Synergistic Effects of Arbuscular Mycorrhizal Fungi and Mycorrhiza Helper Bacteria Alter Cucumber Rhizosphere Fungal Community and Reduce Soil Cadmium Contamination

TL;DR

This study shows that combining fungi and bacteria can reduce cadmium in soil and plants by changing the fungal community around cucumber roots.

Contribution

The study reveals how AMF and MHB co-inoculation alters the cucumber rhizosphere fungal community to reduce soil Cd contamination.

Findings

Co-inoculation with AMF and MHB significantly reduced Cd concentrations in plant tissues and soil.

The treatment increased beneficial fungal phyla like Mortierellomycota and decreased pathogenic Ascomycota.

Fungal diversity and specific OTUs were enriched following AMF–MHB co-inoculation.

Abstract

Cadmium (Cd) contamination in agricultural soils severely impairs plant growth, disrupts microbial communities, and threatens food safety due to its high toxicity and mobility. Conventional remediation methods are often expensive and environmentally unsustainable. In contrast, plant–microbiome interactions offer an eco-friendly solution to reduce Cd accumulation and improve plant growth. Arbuscular mycorrhizal fungi (AMF) and mycorrhiza helper bacteria (MHB) are known to improve plant growth and resilience in Cd-contaminated soils. However, the mechanisms by which AMF and MHB co-inoculation could reduce soil Cd contamination by altering the rhizosphere fungal community remain unclear. This study aimed to evaluate how co-inoculation with AMF (Funneliformis mosseae) and MHB (Alcaligenes faecalis) affects plant Cd uptake and soil Cd content, and how it reshapes the cucumber rhizosphere fungal community. A greenhouse experiment was conducted with four treatments: CK (no inoculation), Fm (AMF inoculation), Af (MHB inoculation), and FA (AMF + MHB co-inoculation). Co-inoculation with AMF and MHB (FA) significantly reduced Cd concentrations in both plant tissues and soil. Fungal communities were profiled using Illumina MiSeq sequencing of the ITS region, and diversity metrics and structural changes were assessed through PCoA and DESeq2. Co-inoculation (FA) significantly reshaped the fungal community, increasing the relative abundances of beneficial phyla such as Mortierellomycota, Basidiomycota and Glomeromycota, while decreasing the abundance of potentially pathogenic Ascomycota. Double inoculation with AMF and MHB also enhanced fungal diversity, as measured by the Simpson index, and enriched specific OTUs. This study uncovers the mechanisms through which AMF–MHB co-inoculation reduces Cd concentrations in both plants and soil by altering the cucumber rhizosphere fungal community composition. These findings demonstrate that AMF–MHB co-inoculation is an effective, biologically driven strategy for remediating Cd-contaminated soils by restructuring cucumber rhizosphere fungal communities.

Figures

Full text

1. Introduction

Soil contamination with heavy metals, particularly cadmium (Cd), has become a critical global environmental issue [1]. Cd is a toxic and persistent metal that accumulates in soils, posing a significant threat to plant health and agricultural productivity [2,3]. At high concentrations, Cd disrupts physiological, biochemical, and molecular processes in plants, entering the food chain through bioaccumulation and biomagnification [4,5,6]. Cd is a non-essential heavy metal that accumulates in the environment primarily through anthropogenic activities, including industrial emissions, mining, atmospheric deposition, production of Ni-Cd batteries, urban sewage sludge, agrochemicals waste incineration, and vehicle exhausts, making it a widespread problem [7,8,9]. The Cd contents in soils typically range from 0.01 to 1 mg kg^−1^, with levels exceeding 3 mg kg^−1^ indicating contamination that warrants remediation [10]. In regions like China, Cd concentrations in agricultural soils often exceed safe limits over 30%, resulting in reduced crop yields, impaired soil fertility, and serious food safety concerns [11]. While Cd contamination is a severe problem in countries like China, where up to 7% of farmland is affected, other regions including Southeast Asia, Europe, India, South America, and South Korea are also facing rising levels of contamination [12,13,14,15]. The high mobility and bioavailability of Cd in soils make traditional remediation techniques, such as chemical or physical methods, costly, inefficient, and often unsustainable [16,17,18].

Phytoremediation specifically uses plants to absorb, sequester, or detoxify pollutants. However, its efficiency is often limited by factors such as slow plant growth rates, low biomass, and limited root depth, all of which hinder the plants’ ability to absorb substantial amounts of heavy metals from the soil [19,20]. Alternatively, bioremediation strategies that utilize microorganisms to degrade, immobilize, or detoxify pollutants have garnered considerable attention. Soil microorganisms, including bacteria, fungi, and AMF, play a key role in the biodegradation of heavy metals in contaminated soils [21,22,23,24]. These microorganisms interact with contaminants through mechanisms such as biosorption, biotransformation, and metal sequestration, which collectively reduce metal bioavailability and mitigate their toxic effects on plants [25,26]. These microbial processes offer a sustainable, cost-effective solution for remediating Cd-polluted soils, enhancing plant growth through improved nutrient availability, soil health and resilience, thus supporting ecological restoration and agricultural productivity.

Among soil microorganisms, AMF such as Funneliformis mosseae and Rhizophagus irregularis are crucial for plant health, particularly in nutrient-deficient soils [27,28]. These fungi form symbiotic relationships with the roots of most terrestrial plants, improving nutrient uptake, particularly phosphorus and nitrogen, and helping plant growth in Cd-contaminated soils [29,30,31,32]. Mycorrhiza helper bacteria (MHB), such as Alcaligenes faecalis and Pseudomonas fluorescens, are known to enhance the colonization of AMF by promoting spore germination and hyphal growth [29,33]. Additionally, MHB are involved in bioremediation processes by facilitating the detoxification of heavy metals and improving soil health [34,35]. Mycoremediation, which leverages the strong enzymatic systems of fungi, is especially effective in breaking down organic pollutants [16]. The plant’s tolerance mechanisms against excessive Cd exposure can be enhanced by AMF such as Funneliformis mosseae, which function as a filter by binding Cd within the plant, hyphae, and roots, a process referred to as metal immobilization [15,36,37]. AMF, particularly Funneliformis mosseae, reduce metal uptake by altering the bioavailability of metals in the soil and sequestering them in plant roots, thereby reducing metal translocation to the aboveground parts of plants and preventing their accumulation in edible tissues [37,38,39,40,41,42]. Recent studies suggest that large populations of native AMF are often found in heavy metal-contaminated soils, indicating that AMF play a crucial role in enhancing plant tolerance to heavy metals and increasing their absorption capacity [39,43]. This symbiosis not only alleviates metal stress but also supports the soil microbial ecosystem, facilitating nutrient cycling and soil fertility [44]. AMF inoculation, such as Funneliformis mosseae, has been shown to alter microbial community structure in the rhizosphere by increasing the abundance of beneficial microorganisms, such as Proteobacteria and Actinobacteria, while suppressing pathogenic species [45,46,47,48]. This shift in microbial diversity contributes to a healthier soil environment, promoting metal detoxification and enhancing plant resilience to heavy metal stress [49]. Furthermore, AMF can influence the types and quantities of organic acids secreted by plant roots, thereby altering the physicochemical properties of rhizosphere soils, improving soil enzyme activities, and affecting the morphology and bioavailability of heavy metals [50,51,52]. The growth enhancement is mediated by AMF-induced nutrient uptake, ultimately leading to the reduction in metal concentrations in plant tissues through dilution [2]. Despite the promising potential of AMF in promoting plant growth in Cd-contaminated soils, the precise mechanisms by which AMF interact with the rhizosphere microbiome to reduce Cd toxicity remain poorly understood. Therefore, we have investigated whether AMF influence the cucumber rhizosphere fungal community in Cd-contaminated soils, thereby reducing Cd toxicity in both plant and soil.

In addition to AMF, MHB such as Alcaligenes faecalis have emerged as important partners in the mycorrhizal symbiosis, enhancing AMF functionality in Cd-contaminated soils [29]. MHB, including Alcaligenes faecalis, a specialized group of plant growth-promoting rhizobacteria, interact with AMF by promoting spore germination, hyphal growth, and symbiotic establishment with plant roots [2,53]. These bacteria significantly contribute to heavy metal detoxification by altering metal speciation in the soil and enhancing metal degradation through microbial metabolic pathways [25,26]. MHB also modulate microbial community structures in the rhizosphere, promoting beneficial microorganisms while suppressing harmful ones, thereby improving the bioremediation process [53,54,55]. MHB enhance AMF functionality by improving nutrient availability and promoting better root colonization. The cooperation between AMF and MHB enhances growth, improves heavy metal uptake, and alters the soil microbial community structure, suggesting that MHB can play a synergistic role with AMF in the remediation of heavy metal-polluted soils [55,56]. The use of microbial consortia consisting of bacteria, algae, and fungi has proven highly effective, as these organisms work synergistically to enhance degradation processes [57]. These consortia are being explored to optimize bioremediation for specific pollutants, demonstrating the benefits of combining different biological systems. Despite these promising outcomes, the exact mechanisms by which AMF and MHB synergistically interact with the cucumber rhizosphere microbiome in Cd-contaminated soils to reduce Cd contamination in both plants and soil remain poorly understood.

The microbial community in the rhizosphere plays a critical role in nutrient cycling, plant health, and the suppression of soil-borne pathogens [58,59,60]. Soil contamination with heavy metals alters microbial community structure and diversity, often leading to a decline in beneficial microorganisms and a shift towards more pathogenic species. Soil microbial communities are significantly influenced by metal concentrations, and both AMF and MHB can modulate these communities to enhance plant resilience and soil health [61]. Based on these studies, we hypothesize: (1) Co-inoculation of AMF and MHB would reduce cadmium (Cd) concentration in both plant and soil, and (2) co-inoculation of AMF and MHB alters the fungal community structure in the cucumber rhizosphere, potentially enriching specific beneficial guilds while modifying the relative abundance of other fungal groups, including those with pathogenic potential. This study aims to investigate the synergistic effects of AMF and MHB co-inoculation on cucumber rhizosphere fungal community structure in Cd-contaminated soils. By employing high-throughput sequencing, this research provides a comprehensive understanding of how AMF and MHB co-inoculation alters fungal community composition, diversity, and structure in the cucumber rhizosphere, and how these changes influence Cd dynamics in both plant and soil. The results help develop more effective and sustainable bioremediation strategies, advancing both agricultural and environmental sustainability.

2. Materials and Methods

2.1. Physicochemical Characteristics and Preparation of Cd-Contaminated Soil

The initial physicochemical characteristics of the soil were as follows: Soil pH: 6.88; EC value: 82.30 μS cm^−1^; organic matter: 30.30 g·kg^−1^; available P: 16.50 mg·kg^−1^; available K: 141.33 mg·kg^−1^; ammonium N: 2.48 mg·kg^−1^; nitrate N: 23.87 mg·kg^−1^; Cd content: 0.16 mg·kg^−1^. To prepare the Cd-contaminated soil, soil was collected from the plow layer (0–20 cm) of Xiangyang Farm, Northeast Agricultural University, and sieved through a 2 mm mesh. Cadmium chloride (CdCl_2_, analytically pure) was added to the soil in the form of an aqueous solution at a concentration of 5 mg·kg^−1^, a level known to be detrimental to plant growth [62]. A total of 1500 mg CdCl_2_ was mixed with 300 kg of soil. The CdCl_2_ was dissolved in deionized water, added to a small portion of the soil, and then thoroughly mixed with the rest of the soil. The soil was maintained at 60% water-holding capacity and incubated for three months at room temperature. At the end of this incubation period, the soil Cd content reached 2.92 mg·kg^−1^. This reduction from the intended 5 mg·kg^−1^ to 2.92 mg·kg^−1^ is likely due to a combination of processes, including adsorption to soil particles (e.g., clay minerals and organic matter), potential precipitation, and possible leaching. These processes can reduce the bioavailability of Cd in the soil. Adsorption to soil particles is a well-documented mechanism that can effectively immobilize heavy metals, reducing their mobility and availability for plant uptake [63]. Leaching, depending on irrigation and soil moisture conditions, might also contribute to a decrease in soil Cd content [64]. Despite this reduction, the final Cd concentration of 2.92 mg·kg^−1^ still represents a moderate stress level, which is appropriate for investigating the effects of bioremediation strategies in Cd-contaminated soils.

2.2. Preparation of AMF and MHB Suspension

The cucumber variety used in this study was Cucumis sativus (Delong C57). The fungal inoculum, collected from pot cultures of Trifolium subterraneum colonized with Funneliformis mosseae, consisted of spores (200 spores/g of soil) and mycorrhizal root pieces. This inoculum was applied during the transplantation of cucumber seedlings [65]. The inoculum was prepared to contain 50 infective propagules per gram of soil, which were quantified using the Most Probable Number (MPN) method, as described by Porter [66]. The inoculum was applied to the roots of the seedlings, after which the surface was covered with soil to ensure the complete colonization of the cucumber seedlings by the AMF agent [65]. The Funneliformis mosseae strain (Nicol. & Gerd.) Gerdemann & Trappe BEG 167 was kindly provided by Dr. Min Li at Qingdao Agricultural University, China.

In addition to the fungal inoculum, Alcaligenes faecalis (OD_600_ = 1) was used for bacterial inoculation. The activated strain of Alcaligenes faecalis (CCTCC M 2024300) was inoculated into a 250 mL triangular flask containing 50 mL of LB liquid medium and incubated at 28 °C with shaking at 180 rpm for 12–16 h. After incubation, the culture was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The bacterial pellet was washed three times with sterile distilled water. The bacterial suspension was then diluted with sterilized distilled water to an OD_600_ of 1 [65]. Alcaligenes faecalis was isolated and identified by our research group, and the strain is preserved at the China Center for Type Culture Collection (preservation number: CCTCC M 2024300).

2.3. Experimental Design

The glasshouse experiment was conducted at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, in a greenhouse at the Horticulture Experimental Station, Northeast Agricultural University, Harbin, China (45°41′ N, 126°37′ E). The current study focused on assessing the potential of AMF and MHB in Cd-contaminated soils to explore their detoxification and remediation properties. The absence of an uncontaminated soil control was intentional, as the primary aim was to simulate real-world contamination scenarios. Four treatments were set up in the experiment: CK: No inoculation; Fm: Single inoculation of Funneliformis mosseae (10 g/pot, about 200 spores); Af: Single inoculation of Alcaligenes faecalis (10 mL/pot, OD_600_ = 1); FA: Double inoculation of Funneliformis mosseae and Alcaligenes faecalis. Each treatment was repeated 3 times, with 15 pots per treatment. The experiment followed a completely randomized design. To ensure the same microbial community structure across treatment groups, when Fm and FA treatments were inoculated with AMF, the CK and Af groups were inoculated with 10 mL AMF filtrate (using a 10 μm filter membrane to filter out AMF) [67]. Similarly, when Af and FA treatments were inoculated with MHB fungal suspension, the CK and Fm groups received 10 mL sterile water. The cucumber plants were grown in a greenhouse setting to control environmental conditions. Each plant was transplanted into an 8 × 8 cm black nutrient pot, and filled with 300 g of sterilized soil. Temperature was maintained at 25 °C with a 16 h light/8 h dark cycle, and relative humidity was kept between 60% and 80%. Irrigation was carried out using groundwater, maintaining soil moisture levels at 60–80% of the maximum field capacity. Regular fertilization was administered to ensure adequate nutrient supply for the plants. These conditions were consistent across all treatments to avoid environmental variability.

2.4. AMF and MHB Inoculation

Cucumber (Cucumis sativus, variety: Delong C57) was used as the test plant, Funneliformis mosseae as the test AMF, and Alcaligenes faecalis as the test MHB. Cucumber seedlings with two cotyledons were planted in 8 × 8 cm black nutrient pots, each filled with 300 g of soil. After 7 days of planting, an MHB suspension was inoculated via root irrigation. The inoculation was supplemented every 7 days, with two applications in total. The same amount of sterile water was applied to the non-inoculated MHB group. Conventional field management practices were followed throughout the experiment. All treatments were irrigated quantitatively with groundwater using the weighing method to ensure that soil moisture was maintained at 60–80% of the maximum field capacity, except during the supplementary application of microbial agents.

2.5. Determination of Cd Concentration in Plant and Soil

To determine cadmium (Cd) concentrations, bulk soil and plant samples (including roots, stems, and leaves) are collected. The plant tissues are rinsed with distilled water, dried at 70 °C to a constant weight, and ground into a fine powder. Similarly, soil samples are dried at 70 °C. A known mass (0.5–1 g for plant samples, 1–2 g for soil) of each sample is digested in a mixture of nitric acid (HNO_3_) and perchloric acid (HClO_4_) in a 4:1 ratio. The samples are heated until digestion is complete, then cooled and diluted to a final volume of 50 mL with distilled water. The digests are filtered to remove any residue. Cadmium concentration is measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [49]. The results, reported in mg/L (ppm), are multiplied by the volume of the digest to calculate the total Cd content in the plant or soil. For plant samples, the Cd content is calculated on a dry weight basis. Quality control is ensured by running blanks and using certified reference materials (CRMs) to validate the results. Replicate analyses are performed to ensure data reproducibility.

2.6. Rhizosphere Soil Sampling

Soil sample collection followed the method described by [65]. Rhizosphere soil was collected from cucumber seedlings grown under different treatments, including CK (no inoculation), Fm (AMF inoculation), Af (MHB inoculation), and FA (AMF + MHB co-inoculation). At 42 days after planting, cucumber seedlings from each treatment group were carefully uprooted. After gently shaking off the loosely adhering soil, the tightly adhering rhizosphere soil was carefully collected using a sterile brush. To obtain sufficient rhizosphere soil for analysis, different replicates were randomly selected, and the rhizosphere soils were pooled to form a composite sample. From this composite, three technical replicates were prepared for DNA extraction and subsequent sequencing. This approach ensured that the microbial community data reflected the variability within each treatment while maintaining biological replication. In total, three biological replicates (composite samples from nine pots per treatment, with three pots per replicate) were used per treatment for microbial community analysis. The rhizosphere soil samples were sieved through a 2 mm mesh sieve to remove stones and roots, thoroughly homogenized, and stored at −80 °C for DNA extraction.

2.7. Soil DNA Extraction

Total DNA was extracted from 0.5 g of frozen cucumber rhizosphere soil (wet weight) using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. DNA extraction was performed in triplicate for each composite soil sample, and the three extracts were pooled to form a composite DNA sample. DNA purity was assessed using a spectrophotometer, and the samples were stored at –80 °C for further analysis.

2.8. High-Throughput Sequencing of Fungi

The composition of soil fungal communities was estimated using the Illumina MiSeq platform. The primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were used to amplify the ITS fungal region [68]. The primers were modified with a unique 6 bp barcode at the 5′ ends to identify each sample. DNA samples were amplified using an ABI GeneAmp^®^ 9700 PCR System (ABI, Waltham, MA, USA) in 25 µL reactions containing 4 µL of 5× FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of forward and reverse primers (5 µM), 0.4 µL of FastPfu Polymerase (Transgen Biotech, Beijing, China), 1.0 µL of template DNA (10 ng), and 16 µL of ddH2O. The PCR conditions were as follows: an initial denaturation for 3 min at 94 °C, followed by 35 amplification cycles consisting of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C, and a final extension for 10 min at 72 °C for the ITS region. Each composite sample was amplified in triplicate. The PCR products were pooled and checked on a 2% agarose gel under UV light, then purified using the AxyPrep™ DNA Gel Purification Kit (AXYGEN, Union City, CA, USA). Based on preliminary agarose gel results, the PCR products were quantified using the QuantiFluor™–ST Blue Fluorescence Quantitation System (Promega, Madison, WI, USA) and mixed according to the sequencing requirements for each sample. The mixture was then paired-end sequenced (2 × 300 bp) on an Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

2.9. Raw Sequence Data Processing of Fungi

The raw sequence data were quality filtered and processed using FLASH [69]. Reads were clustered into Operational Taxonomic Units (OTUs) using the UPARSE pipeline at a 97% similarity threshold [70]. Chimeras were detected and removed using USEARCH 6.1 in QIIME [71]. Fungal taxonomy was assigned using the UNITE 9.0 ITS gene database (https://unite.ut.ee, accessed on 30 April 2024), and OTUs were used for downstream analysis. Negative controls (extraction/PCR) were included and screened for contamination (decontam). To control for sequencing depth bias, all samples were subsampled to the minimum sequencing depth of the study.

2.10. Statistical Analysis

Data were analyzed using Microsoft Excel 2021, SPSS 26.0, and Origin 2021 for mapping. Plant cadmium (Cd) concentration and soil Cd content were subjected to one-way ANOVA at a 5% significance level (p < 0.05). Tukey’s HSD test was applied for pairwise comparisons (p < 0.05). Normality and homogeneity of variance were verified using the Shapiro–Wilk and Levene’s tests, respectively. Soil microbial community alpha diversity, including ACE, Chao, Shannon, and Simpson indices, was calculated using QIIME [71]. A heatmap of the top 50 classified genera across all samples was generated using the gplot package in R [72]. For beta diversity analysis, fungal community composition was evaluated using Principal Coordinates Analysis (PCoA) based on Bray–Curtis dissimilarity metrics. To assess differences in community structure, three complementary non-parametric multivariate statistical tests were employed: Analysis of Similarities (ANOSIM), permutational multivariate analysis of variance (PERMANOVA, implemented via the adonis function), and the multiple response permutation procedure (MRPP). All tests were conducted using Bray–Curtis distance matrices with 999 permutations to ensure robust statistical inference. Taxon accumulation curves, PCoA, ANOSIM, PERMANOVA, and MRPP analyses were carried out using the “vegan” package in R (version 3.3.1) [73]. Differential OTU enrichment was visualized using volcano plots, created with the “ggplot2” and “ggrepel” packages in R [74].

3. Results

3.1. Effects of AMF and MHB Co-Inoculation on Plant Cd Uptake and Soil Cd Content

The plant Cd concentration (mg kg^−1^) in (Figure 1A) shows that the FA treatment significantly reduced Cd accumulation in cucumber plants compared to all other treatments (p < 0.05). In contrast, the CK treatment significantly increased the Cd concentration in plants, confirming that the absence of inoculation leads to greater metal uptake. The Fm treatment (AMF) also decreased plant Cd concentrations, although the reduction was less pronounced than with the FA treatment, emphasizing the synergistic effect of combining AMF and MHB.

The soil Cd content (mg kg), shown in (Figure 1B), reveals that the FA treatment significantly reduced soil Cd levels (p < 0.05), indicating that the co-inoculation of AMF and MHB not only reduces Cd uptake by plants but also decreases Cd availability in the soil. In contrast, the CK treatment exhibited the highest soil Cd content, confirming that, without microbial inoculation, Cd remains bioavailable in the soil. The Fm treatment (AMF) also contributed to a reduction in soil Cd content, but its effect was not as pronounced as that of the FA treatment.

3.2. Effect of AMF and MHB Inoculation on Rhizosphere Fungal Community Composition

High-throughput MiSeq sequencing was used to analyze the fungal communities in cucumber rhizosphere soil, with sequence data clustered at a 97% similarity threshold. A total of eight fungal phyla were identified. The predominant phyla in the cucumber rhizosphere were Ascomycota, Mortierellomycota, and Basidiomycota, collectively comprising over 85% of the fungal sequences, with an average relative abundance exceeding 1%. Specifically, the relative abundance of Ascomycota ranged from 77% to 85%, while Mortierellomycota ranged from 2.34% to 5.23% (Figure 2A). When compared to the control (CK), both the AMF and MHB (FA) co-inoculation and the AMF (Fm) single inoculation treatments significantly increased the relative abundance of Mortierellomycota, Basidiomycota, and Glomeromycota in the cucumber rhizosphere. Notably, the relative abundance of these phyla was significantly higher in the Fm treatment compared to the FA treatment (p < 0.05) (Figure 2B). However, both the FA and Af treatments led to a significant reduction in the relative abundance of Ascomycota (p < 0.05).

At the class level, a total of 18 fungal classes were identified, with nine classes showing an average relative abundance greater than 1% (Figure 2C). The dominant classes, Eurotiomycetes and Sordariomycetes, together accounted for over 59% of the fungal sequences, each with an average relative abundance exceeding 17%. The relative abundance of Eurotiomycetes ranged from 30.01% to 52.42%, while Sordariomycetes ranged from 17.69% to 31.96%. In the MHB (Af) treatment, the relative abundance of Eurotiomycetes in the cucumber rhizosphere was significantly higher compared to the other treatments (p < 0.05). In contrast, the AMF and MHB (FA) treatment significantly increased the relative abundance of Sordariomycetes and Leotiomycetes, while significantly decreasing the abundance of Dothideomycetes, Pezizomycetes, and Paraglomeromycetes (p < 0.05) (Figure 2D).

3.3. Effect of AMF and MHB Inoculation on Rhizosphere Fungal Community Composition at Genus Level

At the genus level, a total of 105 fungal genera were detected. The heatmap illustrates the distribution of the top 50 fungal genera and their relative abundance across treatments (Figure 3), while the table presents the relative abundance of these top 50 genera in the cucumber rhizosphere (Table S1). Among these, Thermomyces, Aspergillus and Mycothermus were the dominant genera, with relative abundances ranging from 11.86% to 36.51%, 8.12% to 11.74%, and 5.56% to 9.30%, respectively. Compared to the control (CK), the AMF and MHB (FA) treatment significantly increased the relative abundance of Mycothermus, Podosphaera, Talaromyces, Albifimbria, Penicillium and Cladorrhinum, while significantly reducing the relative abundance of Thermomyces, Aspergillus, Cladosporium, Lasiobolus, Paraglomus, Poaceascoma and Botryotrichum (p < 0.05). The AMF (Fm) treatment significantly increased the relative abundance of Mortierella, Lasiobolus, Paraglomus and Podospora, with their relative abundances being significantly higher than those in the MHB (Af) and FA treatments (p < 0.05) (Table S1).

3.4. Effect of AMF and MHB Inoculation on Rhizosphere Fungal Community Composition at OTUs Level

A total of 383 fungal OTUs were detected in the cucumber rhizosphere soil. Based on DESeq2 (Benjamini–Hochberg correction, p < 0.01), volcano plots and Manhattan analysis revealed that, compared to the control (CK), the Fm treatment significantly enriched 98 OTUs, primarily concentrated in Basidiomycota, Ascomycota, Glomeromycota, Mortierellomycota, and Chytridiomycota. The significantly enriched OTUs included: OTU41 (0.00% vs. 1.70%), OTU50 (0.00% vs. 1.59%), OTU26 (1.07% vs. 5.10%), OTU57 (0.00% vs. 0.10%), and OTU65 (0.00% vs. 1.03%), which belong to unclassified_Sebacinales, Aspergillus, Paraglomus, Remersonia and unclassified_Sordariales. The significantly depleted OTUs included: OTU28 (Poaceascoma), OTU6 (unclassified Fungi), OTU22 (Cladosporium), OTU116 (Fungi_gen_Incertae_sedis), and OTU39 (Humicola) (Figure 4a,b, Table S2).

Compared to CK, the Af treatment significantly enriched 92 OTUs, mainly concentrated in Ascomycota, Basidiomycota, and Kickxellomycota. The significantly enriched OTUs belong to Botryoderma, unclassified_Sebacinales, Ascobolus, unclassified_Sordariales, and Serendipita (Figure 4c,d, Table S2). Compared to CK, the FA treatment significantly enriched 90 OTUs, mainly concentrated in Ascomycota, Basidiomycota, and Mortierellomycota. The significantly enriched OTUs belong to unclassified_Sordariales, Cladorrhinum, Podosphaera, Botryoderma and Cercophora (Figure 4e,f, Table S2). Compared to the Fm treatment, the FA treatment significantly enriched 86 OTUs, primarily concentrated in Ascomycota, Basidiomycota, and Mortierellomycota. The significantly enriched OTUs belong to Cladorrhinum, unclassified_Sordariales, Ascobolus, Penicillium and Mortierellales_gen_Incertae_sedis (Figure 4g,h, Table S2). Compared to the FA treatment, the Af treatment significantly enriched 93 OTUs, mainly concentrated in Ascomycota, Basidiomycota, and Kickxellomycota. The significantly enriched OTUs belong to Poaceascoma, Fungi_gen_Incertae_sedis, Westerdykella, unclassified_Fungi and Fusarium (Figure 4i,j, Table S2). Compared to the Fm treatment, the Af treatment significantly enriched 94 OTUs, mainly concentrated in Ascomycota, Basidiomycota, and Kickxellomycota. The significantly enriched OTUs belong to Thermomyces, Parabambusicolaceae_gen_Incertae_sedis, Poaceascoma, Penicillium and unclassified_Sordariales (Figure 4k,l, Table S2).

3.5. Effect of AMF and MHB Inoculation on Rhizosphere Fungal Community Diversity and Structural Composition

The fungal community’s ITS rRNA region was analyzed from 12 soil samples using Illumina MiSeq high-throughput sequencing. After quality filtering, a total of 962,564 sequences were obtained, with the shortest sequence length being 74,773. Diversity calculations were performed after normalizing to the minimum sequence count (796). The fungal community coverage in this study exceeded 99%, accurately reflecting the true composition of the fungal community in the soil. AMF and MHB inoculation (FA treatment) significantly reduced the Chao1 index of the cucumber rhizosphere fungal community compared to other treatments (p < 0.05) (Figure 5A). The FA treatment also significantly decreased the OTU count in the cucumber rhizosphere compared to the single inoculation treatments (Fm and Af), with no significant difference observed when compared to the control (CK) (Figure 5B). Compared to CK, all inoculation treatments significantly increased the Shannon and Simpson indices of the cucumber rhizosphere fungal community. The Shannon index was significantly higher for the Fm treatment compared to the other treatments, while the Simpson index was significantly higher for the FA treatment than for all other treatments (p < 0.05) (Figure 5C,D).

Principal Coordinates Analysis (PCoA) revealed a clear separation of soil samples by treatment, with samples from the same treatment grouping together, while distinct separation was observed between the four treatment groups (Figure 5E). Non-parametric multivariate statistical tests further supported these findings, indicating significant differences in the fungal community composition of the cucumber rhizosphere across treatments. Specifically, ANOSIM yielded a strong separation (R = 1.000, p = 0.001), PERMANOVA (adonis) demonstrated a moderate effect size (R^2^ = 0.372, p = 0.003), and MRPP showed a notable difference in group separation (Delta = 0.172, effect size = 0.281, p = 0.001).

4. Discussion

Our results indicate that co-inoculation with Funneliformis mosseae (AMF) and Alcaligenes faecalis (MHB) significantly reduced cadmium concentrations in both plant tissues and soil. This aligns with previous studies showing the role of AMF in heavy metal sequestration [15], and the potential of MHB in enhancing soil microbial communities to aid in metal detoxification [29]. This study highlights the mechanisms through which AMF and MHB interact to alter the rhizosphere microbial community and reduce Cd bioavailability. The findings support both of the hypotheses proposed: (1) Co-inoculation of AMF and MHB reduces Cd concentrations in both plant tissues and soil, and (2) co-inoculation of AMF and MHB alters the fungal community structure in the cucumber rhizosphere, potentially enriching specific beneficial guilds while modifying the relative abundance of other fungal groups, including those with pathogenic potential.

One key mechanism through which AMF and MHB co-inoculation reduces Cd concentrations in both plant tissues and soil is the “dilution effect”. By increasing nutrient availability and water uptake, AMF reduce the toxic effects of Cd on plant tissues [2]. AMF extend their root networks through hyphal filaments, which increase the surface area for nutrient uptake and contribute to phosphorus cycling, particularly under metal stress [62]. AMF also secrete glomalin-related soil proteins (GRSPs), which play a crucial role in metal sequestration and immobilize Cd in the rhizosphere, making it less bioavailable to plants [32,75,76]. These compounds also serve as carbon sources for phosphate-solubilizing bacteria, enhancing phosphatase activity and promoting phosphorus cycling in the rhizosphere [77,78]. Additionally, AMF overcome the inhibition of phosphorus cycling caused by heavy metals by enhancing acid phosphatase (ACP) activity, which helps utilize organic phosphorus, particularly in phosphorus-deficient environments [79]. This creates a positive feedback loop, where increased microbial activity accelerates nutrient availability and contaminant degradation, ultimately reducing Cd translocation to the aboveground plant parts. This finding aligns with other studies showing that AMF mitigate Cd toxicity through metal binding and soil structure enhancement [80,81,82].

The addition of MHB further complements this process by promoting beneficial microorganisms that enhance the microbial community in the rhizosphere [55,56]. MHB contribute to metal detoxification through biosorption and biotransformation processes, where they either adsorb or alter the chemical form of Cd, rendering it less toxic to plants [83,84,85,86]. Additionally, MHB produce phytohormones such as auxins, enhancing the plant’s ability to absorb essential nutrients and reduce the bioavailability of Cd in the rhizosphere [33,87].

AMF reduce the availability of Cd in the soil through various mechanisms such as chelation, immobilization in fungal biomass, organic matter stabilization, and sequestration in fungal vacuoles [32,37,75,76,88]. These processes decrease the availability of Cd in the rhizosphere, limiting its uptake by plants. In addition to the direct metal binding by AMF, the network of AMF hyphal filaments improves soil structure and enhances microbial interactions that further reduce Cd bioavailability [44,49]. MHB also play a vital role in Cd immobilization by secreting extracellular polymeric substances (EPSs), which bind Cd ions, reducing their bioavailability and preventing their uptake by plants [25]. By modifying soil pH and microbial composition, MHB further enhance the soil’s ability to retain Cd in less bioavailable forms, thereby reducing its mobility and minimizing its harmful effects on plant health [45,55,89]. This synergistic interaction between AMF and MHB improves soil quality and microbial resilience, thereby collectively reducing Cd accumulation in both plant tissues and soil.

The co-inoculation of AMF and MHB also reshapes the fungal community in the cucumber rhizosphere. The results not only demonstrate a reduction in Cd levels in both plants and soil, but also highlight the mechanisms by which these microbial inoculants alter the soil fungal community. Co-inoculation significantly increased the relative abundance of Mortierellomycota, Basidiomycota, and Glomeromycota, all of which play crucial roles in nutrient cycling and metal detoxification [45]. While our results indicate that co-inoculation with Funneliformis mosseae (AMF) and Alcaligenes faecalis (MHB) led to changes in the rhizosphere fungal community, we observed that the Fm treatment had a significantly higher relative abundance of Mortierellomycota, Basidiomycota, and Glomeromycota compared to the FA (co-inoculation) treatment. This discrepancy suggests that, while co-inoculation generally improved plant performance, the single AMF inoculation may have provided a more favorable environment for these specific beneficial phyla. The reasons for this could include differences in microbial competition, resource availability, or functional synergy between AMF and MHB under the given experimental conditions. In contrast, the relative abundance of Ascomycota, a potentially harmful group of fungi associated with metal tolerance, was significantly reduced [90]. In this study, we observed that the Ascomycota phylum, which includes well-known pathogenic genera such as Alternaria and Fusarium, was associated with metal tolerance and plant stress [91,92]. These fungi are often linked to plant diseases and can negatively impact plant health, especially under conditions of heavy metal contamination [91,93]. While Ascomycota is a highly diverse phylum, the specific genera detected in our study are pathogenic and contribute to the detrimental effects of Cd on plant growth. Thus, the reduction in Ascomycota abundance in the rhizosphere, especially the Alternaria and Fusarium genera, is likely indicative of improved soil health and a shift away from pathogenic taxa, in line with the detoxification and remediation potential of the microbial inoculants used [94]. This shift in microbial composition suggests that AMF and MHB not only promote beneficial fungi but also suppress pathogenic fungi, as predicted by the second hypothesis. These results are consistent with previous studies showing that microbial inoculants can promote a more balanced and beneficial rhizosphere microbiome under heavy metal stress, ultimately improving soil health [45,46,47]. AMF colonization stimulates the exudation of compounds from plant roots, which can directly chelate contaminants like Cd and enrich beneficial microbial taxa that contribute to better plant growth by reducing Cd accumulation [17,32,76].

In our study, we did not observe any detrimental effects of co-inoculation between Funneliformis mosseae and Alcaligenes faecalis, as both inoculations led to improved cadmium tolerance and reduced Cd uptake by the cucumber plants. While a dedicated co-cultivation experiment was not conducted in this study, the previous literature supports the idea that these organisms often work synergistically to promote plant growth and metal detoxification. For example, studies have reported that AMF and MHB frequently complement each other in enhancing plant resistance to abiotic stress [29]. While the combined inoculation of AMF (Funneliformis mosseae) and MHB (Alcaligenes faecalis) (FA) generally led to improvements in cadmium (Cd) tolerance and microbial community composition, it is important to note that, in some cases, the individual inoculations (Fm and Af) resulted in stronger effects than the co-inoculation treatment. Specifically, the Fm treatment (AMF inoculation) sometimes led to higher increases in fungal diversity and more substantial reductions in Cd levels compared to the FA treatment. These observations may be influenced by several factors, including the specific interactions between Funneliformis mosseae and Alcaligenes faecalis in the rhizosphere, as well as the complexity of the microbial community dynamics. It is possible that the combined inoculation did not achieve a stronger effect in all cases due to competition or other interaction dynamics between AMF and MHB in the rhizosphere. Previous studies have suggested that, while AMF and MHB can act synergistically in some instances, their combined effects may not always exceed the sum of their individual benefits due to factors such as resource competition, differences in optimal growth conditions for each microorganism, or microbial community imbalances. Moreover, the effectiveness of the FA treatment may also depend on the soil type, microbial community composition, and environmental conditions, which could vary between experimental setups and contribute to variability in the results [95,96]. Despite these nuances, the overall trend observed in this study still supports the hypothesis that AMF and MHB can synergistically enhance plant resilience to Cd stress. However, future studies with more rigorous experimental controls and longer experimental durations could provide further insights into the underlying mechanisms and conditions that influence the success of co-inoculation treatments.

The network of hyphal filaments not only extends plant roots but also facilitates microbial interactions, thereby enriching specific microbial taxa that can mitigate Cd toxicity. This selective microbial enrichment promotes microbial functional diversification, enabling the soil ecosystem to better degrade contaminants and rebuild resilience [89]. For example, AMF preferentially enriched the rhizosphere fungal community characteristic of Acorus calamus, to improve Cr resistance [97]. This specialized enrichment promotes microbial functional diversification and improves the metabolic capacity to detoxify harmful substances in the rhizosphere, a mechanism that likely applies to Cd detoxification as well. In the case of Cr stress, AMF inoculation in Iris tectorum promoted the restructuring of the rhizosphere microbial community, with a notable enrichment of Proteobacteria [78]. Similarly, in Iris pseudacorus AMF combined with the remediation of organic pollutants such as hexabromocyclododecane (HBCD), the same pattern of microbial community restructuring was observed, further emphasizing the role of AMF in altering the microbial community in contaminated soils [98]. MHB further enhance the effectiveness of AMF by promoting their colonization and increasing the abundance of beneficial microorganisms in the rhizosphere [55,56]. MHB secrete metabolites that stimulate AMF hyphal growth, which further enhances the microbial community’s ability to promote plant growth by reducing Cd level in both plant tissues and soil [33,87]. By promoting the colonization of these beneficial microorganisms, MHB help to suppress pathogenic species that might otherwise thrive in Cd-contaminated soils, further improving the microbial balance and resilience of the rhizosphere [53,54,55].

Although the co-inoculation with Alcaligenes faecalis did enhance certain fungal taxa, the expected synergy in reducing Cd concentrations was not as clear-cut. It is possible that the competition for nutrients between AMF and MHB in the rhizosphere may have limited the extent of their combined effects. Previous research has shown that, under certain conditions, the presence of multiple microbial species can lead to resource competition rather than complementary benefits [99]. Future studies should explore this aspect further by adjusting inoculum ratios and testing different microbial consortia.

One limitation of our study is that the experimental duration might not have been long enough to fully capture the long-term effects of co-inoculation. While our results demonstrate the initial impacts on Cd concentrations and fungal community structure, longer-term studies are required to assess whether these effects persist or diminish over time, especially in the context of field conditions. Additionally, while we used synthetic Cd contamination, the bioavailability of heavy metals can vary significantly in natural contaminated soils, which could influence the effectiveness of AMF and MHB. To gain deeper insights into the mechanisms of AMF–MHB synergy, future research should consider the bioavailability of heavy metals, as soil characteristics like pH, organic matter content, and microbial community composition can greatly influence microbial activity. Additionally, conducting co-cultivation experiments could shed light on potential antagonistic interactions between AMF and MHB, and whether their combined effects on the rhizosphere are truly synergistic or influenced by external factors.

The richness and diversity of soil microorganisms are key indicators of soil quality and plant adaptability, as a wide range of microorganisms regulate soil function and physicochemical properties [48,100]. Our study demonstrated that the diversity of rhizosphere soil fungal communities was significantly higher in the AMF and MHB co-inoculation treatment compared to the other Cd-exposed treatment. Similar results were obtained in Cd-contaminated soil, where inoculation with AM fungi increased bacterial diversity in the rhizosphere soil of Solanum nigrum [46]. The microbial diversity observed in our study likely reflects an increase in the functional diversity of the microbial community, which improves the stability of soil ecosystems under metal stress [101]. This increase in diversity might contribute to a feedback loop that enhances both the stability of the rhizosphere microbial community and its ability to detoxify contaminants, such as Cd, in the long term. The hyphal network of AMF not only improves nutrient availability but also facilitates the migration of bacteria that contribute to Cd sequestration. Moreover, the EPS produced by MHB not only help in metal binding but also enhance soil fertility by improving soil structure and nutrient availability. This collaborative effect between AMF and MHB leads to a reduction in Cd toxicity and an increase in the stability and diversity of the microbial community in the rhizosphere.

We also found a statistically significant decrease in the Chao1 richness index in co-inoculation treatment (FA) compared to the control (CK). The Chao1 index is sensitive to the number of observed species, and this reduction may indicate that the co-inoculation treatment led to a shift in community structure, favoring certain microbial taxa over others. This could be due to microbial niche competition between AMF, MHB, and native microbes, or a selective enrichment of specific beneficial groups, such as Glomeromycota and Basidiomycota, at the expense of others [46,102]. Furthermore, cadmium contamination may have played a role in this shift, with some microbial species more tolerant of the metal stress induced by the inoculants, while others were suppressed. Although the richness index decreased, the overall shift in community composition suggests that the inoculants are enhancing the resilience and functionality of the rhizosphere, even if it is at the cost of a reduced number of species [43,47,103].

The proposed method of using AMF (Funneliformis mosseae) and MHB (Alcaligenes faecalis) for reducing cadmium contamination in agricultural soils offers a potentially cost-effective alternative to traditional remediation techniques. Traditional methods, such as chemical amendments or physical removal of contaminated soil, are often expensive, energy-intensive, and environmentally unsustainable [16,17,18]. In contrast, bioremediation using microorganisms, particularly through plant–microbiome interactions, presents a more sustainable and economically viable solution [21,22]. The cost of AMF and MHB inoculation is relatively low compared to conventional remediation methods, as the production of these microbial inoculants is well-established, and their application requires minimal infrastructure [23,24]. Furthermore, this method has the added advantage of enhancing plant growth and soil health, which could lead to increased agricultural productivity over time [15]. By improving plant resilience to heavy metal stress, the method could potentially reduce crop losses due to cadmium contamination, resulting in long-term economic benefits for farmers [55,56]. Additionally, the use of bioremediation strategies could reduce the need for costly chemical treatments and mitigate the long-term environmental damage associated with traditional approaches [16]. However, the overall economic feasibility of this method in large-scale agricultural settings depends on factors such as the cost of microbial inoculants, their effectiveness across different soil types, and the scale of contamination [21,22,23,24]. Future studies that include a cost–benefit analysis would help provide a more comprehensive understanding of the economic viability of this approach in real-world applications.

5. Conclusions

This study demonstrates that co-inoculation with AMF and MHB is an effective strategy for reducing cadmium (Cd) contamination in agricultural soils. The dual inoculation significantly lowered Cd concentrations in both cucumber plant tissues and soil. This reduction in Cd levels was accompanied by a shift in the rhizosphere fungal community composition, enriching beneficial phyla (Mortierellomycota, Basidiomycota, and Glomeromycota) and reducing the relative abundance of potentially pathogenic genera within the Ascomycota phylum, such as Alternaria and Fusarium. These changes in the fungal community were associated with reduced bioavailability of Cd in the soil and lower Cd uptake by plants. However, the biochemical mechanisms underlying these effects, including the roles of specific genes, enzymes, or root exudates, were not fully elucidated in this study. Further research is needed to explore these mechanisms in greater detail.