Sequential extraction fractions of Cd.

Author Contributions

Conceptualization, J.G. and M.T.; methodology, J.G., M.T. and L.C.; validation, J.G., M.T., L.W. and C.W.; formal analysis, J.G., Y.Y. (Yin Yi) and J.W.; investigation, J.G., M.T. and X.C.; resources, J.G., J.L. and Y.Y. (Yongsong Yang); data curation, M.T., L.C. and L.W.; writing—original draft preparation, M.T. and J.G.; writing—review and editing, M.T. and K.M.; project administration, J.G.; funding acquisition, J.G. and Y.Y. (Yin Yi). All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Joint Fund of the Natural Science Foundation of China and the Karst Science Research Center of Guizhou Province (Grant No. U1812401), Guizhou Provincial Science and Technology Plan Project (Qian Ke He Cheng Guo [2022] Zhong Dian010), Guizhou forestry scientific research project (Qianlinkehe [2022] No. 28), Karst Rocky Desertification Water-Fertilizer Coupling and Biodiversity Restoration (Guizhou Education Technology [2023]004), Changjiang Scholars and Innovative Research Team in University (IRT_17R50), the Natural Science Foundation of Gansu Province (22JR5RA451, 23JRRA1093), the Fundamental Research Funds for the Central Universities (lzujbky-2021-ey01) in Lanzhou University, and the China Postdoctoral Science Foundation (Grant No. 2023M731470).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. The growth of R. decorum seedings under CK, 2.5 and 5 mM CdCl2 treatments, respectively.
Figure 1. The growth of R. decorum seedings under CK, 2.5 and 5 mM CdCl2 treatments, respectively.
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Figure 2. The effects of the different concentrations of CdCl2 on the physicochemical properties of R. decorum rhizosphere soil. (A) Cd content; (B) EX-Cd: exchangeable Cd; (C) OB-Cd: carbonate-bound Cd; (D) OX-Cd: Fe-Mn oxide-bound Cd; (E) OM-Cd: organic matter-bound Cd; (F) Res-Cd: residual Cd in rhizosphere soil; (G) percentage diagram of Cd forms in rhizosphere soil. (H) pH of rhizosphere soil. The data were expressed as the mean ± standard deviation (mean ± SD); the different letters indicate significantly differences (p < 0.05 by Duncan’s test).
Figure 2. The effects of the different concentrations of CdCl2 on the physicochemical properties of R. decorum rhizosphere soil. (A) Cd content; (B) EX-Cd: exchangeable Cd; (C) OB-Cd: carbonate-bound Cd; (D) OX-Cd: Fe-Mn oxide-bound Cd; (E) OM-Cd: organic matter-bound Cd; (F) Res-Cd: residual Cd in rhizosphere soil; (G) percentage diagram of Cd forms in rhizosphere soil. (H) pH of rhizosphere soil. The data were expressed as the mean ± standard deviation (mean ± SD); the different letters indicate significantly differences (p < 0.05 by Duncan’s test).
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Figure 3. The effects of the different concentrations of CdCl2 on rhizosphere soil properties of R. decorum. (A) TN: soil’s total N, (B) AN: ammonium N, (C) NN: nitrate N, (D) TP: soil’s total P, (E) AP: plant-available P, (F) TK: soil’s total potassium, (G) AK: ammonium potassium, (H) SOC: soil’s organic carbon, (I) catalase, (J) alkaline phosphatase, (K) urease, and (L) invertase enzymatic activities in rhizosphere soils of R. decorum under control and CdCl2 addition treatments. The different letters indicate significantly differences (p < 0.05 by Duncan’s test).
Figure 3. The effects of the different concentrations of CdCl2 on rhizosphere soil properties of R. decorum. (A) TN: soil’s total N, (B) AN: ammonium N, (C) NN: nitrate N, (D) TP: soil’s total P, (E) AP: plant-available P, (F) TK: soil’s total potassium, (G) AK: ammonium potassium, (H) SOC: soil’s organic carbon, (I) catalase, (J) alkaline phosphatase, (K) urease, and (L) invertase enzymatic activities in rhizosphere soils of R. decorum under control and CdCl2 addition treatments. The different letters indicate significantly differences (p < 0.05 by Duncan’s test).
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Figure 4. Bacterial and fungal diversity of rhizosphere soil in R. decorum under the different concentrations of CdCl2. The α diversity of bacterial community showed by the (A) Chao1 index and (B) Shannon index. The α diversity of fungal community showed by the (D) Chao1 index and (E) Shannon index. Principal coordinates analysis (PCoA) of the (C) bacterial and (F) fungal communities. The different letters indicate significantly differences (p < 0.05 by Duncan’s test).
Figure 4. Bacterial and fungal diversity of rhizosphere soil in R. decorum under the different concentrations of CdCl2. The α diversity of bacterial community showed by the (A) Chao1 index and (B) Shannon index. The α diversity of fungal community showed by the (D) Chao1 index and (E) Shannon index. Principal coordinates analysis (PCoA) of the (C) bacterial and (F) fungal communities. The different letters indicate significantly differences (p < 0.05 by Duncan’s test).
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Figure 5. Effects of CdCl2 on the rhizosphere’s microbial community. Relative abundance of (A) bacterial and (C) fungal community composition components at the phylum level. Relative abundance of (B) bacterial and (D) fungal community composition components at the genus level.
Figure 5. Effects of CdCl2 on the rhizosphere’s microbial community. Relative abundance of (A) bacterial and (C) fungal community composition components at the phylum level. Relative abundance of (B) bacterial and (D) fungal community composition components at the genus level.
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Figure 6. The results of redundancy analysis (RDA) showed the correlation between soil physicochemical properties and enzyme activities and bacteria (top 8) and fungi (top 5) at the phylum level, respectively, in rhizosphere soils of R. decorum under different CdCl2 treatment concentrations.
Figure 6. The results of redundancy analysis (RDA) showed the correlation between soil physicochemical properties and enzyme activities and bacteria (top 8) and fungi (top 5) at the phylum level, respectively, in rhizosphere soils of R. decorum under different CdCl2 treatment concentrations.
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Figure 7. Metabolite profiles in rhizosphere soil under different CdCl2 treatments. (A) Principal component analysis of soil metabolites in different treatment groups. (B) The numbers of up- and down-regulated differential metabolites (DMs) in comparative groups of CK vs. 2.5 mM, and CK vs. 5 mM.
Figure 7. Metabolite profiles in rhizosphere soil under different CdCl2 treatments. (A) Principal component analysis of soil metabolites in different treatment groups. (B) The numbers of up- and down-regulated differential metabolites (DMs) in comparative groups of CK vs. 2.5 mM, and CK vs. 5 mM.
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Figure 8. Bacterial and fungal co-occurrence network analysis. Co-occurrence networks were constructed using the rhizosphere soil differential metabolites (top 20), physicochemical properties, and bacteria (top 15 phyla) under (A) CK vs. 2.5 mM treatment and (B) CK vs. 5 mM treatment. Co-occurrence networks were constructed using the rhizosphere soil differential metabolites (top 20), physicochemical properties, and fungi (top 10 phyla) under (C) CK vs. 2.5 mM treatment and (D) CK vs. 5 mM treatment. The size of the nodes is proportional to the abundance of these variables. The yellow and red edges represent positive Pearson correlations, while the pink and blue edges represent negative ones. The top 20 metabolites were selected based on their VIP values.
Figure 8. Bacterial and fungal co-occurrence network analysis. Co-occurrence networks were constructed using the rhizosphere soil differential metabolites (top 20), physicochemical properties, and bacteria (top 15 phyla) under (A) CK vs. 2.5 mM treatment and (B) CK vs. 5 mM treatment. Co-occurrence networks were constructed using the rhizosphere soil differential metabolites (top 20), physicochemical properties, and fungi (top 10 phyla) under (C) CK vs. 2.5 mM treatment and (D) CK vs. 5 mM treatment. The size of the nodes is proportional to the abundance of these variables. The yellow and red edges represent positive Pearson correlations, while the pink and blue edges represent negative ones. The top 20 metabolites were selected based on their VIP values.
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Table 1. Topological features of the co-occurrence networks depicting the interactions between rhizosphere bacterial/fungal taxa, rhizosphere soil physicochemical properties, and metabolites under different Cd stress. Bac: bacteria, Fun: fungi.
Table 1. Topological features of the co-occurrence networks depicting the interactions between rhizosphere bacterial/fungal taxa, rhizosphere soil physicochemical properties, and metabolites under different Cd stress. Bac: bacteria, Fun: fungi.
Total EdgesPositive EdgesNegative EdgesConnectanceAverage DegreeAverage Pathway LengthDiameterClustering Coefficient
BacCK vs. 2.5 mM6303392910.4924.71.314.790.87
CK vs. 5 mM5693771920.4522.31.334.200.90
FunCK vs. 2.5 mM5583052530.5424.30.962.880.90
CK vs. 5 mM5333501830.4922.71.152.870.89
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Tang, M.; Chen, L.; Wang, L.; Yi, Y.; Wang, J.; Wang, C.; Chen, X.; Liu, J.; Yang, Y.; Malik, K.; et al. Comprehensive Analysis of Microbiomes and Metabolomics Reveals the Mechanism of Adaptation to Cadmium Stress in Rhizosphere Soil of Rhododendron decorum subsp. Diaprepes. Horticulturae 2024, 10, 884. https://doi.org/10.3390/horticulturae10080884

AMA Style

Tang M, Chen L, Wang L, Yi Y, Wang J, Wang C, Chen X, Liu J, Yang Y, Malik K, et al. Comprehensive Analysis of Microbiomes and Metabolomics Reveals the Mechanism of Adaptation to Cadmium Stress in Rhizosphere Soil of Rhododendron decorum subsp. Diaprepes. Horticulturae. 2024; 10(8):884. https://doi.org/10.3390/horticulturae10080884

Chicago/Turabian Style

Tang, Ming, Lanlan Chen, Li Wang, Yin Yi, Jianfeng Wang, Chao Wang, Xianlei Chen, Jie Liu, Yongsong Yang, Kamran Malik, and et al. 2024. "Comprehensive Analysis of Microbiomes and Metabolomics Reveals the Mechanism of Adaptation to Cadmium Stress in Rhizosphere Soil of Rhododendron decorum subsp. Diaprepes" Horticulturae 10, no. 8: 884. https://doi.org/10.3390/horticulturae10080884

APA Style

Tang, M., Chen, L., Wang, L., Yi, Y., Wang, J., Wang, C., Chen, X., Liu, J., Yang, Y., Malik, K., & Gong, J. (2024). Comprehensive Analysis of Microbiomes and Metabolomics Reveals the Mechanism of Adaptation to Cadmium Stress in Rhizosphere Soil of Rhododendron decorum subsp. Diaprepes. Horticulturae, 10(8), 884. https://doi.org/10.3390/horticulturae10080884

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