Synergistic roles of ericoid mycorrhizal fungi and mycorrhiza helper bacteria enhance plant stress tolerance and Cd immobilisation

Synergistic roles of ericoid mycorrhizal fungi and mycorrhiza helper bacteria enhance plant stress tolerance and Cd immobilisation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 26 July 2025 V1 Latest version Share on Synergistic roles of ericoid mycorrhizal fungi and mycorrhiza helper bacteria enhance plant stress tolerance and Cd immobilisation Authors : Jing Jiang , Huizhi Zhang , Haifeng Zhu , Yixiao Wang , Qiling Yang , Hongyi Yang [email protected] , and Lili Li Authors Info & Affiliations https://doi.org/10.22541/au.175351878.82508543/v1 306 views 196 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cadmium (Cd) contamination threatens ecosystem safety. Microbial remediation offers an eco-friendly approach to reduce plant Cd accumulation, but synergistic effects between microorganisms, particularly mycorrhizal fungi and mycorrhiza helper bacteria (MHB), are often overlooked. This study revealed the synergistic Cd resistance mechanism in blueberry mediated by ericoid mycorrhizal fungus (ErMF) Oidiodendron maius 143 and MHB Enterobacter sp. R9G through integrated pot and in vitro experiments. ErMF and R9G co-inoculation significantly improved blueberry biomass, root morphology and antioxidant enzyme activities (CAT, POD and SOD) and reduced MDA. Co-inoculation concurrently restricted Cd uptake and translocation and decreased soil Cd bioavailability. Multi-spectral analyses (SEM-EDS, FT-IR, XRD and XPS) demonstrated that co-inoculation synergistically enhanced Cd immobilisation through extracellular polymer secretion, phosphate precipitation (Cd (H 2 PO 4 ) 2 ·2H 2 O) and organic–mineral composite layer formation, improving soil stability. High-throughput sequencing and enzyme activity confirmed that ErMF and R9G restructured soil microbial communities and activated Cd-passivating microorganisms. In vitro reciprocal interactions occurred: ErMF hyphal exudates (e.g., sucrose and proline) enhanced R9G growth, motility, chemotaxis and biofilm formation, while R9G promoted ErMF mycelial growth and sporulation. The ErMF-MHB synergy established a ‘plant physiological regulation–microbial metabolic immobilisation–microbial functional remodelling’ strategy for Cd containment in soil-blueberry systems. 1. Introduction Soil cadmium (Cd) pollution threatens global agricultural sustainability due to its high mobility and biotoxicity, inhibiting plant growth and endangering human health via the food chain (Chen et al., 2020; Zhang and Reynolds, 2019). Traditional physicochemical remediation is costly and disruptive to soil ecosystems (Cao et al., 2025), whereas plant–microbe remediation offers an environmentally sustainable alternative. This approach exploits synergistic root-microbe interactions to immobilize Cd in the rhizosphere, enhance phytoaccumulation or boost plant resistance (Glick, 2003; Wood et al., 2016). Blueberry is a perennial shrub belonging to the Ericaceae family that naturally adapts to acidic soil, allowing it to grow normally in Cd-contaminated areas and providing a foundation for its remediation capacity (Cairney and Meharg, 2010; Read, 1996). Blueberry also exhibits tolerance to Cd stress. Song et al. (2023) reported that blueberry biomass was not only unsuppressed but also dramatically augmented in Cd-stressed soil and effectively immobilised Cd by enhancing its accumulation in the root system. Blueberry elevated chlorophyll content and enhanced activities of resistance enzymes through metabolic regulation under Cd stress (Daghino et al., 2016). Crucially, soil Cd content exhibited no correlation with Cd content in blueberry fruits; that is, even when soil heavy metal concentrations exceed the established maximum threshold limits, corresponding concentrations within the fruit remain below the critical values (Chen et al., 2019; Shorthouse and Bagatto, 1995). The success of Ericaceae plants in heavy metal habitats is attributed to the unique capacity of their ericoid mycorrhizal fungi (ErMF) partners to withstand heavy metal stress and enhance the metal tolerance of host plants (Bradley et al., 1981; Hashem, 1995). Martino et al. (2000) revealed that ErMF Oidiodendron maius isolated from contaminated soils exhibit remarkable tolerance to elevated concentrations of zinc (Zn) and Cd. Specifically, O. maius facilitates the release of Zn from insoluble ZnO and Zn 3 (PO 4 ) 2 by the excretion of extracellular compounds (citric and malic acids) (Martino et al., 2003). Furthermore, O. maius significantly reduces Cd accumulation in host roots, potentially by modulating the expression of metal transport proteins to enhance Cd compartmentalisation or efflux (Casarrubia et al., 2020). Despite these insights, our understanding of the plant protection mechanisms mediated by ErMF remains limited. In mycorrhizal symbiosis, a functional group of bacteria defined as mycorrhiza helper bacteria (MHB) can significantly enhance mycorrhizal fungal colonisation efficiency and symbiotic efficacy by secreting growth-promoting factors that stimulate spore germination, enhance hyphal growth and facilitate fungal cell wall-degrading enzyme production (Duponnois and Garbaye, 1991; Etesami and Glick, 2023). MHB Paenarthrobacter nicotinovorans and Bacillus circulans enhanced the enzymatic activity and gene expression of cell wall-degrading enzymes in EMF, highlighting their potential to promote mycorrhizal colonisation (Yang et al., 2023). Such fungal-bacterial synergism can amplify the ecological functions of fungi and stress resilience (Sangwan and Prasanna, 2021). This field has achieved significant progress in arbuscular mycorrhizal fungi (AMF), but ErMF interactions with MHB remain poorly characterised. The extraradical hyphal network of AMF acts as a ‘biological interface’, which recruits specific functional bacteria by releasing chemical signals, such as organic acids and fructose; this recruitment induces biofilm formation to mitigate soil N₂O emissions and organic phosphorus solubilisation (Li et al., 2023; Zhang et al., 2018b). Under Cd stress, AMF selectively enriched core Cd-tolerant bacteria (e.g., Bacteroidetes and Burkholderiaceae), which effectively reduced Cd bioavailability through extracellular polymer adsorption, metal ion chelation and phosphate-mediated biomineralisation (Wang et al., 2023a; Xing et al., 2020). Although co-inoculation with AM/ECM fungi and MHB has been demonstrated to enhance plant tolerance to Cd (Vivas et al., 2003; Kozdroj et al., 2007). There is a critical gap in research regarding whether analogous protective benefits can be conferred by ErMF–MHB synergy in Ericaceous plants. Based on this, we established a symbiotic model system between blueberry, ErMF and MHB. Blueberry Cd accumulation/translocation patterns, physiological parameters, root system architecture development, rhizosphere soil Cd chemical speciation and microbial community changes were comprehensively measured. The reciprocal relationship between ErMF and MHB in vitro was also characterised. This study is the first to systematically evaluate the regulatory mechanism of ErMF–MHB synergism on the response to Cd stress in blueberry. This work not only addresses a critical knowledge gap concerning the functional mechanisms of ErMF–MHB under Cd stress but also provides a scientific foundation for developing phytoremediation technologies based on plant–microbe symbiotic systems. 2. Materials and methods 2.1 Strain materials The ErMF strain is O. maius 143, isolated from the roots of Vaccinium uliginosum in the Greater Khingan Mountains (Yang et al., 2018). Enterobacter sp. R9G was isolated from Cd-treated blueberry rhizosphere soil, and both were preserved at Northeast Forestry University. 2.2 Pot experimental The two-factor experiment was conducted for microbial inoculation and soil Cd stress on plants. Microbial inoculation treatments included non-inoculation (CK), inoculation with O. maius 143 (ErMF) alone, inoculation with Enterobacter sp. R9G (R9G) alone and co-inoculation applying both O. maius 143 and Enterobacter sp. R9G (ErMF+R9G). Cd levels (Cd0, Cd2 and Cd20 mg kg -1 ) were established by applying CdSO 4 solution. For inoculation, O. maius 143 was cultivated on PDA for 2 weeks, and fungus blocks (5 mm) were then punched into the soil, one centimeter deep, from the roots of sterile blueberry seedlings. The 10 mL of R9G suspension (OD 600 nm =1.0) added to plant roots, while the control group was treated with an equivalent volume of sterilized R9G suspension. The sterile seedlings of the V. uliginosum cultivar ”Legacy” were chosen, and the microcutting and rooting of sterile seedlings were treated as per our previous approach (Jiang et al., 2024). To acclimate to the soil environment, the rooted seedlings were transferred to sterilized soil (charcoal soil: vermiculite = 2:1) for 4 weeks. Eventually, blueberry seedlings with similar growth status and plant height were selected and planted in pots containing 200 g of soil substrate (charcoal soil: vermiculite = 2:1). The soil substrate contained 6.15 g·kg -1 total C, 1.22 g·kg -1 total N, 5.73 g·kg -1 total P and the soil pH was 5.5. The experiment was conducted in a greenhouse at Northeast Forestry University (45°43’ 45.71’ ’ northern latitude, 126°38’11.04’ ’ east longitude, Heilongjiang Province, China) and grown at 25-30°C under a 12 h light pattern. O. maius 143 was first inoculated on blueberry roots for 6 weeks until root colonisation exceeded 15%. Subsequently, CdSO 4 solutions were applied to achieve soil Cd concentrations of 0, 2 and 20 mg kg -1 . The 10 mL R9G suspension was added to the soil. After 8 weeks, the blueberry plant and rhizosphere soil were analysed. 2.3 Plant growth parameters and fungal colonisation After 8 weeks of Cd stress, the plant was dried at 105°C for 30 min and 70°C for 48 h to determine the biomass and root biomass/shoot biomass (R/S). Root architecture parameters (length, surface area, volume, etc.) were quantified using the Epson Expression 10000XL scanner and WinRHIZO software. Root mass ratio (RMR, root dry weight/whole plant dry weight, g·g -1 ), root fineness (RF, root length/root volume, cm·cm -3 ), root tissue density (RTD, root dry weight/root volume, g·cm -3 ), specific root length (SRL, root biomass/total root length, g·cm -1 ) and specific root area (SRA, root biomass/root surface area, g·cm 2 -1 ) were calculated according to Ryser and Lambers et al (1995). The root samples were processed to evaluate the level of mycorrhizal colonisation: fixed in FAA (formalin-acetoacetanol), cleared with KOH and stained with trypan blue solution. Mycorrhizal colonisation rate was evaluated in a binocular stereomicroscope (Olympus SZ-PT) (Phillips, 1970). 2.4 Plant stress physiology biochemical analyses Leaf antioxidant enzymes were extracted from 0.5 g of fresh tissue in pre-cold PBS (0.05 M, pH 7.8) and centrifuged (8000 g, 20 min). Based on the method of Han et al. (2021), superoxide dismutase (SOD) activity was quantified by NBT photochemical reduction inhibition at 560 nm, catalase (CAT) activity was determined by H 2 O 2 decomposition kinetics at 240 nm, peroxidase (POD) activity assessed guaiacol oxidation at 470 nm, malondialdehyde (MDA) content was measured using thiobarbituric acid reaction after TCA extraction at 532 nm. Proline was extracted with sulfosalicylic acid, reacted with acid ninhydrin and quantified at 520 nm by toluene phase separation (Bates et al., 1973). Soluble sugar content was measured using the anthrone-sulfuric acid method at 520 nm (Liu et al., 1973). 2.5 Plant element absorption, Cd bioconcentration factor and translocation factor The manganese (Mn), zinc (Zn), magnesium (Mg), calcium (Ca), copper content (Cu), potassium (K) and iron (Fe) content of plant leaves were determined by atomic absorption spectrophotometer (NovAA400P, Germany) according to You et al.’s method (2025). For Cd content, the 0.2 g (dry weight) of soils, plant leaves and roots were added into the mixed digestion solution of HNO 3 and HClO 4 (7:3, v/v) for full digestion in a graphite digester at 220°C. At the end of the digestion, the 10 mL of HNO 3 was added and filtered through a 0.22 µm filter membrane. Cd content was determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent, 7900). The Cd bioconcentration factor (BCF) and transport factor (TF) were calculated as follows: BCF = Cd concentration in leaves/Cd concentration in soils; TF = Cd concentration in leaves/Cd concentration in roots (Wang et al., 2021a). 2.6 Extraction of chemical forms of soil Cd The modified Tessier sequential extraction method was used to identify the chemical form of soil Cd in accordance with Zhu et al.’s method (2018), and the concentration of Cd in the five extracted chemical fractions was detected by ICP-MS. 2.7 Soil physicochemical properties and enzyme activity analysis Soil pH was measured with a soil-to-water ratio of 5:2 by using a pH meter. Soil organic carbon (SOC) was determined according to the dichromate oxidation method (Fang et al., 2017). For the measurement of soil total P (TP) and available P (AP), soil was extracted by hydrofluoric acid-perchloric acid (HF-HClO 4 ) and sodium bicarbonate (NaHCO 3 ), and then measured by the molybdenum blue method at 700 nm (Song et al., 2007). Total nitrogen (TN) was tested using an elemental analyser (Vario EL Cube, Elementar Corp., Germany). The assay of the available N (AN) was based on the chlorate inhibition method (Inselsbacher and Nsholm, 2012). Based on the research method of Prisch et al. (2011), the activities of β-glucosidase (BG), β-cellobiosidase (BX), β-xylosidase (CBH), L-leucine aminopeptidase (LAP), β-N-acetylglucosaminidase (NAG) and acid phosphomonoesterase were measured by fluorescence values under an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Urease and invertase activities were quantified by indophenol blue colorimetric and 3, 5-dinitrosalicylic acid colorimetry methods as Wang et al. (2019) and Xu et al. (2020) described. 2.8 Microscale soil surface morphology and element analysis Rhizosphere soils post-treatment were lyophilized and homogenized for Cd-stress remediation analysis. Scanning electron microscopy (SEM, ApreoS) and energy dispersive spectrometry (EDS, Aztec X-Max80) characterized surface morphology and composition (Huse et al., 2008). Fourier transform infrared spectroscopy (FTIR, INVENIO R) detected functional groups (500-4000 cm⁻¹) (Tan et al., 2019). X-ray diffraction (XRD, D8-Advance, Bruker) and X-ray photoelectron spectroscopy (XPS, ESCALAB250Xi, Thermo Fisher-VG Scientific) analyzed crystal structures and surface chemistry respectively (He et al., 2022). 2.9 DNA extraction, HiSeq sequencing and bioinformatics analysis The rhizosphere soil genomic DNA was isolated with the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Amplification of bacterial 16S rRNA (V3-V4 region) employed primers 343F/798R, while fungal ITS regions utilised ITS1F/ITS2R (Gardes and Bruns, 2010; Huse et al., 2008). PCR amplification system: 4 μL 5×FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL 5 μM each primer, 10 ng genomic DNA, and finally add ddH 2 O to make up to 20 μL. The whole PCR conditions were as follows: 95°C for 3 min, 30 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and finally extension at 72°C for 10 min. The amplified fragments were pooled in equimolar and paired-end sequenced by using the Illumina HiSeq 2500 platform (Biomker Technologies Corporation, Beijing, China). 2.10 Enterobacter sp. R9G metabolites on ErMF growth analysis R9G was cultured in LB medium (30°C, 180 rpm, 24 h). The fermented product was centrifuged (10000 g, 15 min, 4°C), filtered through a 0.22 μm filter membrane. ErMF grown on PDA plates supplemented with 2% R9G fermentation product (25°C, 10 d), while controls received equivalent LB medium. Colony diameters and spore counts were recorded. For biomass, three 5-mm mycelial plugs were inoculated in liquid PDA (28°C, 150 rpm, 10 d), then dried (85°C, 24 h) and weighed. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf 2.11 Hyphal exudates for Enterobacter sp. R9G growth and biofilm formation For growth assay, experiments in 96-well microplates added 180 μL of hyphal exudates and 20 μL of R9G suspension (OD 600 nm =1.0), 180 μL of M medium (without sucrose, EDTA and vitamins) and 20 μL of R9G suspension (OD 600 nm =1.0) were added as a control. The absorbance value of OD 600 nm was monitored every 4 h at 30°C. Hyphal exudate effects on R9G biofilm formation were quantified via crystal violet assay (Zhang et al., 2020). R9G suspension (OD 600 nm =1.0) and exudates were mixed in 96-well plates (1:9, v/v), with modified M medium (without sucrose, vitamin and EDTA) as control. After 48 h incubation at 30℃, cells were washed three times with phosphate buffer (PBS), stained with 1% crystal violet (20 min), rinsed and solubilised in 33% acetic acid for OD 595 nm measurement. For extracellular polysaccharide and protein contents assay, R9G suspension (OD 600 nm =1.0) was mixed with hyphal exudates (1:9, v/v) and incubated (30°C, 200 rpm) for 36 h, settled for 48 h. After measuring OD 595 nm , samples were centrifuged (4°C, 6000 g) for 20 min, and washed three times with PBS. The pellet was resuspended in 1 mL of 0.01 M KCl following sonication (5 s each time, 5 s gap, 5 cycles), centrifugation (4°C, 4000 g, 20 min), and 0.22 μm filtration. Extracellular polysaccharides and protein were quantified using the phenol-sulfuric acid method and folin-ciocalteu method, respectively (Wang et al., 2022b). 2.12 Swarming and swimming motility assay of Enterobacter sp. R9G According to Liu (2021) and Mei et al.’s (2024) method, the swimming (1% tryptone, 0.5% NaCl, 0.25% glucose and 0.3% agar) and swarming medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.5% glucose and 0.5% agar) supplemented with 0.2 mg·mL -1 hyphal exudates were inoculated with 2 μL R9G suspension (OD 600 nm =0.4). After incubation at 30°C for 8 h (swimming) and 20 h (swarming). Deionised water served as the control. The diameters of swimming rings and cluster rings were measured by the crossing method. 2.13 Chemotaxis assay of Enterobacter sp. R9G Based on our previous identification in O. maius 143 hyphal exudates (Yang et al., 2023), the relatively high expression content of sugars (sucrose, fructose and arabinose), organic acids (succinic acid, salicylic acid and α-ketoglutaric acid), and amino acids (alanine, proline and cysteine) were selected as chemotaxis candidates. The chemotaxis was measured in a soft-agar plate and capillary system (Caetano-Anollés et al., 1988). For agar assays, the 2 μL of R9G suspension (OD 600 nm =1.0) was placed on agar medium containing 0.3 mM of the candidate substances and incubated at 30°C for 3-7 d, and the turbidity around the bacterial solution was observed. For capillary assays, capillaries filled with 50 μM candidate compound were immersed in a 500 μL R9G suspension (OD 600 nm =1.0). The colony-forming units (CFUs) were counted after static incubation at 28°C for 30 min. The controls lacked candidate substances. The relative chemotaxis index (RCI) was calculated as the ratio of bacteria under candidate treatment to control, with RCI≥2 considered significant (Felipe et al., 2010). 2.14 Key metabolites for Enterobacter sp. R9G growth and biofilm formation R9G suspensions (OD 600 nm =0.1) were inoculated (1%, v/v) into M9 medium supplemented with 0.8% of different key metabolites of hyphal exudates. Controls included: C1 (M9 (-C) + H 2 O) and C2 (M9 + 0.8% glucose + H₂O). The mixture was dispensed into 96-well plates and incubated at 30°C for 48 h. The OD 600 nm was monitored every 4 h to evaluate R9G growth in response to the metabolites. To assess the key metabolite of hyphal exudates effects on R9G biofilm production. The R9G suspension (OD 600 nm =0.1) was inoculated at 1% into TSB liquid medium containing key metabolites (0, 0.05 mM, 0.1 mM and 1 mM). The mixture 2 mL was dispensed into 24-well plates and incubated at 30°C for 48 h, with biofilms processed per section 2.4. 2.15 Statistical analysis The data were analyzed using SPSS 25.0 and Origin 8.0. Significant differences were analyzed using T-tests and Duncan’s test. Two-way ANOVA was performed to evaluate the effects of Cd, inoculation, and their interaction. Non-metric multidimensional scaling (NMDS) was used to visualize microbial communities, and Mantel tests analyzed correlations between plant physiology, soil factors and microbial communities under Cd stress. 3. Results 3.1 Effects of co-inoculation on blueberry biomass, root economic strategy, element uptake and Cd accumulation The Cd stress reduced plant shoot biomass, root biomass and root biomass/shoot biomass (R/S), whereas microbial inoculation restored these parameters (Fig. 1A). The shoot biomass, root biomass and R/S of all inoculated plants were 1.50-, 1.90- and 0.77-fold higher than those of the non-inoculated plants at different Cd levels. The shoot and root dry weights of ErMF+R9G co-inoculated plants were higher than those of the other inoculated plants. Concurrently, Cd stress suppressed ErMF colonisation, but R9G alleviated the inhibition, enhancing the colonisation rates by 33.70%–15.31% across Cd0–Cd20 levels. The root economic spectrum reflected a trade-off between nutrient acquisition (high SRL and SRA, thin elongated roots, low branching) and conservative strategies (low SRL and SRA, thick roots, high branching) (Mccormack et al., 2012; Zhou et al., 2022). Under Cd stress, non-inoculated plants transitioned from an acquisitive to a conservative strategy to enhance survival probability and resource use efficiency, and ErMF+R9G co-inoculation partially restored the acquisitive strategy, as demonstrated by the PCA alignment of root developmental indices (Fig. 1B). This effect may be attributed to plant–microbe symbiosis, which enhances the overall resource utilisation efficiency (e.g., the fungal hyphal network potentially supplanting fine root functions), thereby reallocating plant resources toward root expansion rather than defensive investments (Wang et al., 2024). The presence of ErMF and R9G improved the developmental status of the plant root system under Cd stress by decreasing the root length and surface area and increasing the root volume, root diameter and number of root tips and forks, which shifted the root system toward a conservative strategy (Table S1). Notably, ErMF+R9G co-inoculation outperformed single inoculants in root tips and branches promotion. These structural modifications likely expanded ecological niches for ErMF colonisation (Kong et al., 2019), consistent with R9G promoted ErMF colonisation rate. Inoculation and Cd stress and their interaction had significant effects on plant Cd distribution ( P < 0.001, Fig. 1C). Compared with those in non-inoculated plants, the ErMF, R9G and ErMF+R9G co-inoculation significantly reduced the Cd concentration in the leaves by 61.1%–74.0% (Cd2) and 50.72%–80.33% (Cd20), while increasing root Cd accumulation by 42.38%–91.14% (Cd2) and 137.93%–234.76% (Cd20) ( P < 0.05). The results indicated that inoculation reduced the transport of Cd from the roots to the aboveground of the plants, among which ErMF+R9G co-inoculation had the best effect. Inoculation also significantly decreased BCF and TF ( P < 0.05, Fig. 1D), particularly in ErMF+R9G co-inoculated plants (54.99%–71.01% reduction) compared with those in non-inoculated plants. Concurrently, Cd stress significantly reduced the concentrations of Fe, Mn, Zn, K and Mg in plant leaves but did not affect Ca ( P < 0.05, Fig. 1E). Compared with the lack of inoculation, ErMF inoculation and ErMF+R9G co-inoculation enhanced the concentrations of Mn, Mg, Ca, Fe and Zn in plants with or without Cd stress. Fig. 1. (A) Plant physiological parameters. (B) Principal components analysis of plant resource allocation strategies, (C) Cd accumulation in plant roots and shoots, (D) Cd bioconcentration factor (BCF) and translocation factor (TF), (E) Mg, Mn, Zn, Fe, Ca and K content in plant, (F) MDA, CAT, POD, SOD, proline and soluble sugar level in plant. Different lowercases letters represent significant differences among different inoculation treatment at the same Cd level ( P < 0.05), and different uppercases represent significant differences among different Cd level by Duncan test ( P < 0.05). P -values of two-way ANOVAs of Cd, inoculation and their interaction (Cd×inoculation) are indicated *** P < 0.001, ** P < 0.01, * P < 0.05, ns indicates differences no significant. 3.2 Effects of co-inoculation on antioxidant enzymes and osmoprotectant synthesis of blueberry under Cd stress As shown in Fig. 1F, Cd stress dose-dependently increased the levels of MDA, proline and soluble sugars, while inoculation treatments collectively reduced MDA content and increased proline and soluble sugar contents. ErMF+R9G co-inoculation significantly decreased MDA content by 106.58% and increased proline and soluble sugar contents by 51.11% and 24.31% under Cd20 ( P < 0.05), respectively, compared with other treatments. Antioxidant enzymes (SOD, POD and CAT) also exhibited a dose-dependent effect on Cd concentration. ErMF+R9G co-inoculation showed synergistic enhancement, which increased the levels of SOD, POD and CAT by 21.80, 100.33 and 171.43%, respectively, compared with CK under Cd20. These responses demonstrated that microbial inoculants, particularly ErMF+R9G, synergistically alleviated Cd-induced oxidative damage and regulated the synthesis of compatible solutes through enzyme resistance mechanisms. 3.3 Effects of co-inoculation on extraction of chemical forms, physicochemical properties and enzyme activities of soil Cd The species distribution of Cd in blueberry rhizosphere soil was as follows: CdEx (54%–69%), CdCar (19%–27%), CdFeOx+MnOy (6%–10%), CdOM (1%–11%) and CdRes (0–9%) (Fig. 2A). Inoculation treatments differentially modulated Cd speciation. ErMF+R9G co-inoculation maximally reduced CdEx (26.40%) and CdCar (32.50%) but increased CdOM (641.67%) and CdRes (427.04%) under Cd20 compared with those in the non-inoculated control. Hence, microbial inoculation plays a role in Cd stabilisation. The physicochemical properties and enzyme activities (except for SOC and CBH) of rhizosphere soil were significantly and interactively affected by Cd stress and inoculation treatments ( P < 0.05, Fig. 2B-I, Table S2). Increasing Cd levels elevated SOC, TN and TP while reducing AN, AP and pH, and this effect was counteracted by inoculation treatments, especially co-inoculation with ErMF+R9G. Moreover, Cd stress suppressed the activities of β-glucosidase (BG), β-xylosidase (BX), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP) and invertase while enhancing urease activity. Microbial inoculation (ErMF and ErMF+R9G co-inoculation) regulated soil enzyme activities. Among them, ErMF+R9G co-inoculation significantly increased the enzyme activity of BG (12.93-fold), BX (0.70-fold), CBH (3.14-fold), acid phosphomonoesterase (PHO) (2.43-fold), LAP (1.47-fold), invertase (4.95-fold), NAG (1.06-fold) and urease (0.64-fold) under Cd20 ( P < 0.05). Overall, ErMF+R9G co-inoculation increased the rhizosphere soil nutrient concentration by promoting the activity of extracellular enzymes to cope with Cd stress. Fig. 2. Cd fractionation and enzymatic activity profiles in rhizosphere soils under different inoculation treatments. (A) Chemical speciation of Cd fractions, (B) β-glucosidase (BG), (C) β-xylosidase (BX), (D) β-cellobiosidase (CBH), (E) acetylglucosidase (NAG), (F) leucine aminopeptidase (LAP), (G) acid phosphomonoesterase (PHO), (H) urease and (I) invertase. Different lowercases letters represent significant differences among different inoculation treatment at the same Cd level ( P < 0.05), and different uppercases represent significant differences among different Cd level by Duncan test ( P < 0.05). P -values of two-way ANOVAs of Cd, inoculation and their interaction (Cd×inoculation) are indicated *** P < 0.001, ** P < 0.01, * P < 0.05, ns indicates differences no significant. 3.4 Effects of co-inoculation on soil chemical composition and structural change The SEM-EDS analysis revealed microstructural changes in Cd-stressed blueberry rhizosphere soil following microbial inoculation (Fig. 3A). The CK group exhibited loose lamellar structures with high O, Si and Al contents (He et al., 2022). ErMF-treated soils formed biofilm-coated honeycomb aggregates with suppressed cracking by carboxyl/hydroxyl-rich EPS, while decomposing organic matter via carbohydrate active enzymes (CAZy) (Guo et al., 2010; Martino et al., 2018; Silvia et al., 2018). R9G-treated soils promoted dense graphitic layer formation via EPS and organic acid secretion, high Cd concentration (Cd20) reduced triggered microcracking. ErMF+R9G co-inoculation synergistically optimised soil structure and formed organic–mineral composite layers (C: 55.63%–71.30%; Si: 3.53%–5.34%) without visible cracks but with enhanced stability. The Cd content was not detected due to the detection limit of EDS. Meanwhile, the ErMF+R9G co-inoculation treatment achieved the highest relative atomic content of soil phosphorus (0.14%–0.17%), suggesting that ErMF and R9G synergistically enhanced organic phosphorus mineralisation. FTIR analysis revealed changes in functional groups of Cd-stressed rhizosphere soil following microbial inoculation (Fig. 3B). The 3420 cm -1 peak (O-H/N-H) intensity decreased under Cd stress due to Cd 2+ –hydroxyl binding but was restored by inoculation (especially ErMF+R9G) (Sene et al., 1994; Wang et al., 2020). The aliphatic C-H stretching vibrations (2903–2850 cm -1 ) exhibited increased intensity in microbial inoculated soils, the ErMF+R9G co-inoculation led to the strongest intensity, indicating microbial stabilisation of organic matter (Fang et al., 2024; Gao et al., 2021). The 1632 cm -1 peak (aromatic C=C, carboxylate C=O) decreased in non-inoculated soil from the Cd–carboxylate, whereas microbial inoculation may increase the carboxyl group content through EPS secretion (Lin et al., 2025; Liu et al., 2024b). The P-O vibrations (1000–1100 cm -1 ) intensified maximally in co-inoculation, suggesting enhanced soil phosphate processes (Yang et al., 2022). XRD analysis revealed Cd–phosphate precipitation (Cd(H 2 PO 4 ) 2 2H 2 O) across all treatments, with peak intensities enhanced in microbial-inoculated soils (Fig. 3C). ErMF+R9G co-inoculation exhibited the highest Cd–phosphate complexation efficiency, indicating the synergistic activation of phosphate ions by secretion of organic acids/phosphatase. Diffraction peaks of silicate minerals (e.g., SiO 2 , NaAlSi 3 O 8 and K(Si 3 Al)O 8 )) intensified in inoculated treatments while maintaining structural integrity under Cd stress. The chemical composition and coordination states of Cd-stressed rhizosphere soil under inoculation treatments were further investigated by XPS (Fig. 3D). Control soils showed dominant C-C bonds (284.8 eV), while inoculation reduced C-C intensity, indicating organic oxidation. The emergent C-O bonds (286.23–286.53 eV) appeared in all inoculated treatments, except Cd20-R9G, the maximum peak intensity under ErMF+R9G co-inoculation due to synergistic EPS-enhanced Cd adsorption. The absence of C-O signals in Cd20-R9G may reflect Cd-mediated suppression of bacterial metabolic activity. The co-inoculation maximized O-C=O intensity, suggesting enhanced organic acid and phosphatase secretion for Cd complexation. The peaks of Cd 3d at 407.89–416.73 eV (Cd 3d₅/₂, Cd 3d₃/₂) and P 2p at 133.00–134.10 eV (P 2p₃/₂, P 2p₁/₂) corresponding to phosphate species, such as H 2 PO 4 − (Fig. S1) (Shahid et al., 2016; Chen et al., 2023). Fig. 3. (A) Scanning electron microscopy and energy dispersive spectrometry analysis, (B) The fourier transform infrared spectroscopy analysis, (C) X-ray diffraction analysis (D) X-ray photoelectron spectroscopy C 1s spectrum of different inoculation treatments exposed to 2 and 20 mg kg -1 of Cd. 3.5 Effects of co-inoculation on rhizosphere microbial community Microbial α-diversity (Ace, Chao1, Simpson and Shannon) responded differently to Cd and inoculation treatments (Fig. S2). Cd stress reduced bacterial diversity and richness to a certain extent. Compared with the CK, the inoculation treatments had no significant difference in bacterial richness, and ErMF inoculation reduced Simpson and Shannon. Co-inoculation with ErMF+R9G maintained bacterial diversity. Under Cd stress, the fungal community maintained considerable richness and diversity. However, ErMF+R9G co-inoculation significantly increased the fungal diversity under Cd stress compared with that under Cd-free conditions ( P < 0.05), thereby maintaining fungal diversity while restoring diversity parameters compromised by Cd toxicity. At the genus level (Fig. 4A), the dominant bacterial genera included Burkholderia , Dyella , Pseudolabrys and 67-14. Compared with CK, Cd stress significantly reduced the relative abundance of Pseudolabrys and Rudaea and increased that of 67-14 and Dongia ( P < 0.05); no significant difference in the abundance of Burkholderia was observed. Compared with CK, ErMF+R9G co-inoculation significantly increased the relative abundance of 67-14 and Dongia but reduced that of Pseudolabrys under both Cd-free and Cd-stressed conditions and significantly increased the relative abundance of Rudaea under Cd stress ( P < 0.05). Notably, the abundance of Burkholderia exhibited Cd-dependent regulation, ErMF+R9G co-inoculation increased the relative abundance of Burkholdera (1.08- fold) without Cd, and ErMF inoculation achieved a 2.46-fold increase under Cd stress. The genus-level composition of the fungal community included Fibulochlamys , Talaromyces , Exophiala , Kendrickiella , Penicillium , Scolecobasidium and Ochroconis (Fig. 4B). In CK, Fibulochlamys emerged as the dominant genus, with Epicoccum , Kendrickiella and Rhinocladiella also exhibiting relatively high abundance. Notably, ErMF inoculation significantly enhanced the relative abundance of Fibulochlamys under Cd-free and Cd-stressed conditions ( P < 0.05). Conversely, R9G decreased the abundance of Fibulochlamys , but increased Exophiala under Cd stress ( P < 0.05). ErMF+R9G co-inoculation significantly suppressed the relative abundance of Fibulochlamys , Talaromyces , Kendrickiella and Penicillium while enriching Exophiala and Ochroconis ( P < 0.05), with the latter increasing 58% under Cd stress versus Cd-free conditions. The NMDS analysis revealed that fungal and bacterial communities were significantly separated across treatments, indicating that Cd stress and inoculation treatments imposed specific selective pressure on microbial communities ( P <0.05, Fig. 4C-D). LEfSe identified treatment-specific bacterial biomarkers (Cd0-CK:3, Cd0-ErMF:11, Cd0-R9G:16, Cd0-ErMF+R9G:3, Cd20-CK:1, Cd20-ErMF:5, Cd20-R9G:3) and fungal biomarkers (Cd0-CK:14, Cd0-ErMF:8, Cd0-R9G:17, Cd0-ErMF+R9G:7, Cd20-CK:6, Cd20-ErMF:1, Cd20-R9G:17, Cd20-ErMF+R9G:6) (Fig. S3). The analysis of microbial community assembly revealed that Cd0-CK, Cd0-R9G, Cd0-ErMF+R9G, Cd20-R9G and Cd20-ErMF+R9G were deterministic processes (|βNTI|>2), which were dominated by homogeneous selection (Fig. 4E). Cd0-ErMF, Cd20-CK and Cd20-ErMF followed stochastic processes (|βNTI|>2). Moreover, high Cd concentration weakened the deterministic effect of all treatments, suggesting that heavy metal stress may disrupt microbial interactions. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Fig. 4. Rhizosphere microbiota composition and assembly processes. (A) Bacterial community composition (genus level), (B) fungal community composition (genus level), (C) non-metric multidimensional scaling (NMDS) of bacterial communities, (D) NMDS of fungal communities, (E) Relative contributions of deterministic versus stochastic processes to community assembly. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf 3.6 Correlation analysis of plant physiology, Cd form, soil properties and microbial communities The Mantel test revealed significant correlations between soil Cd fractions (available Cd and organic-bound Cd), plant Cd accumulation parameters (aboveground Cd and underground Cd, TF and BCF) and physiological indicators (POD, proline content and RTD) ( P < 0.05). Soil nutrients (SOC, TP, AP, TN and AN) were significantly associated with plant biomass, micronutrient contents (Mn, Fe, Zn, Mg and Ca), MDA, POD and PAA levels ( P < 0.05, Fig. 5A). Specific enzymes regulated Cd dynamics: NAG and AN influenced available Cd; urease and TP affected organic-bound Cd; and CBH, NAG, LAP, PHO and invertase governed Cd migration ( P < 0.05, Fig. 5B). These findings demonstrated that Cd speciation governs its accumulation, translocation, and stress responses in plants, highlighting the roles of soil nutrient cycling in regulating Cd form and migration. Notably, bacterial communities correlated with CBH, PHO, invertase and SOC, whereas fungal communities associated with CBH, invertase and SOC, indicating microbial regulation of key enzymes potentially influences Cd fractions. Fig. 5. (A) Relationship between soil Cd form, nutrient and plant physiological responses, (B) relationship between soil properties and bioavailability of Cd, (C) relationship between soil properties and microbial community structure. * P < 0.05. 3.7 Effect of reciprocal metabolite exchange between Enterobacter sp. R9G and ErMF on fungal biomass/sporulation and bacterial growth/motility/biofilm formation/chemotaxis Following enhanced Cd tolerance and soil microenvironment remodeling from ErMF-MHB co-inoculation in blueberry, we characterized their mutualistic interactions in vitro to elucidate underlying synergistic mechanisms. ErMF growth and sporulation in R9G metabolites were determined (Fig. 6A). Compared with the control group, the R9G metabolites significantly increased the dry weight and colony diameter of ErMF by 36.98% and 49.46%, respectively, and the sporulation capacity by 216.00% ( P < 0.01). The secretion of mycorrhizal fungi can recruit bacteria and stimulate their growth, thereby mediating their interaction (Jiang et al., 2021; Zhang et al., 2020). The hyphal exudates of ErMF significantly promoted the growth of R9G, and the effect was more pronounced compared with other control groups (Fig. S5A). The effect of hyphal exudates on bacterial swimming and swarming mobility was explored to represent the ability of bacteria to attach to surfaces (Liu et al., 2021); the diameter of the halo of R9G significantly increased by 166.97% and 104.63% in the presence of hyphal exudates compared with the control ( P < 0.05, Fig. 6B). Furthermore, hyphal exudates significantly promoted biofilm formation and extracellular polysaccharide/protein production in R9G ( P < 0.05, Fig. S5B; Fig. 6C). The specific substances in hyphal exudates were analyzed. Growth assays revealed that R9G preferentially utilised carbohydrates (sucrose, fructose and arabinose), followed by amino acids (alanine, proline and cysteine), and had limited organic acid (succinic, salicylic and α-ketoglutaric) assimilation efficiency (Fig. 6D). Chemotaxis analysis demonstrated strong attraction (RCI≥2) toward sucrose, proline, arabinose, fructose, alanine and cysteine, while salicylic and α-ketoglutaric acids had no chemotactic effect on R9G (Fig. 6E, Fig. S6). Biofilm assays indicated that sucrose, proline, arabinose, fructose, alanine and cysteine significantly enhanced biofilm formation ( P < 0.05, Fig. 6F). Sucrose, arabinose, alanine and cysteine showed a tendency to enhance biofilm formation and then diminish it as their concentrations increased. Fructose and proline dose-dependently enhanced the biofilm formation, whereas succinic acid, salicylic acid and α-ketoglutaric acid inhibited it. These findings confirmed sucrose, fructose, arabinose, alanine, proline and cysteine as key metabolites involved in recruitment, colonisation and growth promotion. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Fig. 6. Reciprocal interactions between Enterobacter sp. R9G and O. maius 143. (A) Growth response of ErMF to R9G metabolites, (B) swimming and swarming motility of R9G mediated by hyphal exudates, (C) extracellular polysaccharide and protein contents of R9G mediated by hyphal exudates, (D) growth curves of R9G mediated by key hyphal exudates, (E) chemotactic behavior of R9G mediated by key hyphal exudates, (F) biofilm formation of R9G mediated by key hyphal exudates. Denote significant differences between treats according to paired samples t-test, *** P < 0.001, ** P < 0.01, * P < 0.05. Different lowercases letters represent significant differences among different treatment by Duncan test ( P < 0.05). 4. Discussion 4.1 Co-inoculation regulates blueberry physiological responses and Cd translocation under Cd stress Cd stress significantly reduced biomass in non-inoculated plants ( P < 0.05). Inoculation, particularly EMF+R9G co-inoculation, effectively counteracted Cd-induced growth inhibition. The elevated R/S indicated that plants allocated more resources to the root system than to the aboveground parts to enhance adaptation to Cd stress. Notably, EMF+R9G co-inoculation increased root branching and root tip proliferation in blueberry plants, accompanied by reduced SRL and SRA (negatively correlated with R/S, Fig. 5A). This morphological adjustment reflected the strategy of ‘main root stress resistance + lateral root symbiosis’ to balance Cd tolerance and ErMF symbiosis under Cd stress. Moreover, R9G significantly increased the colonisation rate of ErMF under Cd stress ( P < 0.05), aligning with established MHB processes that promote mycorrhization (Yang et al., 2023; Zhang et al., 2024; Wang et al., 2021c). Thus, R9G may optimise the symbiotic relationship between blueberry and ErMF by optimising root development and promoting mycorrhizal colonisation to enhance plant resistance to Cd. Cd accumulation was significantly higher in blueberry roots than shoots ( P stem > leaf BCF patterns reported by Song et al. (2023). ErMF+R9G co-inoculation significantly increased root Cd content but decreased BCF and TF versus controls ( P < 0.05), indicating co-inoculation effectively impeded the occurrence of Cd-induced toxicity symptoms in aboveground parts, thereby contributing to the stabilisation of the plant (Joner and Leyval, 1997). The reason might be the impacts of mycorrhizal symbiosis on heavy metal accumulation across various plant species and organs (Shi et al., 2018; Zhang et al., 2018a). The hyphae of mycorrhizal fungi (AMF/ECM) act as a Cd pool by adsorbing and binding metals, thereby immobilising Cd within root systems and effectively reducing the translocation of Cd and protecting the branches from damages (Han et al., 2021; Luo et al., 2014). Casarrubia et al. (2020) found that the Cd content in the mycorrhizal roots of blueberry was significantly lower than that in non-mycorrhizal roots. However, our study found ErMF inoculation and ErMF+R9G co-inoculation significantly enriched Cd in roots ( P < 0.05), suggesting that ErMF may play an important role in Cd immobilisation in roots. The observed discrepancies may be attributed to the growth of V. myrtillus seedlings in MMN medium containing 1 μM Cd, where variations in cultivation conditions and growth status of blueberry resulted in differential protective effects conferred by ErMF under Cd stress. Meanwhile, R9G addition enhanced mycorrhizal colonisation, consequently leading to greater root Cd retention in co-inoculated plants. BCF and TF were positively correlated with aboveground Cd content and negatively correlated with R/S (Fig. 5A). As such, plants will allocate more resources to the root system, favouring Cd immobilisation in cooperation with ErMF and reducing the transport and enrichment of aboveground Cd. The increase in biomass under EMF+R9G treatment may also cause the ‘dilution effect’ (Bai et al., 2008; Nielsen and Jensen, 1983); that is, even though the total Cd content may be higher under ErMF+R9G co-inoculation, the higher biomass will dilute the Cd content and result in a lower concentration. Under Cd stress, blueberry exhibited enhanced resistance by increasing R/S and optimising root conformation, and the co-inoculation synergistically alleviated Cd toxicity by promoting mycorrhizal colonisation, enhancing biomass and reinforcing Cd immobilisation in roots. Additionally, ErMF+R9G co-inoculation significantly increased shoot Mn, Fe, Zn, Mg, Ca, and K content versus other treatments ( P < 0.05); this finding indicates that Mn, Fe, Zn and Cd may reduce the accumulation and transport of Cd by competing for absorption sites or regulating the expression of transport proteins (e.g., ZIP family proteins) (Gao et al., 2024; Haider et al., 2021), this process likely contributes to reduced Cd bioavailability by decreasing the exchangeable Cd fraction in the soil (Arinzechi et al., 2025; Ma et al., 2024). Co-inoculation also reinforced antioxidant defence by upregulating CAT, POD and SOD activities and reducing MDA content while promoting proline and soluble sugar synthesis ( P < 0.05). These synergistic mechanisms–enhancing antioxidant capacity, osmotic regulation and competitive nutrient uptake–support microbial co-inoculation as a promising strategy for improving phytoremediation efficacy under heavy metal stress, consistent with observations in other environmental stressors (Jian et al., 2019; Wang et al., 2021b). For instance, Ben et al. (2020) claimed that the combined application of AMF and rhizobium could alleviate the negative effects of high salt content by stimulating plant growth, mineral absorption, antioxidant systems and compatible solute synthesis. 4.2 Regulatory strategies of co-cultivation on Cd removal mechanisms in blueberry rhizosphere soil The effectiveness of Cd depends on its active form in the substrate (Chen et al., 2014; Zhong et al., 2011). ErMF+R9G co-inoculation effectively converted the Cd of high-mobility and biotoxicity components (CdEx and CdCar) into poor bioavailability components (CdOM and CdRes). The ErMF+R9G co-inoculation also significantly increased the activities of BG, BX, CBX, PHO, LAP, NAG, invertase and urease, which in turn increased the contents of soil SOC, TP, AP, TN and AN ( P <0.05). In particular, NAG and AN significantly affected available Cd; urease and TP significantly affected organically bound Cd; and CBH, NAG, LAP, PHO and invertase significantly affected Cd migration ( P <0.05). The increase in PHO activity may release inorganic phosphates, which produce Cd–phosphate precipitates to promote Cd immobilisation (Huang et al., 2024; Lin et al., 2025). The increased invertase and urease activities may stimulate microbial biomass turnover and promote Cd–organic matter (CdOM) complexation (Yan et al., 2024). These enzyme kinetics emphasise the role of co-ErMF+R9G in improving soil nutrients and indirectly affecting soil Cd morphology. Multi-spectral analyses demonstrated that ErMF+R9G co-inoculation immobilised Cd through synergistic organo-metal complexation, phosphate precipitation and silicate stabilisation. Specifically, the ErMF+R9G co-inoculation induced pronounced stretching vibrations in EPS-associated functional groups, accompanied by increased peak intensity of C-O bonds in the XPS spectra. ErMF may release organic carbon by secreting CAZy enzymes that bind to mycelium, agglomerates and EPS to immobilise Cd (Perotto et al., 1995; Read, 1991), while R9G contributed through bacterial cell wall adsorption and EPS complexation (Yan et al., 2024; Huang et al., 2024). The enhanced secretion of fungal extracellular metabolites and bacterial EPS synergistically amplified Cd adsorption, indicating the superior efficacy of co-inoculation. ErMF+R9G co-inoculation also significantly elevated soil phosphorus levels, as evidenced by an increased relative atomic percentage in EDS analysis, coupled with the increased phosphatase activity and soil available phosphorus content; meanwhile, the XRD data of the Cd(H 2 PO 4 ) 2 ·2H 2 O precipitate and the maximum intensity of the P 2p peak (133.0/134.1 eV) and the O-C=O peak in XPS provide further convincing evidence. Overall, ErMF and R9G may synergistically release PO 4 3 ⁻ through phosphatase secretion and organic acid exudation to promote Cd–PO 4 precipitation. Moreover, ErMF+R9G co-inoculation promoted silicate stabilisation by forming organo-mineral composite layers (C: 55.63%–71.30%; Si: 3.53%–5.34%). The results revealed no lack of peak shift in soil matrix minerals (e.g., NaAlSi₃O₈ and SiO₂) and no significant disruption in soil morphology or elemental distribution. Hence, ErMF+R9G co-inoculation for the remediation of soil Cd does not impose a burden on the soil and can be used as a safe and effective strain for soil remediation. 4.3 Modulation of microbial community assembly in blueberry rhizosphere soil by co-inoculation In this study, Cd stress reduced microbial diversity. Microbial inoculation enhanced fungal diversity in blueberry rhizosphere under Cd stress while decreasing bacterial diversity. This phenomenon may be attributed to Cd and introduced microorganisms altering the soil microenvironment, simplifying bacterial community structure, changing the dominant bacterial groups and reducing overall diversity. Previous studies documented that AMF can selectively recruit distinct bacteria, and functional microorganisms play a dominant role when encountering stress (Herren and Mcmahon, 2018). The combination of AMF and Cd formed a functional module of heavy metal-resistant microbial communities, where recruited functional microbes, including Burkholderia and Dyella , were identified (Wang et al., 2023a). This research found that Cd stress reduced the abundance of Burkholderia , whereas ErMF inoculation reversed this trend, indicating that ErMF may increase the recruitment of heavy metal-tolerant microorganisms by providing ecological niches or secretions through mycelial networks. However, ErMF+R9G co-inoculation did not significantly enhance Burkholderia abundance under Cd stress, which might have caused competition among microorganisms (Li et al., 2024). Furthermore, ErMF+R9G co-inoculation significantly enriched Dongia and strain 67-14 but reduced the abundance of Pseudolabrys in the presence or absence of Cd stress ( P < 0.05). Significant correlations were observed between bacterial communities and levels of CBH, PHO, sucrase and SOC ( P < 0.05), with Burkholderia exhibiting strong positive linkages with CBH, sucrase and PHO activities, suggesting its potential role in indirectly mediating Cd bioavailability through carbon source metabolism and phosphate activation (Yan et al., 2024). Dongia was positively correlated with PHO and urease activities, and its enrichment may strengthen plant Cd tolerance by activating phosphorus. The diminished Pseudolabrys population could be attributed to Cd-triggered intensification of carbon source competition, as evidenced by its negative correlations with CBH and invertase activities (Fig. S4A) (Li et al., 2024). These enzymatic activities exhibited strong associations with Cd migration, suggesting the occurrence of microbial regulation of Cd fixation through organic carbon decomposition (e.g., cellobiose hydrolysis) to release chelating groups or modulate phosphate metabolism. The present study found that inoculation treatments exerted modulatory effects on fungal community composition. The ErMF+R9G treatment induced a diversified fungal community structure by suppressing the relative abundance of Fibulochlamys , Talaromyces , Kendrickiella and Penicillium and significantly enhancing the prevalence of Exophiala and Ochroconis ( P < 0.05). Significant correlations between fungal communities and levels of CBH, sucrase and SOC ( P < 0.05), with Ochroconis exhibited a strong positive correlation with urease activity, and a markedly negative correlation was found between Simplicillium and PHO activity (Fig. S4C). Moreover, urease significantly affected the organic binding Cd, and PHO significantly affected the Cd migration ( P < 0.05). In summary, the inoculation treatments optimised enzyme activities and nutrient cycling in the rhizosphere microenvironment by regulating the abundance of specific microbiota (e.g., Burkholderia and Ochroconis ), thereby directly or indirectly influencing soil properties and Cd migration to alleviate Cd toxicity. Microbial community assembly revealed that Cd stress and inoculation treatments exerted selective pressures on microbial communities, as evidenced by the separated fungal and bacterial communities in the NMDS analysis. Treatment-specific microbial biomarkers identified via the LEfSe highlighted unique microbial characteristics under different conditions. Simultaneously, the Cd0-CK, Cd0-R9G, Cd0-ErMF+R9G, Cd20-R9G and Cd20-ErMF+R9G were deterministic processes, this pattern was attributed to the synergy of microbial functions or the strengthened deterministic process caused by environmental filtering. Specifically, the synergistic effects of ErMF+R9G contributed to the formation of a stable functional network through metabolic complementarity, thereby enhancing deterministic environmental selection (Zhu et al., 2024). By contrast, Cd0-ErMF, Cd20-CK and Cd20-ErMF followed stochastic processes. For ErMF, Cd stress likely constrained mycorrhizal network functionality, and homogenising dispersal was regarded as the dominant mechanism. For Cd20-CK, high Cd concentrations potentially suppressed environmental microbial regulation, resulting in ecological drift as the primary driver and increased stochasticity. These insights into the mechanisms underlying microbial-mediated Cd immobilisation provide a sustainable framework for plant management in contaminated ecosystems. 4.4 The interaction between ErMF and Enterobacter sp. R9G in vitro The synergistic detoxification by microbial symbionts in Cd-contaminated soil have been several studies (Asif et al., 2025; Wang et al., 2021b). However, the coexistence and functional interactions between mycorrhizal fungi and MHB under Cd exposure remain largely unexplored. Pre-stress metabolic reciprocity–including chemotactic recognition, nutrient exchange, and biofilm colonisation–is essential for symbiotic stress resistance (Manzoor et al., 2025). Therefore, we further analyzed the mutualistic interactions between ErMF and MHB through in vitro experiments. It was found that ErMF secretions significantly enhanced the growth, biofilm formation, and motility of R9G ( P < 0.05). R9G preferentially assimilated carbohydrates (sucrose, fructose and arabinose) from the hyphal exudates as efficient energy sources, consistent with bacterial central carbon metabolism (Gralka et al., 2023). Sucrose, proline, arabinose, fructose, alanine, cysteine and succinate were identified as key chemotactic substances. Relevant studies indicated that amino acids generally serve as stronger attractants than carbohydrates (Brunet et al., 2025). Proline is a potent bacterial attractant that induces maximal chemotactic responses at low concentrations and enhances biofilm formation in a dose-dependent manner (Wang et al., 2023b), consistent with our findings. The chemotactic effect of sucrose was not significantly different from that of proline, which may be due to its energy production indirectly enhancing chemotaxis. Reciprocally, R9G fermentation products significantly increased ErMF hyphal biomass and sporulation ( P < 0.05). This effect aligns with the documented mechanism of MHB, such as Paenarthrobacter nicotinovorans and Bacillus circulans , which promote fungal growth of ErMF by providing carbon sources and growth factors (Yang et al., 2023). These bacteria augment fungal nutrient acquisition and colonisation efficiency to increase mycorrhizal colonisation rates in host plants and reinforce plant stress tolerance (Manzoor et al., 2025; Zhang et al., 2024). Metabolic interactions exist between ErMF and R9G, and they jointly promote cadmium immobilization through interrelated mechanisms, as illustrated in conceptual diagram Fig. 7. The increased hyphal biomass and sporulation induced by R9G may contribute to the formation of mycelial networks and enhance the fixation ability of Cd. Substances, such as sucrose and proline, secreted by ErMF recruit R9G as chemotactic signals, and the hyphal network provided migration channels and attachment sites for R9G colonisation. Jiang et al. (2021) and Zhang et al. (2020) reported that phosphate-solubilising bacteria and rhizobia migrating along AMF hyphae might be involved in the process. Carbohydrates and amino acids in hyphal exudates serve as carbon sources for R9G to potentially alleviate Cd toxicity to bacteria, while hyphal exudates might facilitate Cd immobilisation by enhancing R9G synthesis of EPS and increasing phosphatase activity. As reported by Zhang et al. (2018b), AMF can enhance bacterial phosphatase activity, with fructose serving as a key metabolite to stimulate microbial phosphate-solubilising functions. Mei et al. (2024) found that S. grevillea chemotaxis-assisted C. lapagei adhere to the surface of S. grevillea , thereby increasing the biomass of C. lapagei and upregulating genes related to chemotaxis, colonisation and proliferation; in addition, C. lapagei promotes the phosphatase and phytase activities of S. grevillea . Current understanding of ErMF-mediated phosphorus activation remains limited. Therefore, the specific effects of ErMF and R9G on the morphological transformation of Cd should be comprehensively analysed by techniques such as X-ray Absorption Near Edge Structure. In particular, the mechanism of phosphatase and organic acid secretion dynamics in their interaction and the morphological transformation of Cd remains to be clarified. Fig. 7. Conceptual diagram depicting the synergistic effects of O. maius 143 and Enterobacter sp. R9G on blueberry physiology, Cd transformation and microbial community in Cd-contaminated soil. 5. Conclusions This study systematically elucidated mechanisms underlying the synergistic enhancement of Cd resistance in blueberry by ErMF and Enterobacter sp. R9G through a multi-level approach. ErMF+R9G co-inoculation effectively mitigated Cd toxicity and reduced transport within the host plant. Co-inoculation also improved soil Cd bioavailability, enhanced Cd stabilisation through EPS secretion and phosphate precipitation, and strengthened soil remediation by promoting the growth and activity of beneficial microbial communities. Crucially, chemical signaling drives mutualism: hyphal exudates promoted bacterial recruitment and colonization, while bacteria stimulated fungal growth. In summary, the synergistic interaction between ErMF and R9G enhances Cd tolerance in blueberry, thereby providing valuable insights for the application of plant–microbe synergistic phytoremediation of Cd-stressed soils. Author contribution Jing Jiang conceived, designed the research and wrote the original draft. Huizhi Zhang performed the plant culture and chemical analysis. Haifeng Zhu analysed the data. Yixiao Wang and Qiling Yang revised the manuscript. Hongyi Yang and Lili Li contributed new reagents and acquired funding. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Research Funds for Research Institutes of Heilongjiang Province (Grant number CZKYF2025-1-A011), the Natural Science Foundation of Heilongjiang Province (Grant number LH2023C044), and the National Natural Science Foundation of China (Grant numbers 32071806 and 31971694). Data availability The datasets supporting this study are included in the manuscript and supplementary information. Additional raw data are available from the corresponding author upon reasonable request. The Illumina MiSeq sequence datasets are available at the NCBI Sequence Read Archive BioProject ID PRJNA1285493. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf References Arinzechi, C., et al., 2025. 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Community assembly of organisms regulates soil microbial functional potential through dual mechanisms. Global Change Biology 30(2), e17160. https://doi.org/10.1111/gcb.17160. Information & Authors Information Version history V1 Version 1 26 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords blueberry cadmium development ericoid mycorrhizal fungi growth mycorrhiza helper bacteria Authors Affiliations Jing Jiang Northeast Forestry University View all articles by this author Huizhi Zhang Northeast Forestry University View all articles by this author Haifeng Zhu Northeast Forestry University View all articles by this author Yixiao Wang Northeast Forestry University View all articles by this author Qiling Yang Northeast Forestry University View all articles by this author Hongyi Yang [email protected] Northeast Forestry University View all articles by this author Lili Li Heilongjiang Academy of Forestry View all articles by this author Metrics & Citations Metrics Article Usage 306 views 196 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jing Jiang, Huizhi Zhang, Haifeng Zhu, et al. Synergistic roles of ericoid mycorrhizal fungi and mycorrhiza helper bacteria enhance plant stress tolerance and Cd immobilisation. Authorea . 26 July 2025. 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