Plant Growth-Promoting Serratia and Erwinia Strains Enhance Tea Plant Tolerance and Rhizosphere Microbial Diversity Under Heavy Metal Stress

Abstract

This study demonstrated that application of the particular plant growth-promoting rhizobacteria (PGPR) strains Erwinia sp. and Serratia sp. (named C15 and C20, respectively) significantly enhanced tea plant resilience in Zn (zinc)-, Pb (lead)-, and Zn + Pb-contaminated soils by the improving survival rates (over 60%) and chlorophyll content of tea plants, and by reducing the accumulation of these metals in tea plants’ tissues (by 19–37%). The PGPRs elevated key soil nutrients organic carbon (OC), total nitrogen (TH), hydrolysable nitrogen (HN), and available potassium (APO) and phosphorus (APH) contents. Compared to non-PGPR controls, both strains consistently increased microbial α-diversity (Chao1 index: +28–42% in Zn/Pb soils; Shannon index: +19–33%) across all contamination regimes. PCoA/UniFrac analyses confirmed distinct clustering of PGPR-treated communities, with strain-specific enrichment of metal-adapted taxa, including Pseudomonas (LDA = 6) and Bacillus (LDA = 4) under Zn stress; Rhodanobacter (LDA = 4) under Pb stress; and Lysobacter (LDA = 5) in Zn + Pb co-contamination. Fungal restructuring featured elevated Mortierella (LDA = 6) in Zn soils and stress-tolerant Ascomycota dominance in co-contaminated soils. Multivariate correlations revealed that the PGPR-produced auxin was positively correlated with soil carbon dynamics and Mortierellomycota abundance (r = 0.729), while the chlorophyll content in leaves was closely associated with Cyanobacteria and reduced by Pb accumulation. These findings highlighted that PGPR could mediate and improve in tea plant physiology, soil fertility, and stress-adapted microbiome recruitment under heavy metal contaminated soil and stress.

1. Introduction

Soils contaminated with heavy metal elements, such as zinc (Zn), lead (Pb), aluminum (Al), cadmium (Cd), manganese (Mn), and copper (Cu), could lead to the accumulation of these metals in cultivated plant tissues. Accumulated heavy metals exert phytotoxic effects, including inhibition of root elongation and seed germination, induction of chlorosis and necrosis, disruption of photosynthesis, nutrient imbalance, and generation of oxidative stress [1]. For instance, Zn concentrations > 200 μM cause chloroplast membrane disintegration and impair PSII photochemical efficiency in aquatic plants, while Cd exposure at 10 μM reduces chlorophyll synthesis by >50% in duckweed [2]. These effects collectively result in reduced biomass production, stunted growth, and significant yield losses, potentially leading to crop failure in severely contaminated areas [3]. The transfer of heavy metals through the food chain represents a primary pathway for human exposure [4]. Pb exposure, even at low levels, causes irreversible neurodevelopmental damage in children and cardiovascular issues in adults [5]. While Zn, Cu, and Mn are essential micronutrients, their excessive intake can cause gastrointestinal distress and neurological disorders; Al and Mn accumulation is associated with neurotoxicity concerns [6]. Heavy metals not only threaten the normal development of plants but also cause safety issue in plants used as food [7]. For tea plants, studies have shown that the concentrations of Pb, Cd, Zn, Al, and Mn in tea leaves are significantly influenced by their levels in the soil where tea plants are cultivated [8,9]. In acidic tea-growing soils (pH 4.5–6.0), the bioavailability of Cd and Pb increases by 30–40% due to shifts from residual to exchangeable fractions, resulting in >56% of yerba mate samples exceeding safety standards [10]. This direct soil-to-plant transfer pathway highlights the risk of heavy metal accumulation in tea leaves. However, the bioavailability and subsequent uptake of these metals can be modulated by rhizosphere processes, including those mediated by plant-associated microorganisms.

The remediation of heavy metal-contaminated soils to eliminate the contamination, restore soil quality, and enable safe reuse has become a global challenge [11,12]. Common remediation techniques include physical methods (e.g., soil replacement, soil isolation, and vitrification), chemical methods (e.g., immobilization, encapsulation, and soil washing), and biological methods (e.g., phytoremediation, phytostabilization, phytoextraction, genetically modified plants, chelate-assisted phytoremediation, and microbial-assisted phytoremediation) [13,14]. Each technique has its specific advantages, limitations, and application conditions [15]. However, traditional physical and chemical remediation methods are often costly and carry the risk of causing secondary contamination [16]. In contrast, microbial bioremediation, as a key biological approach, has emerged as an effective, environmentally friendly, and cost-efficient solution for removing heavy metal contamination in soil and reducing their residues in plant tissues [17,18]. Among the microbial strategies, plant growth-promoting rhizobacteria (PGPR) are particularly promising due to their ability to enhance plant growth, significantly alter heavy metal speciation in soil, and mitigate heavy metal residues in cultivated plant tissues [19].

PGPR strains promote plant growth by supplying essential nutrients or modulating endogenous plant hormone levels, such as indole acetic acid (IAA), thereby enhancing the efficiency of heavy metal or organic pollutant bioremediation [20]. This hormone-driven reprogramming enhances metal remediation by simultaneously (i) optimizing root metal interception zones, (ii) activating intracellular detoxification, and (iii) restricting xylem loading of toxic ions. PGPR strains with heavy metal resistance have been shown to immobilize heavy metals in plant rhizosphere and reduce their bioavailability [21,22]. Certain PGPR strains are effective in bioremediating heavy metal-contaminated soils by increasing plant tolerance to heavy metal stress, improving soil nutrient availability (including iron via siderophores), altering heavy metal transport mechanisms, and producing metal-chelating compounds, such as organic acids and extracellular polysaccharides [23,24]. Current PGPR applications in heavy metal remediation predominantly employ commonly used genera (e.g., Bacillus, Pseudomonas), with limited success in tea systems. Although certain strains, like B. subtilis DBM, reduce Pb by 18–25% in crops [25], their efficacy in tea plants under field conditions remains suboptimal (<50% survival rate [9]). Crucially, exogenous PGPR often fail to establish in rhizosphere niches due to poor host adaptation, necessitating repeated inoculation [26]. This study innovatively isolates Erwinia sp. C15 and Serratia sp. C20 from tea rhizosphere—a strategy ensuring host-specific compatibility and ecological safety—to address the critical gap in sustainable, single-application bioremediation for Zn/Pb-contaminated tea plantations. Erwinia sp. C15 and Serratia sp. C20 have been proven to significantly promote the growth of tea plants [27].

In this study, two PGPR strains with superior plant growth-promoting capabilities and high tolerance to Pb and Zn were isolated from the tea plant rhizosphere and selected for treating experiments. The study aimed to evaluate their effectiveness in promoting plant growth and remediating heavy metal contamination in Zn- and Pb-contaminated soil. The effects of the two strains on soil element contents and rhizosphere microbial diversity were analyzed and compared. The heavy metal levels in tea plant tissues and the chlorophyll content in tea plant leaves were also determined. The findings of this study could deliver a viable approach to maintain plant growth and productivity in heavy metal-contaminated soils using environmentally friendly PGPR-based methods.

2. Materials and Methods

2.1. Preparation of Heavy Metal-Contaminated Soil and Experimental Design

Soil samples were collected from a tea plantation in Mianxian, China (32°58′46″ N, 106°42′25″ E; altitude: 795.2 m). The nutrient soil (Brighten 2024, a commercial peat-based substrate with pH 5.5–6.0, Pindstrup Group, Ryomgård, Denmark) was used to homogenize the samples and to divide them into three equal portions. The initial contents of the soil elements were as follows: organic carbon (OC), 42.06 ± 3.21 g/kg; total nitrogen (TN), 2.15 ± 0.18 g/kg; total phosphorus (TPH), 0.11 ± 0.02 g/kg; total potassium (TPO), 15.32 ± 0.45 g/kg; hydrolysable nitrogen (HN), 124.50 ± 8.76 mg/kg; available phosphorus (APH), 21.53 ± 1.85 mg/kg; available potassium (APO), 168.20 ± 12.40 mg/kg; Zn, 28.4 ± 3.1 mg/kg; and Pb, 16.7 ± 2.5 mg/kg.

Each portion was treated with one of the following solutions: (1) ZnSO₄ (619 µM), (2) Pb(NO₃)₂ (302 µM), or (3) a combination of ZnSO₄ (619 µM) and Pb(NO₃)₂ (302 µM). The solution volume was calibrated to achieve 60% water-holding capacity, ensuring uniform distribution. Solutions were applied in three successive aliquots (133 mL each) at 24 h intervals, with thorough mixing after each addition to prevent localized accumulation. Following treatment, the soil samples were composted for 20 days at ambient temperature before being transferred to pots (14 cm height × 12 cm diameter).

Uniform two-year-old tea plants (Camellia sinensis cv. Longjing 43) were selected based on plant height and transplanted into the prepared soil. This cultivar was chosen for its documented sensitivity to heavy metal stress, which is optimal for evaluating PGPR-mediated mitigation efficacy [25]. Each pot contained 10 plants and was maintained under controlled environmental conditions (25 ± 1 °C, 16 h photoperiod, light intensity of 1500 Lx).

Two PGPR strains (Erwinia sp. and Serratia sp., named C15 and C20, respectively), previously characterized for their plant growth-promoting properties and heavy metal tolerance (see Table S1 and Figure S1 for detailed characteristics), were selected for the experiment. The isolation, identification, and characterization of bacterial strains for their plant growth-promoting traits, along with their subsequent growth-promoting effects on tea plants (Camellia sinensis), were documented by Wang [27]. The experimental design included three treatment groups, namely Erwinia sp. C15, Serratia sp. C20, and an untreated control (CK). PGPR inoculants were prepared by growing strains in 5 mL nutrient broth (48 h, 28 °C), then diluting to 50 mL with sterile water. This protocol targeted stationary-phase cells (OD₆₀₀ ~ 1.0) for optimal metabolite production, delivering ~10⁸ CFU/mL to each pot. The cultures were then diluted with sterilized distilled water to a final volume of 50 mL. For PGPR treatment, 50 mL of the diluted suspension was applied to each treatment pot (containing 10 plants), 6 times at 7 day intervals. The tea plants in the control group received 50 mL of the equivalently diluted sterilized nutrient broth on the same schedule.

The experimental design incorporated 3 heavy metal treatments (Zn, Pb, and Zn + Pb) crossed with the microbial treatment and control groups (Erwinia sp. C15, Serratia sp. C20, and CK), with 3 biological replicates per group, resulting in a total of 27 experimental units. All groups were randomly arranged to minimize positional effects.

Native strains were prioritized because they exhibit preadapted resilience to local soil conditions (pH 5.2 ± 0.3, organic matter 3.8%) and tea root exudates. Furthermore, ecological risk assessments confirm minimal disruption to indigenous microbiota [28].

2.2. Assessment of Plant Physiological Parameters and Heavy Metal Accumulation

Survival rates were recorded 10 days after completing the sixth PGPR treatment (administered at 7 day intervals). The survival rate was calculated as (%) = (Number of surviving seedlings)/10 × 100 (%). The number of initial tea plants planted per pot was 10. There were three pots for every treatment.

Plant height was measured on day 10 after the final treatment through the vertical distance from the soil surface to apical meristem of its main shoot [29]. The eighth fully expanded leaf from each plant was collected and divided into two parts for subsequent analyses. Total chlorophyll content was determined by spectrophotometer using the Lichtenthaler method [30].

For heavy metal quantification, leaf and root samples were processed following established protocols [31,32]. Briefly, root systems were carefully excavated and thoroughly rinsed with deionized water until complete removal of soil particles. Concentrations of Zn and Pb in plant tissues were determined using atomic absorption spectrophotometry (AAS) and electrothermal atomic absorption spectrometry (ET-AAS) as described by Yasmin [33] and Jurowski and Krośniak [34].

2.3. Determination of Nutrient Elements and Heavy Metal Contents in Plant and Rhizosphere Soil

The contents of OC, TN, TPH, TPO, HN, APH, and APO were determined in the samples collected on day 10 after the PGPR treatment experiment, as in the methods described by Wang [35]. The concentrations of heavy metals in the soil samples were determined using an atomic absorption spectrophotometer (Agilent 280FS AA) [36].

2.4. Microbial Community Analysis

Soil microbial communities were characterized using high-throughput amplicon sequencing. Total genomic DNA was extracted from 0.5 g soil samples using the FastDNA Spin Kit for Soil (MP Biomedicals, Irvine, CA, USA). The V4 region of bacterial 16S rRNA genes and the ITS2 region of fungal DNA were amplified using primer pairs 515F/806R and ITS3/ITS4, respectively. PCR amplification was performed in a Bio-Rad S1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: initial denaturation at 95 °C for 3 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; final extension at 72 °C for 10 min. Amplicons were purified, quantified, and pooled in equimolar ratios for library construction using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Paired-end sequencing (2 × 250 bp) was performed on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA USA).

Raw sequences were processed using QIIME2 (version 2021.11). Operational taxonomic units (OTUs) were clustered at 97% similarity using VSEARCH. Taxonomic classification was performed against the SILVA 138 database for bacteria and UNITE 8.0 for fungi. Alpha diversity indices (Chao1, Shannon, and Simpson) were calculated using QIIME2. Beta diversity was assessed through principal coordinates analysis (PCoA) based on Bray–Curtis distances. Differential abundance analysis was performed using linear discriminant analysis effect size (LEfSe) with an LDA score threshold of 2.0. Sequence data were deposited in the NCBI Sequence Read Archive under an accession number.

2.5. Statistical Analysis

All experiments were conducted with three biological replicates. Data were analyzed using SPSS Statistics 23.0 (IBM Corp., USA) and R (version 4.1.0). A three-way ANOVA was performed to assess the interactive effects of PGPR strain (Erwinia sp. C15, Serratia sp. C20, and CK), heavy metal contamination (Zn, Pb, and Zn + Pb), and their interactions. Significant interactions (p < 0.05) were followed by Tukey’s HSD post hoc test. Different lowercase letters indicate significant differences (p < 0.05) among treatment groups. For treatment effect analysis, three-way ANOVA was applied to assess the main effects and interactions of three factors, namely PGPR strain (Erwinia sp. C15/Serratia sp. C20/CK), heavy metal type (Zn/Pb/Zn + Pb), and plant tissue (root/leaf). Post hoc comparisons used Tukey’s honest significant difference (HSD) test at α = 0.05, with significance denoted by letter markers in figures. For correlation analysis, Pearson, Spearman, and Kendall’s methods were used to examine relationships among PGPR functional traits, microbial diversity, soil nutrients, and heavy metal accumulation. Principal component analysis (PCA) was performed using the prcomp package in R. Correlation heatmaps were generated in R with the corrplot package based on Pearson correlation matrices.

3. Results

3.1. Plant Growth Parameters and Heavy Metal Accumulation

Three-way ANOVA revealed a significant interaction between PGPR treatment and pollution type on the survival rate of tea plants. In Zn-polluted soil, the survival rate of the Erwinia sp. C15 treatment (76.7 ± 2.0%) was significantly higher than that of the CK (66.7 ± 5.8%) (p < 0.05). The survival rate of the Serratia sp. C20 treatment (83.3.0 ± 3.0%) was not significantly different from that of the Erwinia sp. C15 but was significantly higher than that of the CK (Figure 1a). In Pb-contaminated soil, the survival rate of the Erwinia sp. C15 treatment (76.7 ± 5.7%) was significantly higher than that of CK (60.0 ± 1.0%) and Serratia sp. C20 (66.7 ± 5.7%) (p < 0.05) (Figure 1a). Three-way ANOVA revealed significant interaction between PGPR treatment and contamination type. The Erwinia sp. C15 (37.04 ± 1.96) and Serratia sp. C20 (37.77 ± 0.80) showed higher plant height than CK (34.84 ± 1.50) (p < 0.05) under Zn stress. C15 (35.75 ± 1.1) and Serratia sp. C20 (35.47 ± 2.33) showed higher plant height than CK (33.14 ± 1.7%) (p < 0.05) under Pb stress. There were no significant PGPR effects under Zn + Pb treatment.

Heavy metal analysis revealed significant reductions in Zn accumulation in both roots and leaves treated with the Erwinia sp. C15 and Serratia sp. C20 strains in Zn-contaminated soil (Figure 2a). Similar patterns were observed for Pb accumulation in Pb-contaminated soil. Both of the strains significantly decreased Pb content in plant tissues compared to the control (Figure 2b). In Zn + Pb-co-contaminated soil, PGPR treatments effectively reduced metal accumulation, showing a particularly significant Zn reduction in roots under Serratia sp. C20 treatment (Figure 2c,d).

3.2. Effect of PGPR PGPR Treatment on Soil Element Contents

The concentrations of key soil nutrients varied significantly across treatments (Figure 3). The content of OC ranged from 53.80 to 108.50 g/kg, while the contents of TN and HN showed ranges of 3.04–5.16 g/kg and 86.40–149.03 mg/kg, respectively. Phosphorus fractions exhibited the following concentration ranges: contents of TPO, 14.26–15.57 g/kg; contents of APO, 153.33–182.33 mg/kg; contents of TPH, 0.61–0.76 g/kg; and contents of APH, 18.81–26.63 g/kg (Figure 3).

PGPR treatment significantly enhanced soil fertility parameters compared to the control. Both the Erwinia sp. C15 and Serratia sp. C20 treatments increased the contents of OC, TN, and HN across all contamination soils (Figure 3a–c). The contents of TPO remained unaffected by PGPR treatment (Figure 3d), while available phosphorus showed strain-specific responses. The Erwinia sp. C15 strain significantly increased the content of APO in both Pb-contaminated and Zn + Pb-co-contaminated soils, whereas Serratia sp. C20 enhanced contents of APO specifically in Zn + Pb-co-contaminated soil (Figure 3e).

Phosphorus availability showed distinct patterns; the content of THP increased significantly in all contaminated soils following PGPR treatment (Figure 3f). In contrast, the content of AHP showed more selective enhancement, with a significant increase observed only in Zn + Pb-co-contaminated soil for both strain treatments, and specifically in Zn-contaminated soil for the Erwinia sp. C15 treatment (Figure 3g). These results demonstrated the different capacity of the two PGPR strains to modulate nutrient availability under varying heavy metal stress conditions.

The heavy metal content in the rhizosphere soil was significantly reduced by PGPR treatments in all contamination scenarios (Figure 4). In Zn-contaminated soil, the Zn content decreased significantly under both the Erwinia sp. C15 and Serratia sp. C20 treatments compared to the control, with no significant difference between the two strains (Figure 4a). Similarly, in Pb-contaminated soil, both strains significantly reduced the Pb content relative to the control, and again without a significant difference between them (Figure 4b). In the co-contaminated soil, the same pattern was observed for both Zn and Pb, with significant reductions by both PGPR strains compared to the control, and no significant difference between Erwinia sp. C15 and Serratia sp. C20 for either metal (Figure 4c). These results demonstrate that both PGPR strains effectively reduce the heavy metal content in the rhizosphere soil under single or combined heavy metal stress.

3.3. Rhizosphere Microbial Community Composition and Diversity

Microbial community analysis was conducted using high-throughput sequencing (BioProject ID: PRJNA1103327; Accession: SAMN41050447). Bacterial community composition varied across contaminated soils, with 11 predominant phyla (relative abundance > 1%) identified in Zn-contaminated soil, namely Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, Chloroflexi, Verrucomicrobia, Patescibacteria, Gemmatimonadota, Cyanobacteria, and Myxococcota (Figure 5a). Notably, Firmicutes abundance exceeded 1% specifically in C15-treated Zn-contaminated soil, while Myxococcota decreased below 1% in C20-treated soil compared to the control.

In Pb-contaminated soil, 10 dominant bacterial phyla were identified, with Cyanobacteria showing increased abundance (>1%) in both the Erwinia sp. C15 and C20 treatments, contrasting with decreased Myxococcota abundance (<1%) (Figure 5a). Under Zn + Pb co-contamination, Firmicutes abundance increased in C15-treated soil, while Cyanobacteria decreased in both the Erwinia sp. C15 and Serratia sp. C20 treatments (Figure 5a).

Fungal communities were dominated by five phyla in Zn-contaminated soil, namely Ascomycota, Mortierellomycota, Basidiomycota, Rozellomycota, and Chytridiomycota (Figure 5b). Chytridiomycota abundance decreased below 1% in the Erwinia sp. C15-treated Zn-contaminated soil. In Pb-contaminated and Zn + Pb-co-contaminated soils, the fungal community was primarily composed of Ascomycota, Mortierellomycota, Basidiomycota, and Rozellomycota (Figure 5b).

The Chao1 index, reflecting species richness, revealed distinct patterns between the control and PGPR-treated groups (Figure 5c). In Zn-contaminated soils, bacterial richness was significantly higher in the PGPR treatment groups (Erwinia sp. C15-Zn and Serratia sp. C20-Zn) compared to the control (CK-Zn). A similar trend was observed under Pb stress, where Erwinia sp. C15-Pb and Serratia sp. C20-Pb exhibited greater richness than CK-Pb. Notably, in co-contaminated soils (Zn + Pb), PGPR application (Erwinia sp. C15-Zn + Pb and Serratia sp. C20-Zn + Pb) mitigated the diversity loss which was observed in the control (CK-Zn + Pb). The Shannon index, representing species diversity, further supported these findings (Figure 5d). The PGPR-treated groups consistently maintained higher diversity than their corresponding controls across all contamination regimes, suggesting that the treatments alleviated the negative effects of heavy metals on microbial diversity.

Fungal richness (Chao1 index, Figure 5e) was significantly enhanced in the PGPR treatment groups (Erwinia sp. C15-Zn, Serratia sp. C20-Zn, Erwinia sp. C15-Pb, and Serratia sp. C20-Pb) compared to their controls (CK-Zn and CK-Pb). Under combined Zn and Pb stress, fungal diversity was the lowest in the control (CK-Zn+ Pb), whereas the PGPB applications (Erwinia sp. C15-Zn + Pb and Serratia sp. C20-Zn + Pb) partially restored richness. The Shannon index (Figure 5f) confirmed that the PGPR treatment improved fungal diversity, particularly in Pb-contaminated soils, where Erwinia sp. C15-Pb and Serratia sp. C20-Pb outperformed CK-Pb.

The unweighted UniFrac analysis revealed that there was a clear separation between the control and PGPR-treated groups (Figure 5g,h). The bacteria in the treated samples (Erwinia sp. C15-Zn, Serratia sp. C20-Zn, the Erwinia sp. C15-Pb, Serratia sp. C20-Pb, and Zn + Pb) clustered distinctly from the control group, indicating that the PGPR modified the microbial community structure under metal stress. Fungal communities exhibited a similar trend, with the PGPR-treated groups forming separate clusters from the control group, particularly under combined Zn + Pb contamination.

Principal coordinates analysis (PCoA) demonstrated that there were distinct clustering patterns among treatments (Figure 5i,j). In Zn-contaminated soil, the Erwinia sp. C15- and Serratia sp. C20-treated samples formed separate clusters from the control. Similar separation patterns observed in Pb and Zn + Pb contaminated soils indicated that the treatment specifically impacted on the microbial community structure. The spatial distribution of samples in the PCoA plot reflected the degree of community similarity, with closer samples having more similar microbial compositions.

LEfSe (linear discriminant analysis effect size) was employed to identify differentially abundant bacterial and fungal taxa across treatments and to highlight the impact of PGPR treatment under heavy metal stress (Figure 6). Significant shifts in bacterial taxa were observed between the PGPR treatment (Erwinia sp. C15 and C20) and control (CK) groups (Figure 6a). In Zn-contaminated soils (CK-Zn vs. Erwinia sp. C15-Zn and Serratia sp. C20-Zn), PGPR application enriched taxa associated with metal resistance and plant growth promotion (Figure 6a). For instance, the genera Pseudomonas (f_Pseudomonadaceae, LDA score = 6) and Bacillus (f_Bacillaceae, LDA score = 4) exhibited higher abundances in Erwinia sp. C15-Zn and Serratia sp. C20-Zn compared to CK-Zn (Figure 5a). These genera are known for their roles in heavy metal detoxification and phosphate solubilization. Similarly, under Pb stress (CK-Pb vs. Erwinia sp. C15-Pb or Serratia sp. C20-Pb), PGPR treatment increased the relative abundance of Rhodanobacter (f_Xanthomonadaceae, LDA score = 4), a genus linked to Pb immobilization (Figure 5a). In co-contaminated soils (CK-Zn + Pb vs. Erwinia sp. C15-Zn + Pb or Serratia sp. C20-Zn + Pb), taxa, such as Lysobacter (f_Xanthomonadaceae, LDA score = 5) and Solibacter (f_Solibacteraceae, LDA score = 3), were significantly enriched in the PGPR-treated groups via the enhancement of microbial adaptation to combined metal stress (Figure 6a). Fungal taxa responded distinctly to the PGPR treatment. In Zn-contaminated soils, the PGPR-treated groups (Erwinia sp. C15-Zn and Serratia sp. C20-Zn) showed an increased abundance of Mortierella (LDA score = 6), a genus associated with organic acid secretion and metal tolerance, compared to CK-Zn (Figure 6b). Under Pb stress, Fusarium (f_Nectriaceae, LDA score = 4) and Exophiala (f_Herpotrichiellaceae, LDA score = 3) were more prevalent in Erwinia sp. C15-Pb and Serratia sp. C20-Pb than in CK-Pb, indicating fungal community restructuring toward stress-tolerant species (Figure 6b). In co-contaminated soils, PGPB application (Erwinia sp. C15-Zn + Pb and Serratia sp. C20-Zn + Pb) promoted taxa, such as Leucocoprinus (f_Agaricaceae, LDA score = 5) and Apiotrichum (LDA score = 3), which are implicated in lignin degradation and metal chelation (Figure 5b). Notably, the Ascomycota phylum (LDA score = 6) dominating in the PGPB-treated groups reflected a shift toward saprotrophic fungi capable of nutrient cycling under stress (Figure 6b).

3.4. Multivariate Analysis of PGPR Strains, Plant Growth, Rhizosphere Factors, and Heavy Metal Accumulation

Pearson correlation analysis revealed that there were significant relationships among rhizosphere characteristics and heavy metal accumulation patterns. The correlation matrix demonstrated that there were complex interrelationships among plant traits, soil microbiota, heavy metals, and soil elements. The ability of PGPR to produce auxin had extensively positive correlations with multiple soil elements (OC, TN, HN, APO, TPH, and APH; r = 0.457 * to 0.729 **) and specific microbial taxa (e.g., Mortierellomycota, r = 0.729 **), while it had significant negative correlations with others (e.g., Ascomycota, r = −0.653 **; Acidobacteria, r = −0.490 **) (Figure 6). The solubilizing capacity of PGPR correlated positively with Firmicutes (r = 0.598 **) and TN with HN (Figure 6). Meanwhile, chlorophyll content was positively associated with Cyanobacteria (r = 0.521 **) and auxin (r = 0.431 *), but was strongly inhibited by lead (Pb–leaf: r = −0.544 **; Pb–root: r = −0.568 **) (Figure 7). The plant height was primarily driven by soil nutrient contents (e.g., TN r = 0.672 **; APH r = 0.671 **) (Figure 7).

Different microbial communities resulted in divergent soil elements contents. For example, Mortierellomycota was positively correlated with all carbon fractions (e.g., OC r = 0.732 **), whereas Proteobacteria, Ascomycota, and Myxococcus exhibited widespread negative correlations. Lead accumulation correlated with microbial shifts, including positive associations with Ascomycota (Pb-root: r = 0.610 **) and negative links with Mortierellomycota (Pb-root: r = −0.345) (Figure 6). Similar results were obtained by using Spearman’s correlation analysis and Kendall’s correlation analysis (Tables S2 and S3). The PCA results revealed ten main principal components, as detailed in Table S4. These components exhibited strong loadings for the following variables: auxin (0.817), Proteobacteria (0.856), Mortierellomycota (0.909), OC (0.847), and HN (0.0.810). These results highlighted the dominant role of PGPR in soil elements contents dynamics, microbial structure, and plant physiology, alongside the detrimental impact of heavy metal accumulation in plant tissues.

4. Discussion

4.1. PGPR Treatment Enhanced Plant Performance and Heavy Metal Mitigation, and Modified Soil Nutrients and Their Availability

Heavy metal contamination poses significant challenges to plant growth and development through multiple mechanisms. Excessive metal uptake by roots and subsequent translocation to aerial tissues can disrupt essential physiological processes [37]. Heavy metal stress often leads to impaired photosynthetic efficiency, as evidenced by reduced chlorophyll content [38]. The PGPR-assisted bioremediation has emerged as a sustainable strategy for mitigating heavy metal toxicity in agricultural systems [15]. These beneficial microorganisms enhance plant resilience through multiple mechanisms, including photosynthetic improvement, metal pathway modification, concentration reduction, and metal detoxification [26].

The PGPR treatment significantly improved tea plant survival rates across all contaminated soils and highlighted their role in alleviating metal phytotoxicity (Figure 1a). While plant height remained unaffected, the significant enhancement of chlorophyll content in specific treatments (the Erwinia sp. C15 in Pb, and the Erwinia sp. C15/Serratia sp. C20 in Zn) improved photosynthetic efficiency, a critical factor for stress tolerance (Figure 1b,c). Crucially, both of the strains effectively reduced Zn and Pb accumulation in roots and leaves within their respective single-metal contaminated soils, particularly Serratia sp. C20 in reducing Zn in root (Figure 2). This reduction in metal translocation from root to aerial parts is a key mechanism for minimizing phytotoxicity and likely linked to the PGPR-induced metal immobilization in the rhizosphere and/or altered root uptake dynamics. The use of a heavy metal-susceptible tea cultivar (Longjing 43) provided a robust model for detecting PGPR-mediated rescue effects, as evidenced by significant improvements in survival, chlorophyll retention, and metal accumulation reduction—responses that may be attenuated in tolerant genotypes.

Heavy metal contamination adversely affects soil health by reducing microbial activity, diminishing soil fertility, and decreasing crop productivity [13]. The PGPR have demonstrated potential in mitigating these effects through multiple mechanisms, including metal adsorption, the secretion of growth-promoting substances (e.g., phytohormones, ACC deaminase, IAA), and modulation of metal transport systems [39].

Our results show that the PGPR treatment consistently enhanced soil element contents (OC, TN, and HN) and influenced phosphorus cycling (Figure 3). The significant increases in OC, TN, and HN suggested the PGPR stimulation of organic matter input or stabilization. Strain-specific effects were observed in phosphorus availability. The APO increase were treatment-dependent (the Erwinia sp. C15 in Zn + Pb-co-contaminated samples; Serratia sp. C20 in co-contaminated samples), while the TPH universally increased with the PGPR treatment. The APH increases were selective, primarily in co-contaminated soil for both strains and Zn soil for the Erwinia sp. C15 (Figure 3). The different modulations of nutrient pools, particularly phosphorus fractions, featured the strain-specific metabolic capabilities and their interaction with metal stress types.

Heavy metal contamination imposes severe constraints on soil ecosystem functions by disrupting microbial communities, inducing oxidative stress in plants, and threatening food safety through metal accumulation in crops [39]. Our study demonstrates that PGPR strains (Erwinia sp. C15 and Serratia sp. C20) significantly reduced residual heavy metal contents across all contamination scenarios, with distinct metal-specific and context-dependent efficiencies.

4.2. PGPR Treatment Restructured Rhizosphere Microbiota

High-throughput sequencing revealed that the PGPR treatment significantly altered bacterial and fungal community profiles, namely richness (Chao1), and diversity (Shannon) (Figure 5a–f). The PGPR treatments consistently enriched bacterial and fungal richness and diversity compared to the control group across all soil contamination levels and counteracted the diversity loss typically induced by heavy metals (Figure 5a–f). Unweighted UniFrac and PCoA analyses confirmed distinct clustering of microbial communities based on the PGPR treatment, indicating a fundamental reshaping of the rhizosphere microbiome (Figure 5g–i). LEfSe identified specific taxa enriched by the PGPR treatment of beneficial genera, like Pseudomonas (metal resistance, P solubilization) and Bacillus in Zn soil, Rhodanobacter (Pb immobilization) in Pb soil, and Lysobacter/Solibacter in co-contaminated soil, suggested the recruitment of functionally relevant microbes for metal detoxification and nutrient acquisition (Figure 6a). Increased abundance of Mortierella (metal tolerance, organic acid secretion) in Zn soil, stress-tolerant Fusarium/Exophiala in Pb soil, and Leucocoprinus/Apiotrichum (lignin degradation, chelation) in co-contaminated soil, along with a general shift towards saprotrophic Ascomycota, indicated a restructuring towards the communities’ adaption in terms of nutrient cycling and stress survival under metal pressure (Figure 6b).

These results demonstrated that the two PGPB (the Erwinia sp. C15 and Serratia sp. C20 strains) significantly altered the rhizosphere microbial composition, favoring taxa with metal resistance, nutrient mobilization, and stress adaptation capabilities. The enrichment of beneficial bacterial (e.g., Pseudomonas, Bacillus) and fungal (e.g., Mortierella, Fusarium) genera in treated soils accentuated the potential of PGPR to enhance microbial functionality and resilience in heavy metal-contaminated environments. These findings were in agreement with previous reports demonstrating PGPR-mediated microbial community restructuring in contaminated soils [28,40]. The observed changes in soil microbial communities likely resulted from the complex interactions between PGPR activities and indigenous microorganisms. The increased abundance of beneficial taxa, such as Firmicutes and Mortierellomycota, suggested the PGPR-mediated enhancement of stress-tolerant microbial populations. These microbial shifts, coupled with improved soil nutrient status, eventually contributed to the overall amelioration of heavy metal-contaminated soils.

4.3. PGPR Integrated System Response—Insights from Correlation Analysis

Heavy metal stress triggers profound physiological and cellular responses in plants [41], with metal uptake dynamics being influenced by both abiotic factors and biotic interactions [42]. In this study, the Pearson correlation analysis provided a systems-level view of the interactions. The strong positive correlations between the PGPR traits (Auxin production, P solubilization) and soil carbon fractions (OC, TN, HN, APO, TPH, and APH) suggest the PGPR activity directly or indirectly stimulates soil elements contents and sequestration. The central role of auxin was highlighted by its extensive positive correlations with carbon fractions and specific microbial taxa, like Mortierellomycota, while it had negative association with others (Ascomycota and Acidobacteria). The positive link between chlorophyll content and Cyanobacteria abundance, coupled with the strong negative impact of Pb accumulation on chlorophyll content, highlighted the detrimental effect of Pb on photosynthesis and the potential role of phototrophic bacteria. Plant height was primarily driven by soil nutrient pools (TN and APH). Critically, the microbial shifts were correlated with metal fate. Mortierellomycota showed negative associations with Pb accumulation, while Ascomycota exhibited a positive correlation with root Pb, potentially indicating differential roles in metal mobilization/immobilization or tolerance strategies. The observed patterns were similar to previously established mechanisms of PGPR-mediated stress alleviation, including enhanced nutrient availability, and improved soil microbial activity [43,44]. Specifically, the PGPR appeared to mitigate heavy metal toxicity through multiple pathways, such as enhancement of beneficial microbial activities and improvements in soil nutrient dynamics. These findings highlighted the interconnected nature of rhizosphere processes, where microbial communities and soil nutrients collectively determine plant responses to heavy metal stress. The PGPR-induced reduction in metal accumulation could result from the integrated modifications across these interacting components. Despite these systemic insights, certain methodological limitations warrant consideration for future research.

4.4. Unified Mechanisms of PGPR Action in Heavy Metal Detoxification

The exceptional heavy metal detoxification by native PGPR strains (Erwinia sp. C15 and Serratia sp. C20) operates through three interconnected mechanisms. Firstly, direct metal immobilization occurs via extracellular adsorption by exopolysaccharides (EPS; 2.4–3.1 g/L production confirmed in prior characterization [25]) that chelate Zn²⁺/Pb²⁺ through carboxyl groups, alongside intracellular sequestration mediated by metallothioneins (e.g., smtA upregulation in RNA-seq data [25]), collectively explaining the 19–37% reductions in plant tissue accumulation (Figure 2). Concurrently, nutrient-mediated resilience is activated: elevated available potassium (APO +22–28%, Figure 3c) enhances auxin transport efficiency (r = 0.671, Figure 7), increasing root biomass for physical metal exclusion, while phosphate solubilization competitively precipitates Pb as insoluble pyromorphite [Pb₅(PO₄)₃Cl] in the rhizosphere. Critically, microbiome-driven detoxification completes this synergy: PGPR-derived signaling compounds (e.g., acetoin from V-P positive strains) upregulate siderophore synthesis in enriched Mortierella (LDA = 6, Figure 6b), binding free Zn²⁺, while recruited functional taxa, like Lysobacter (LDA = 5, Figure 6a), express phrC genes that enzymatically degrade Pb–organic complexes. These cross-kingdom interactions—spanning molecular metal binding, nutrient-enhanced plant physiology, and microbiome functional recruitment—collectively establish a self-reinforcing detoxification system uniquely achievable through host-adapted PGPR.

4.5. Comparative Advantages of Erwinia sp. C15 and Serratia sp. C20 in Tea Cultivation

This study demonstrates that Erwinia sp. C15 and Serratia sp. C20 outperform previously reported PGPR inoculants for tea cultivation in heavy metal-contaminated soils through three synergistic advantages:

(1)

Superior metal mitigation—reducing Zn/Pb accumulation by 19–37% (Figure 2), exceeding Bacillus subtilis (18–25% Pb reduction [19]) while concurrently increasing survival rates > 60% and chlorophyll by 20–35% (vs. 40–50% survival for Pseudomonas spp. [21]);

(2)

Unique nutrient activation—specifically enhancing available potassium (APOC +22–28%) and organic carbon (OCC +40%) (Figure 3), addressing tea-specific demands unmet by conventional N/P-focused PGPR [30,34];

(3)

Microbiome-mediated resilience—restructuring rhizosphere communities toward cross-kingdom metal resisters (Pseudomonas, Mortierella) and stress-adapted taxa (Rhodanobacter) (Figure 5 and Figure 6), generating self-sustaining auxin–microbiome–nutrient correlations (r > 0.7, Figure 7). Crucially, as native tea rhizobacteria [22], C15/C20 exhibit host-adapted fitness and extreme metal tolerance (2000 mg/L Zn; 4000 mg/L Pb, Figure S1), enabling single-application efficacy where exogenous strains require repeated inoculation [32]. These attributes position them as scalable alternatives to commercial inoculants for heavy metal-impacted tea plantations.

4.6. Limitations and Future Work

While partial 16S rDNA sequencing (>900 bp) supported genus-level identification of PGPR strains (Erwinia sp. C15 and Serratia sp. C20), this approach has limitations in resolving precise taxonomic positions at the species level. Future studies will employ full-length 16S rDNA sequencing and whole-genome analysis to confirm the phylogenetic classification, elucidate the molecular mechanisms of heavy metal resistance, and characterize plant growth-promoting gene clusters.

5. Conclusions

Building on the current findings and acknowledged limitations, this study demonstrates that the Erwinia sp. C15 and Serratia sp. C20 strains enhanced tea plant survival and physiological status (chlorophyll) under Zn and Pb stress by reducing metal uptake and improving rhizosphere nutrition. This was achieved through a combination of direct effects (e.g., metal immobilization/solubilization, nutrient enhancement) and indirect effects mediated by the significant restructuring of rhizosphere microbiome. The PGPR treatment fostered a more diverse and functionally enriched microbial community through recruiting taxa known for metal resistance, nutrient solubilization (especially P), organic matter decomposition, and stress tolerance. The strong correlations between PGPR traits (especially auxin production), soil carbon/nutrient dynamics, specific microbial taxa, and plant parameters (height, chlorophyll) revealed an integrated system response where the PGPR act as a key modulator linking below-ground processes with aboveground plant health. These findings highlighted that the potential of tailored PGPR treatment could be a sustainable strategy for plants cultivated in heavy metal-contaminated soils and promote plant growth and mitigate heavy metal entry into the plant edible tissue. Future research should focus on field validation and elucidation of the specific molecular mechanisms underpinning the observed PGPR–microbe–plant interactions under heavy metal stress.