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Article

Regulatory Mechanisms of Plant Growth-Promoting Bacteria in Alleviating Microplastic and Heavy Metal Combined Pollution: Insights from Plant Growth and Metagenomic Analysis

by
Yan Chen
1,2,
Han Liu
3,
Yu-Bo Sun
3,
Meng Wang
3,
Yue-Liang Meng
3,
Yong-Qi Liu
3,
Xue-Min Ren
3,
Bai-Lian Larry Li
3,
Xue-Mei Han
4,
Ling Liu
1,* and
Zhao-Jin Chen
3,*
1
College of Agriculture, Henan University of Science and Technology, Luoyang 471023, China
2
School of Civil and Architectural Engineering, Nanyang Normal University, Nanyang 473061, China
3
College of Water Resource and Modern Agriculture, Nanyang Normal University, Nanyang 473061, China
4
College of Life Sciences, Hainan Normal University, Haikou 571158, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(9), 2168; https://doi.org/10.3390/agronomy15092168
Submission received: 3 August 2025 / Revised: 5 September 2025 / Accepted: 9 September 2025 / Published: 11 September 2025
(This article belongs to the Special Issue Microplastics and Nanoplastics in Agroecosystems: Strategies to Mitigate Plastic Pollution for Sustainable Agriculture)
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Abstract

The co-occurrence of microplastics and heavy metals in soil can lead to synergistic interactions that may exert more pronounced toxic effects on plant growth. Previous studies have demonstrated the promising potential of plant growth-promoting bacteria (PGPB) in mitigating the combined toxicity of microplastics and heavy metals. However, the rhizosphere microbial mechanisms underlying this alleviation remain unclear. Metagenomic sequencing offers significant advantages for microbial functional analysis, yet it has been underutilized in studies involving combined microplastic and heavy metal contamination. In this study, a pot experiment was conducted to evaluate the effects of inoculating sorghum with two plant growth-promoting bacterial (PGPB) strains, Bacillus sp. SL-413 and Enterobacter sp. VY-1, on plant tolerance to co-contamination with 13 μm polyethylene (PE) microplastics (0.5%, w/w) and cadmium (Cd, 10 mg kg−1). The impact on rhizosphere microbial community structure and function was assessed using metagenomic analysis. The results showed that PE-Cd co-contamination, compared to Cd alone, caused varying degrees of reduction in sorghum height and biomass, indicating an enhanced toxic effect due to the combined pollutants. Inoculation with PGPB effectively alleviated the PE-Cd combined toxicity, resulting in increases in sorghum height by 4.81–12.50%, biomass by 0.43–38.40%, and Cd accumulation by 6.20–38.07%. Both Cd and PE-Cd treatments, as well as PGPB inoculation, significantly altered the composition of rhizosphere soil bacterial communities, particularly affecting the relative abundances of Ramlibacter, Solirubrobacter, and Streptomyces. Metagenomic analysis further revealed that PE-Cd co-contamination suppressed microbial functional potential in the rhizosphere. However, inoculation with Bacillus sp. SL-413 and Enterobacter sp. VY-1 alleviated the functional stress induced by PE-Cd co-contamination and significantly enhanced microbial gene functions in the soil. Specifically, genes involved in nitrogen and phosphorus cycling increased by 3.35–5.32% and 2.26–7.38%, respectively, compared to the PE-Cd treatment without inoculation. This study provides fundamental data and scientific evidence for understanding the ecotoxicological effects of microplastic and heavy metal co-contamination, as well as the potential for microbial remediation using PGPB.

1. Introduction

Cadmium (Cd), a non-essential element, poses significant toxic effects on soil ecosystems when excessively accumulated [1,2]. Remediation of Cd-contaminated soils is therefore crucial for promoting sustainable agriculture, ensuring the yield and quality safety of agricultural products, and safeguarding human health [3,4]. Concurrently, microplastics (MPs) have emerged as a new class of soil contaminants, primarily introduced through sewage irrigation, agricultural mulching films, biosolids, and organic fertilizers [5,6,7]. Weber et al. [8] reported that a survey of biogas plants showed the median concentration of microplastics in soils treated with digestates derived from various organic wastes was 6400 p kg−1, with a range of 800–33,800 p kg−1. Notably, areas affected by microplastic pollution often overlap with those contaminated by heavy metals, particularly cadmium, thereby increasing the likelihood of their co-occurrence and interaction [9,10].
Microplastics possess unique physicochemical properties such as high specific surface area, hydrophobicity, and surface charge, enabling them to interact with other pollutants in soil and serve as vectors, thus complicating their environmental impacts [11]. High concentrations of microplastics (MPs) combined with heavy metals may exert synergistic toxic effects by promoting heavy metal bioavailability and accumulation in plants, ultimately suppressing plant growth. [9,11]. For instance, Wang et al. [12] reported that the addition of MPs such as PS and PLA to soil increased the bioavailability of Cd, ultimately altering plant growth and microbial community structure. Similarly, Feng et al. [13] found that the coexistence of MPs and heavy metals may affect the mobility of heavy metals, soil fertility, and microbial diversity and functionality.
Plant growth-promoting bacteria (PGPB) can alleviate heavy metal stress and enhance plant growth by producing phytohormones (e.g., IAA) and ACC deaminase. Additionally, PGPB improve plant nutrient acquisition by solubilizing otherwise insoluble phosphorus and potassium in soil [14,15,16]. These bacteria have been widely applied in the remediation of complex pollution scenarios involving heavy metals and organic pollutants, or heavy metals and salinity, with promising outcomes [17,18]. Our previous studies demonstrated that PGPB can mitigate the combined stress of microplastics and Cd in sorghum under hydroponic and pot conditions by modulating gene expression, enhancing antioxidant enzyme activity, and mobilizing soil nutrients, thus showing potential for remediating mixed MPs and heavy metal contamination [19,20,21].
Soil microbial communities, often described as the plant’s “second genome,” represent the most dynamic biological components in the soil environment and are essential for supporting plant development and regulating biogeochemical processes [22,23]. The advent of metagenomics—a high-throughput next-generation sequencing (NGS) approach—enables direct extraction and analysis of microbial DNA from environmental matrices, thereby uncovering the taxonomic structure, functional potential, and ecological roles of microorganisms [24]. This technique has emerged as a critical method for advancing our understanding of plant–microbe interactions in the context of phytoremediation [25,26,27]. Despite its potential, there remains a paucity of studies employing metagenomic tools to elucidate how PGPB influence sorghum adaptation under simultaneous exposure to MPs and Cd contamination. In the present work, a pot-based experiment was established to examine the role of PGPB inoculation in enhancing sorghum resistance to combined stress induced by polyethylene (PE, 13 μm) microplastics and cadmium. To amplify the ecotoxicological effects of polyethylene (PE) microplastics, a concentration of 0.5% (w/w) was selected for this experiment, following the approach used in previous studies [28,29,30]. Metagenomic sequencing was utilized to investigate shifts in rhizosphere microbial community structure and associated functional gene profiles, with the objective of offering mechanistic insights and technical foundations for the development of PGPB-assisted phytoremediation strategies in MPs and heavy metal co-contaminated soils.

2. Materials and Methods

2.1. Experimental Materials

In this experiment, two strains of plant growth-promoting bacteria (PGPB), namely Enterobacter sp. VY-1 and Bacillus sp. SL-413, were employed. These microbial strains, previously isolated and cryopreserved in our laboratory, are characterized by their ability to mobilize phosphorus and potassium, produce siderophores and indole-3-acetic acid (IAA), and exhibit substantial cadmium resistance, as reported by Liu et al. [19]. The test sorghum used was Kyoto Ferro 100, certified sorghum seeds were obtained from Shuyang Tuojing Horticultural Co., Ltd. The soil used for pot trials was collected from a non-contaminated site located in the Pomegranate Garden at Nanyang Normal University. Comprehensive information on the initial physicochemical attributes of the soil can be found in Liu et al. [19]. Prior to use, the soil was manually cleared of coarse materials such as stones and plant fragments, followed by natural air-drying and sieving through a 20-mesh screen. The experimental design is detailed in Table 1. To simulate Cd contamination, cadmium sulfate octahydrate (CdSO4·8H2O) was introduced to yield a final concentration of 10 mg per kilogram of soil. Additionally, polyethylene microplastics with an average particle diameter of 13 μm were incorporated at 0.5% (w/w). The treated soil was mixed uniformly and left to equilibrate for 30 days under ambient conditions.

2.2. Pot Experiment

Pots were each filled with 0.75 kg of prepared soil, with three replicates assigned to each experimental treatment. Uniform, healthy sorghum seeds were sown, and the pots were randomly distributed within a greenhouse to maintain uniform growth conditions. When seedlings attained approximately 5 cm in height, thinning was performed to retain three plants per pot. The bacterial strains were cultivated in liquid LB medium until reaching the logarithmic growth phase, then harvested by centrifugation and resuspended in sterile deionized water to a concentration of 1 × 108 colony-forming units (cfu) per milliliter. Thirty days after seed germination, each plant was inoculated with 10 mL of the bacterial suspension, while control groups received the same volume of sterile water. The pot experiment was conducted between April and June 2023. After 90 days of cultivation, sorghum plants and rhizosphere soil samples were harvested for further analysis.

2.3. Determination of Cd Content and Soil Properties

Plant tissues were oven-dried at 80 °C until constant weight was achieved, then ground to a fine powder. Approximately 0.1 g of the dried material was digested in a PTFE crucible using a microwave-assisted digestion system with a mixture of HCl, HNO3, HF, and HClO4. The resulting solutions were filtered and analyzed for cadmium concentrations via inductively coupled plasma optical emission spectrometry (ICP-OES). For assessing bioavailable Cd in soil, 5.0 g of soil was extracted with DTPA solution (pH 7.30 ± 0.05), followed by shaking, centrifugation, and filtration through a 0.22 μm membrane before ICP-OES measurement. Soil pH was determined potentiometrically using a 1:5 soil-to-water ratio. Available phosphorus was quantified through 0.5 mol·L−1 NaHCO3 extraction combined with molybdenum-antimony colorimetric analysis. Available potassium content was measured by flame photometry, and alkaline hydrolyzable nitrogen was evaluated using the alkali diffusion method.

2.4. Metagenomic Analysis

Total DNA was extracted from 0.5 g of rhizosphere soil using the FastDNA SPIN Kit for Soil (MP Biochemicals, Solon, OH, USA). DNA purity and concentration were determined via ultraviolet spectrophotometry. Sequencing was conducted on the Illumina NovaSeq platform. Clean reads obtained were assembled using MEGAHIT version 1.1.2, retaining contigs of at least 300 base pairs. Open reading frames (ORFs) were predicted using Prodigal with a minimum length cutoff of 100 bp and subsequently translated into amino acid sequences. Gene clustering was performed using CD-HIT v4.6.1 with thresholds of 95% sequence identity and 90% coverage; the longest sequence from each cluster was selected to build a non-redundant gene catalog. High-quality reads from each sample were mapped to the gene catalog via SOAPaligner at 95% identity to calculate gene abundances. Functional and taxonomic annotations were assigned based on NR, EggNOG, and KEGG databases.

2.5. Data Analysis

Statistical analyses of soil physicochemical properties, heavy metal levels, and sequencing data were conducted using SPSS version 17.0. Differences among groups were evaluated through Student’s t-test and one-way analysis of variance (ANOVA).

3. Results

3.1. Effects of Different Treatments on Sorghum Growth

The effects of various treatments on sorghum growth are shown in Figure 1A,B. The addition of Cd significantly inhibited sorghum shoot and root length as well as biomass accumulation. Compared to the control (ctrl), Cd treatment reduced shoot, root, and total length by 13.94%, 22.34%, and 16.23%, respectively, and decreased shoot, root, and total dry weight by 19.13%, 29.15%, and 21.84%, respectively. These results indicate that 10 mg kg−1 of Cd in the soil imposes significant stress on sorghum growth. Under the combined PE-Cd contamination, plant height and biomass decreased further compared to the Cd-only group, with shoot and total length reduced by 3.70% and 2.77%, and shoot, root, and total dry weight decreased by 1.33%, 1.15%, and 0.70%, respectively, suggesting enhanced toxicity from the combined PE-Cd exposure.
Inoculation with plant growth-promoting bacteria significantly promoted sorghum growth under PE–Cd stress. Specifically, Bacillus sp. SL-413 increased shoot, root, and total length by 4.81%, 12.33%, and 6.76%, respectively, while Enterobacter sp. VY-1 enhanced shoot and total length by 12.50% and 6.76%, respectively. In terms of biomass, Bacillus sp. SL-413 elevated shoot, root, and total dry weight by 20.75%, 20.07%, and 20.61%, whereas Enterobacter sp. VY-1 increased them by 38.40%, 0.43%, and 30.76%, respectively.

3.2. Effects of Different Treatments on Cd Accumulation in Sorghum

The co-contamination of PE and Cd produced differential effects on Cd levels in sorghum’s shoot and root tissues. Relative to the Cd-only treatment, shoot Cd concentration decreased by 3.91%, whereas Cd content in roots exhibited a 3.15% increase (Figure 2). When inoculated with either bacterial strain, Cd uptake by the plants was enhanced. Specifically, Bacillus sp. SL-413 and Enterobacter sp. VY-1 elevated Cd concentration in shoots by 10.47% and 10.76%, respectively, and in roots by 23.42% and 30.61%, compared to plants exposed solely to the PE-Cd mixture. Under PE-Cd treatment, Cd accumulation rose by 7.71% in shoots and 25.67% in roots. Further inoculation with Bacillus sp. SL-413 resulted in an increase in shoot Cd accumulation by 19.10% and root accumulation by 43.61%, whereas Enterobacter sp. VY-1 enhanced shoot and root Cd levels by 38.07% and 6.20%, respectively, relative to the PE-Cd treatment.

3.3. Effects of Different Treatments on the Physicochemical Properties of Sorghum Rhizosphere Soil

Soil pH, available potassium (AK), available phosphorus (AP), and alkaline hydrolyzable nitrogen (AN) exhibited similar trends across treatments, with values decreasing in the order ctrl > Cd > PE-Cd, suggesting that both Cd and PE-Cd contamination reduced the availability of essential soil nutrients (Table 2). Inoculation with Bacillus sp. SL-413 or Enterobacter sp. VY-1 significantly improved AK, AP, and AN levels compared to the PE-Cd treatment. Specifically, Bacillus sp. SL-413 increased AK, AP, and AN by 3.57%, 16.48%, and 75.00%, respectively, while Enterobacter sp. VY-1 increased them by 3.38%, 10.99%, and 53.57%, respectively. DTPA-extractable Cd (DTPA-Cd) was reduced by PE addition. Enterobacter sp. SL-413 inoculation further reduced DTPA-Cd compared to PE-Cd alone, while Enterobacter sp. VY-1 inoculation increased DTPA-Cd slightly.

3.4. Effects of Inoculated Strains on Bacterial Community Composition

Metagenomic sequencing at the genus level identified 4004 genera in total across four treatment groups, with 3469 shared genera. The number of unique genera in the Cd, PE-Cd, PE-Cd–SL-413, and PE-Cd–VY-1 groups was 71, 35, 60, and 71, respectively. Principal coordinate analysis (PCoA) was conducted using the R package vegan (v2.5-3) to assess the impact of inoculation under PE-Cd stress on bacterial community composition. As shown in Figure 3, PC1 and PC2 explained 14.26% and 11.09% of the variation, respectively. Control samples clustered in the lower-left quadrant, while PE-Cd samples were located in the upper-left quadrant, indicating a clear shift in community composition due to PE-Cd stress. Inoculation with Bacillus sp. SL-413 and Enterobacter sp. VY-1 resulted in overlapping but distinct distributions, suggesting that both strains influenced community structure. Subsequent ANOSIM analysis revealed a significant difference between inoculated and non-inoculated groups (p = 0.01).
Using LEfSe (Linear Discriminant Analysis Effect Size) analysis, 26 taxa showed significantly different relative abundance (LDA score ≥ 2) across treatments (Figure 4). In the Cd group, enriched taxa included Gemmatimonadaceae, Ilumatobacteraceae, Methylobacteriaceae, Trichocoleusaceae, Microvirga, Ramlibacter, and Trichocoleus. In the PE-Cd group, Solirubrobacteraceae and Solirubrobacter were significantly enriched. In Bacillus sp. SL-413-treated samples, the most abundant taxa were Acidimicrobiia, Acidimicrobiales, and Hydrocarboniphaga. In Enterobacter sp. VY-1-treated samples, enriched taxa included Streptomycetales, Streptomycetaceae, and Streptomyces.
To investigate the relationships between soil bacterial communities and environmental variables, redundancy analysis (RDA) was performed. The first two RDA axes accounted for 26.90% and 25.01% of the total variance, respectively (Figure 5). According to the Envfit test, available potassium (AK; r2 = 0.532, p = 0.029) and DTPA-extractable Cd (r2 = 0.661, p = 0.018) were identified as key factors significantly associated with shifts in bacterial community composition (p < 0.05).

3.5. Functional Profiling of Metagenomes

3.5.1. KEGG Functional Annotation

As shown in Figure 6, non-redundant (Nr) genes were annotated against the KEGG database, which classifies functions into six major categories: Metabolism, Environmental Information Processing, Cellular Processes, Genetic Information Processing, Human Diseases, and Organismal Systems. Among the first-level KEGG metabolic pathways, metabolism exhibited the highest gene abundance in the sorghum rhizosphere microbiome, representing the dominant functional category. This was followed by Environmental Information Processing, Genetic Information Processing, Cellular Processes, Human Diseases, and Organismal Systems, with the latter showing the lowest abundance.
Comparative analysis revealed that the gene abundance in the PE-Cd co-contamination group was consistently lower than that in the Cd-only treatment, with a reduction ranging from 6.95% to 8.26%. Conversely, inoculation with PGPB increased gene abundance relative to the PE-Cd group, with Bacillus sp. SL-413 and Enterobacter sp. VY-1 treatments enhanced abundance by 2.89–3.78% and 5.24–7.35%, respectively.
At the second KEGG hierarchical level within the metabolism category, the most abundantly annotated pathways included Global and Overview Maps, Carbohydrate Metabolism, Amino Acid Metabolism, Energy Metabolism, Metabolism of Cofactors and Vitamins, Membrane Transport, Cellular Community—Prokaryotes, and Signal Transduction. Consistent with the first-level results, the PE-Cd group exhibited a 7.38% lower gene abundance compared to the Cd-only group. The PGPB-inoculated groups showed increased gene abundance relative to PE-Cd, with Bacillus sp. SL-413 and Enterobacter sp. VY-1 increasing abundance by 3.33% and 6.01%, respectively.

3.5.2. Functional Differences in Rhizosphere Soil Microbial Communities

To identify significantly different functional and metabolic pathways among treatments, t-tests were conducted on third-level KEGG pathways comparing PE-Cd co-contamination and PGPB inoculations. As illustrated in Figure 7 and Supplementary Table S1, compared to Cd treatment alone, PE-Cd co-contamination induced significant changes (p < 0.05) in 26 metabolic pathways, including Thyroid Hormone Signaling Pathway, Primary Bile Acid Biosynthesis, Steroid Degradation, Glycosylphosphatidylinositol (GPI)-Anchor Biosynthesis, GABAergic Synapse, O-Antigen Repeat Unit Biosynthesis, Calcium Signaling Pathway, Glutamatergic Synapse, and Glutathione Metabolism. Inoculation with Bacillus sp. SL-413 (PE-Cd-SL-413) significantly altered 15 pathways relative to PE-Cd treatment, such as Citrate Cycle (TCA Cycle), Nitrotoluene Degradation, Arabinogalactan Biosynthesis (Mycobacterium), Cell Cycle, O-Antigen Repeat Unit Biosynthesis, Viral Carcinogenesis, and Melanogenesis. Similarly, Enterobacter sp. VY-1 inoculation (PE-Cd-VY-1) led to significant differences in 15 pathways, including Biotin Metabolism, Human Papillomavirus Infection, Type II Diabetes Mellitus, Viral Carcinogenesis, Phenylalanine, Tyrosine and Tryptophan Biosynthesis, Vascular Smooth Muscle Contraction, and Pantothenate and CoA Biosynthesis. Comparing the two inoculants, PE-Cd-VY-1 and PE-Cd-SL-413 differed significantly in 15 pathways, such as O-Antigen Repeat Unit Biosynthesis, Arabinogalactan Biosynthesis (Mycobacterium), NF-kappa B Signaling Pathway, Folate Biosynthesis, Peroxisome, Biotin Metabolism, and Riboflavin Metabolism.

3.5.3. Differences in Nitrogen and Phosphorus Cycling Functional Genes

To analyze variations in nitrogen and phosphorus cycling functional genes among treatments, 38 KEGG orthology (KO) genes related to nitrogen metabolism were examined. These genes are primarily involved in pathways such as Nitrogen Fixation, Nitrification, Denitrification, Assimilatory Nitrate Reduction (ANRA), Dissimilatory Nitrate Reduction to Ammonium (DNRA), Organic Nitrogen Metabolism, and Nitrogen Transport (Figure 8).
The total abundance of these nitrogen cycle-related KO genes was 37,354 in the Cd treatment, 34,840 in the PE-Cd group, 36,007 in PE-Cd-SL-413, and 36,692 in PE-Cd-VY-1. PE-Cd co-contamination reduced gene abundance by 6.73% compared to Cd treatment alone. Inoculation with Bacillus sp. SL-413 and Enterobacter sp. VY-1 increased gene abundance by 3.35% and 5.32%, respectively, relative to PE-Cd (Figure 8A). Hierarchical clustering heatmaps showed that the Cd group clustered distinctly from the other groups, indicating that PE-Cd co-contamination was the primary factor affecting nitrogen cycling genes (Figure 8A). Compared to PE-Cd, PE-Cd-SL-413 showed increased abundance in 23 genes, with napA (K02567), nirD (K00363), gltD (K00266), pmoC-amoC (K10946), and nirB (K00362) increasing by over 10%. PE-Cd-VY-1 showed increases in 28 genes relative to PE-Cd, with napB (K02568), gudB rocG (K00260), napA (K02567), nirD (K00363), gdhA (K00262), pmoA-amoA (K10944), nosZ (K00376), norC (K02305), gltD (K00266), pmoB-amoB (K10945), nasB (K00360), E3.5.1.49 (K01455), and nirB (K00362) exceeding a 10% increase.
Phosphorus cycling gene involving Organic P Mineralization, Inorganic P Solubilization, Regulatory pathways, Transporters, Polyphosphate Synthesis, and Polyphosphate Degradation (Figure 8B). The total abundance of phosphorus cycling genes decreased by 8.72% in the PE-Cd group compared to Cd alone. PGPB inoculation increased gene abundance by 2.26% to 7.38% relative to PE-Cd. Heatmap clustering showed Cd treatment separating from other groups, again indicating PE-Cd co-contamination as the major influencing factor (Figure 8B). PE-Cd-SL-413 showed increased abundance in 21 genes compared to PE-Cd, including ppaX (K06019, +160%), phnK (K05781, +105%), phnW (K03430, +160%), phnH (K06165, +47.22%), and phoQ (K07637, +40%). Among these, 8 KOs increased by more than 10%. Furthermore, 17 KOs such as ppaX (K06019, +100%), phnJ (K06163, +60.83%), phoQ (K07637, +60%), and phnM (K06162, +46.44%) increased by more than 10%.

4. Discussion

When introduced independently into soil systems, both microplastics and heavy metals have been shown to adversely affect plant development by modifying soil chemical and physical characteristics or by exerting direct toxic pressure on vegetation [9,31,32,33,34]. In the soil–plant interface, heavy metals are primarily accumulated through root uptake and subsequent subcellular and long-distance transport within plant tissues, which ultimately impairs growth and decreases total biomass [35,36,37]. In the current investigation, sorghum subjected to cadmium stress (10 mg·kg−1) demonstrated notable reductions in both shoot and root length by 14–22%, along with declines in dry biomass ranging from 19% to 29%, relative to the untreated control group. These results indicate pronounced phytotoxicity of cadmium exposure and align with observations reported by Duan et al. [33]. Existing literature has documented that the coexistence of microplastics and heavy metals in terrestrial environments may lead to compound toxicity due to their interactive behavior, with microplastics potentially serving as vectors for heavy metals [19,38]. In this study, combined exposure to PE–Cd treatment exacerbated the inhibitory effects on plant growth compared with cadmium alone. Specifically, shoot length and total plant height were further reduced by 4% and 3%, respectively, while the dry mass of aboveground and root tissues decreased by 3–4% and 0.7–1.3%. These findings are consistent with Liu et al. [19], who also reported intensified phytotoxicity under combined PE and Cd contamination. Microplastics are known to interfere with soil nutrient dynamics; for example, Han et al. [39] demonstrated that microplastic contamination diminishes the availability of key nutrients in rhizosphere soils, particularly phosphorus and potassium. Correspondingly, our results revealed that PE addition significantly lowered the concentrations of available potassium, available phosphorus, and alkali-hydrolyzable nitrogen in soil [19,25]. This decline in essential nutrient availability likely represents a critical factor constraining sorghum growth under composite pollutant stress.
In environments co-contaminated with microplastics and heavy metals, plant growth-promoting bacteria (PGPB) play a critical role in enhancing plant resilience to environmental stress, promoting nutrient uptake, and maintaining soil ecological health [19,20,40]. The strains used in this study, Bacillus sp. SL-413 and Enterobacter sp. VY-1, were isolated from soil co-contaminated with microplastics and heavy metals [19,41]. These strains possess multiple plant growth-promoting traits, including phosphate solubilization and potassium mobilization [19]. Under PE-Cd stress conditions, both strains significantly increased the availability of K, P, and N in the soil, with improvements ranging from 3% to 75%, effectively mitigating the nutrient deficiencies induced by PE. Similar trends were reported by Zhang et al. [20]. Additionally, Bacillus sp. SL-413 and Enterobacter sp. VY-1 can produce plant hormones such as IAA, which are known to play important roles in alleviating compound pollution stress [42,43,44]. Pot experiment results confirmed that bacterial inoculation significantly enhanced sorghum growth under PE-Cd stress, increasing plant length by 5–13% and biomass by 0.4–38.4%, thus alleviating the enhanced phytotoxicity caused by PE-Cd co-contamination.
Inoculating PGPB into the plant rhizosphere not only exerts direct effects on plant growth but also influences the composition and structure of rhizosphere microbial communities. Changes in the species composition of rhizosphere microbial communities are key indicators of soil health and plant adaptability and play a vital role in determining plant productivity [45,46]. Previous research has shown that PGPB can reshape the composition and function of soil bacterial communities, helping mitigate the stress imposed by microplastic-heavy metal co-contamination and thereby improving plant growth and remediation efficiency [25,40]. PCoA analysis in this study revealed that inoculation with Bacillus sp. SL-413 and Enterobacter sp. VY-1 altered the structure of bacterial communities. According to Habibollahi et al. [47], various bacterial phyla such as Proteobacteria, Firmicutes, Acidobacteria, and Actinobacteria are critical to plant development. Our findings confirm that these dominant taxa are present in soils affected by PE-Cd stress and in inoculated treatments, suggesting their potential involvement in stress alleviation and plant growth promotion under co-contamination. LEfSe analysis further showed that bacterial groups such as Acidimicrobiales and Streptomyces were significantly enriched by PGPB inoculation. Notably, Streptomyces species are well-documented for their roles in microbe-assisted phytoremediation of heavy metal-contaminated soils [48]. Similarly, Ren et al. [49] reported increased abundance of Acidimicrobiales following PGPB inoculation. These results suggest that PGPB alleviate stress by reshaping the functional microbial community structure under PE-Cd pollution.
Microplastic-heavy metal co-contamination can also alter microbial functions in soil [31,34,50]. Metagenomic sequencing provides a powerful approach for functional analysis, yet it has rarely been applied in studies of such composite pollution [26,50,51]. Duan et al. [33] used metagenomics to analyze the effects of PS, PE, and Cd co-contamination on rhizosphere microbial function in sorghum and found that co-contamination altered the abundance of microbial taxa and functional genes associated with metabolic pathways, varying with microplastic type, particle size, and concentration. Our metagenomic analysis revealed that PE-Cd contamination reduced gene abundance in KEGG Level 1 and Level 2 functional categories by 7–8%, with 26 metabolic pathways significantly affected. These findings indicate that PE-Cd composite pollution more severely impairs soil microbial function compared with Cd alone. In contrast, PGPB inoculation increased the abundance of functional microbes and restored microbial functionality. In our study, gene abundance in inoculated groups increased by 3–4% and 5–7% compared with the PE-Cd group, and 26 metabolic pathways were significantly enriched, highlighting the ability of Bacillus sp. SL-413 and Enterobacter sp. VY-1 to alleviate functional stress and enhance microbial performance under PE-Cd conditions.
Microbial-driven cycling of soil mineral nutrients plays a vital role in supporting plant growth [52,53]. Metagenomic analysis of nitrogen and phosphorus cycling gene composition revealed that PE-Cd co-contamination reduced gene abundance by 7% and 9%, respectively, compared to Cd treatment alone. This decline aligned with observed decreases in soil available phosphorus and alkali-hydrolyzed nitrogen, indicating that PE impairs microbially mediated N and P cycling [30,54]. Bacillus sp. SL-413 and Enterobacter sp. VY-1 exhibit strong phosphate solubilization and potassium mobilization capacities [19]. Inoculation increased the abundance of N- and P-cycling genes by 3–5% and 2–7%, respectively, compared with PE-Cd treatment. Notably, 13 key nitrogen cycling genes (e.g., K02568 napB, K00260 gudB/rocG, K02567 napA, K00363 nirD) and 25 phosphorus cycling genes (e.g., K06019 ppaX, K05781 phnK, K03430 phnW, K06165 phnH) increased by more than 10%. These findings suggest that PGPB enhance soil microbial functional gene abundance, promote mineral nutrient availability, and represent an important mechanism for alleviating composite pollution stress under PE-Cd contamination.

5. Conclusions

The results showed that the combined stress of Cd and microplastics had significant negative effects on plant growth, Cd accumulation, and soil physicochemical properties, whereas inoculation with PGPB effectively mitigated this toxicity. PGPB significantly enhanced plant biomass and root activity and increased Cd accumulation in both roots and shoots. It improved soil physicochemical properties by regulating the availability of key nutrients such as N and P. Metagenomic analysis further revealed that PGPB exerted its beneficial effects by modulating the composition and functional responses of rhizosphere microbial communities, including enhancing microbial diversity and increasing the abundance of key functional genes involved in N fixation, nitrification, and P transformation. This study elucidates the potential mechanisms by which PGPB alleviates the combined stress of microplastics and Cd, providing a theoretical basis for developing novel microbe-based remediation strategies. It should be noted that the 0.5% PE microplastic concentration used here likely exceeds levels typically found in agricultural soils. Further experiments under real-world conditions are required to assess the practical potential of PGPB in mitigating combined microplastic and Cd stress.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15092168/s1: Table S1: Significantly different metabolic pathways at KEGG Level 3 among different treatments.

Author Contributions

Conceptualization, B.-L.L.L., X.-M.H., L.L. and Z.-J.C.; Methodology, Y.C., H.L., Y.-B.S., M.W., Y.-Q.L., X.-M.R., B.-L.L.L., X.-M.H., L.L. and Z.-J.C.; Software, Y.C., H.L., Y.-B.S., M.W., Y.-L.M., L.L. and Z.-J.C.; Investigation, Y.C., H.L., Y.-B.S., M.W., Y.-L.M., Y.-Q.L. and X.-M.R.; Resources, L.L. and Z.-J.C.; Data curation, Y.C., H.L., Y.-B.S., M.W., Y.-L.M., Y.-Q.L. and X.-M.R.; Writing—original draft, Y.C. and L.L.; Writing—review and editing, Y.C., X.-M.R., B.-L.L.L., X.-M.H., L.L. and Z.-J.C.; Visualization, Y.C., L.L. and Z.-J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant Nos. U2004145, 32360323), and the Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 23HASTIT018), the Key Scientific and Technological Project of Henan Province (Grant No. 252102111030), The National Natural Science Foundation of China Cultivation Project of Nanyang Normal University (Grant No. 2025PY038).

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Acknowledgments

We acknowledged the contribution of AI-powered tools in facilitating grammar checks during the proofreading process.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Agronomy 15 02168 g001
Figure 1. Length (A) and dry weight (B) of aboveground and underground parts of sorghum under different treatments. Different letters indicate significant differences between the treatments (p < 0.05). Every measurement was performed with three replicates (n = 3).
Figure 1. Length (A) and dry weight (B) of aboveground and underground parts of sorghum under different treatments. Different letters indicate significant differences between the treatments (p < 0.05). Every measurement was performed with three replicates (n = 3).
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Figure 2. Cd content (A) and accumulation (B) in aboveground and underground parts of sorghum under different treatments. Different letters indicate significant differences between the treatments (p < 0.05). Every measurement was performed with three replicates (n = 3).
Figure 2. Cd content (A) and accumulation (B) in aboveground and underground parts of sorghum under different treatments. Different letters indicate significant differences between the treatments (p < 0.05). Every measurement was performed with three replicates (n = 3).
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Figure 3. PCoA of rhizosphere bacterial communities under different treatments.
Figure 3. PCoA of rhizosphere bacterial communities under different treatments.
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Figure 4. LEfSe analysis showing differentially abundant bacterial taxa across treatments. Different node colors indicate that bacterial taxa are enriched and significant differences in corresponding groups, while light yellow nodes indicate no significant difference.
Figure 4. LEfSe analysis showing differentially abundant bacterial taxa across treatments. Different node colors indicate that bacterial taxa are enriched and significant differences in corresponding groups, while light yellow nodes indicate no significant difference.
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Figure 5. Ordination biplot generated by RDA illustrating the associations between bacterial taxa and measured environmental parameters.
Figure 5. Ordination biplot generated by RDA illustrating the associations between bacterial taxa and measured environmental parameters.
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Figure 6. KEGG primary functional categories of rhizosphere microorganisms under different treatments and relative abundance of major metabolic pathways.
Figure 6. KEGG primary functional categories of rhizosphere microorganisms under different treatments and relative abundance of major metabolic pathways.
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Figure 7. Significantly different (* p < 0.05, ** p < 0.01, *** p < 0.001) metabolic pathways at KEGG Level 3 among different treatments.
Figure 7. Significantly different (* p < 0.05, ** p < 0.01, *** p < 0.001) metabolic pathways at KEGG Level 3 among different treatments.
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Figure 8. A heatmap showing the hierarchical clustering of the N (A) and P (B) metabolism related KOs based on predicted gene copies.
Figure 8. A heatmap showing the hierarchical clustering of the N (A) and P (B) metabolism related KOs based on predicted gene copies.
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Table 1. Design of the experiment.
Table 1. Design of the experiment.
Experimental
Grouping		MPs
Class		MPs Particle
/μm		MPs Concentration
/%		Cd Concentration
/(mg·kg−1)		PGPB
ctrl--00-
Cd--010-
PE-CdPE130.510-
PE-Cd-SL-413PE130.510Bacillus sp. SL-413
PE-Cd-VY-1PE130.510Enterobacter sp. VY-1
Table 2. Physicochemical properties of rhizosphere soil under different treatments (means ± SEs).
Table 2. Physicochemical properties of rhizosphere soil under different treatments (means ± SEs).
TreatmentpHAK (mg·kg−1)AP (mg·kg−1)AN (mg·kg−1)DTPA-Cd (mg·kg−1)
ctrl6.52 ± 0.1 a84.93 ± 1.33 c4.00 ± 0.50 c28.47 ± 0.81 e-
Cd6.14 ± 0.11 b73.90 ± 0.52 b3.20 ± 0.10 a17.27 ± 0.81 b4.84 ± 0.12 d
PE-Cd6.14 ± 0.01 b70.93 ± 0.57 a3.03 ± 0.12 a13.07 ± 0.81 a3.89 ± 0.12 b
PE-Cd-SL-4136.14 ± 0.05 b73.47 ± 0.74 b3.53 ± 0.55 ab22.87 ± 1.62 d3.55 ± 0.06 a
PE-Cd-VY-16.20 ± 0.08 b73.33 ± 2.18 b3.37 ± 0.38 ab20.07 ± 0.81 c4.18 ± 0.07 c
Different letters indicate significant differences between the treatments (p < 0.05). Every measurement was performed with three replicates (n = 3).
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Chen, Y.; Liu, H.; Sun, Y.-B.; Wang, M.; Meng, Y.-L.; Liu, Y.-Q.; Ren, X.-M.; Li, B.-L.L.; Han, X.-M.; Liu, L.; et al. Regulatory Mechanisms of Plant Growth-Promoting Bacteria in Alleviating Microplastic and Heavy Metal Combined Pollution: Insights from Plant Growth and Metagenomic Analysis. Agronomy 2025, 15, 2168. https://doi.org/10.3390/agronomy15092168

AMA Style

Chen Y, Liu H, Sun Y-B, Wang M, Meng Y-L, Liu Y-Q, Ren X-M, Li B-LL, Han X-M, Liu L, et al. Regulatory Mechanisms of Plant Growth-Promoting Bacteria in Alleviating Microplastic and Heavy Metal Combined Pollution: Insights from Plant Growth and Metagenomic Analysis. Agronomy. 2025; 15(9):2168. https://doi.org/10.3390/agronomy15092168

Chicago/Turabian Style

Chen, Yan, Han Liu, Yu-Bo Sun, Meng Wang, Yue-Liang Meng, Yong-Qi Liu, Xue-Min Ren, Bai-Lian Larry Li, Xue-Mei Han, Ling Liu, and et al. 2025. "Regulatory Mechanisms of Plant Growth-Promoting Bacteria in Alleviating Microplastic and Heavy Metal Combined Pollution: Insights from Plant Growth and Metagenomic Analysis" Agronomy 15, no. 9: 2168. https://doi.org/10.3390/agronomy15092168

APA Style

Chen, Y., Liu, H., Sun, Y.-B., Wang, M., Meng, Y.-L., Liu, Y.-Q., Ren, X.-M., Li, B.-L. L., Han, X.-M., Liu, L., & Chen, Z.-J. (2025). Regulatory Mechanisms of Plant Growth-Promoting Bacteria in Alleviating Microplastic and Heavy Metal Combined Pollution: Insights from Plant Growth and Metagenomic Analysis. Agronomy, 15(9), 2168. https://doi.org/10.3390/agronomy15092168

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