Microbiome–metabolite signaling drives aluminum stress alleviation in soybean under intercropping and selenium nanoparticle application

ABSTRACT

Aluminum (Al) toxicity and soil-borne pathogens severely constrain legume productivity in acidic soils, yet the signaling mechanisms underlying intercropping-mediated stress alleviation remain insufficiently understood. Here, we investigated whether soybean-sorghum intercropping under Al stress (SSAl), alone or combined with selenium nanoparticles (SSAl + Se), modulates rhizosphere signaling networks and plant defense responses. Integrated 16S rRNA microbiome sequencing and rhizosphere metabolomics were employed to decipher microbe‒metabolite interactions associated with Al detoxification and disease suppression. Compared with monoculture soybean under Al stress (MSAl), SSAl and SSAl + Se significantly reduced Al accumulation (16.9% and 57.4%, respectively) and Fusarium wilt incidence (10.9% and 34.4%, respectively), accompanied by enhanced root growth. These treatments attenuated oxidative stress, as evidenced by decreased O₂⁻, H₂O₂, and malondialdehyde (MDA) levels, while stimulating antioxidant enzyme activities (SOD, POD, and APX), indicating reinforcement of redox homeostasis. In the rhizosphere, NH₄⁺–N and available K levels increased, with NH₄⁺–N positively correlated with urease activity and negatively correlated with Al accumulation, suggesting nitrogen-mediated modulation of Al dynamics. Microbiome analysis revealed enrichment of beneficial taxa, including Streptomyces, Intrasporangium, and Sphingomonas, which are positively associated with antimicrobial and stress-related metabolites such as 15-methyl palmitate, lactucin, and sordarin. These coordinated shifts in the microbial community structure and metabolite profiles indicate that the activation of rhizosphere chemical signaling restricts pathogen proliferation and enhances Al detoxification. Collectively, our findings demonstrate that selenium nanoparticles potentiate intercropping-induced rhizosphere reprogramming, linking redox regulation, nitrogen transformation, and microbiome–metabolite signaling to improve aluminum stress tolerance and disease resistance in soybean. This study provides mechanistic insight into how nano-enabled agronomic strategies influence plant signaling networks under edaphic stress.

Graphical abstract

Introduction

Soil is essential for sustaining crop vitality and guaranteeing the consistent output of agricultural goods. Aluminum (Al) contamination in cropland and soil-borne pathogens are prevalent biotic and abiotic stressors that impact agriculture worldwide.¹ The main sources of Al in agricultural soil are soil acidification, acid rain, and overuse of N fertilizers.² Plants can efficiently absorb and build up Al via their root system, even at lower levels, resulting in detrimental impacts on plant growth and threatening farming and food safety.³ Furthermore, Fusarium can readily build in soil subjected to continual cropping, resulting in frequent cases of soil-borne disorders in diverse crops, including cucumber, beans, and tomato.^4,5 With the growth of the global population and the progression of urbanization, the supply of arable land has become progressively constrained, resulting in extensive intensive cultivation. Consequently, to tackle these difficulties, sustainable agricultural practices, including intercropping, have garnered considerable attention because of their capacity to improve crop tolerance and soil health.

Soybean is a significant and extensively farmed leguminous crop worldwide, principally owing to its substantial nutritional value and adaptability. Abundant in proteins, oils, and vital amino acids are pivotal for fulfilling global demands for food and feed.⁶ As a vital source of protein and vegetable oil, soybean is essential for human and animal nutrition. Its cultivation is essential for global food security, particularly in regions such as Asia, where it underpins both direct consumption.⁷ Soybeans are a crucial commodity for biofuel production and have various industrial applications, including the manufacture of biodiesel, polymers, and pharmaceuticals. The global significance of soybeans is escalating due to rising demand driven by population growth, dietary changes, and sustainable agricultural methods.^8,9 While soybean is not a conventional hyperaccumulator of metals and can significantly impede its development, manufacturing efficiency, and sustainability. Prior research indicates that intercropping can efficiently manage Fusarium wilt in beans and increase crop output.^10,11 Moreover, techniques using advantageous components, such as selenium, may contribute to mitigating this strain. Nonetheless, there are no documented findings about the potential of intercropping soybean with sorghum under Al stress to mitigate Al toxicity and manage soybean wilt to promote growth.

While selenium is not classified as an essential element for crops, it is acknowledged as a useful component in the majority of plants.^12,13 Plants exhibit selenium stress resistance through their capacity to alleviate abiotic stress, including metal toxicity, as well as their distinctive protective mechanisms and robust adhesion features. Selenium nanoparticles (SeNPs) have been used in agriculture because of their diminutive size, extensive SSA, biocompatibility and stability, in contrast to traditional selenium products.¹⁴ Research has shown that SeNPs not only stimulate crop growth but also augment seed vitality, elevate the activity of enzymes, enhance root development, improve fertilizer and WUE, accelerate metabolic processes, and bolster resilience to pests and diseases, thus increasing crop quality and yield. These data indicate that SeNPs are effective alternatives for mitigating Al toxicity and enhancing plant development. Considering that plants subjected to abiotic and biotic challenges frequently demonstrate inhibited development and diminished systemic resilience, it is essential to investigate the potential of SeNPs to alleviate these impacts, especially regarding Fusarium wilt and Al toxicity. However, SeNPs ability to simultaneously influence metabolic pathways in soybean and microbes in rhizosphere, thus alleviating Al toxicity and improving resilience to Fusarium wilt, remains inadequately understood. The potential of exogenous SeNPs to improve interspecific root contact in intercropped soybean and subsequently stimulate its growth remains questionable. Moreover, the mechanisms via which rhizosphere microbe metabolite interactions may contribute to these processes remain unclear.

This work sought to clarify the impacts of SeNPs and intercropping on Al toxicity and resilience to soybean Fusarium wilt under Fusarium and aluminum stress, along with interaction mechanisms among rhizosphere microbes and metabolites. A pot study was conducted with foliar application of SeNPs to ascertain that (a) the application of SeNPs and intercropping mitigated Al toxicity and the incidence of Fusarium wilt in soybean, (b) intercropping and SeNPs augmented the systemic defense mechanisms and antioxidant capacity of soybean, (c) intercropping and SeNPs elevated the NH₄⁺−N concentration and associated enzymatic activities in the soybean rhizosphere, and (d) the rhizosphere association of the microbiota and metabolome in soybean elucidated the significant impacts of SeNPs application and intercropping. These outcomes establish a theoretical foundation for employing SeNPs and intercropping in farming to increase soil fertility, manage disease, and augment output.

Materials and methods

Experimental site and materials

An experiment using soil-filled pots was conducted in the greenhouse of Yunnan University, which is located at 25.0533° N and 102.7035° E. This study was carried out under natural light conditions with an approximate photoperiod of 12 hours. The temperature inside the greenhouse ranged from 19 °C to 29 °C, while the relative humidity was maintained between 45% and 55%. The experiment ran from late September 2024 to late December 2025. Artificially cultivated soil was used to ensure uniformity and control over the soil properties. The experimental site has a long history of soybean cultivation, which has led to the accumulation of soil-borne Fusarium pathogens and the frequent occurrence of Fusarium wilt.

Before the experiment, soil was collected from the top 0 to 20 cm layer, air dried, cleared of debris, and passed through a 2 mm sieve to ensure homogeneity. The soil physicochemical properties were analyzed before use. The soil had a pH of 7.2, total nitrogen content of 1.7 g kg⁻¹, organic matter content of 23.9 g kg⁻¹, total phosphorus content of 0.7 g kg⁻¹, and total potassium content of 13.1 g kg⁻¹. The available nutrient fractions included alkali nitrogen at 99.7 mg kg⁻¹, Olsen phosphorus at 17.1 mg kg⁻¹, and available potassium at 120.3 mg kg⁻¹. The baseline aluminum concentration was 0.080 mg kg⁻¹ in the form of Al³⁺. To ensure a consistent nutrient supply across treatments, fertilizers were applied uniformly at the time of pot preparation using potassium sulfate with 50% K₂O, superphosphate with 16% P₂O₅, and urea with 47% nitrogen content.

Aluminum stress was imposed by adding aluminum chloride to the soil at a controlled rate to simulate Al-contaminated conditions. Selenium nanoparticles were synthesized using a microwave-assisted method, which produced particles with high stability, uniform dispersion, and strong bioavailability. These nanoparticles were selected because of their known ability to interact efficiently with plant systems and soil environments. The SeNPs were evenly mixed into the soil prior to pot filling to ensure a uniform distribution.

Two crop species were used in this study. The soybean cultivar NARC II was selected as the test crop because of its agronomic importance and sensitivity to both aluminum stress and Fusarium wilt. The sorghum cultivar sorghum (2011) was used as the intercrop species because of its known compatibility with soybean and its ability to influence rhizosphere interactions. Seeds of both crops were carefully selected for uniform size, shape, and health and then surface sterilized and pregerminated before sowing to ensure consistent plant establishment across all the treatments.

All the pots were filled with equal amounts of prepared soil and arranged in a completely randomized design inside the greenhouse. The soil moisture was maintained at approximately 60% to 70% of the field capacity through regular irrigation with demineralized water. Pots were repositioned periodically to minimize positional effects within the greenhouse. Standard agronomic practices were followed throughout the experiment to maintain uniform growth conditions. This controlled setup allowed precise evaluation of aluminum stress, intercropping effects, and selenium nanoparticle application on soybean growth, rhizosphere processes, and disease resistance.

Crop management and experimental setup

The experiment was designed using a single-factor layout with three treatment systems to evaluate the effects of intercropping and selenium nanoparticles under aluminum stress. The treatments included monocropped soybean under aluminum stress (MSAl), soybean sorghum intercropping under aluminum stress (SSAl), and soybean sorghum intercropping under aluminum stress combined with selenium nanoparticles (SSAl plus Se). In the monocropping system, each pot contained three rows of soybean with three plants per row. Plant spacing was maintained at 10 cm between plants and 8 cm between rows to ensure uniform growth conditions. In the intercropping system, a strip arrangement was adopted where one row of soybean with three plants was followed by three rows of sorghum with six plants per row and then another row of soybean with three plants. This configuration was selected to maximize root interaction and rhizosphere overlap between the two species. Each treatment was replicated three times, resulting in a total of nine experimental pots arranged in a completely randomized design.

Topsoil from 0 to 20 cm layer was collected from the field, air dried in the greenhouse for one week, and cleared of debris such as stones and plant residues. The soil was then sieved through a 2 mm mesh to ensure homogeneity. Each pot was filled with 9 kg of prepared soil. Aluminum stress was imposed by adding aluminum chloride at a concentration equivalent to 2 mg kg⁻¹ Al³⁺. For each pot, 36.61 mg of AlCl₃ was thoroughly mixed into the soil to achieve a uniform distribution. After mixing, the soil was incubated for four weeks at approximately 60% of field capacity to allow equilibration and stabilization of aluminum within the soil matrix, thereby simulating realistic aluminum-contaminated conditions.

Selenium nanoparticles were prepared in demineralized water and evenly incorporated into the soil at a concentration of 250 mg kg⁻¹ prior to pot filling in the designated treatment. Care was taken to ensure uniform dispersion of the nanoparticles throughout the soil to avoid localized concentration effects. To eliminate variability arising from nutrient availability, all the treatments received the same basal fertilization at the time of pot preparation. Nitrogen, phosphorus, and potassium were applied at equal rates of 100 mg kg⁻¹ using urea, superphosphate, and potassium sulfate, respectively.

Soybean and sorghum seeds were carefully selected for uniform size, shape, and physical integrity to ensure consistent germination. Seeds were surface sterilized using 10% hydrogen peroxide for 30 minutes to remove surface contaminants, followed by thorough rinsing with demineralized water. The sterilized seeds were then pregerminated in darkness for 3 d to achieve uniform emergence before sowing.

All the pots were maintained under controlled greenhouse conditions with temperatures ranging from 19 °C to 29 °C and a relative humidity of around 50%. Pots were randomly arranged and repositioned every 3 d to minimize positional effects and ensure equal exposure to environmental conditions. Irrigation was carried out twice daily using demineralized water, with 200 to 250 mL applied per pot to maintain the soil moisture at 60% to 70% of the field capacity. Throughout the experiment, standard agronomic practices were followed to maintain consistent growth conditions and minimize external variability. This controlled experimental setup allowed precise evaluation of treatment effects on plant growth, rhizosphere processes, and stress responses under aluminum toxicity.

Soil and plant sampling

Following 3 months of post-cultivation, 2 plants were chosen for destructive testing. The whole soybean plant was meticulously extracted from pot, and the agronomic characteristics of the aerial components were promptly documented, alongside assessment of soybean Fusarium wilt as well as biomass assessment (dried at 105 °C for 20 minutes and subsequently at 70 °C until a consistent weight was achieved). Rhizosphere soil was obtained with “shaking soil technique.”¹⁵ Plant roots were initially cleaned with demineralized water, and crops were subsequently segmented into distinct organs (aerial portions and roots) utilizing scissors. Meticulous attention was devoted to preserving root structure integrity. The roots were subsequently immersed in 20 mM EDTA-2Na for 15 minutes to eliminate the Al and Se ions adhering to the surface, followed by extensive washing with demineralized water. Roots were subsequently created for root scan examination and assessment of defense chemicals and enzymatic activities. The collected rhizosphere soil specimen was sieved using 2-mm mesh and partitioned into 2 segments. One portion was utilized to assess conventional physicochemical parameters and the aluminum concentration of the soil, with the remaining portion was preserved in a sterilized container at −75 °C for metabolomic and bacterial investigations. The collection technique adhered rigorously to sterilized operating protocols to avert pollution.

Indicator measurement and techniques

Analysis of soybean Fusarium wilt

An analysis of wilt disease was carried out on all soybean plants following the removal of their above-ground portions. The soybean wilt categorization conditions according to five-tier classification criteria.¹⁶

$$\mathrm{Disease}\mathrm{index}={\sum }_{}(\mathrm{No}.\mathrm{of}\mathrm{diseased}\mathrm{plants}\mathrm{at}\mathrm{every}\mathrm{level}\times \mathrm{level}\mathrm{value}/\mathrm{total}\mathrm{No}.\mathrm{plants}\mathrm{studied}\times \mathrm{highest}\mathrm{level}\times 100.$$ (1)

Assessment of physicochemical parameters of rhizosphere soil

The collected soil specimens underwent sieving and examined utilizing the subsequent methodologies: OM was quantified through the potassium dichromate oxidation technique, and nitrate N and ammonium N were evaluated via indigo N, N-Dimethylindoaniline colorimetric technique and UV-spectrophotometer. Available P was analyzed with sodium bicarbonate and quantified via the Mo‒Sb colorimetric technique, whereas total P was assessed by the perchloric acid‒sulfuric acid digestion technique. The flame photometry technique employed to ascertain accessible and total K concentrations. All the aforementioned physicochemical properties were assessed by methodology outlined via Bao.¹⁷ Rhizosphere soil enzymatic activity assessment was conducted with dry soil (5 g; screened through a 1 mm screen), which was subsequently deposited in a centrifuge tube (50 mL). The buffer and extraction solutions were then mixed according to the producer's recommendations for invariant-temperature farming. The levels of neutral phosphatase (NPase), catalase, urease (URE), dehydrogenase, sucrase (SUC), and cellulase (cell) in the soil were quantified utilizing soil enzymatic activity test kits, following the comprehensive protocols outlined in the manufacturer's guidelines.¹⁸ The bioavailable Al level in the rhizosphere was extracted utilizing DTPA, while total Al in the plant leaves and soil was determined through a mixture of nitric acid, perchloric acid, and hydrofluoric acid (v/v/v, 5:1:1) and nitric acid digestion, subsequently followed by ICP-MS. Standard test findings decreased within the permissible error margin, exhibiting 90%–105% recovery rate.

Root morphological traits analysis

Following the cleaning of the gathered and detached roots with demineralized water, the samples were positioned on the root plates of the root scanner, infused with a suitable volume of demineralized water, and imaged utilizing predetermined methods. The scanned images were analyzed with software for image processing, and root morphology metrics, including the root length, surface area, perimeter, average diameter, and ratio of the root length-diameter obtained via dedicated root analysis programs.

Assessment of antioxidant properties and defensive enzymatic activities in soybean

0.1 g of fresh leaf obtained, and extraction solution (1 mL) was mixed to homogenize the mixture in an ice bath. Subsequent to homogenization, the supernatant was subjected to centrifugation at 12000 × g for 8 minutes at 4 °C, collected, and maintained on ice for the assessment of superoxide anion, hydrogen peroxide, and MDA levels and POD, APX, and SOD levels. The concentrations of superoxide anions, hydrogen peroxide and malondialdehyde were quantified using TCA-TBA, hydroxylamine, and titanium sulfate techniques in accordance with the protocols established by Tewari et al.¹⁹. The SOD level was quantified following the methodology of Weisany et al.,²⁰ APX level was assessed according to Esfandiari et al.,²¹ and the POD level was evaluated using the guaiacol technique as outlined by Alici and Arabaci.²²

Rhizosphere soil microorganism’s assessment

The total DNA from the soil microbe genomes was obtained with a soil DNA kit from frozen-dried soil specimen (0.5 g). An AxyPrep DNA Plate Separation Kit employed to purify the isolated product. An agarose gel electrophoresis (AGE) was utilized to evaluate extracted DNA quality, while NanoDrop2000 applied to quantify the DNA level and determine purity. Genomic DNA for relative quantitative detection of high divergent V3-V4 area of 16S-rRNA gene, designated for soil bacterium sequencing. The primers employed were 806R (5-GACTACHVGGGTWTCTAAT-3) and 338F (5-ACTCCTACGGGAGGCAGCAG-3). The PCR apparatus and methodology are detailed in the Supplementary material. The processing, OTU clustering, sequencing, and microbial species identification of PCR outputs were performed on a cloud system. The raw sequence results were initially archived in the Sequence Read Archive of NCBI and subsequently analyzed and processed via the QIIME program.

Rhizosphere soil metabolomics analysis

Preparation of samples

Initially, a freeze-dried rhizosphere soil specimen (5 mg) was precisely measured and placed into a 1.5 mL spin tube, to which a 5 mm grinder bead was placed. After that, an extraction solution (1000 µL) comprising of L-2-chloroalanine (0.02 mg/mL) was introduced for metabolite extraction. The specimen was processed with a freeze-dried tissue grinder for 5 minutes at −8 °C and 50 Hz, thereafter underwent Hz and subsequently subjected to lower-temperature ultrasonic removal for 25 minutes at 4 °C and 50 kHz. The specimen was thereafter stored at −18 °C for 25 minutes and centrifuged for 20 minutes at 5 °C and 12,500 × g, and the resultant liquid was transferred to an injecting vial containing a tube inside for testing. Furthermore, the supernatant (20 µL) from every specimen was combined to formulate a quality reference specimen. A QC specimen was introduced after each of the three specimens to assess the reproducibility of every step during the evaluation.

Liquid chromatography-mass spectrometry detection

Detection was conducted utilizing ultra-HPLC Fourier transform mass spectrometer apparatus. The chromatographic column utilized was an ACQUITY-UPLC-HSS T3. Mobile stage A comprised 5% acetonitrile and 95% water supplemented with formic acid (0.1%), whereas mobile stage B included 5% water, 47.5% isopropanol, and 47.5% acetonitrile, which also contained formic acid (0.1%). The injection volume was 3 µL, the column temperature was maintained at 35 °C, and 0.4 mL/min was the total flow rate. The gradient elution protocol was as follows: 0–3.4 minutes, 0–24.5% B; 3.5–5 minutes, 24%–65% B; 5–5.5 minutes, 65%–100% B; 5–7.4 minutes, 100% B, 0.4–0.6 mL per minute; 7–7.6 minutes, 100%–51.5% B; 7–7.8 minutes, 51%–0% B, 0.6–0.5 mL per minute; 7.8–9 minutes, 0% B, 0.5–0.4 mL per minute; and 9–10 minutes, 0% B, 0.4 mL per minute. The mass spectrometer parameters were an electrospray ionization source at 430 °C and mass spectra voltages of 3500-V and −3500-V, along with a mass scan assortment of 70 m/z-1050 m/z.

Data processing in computing and multivariate metabolite assessment

The raw data were transferred into the metabolomics analysis software Progenesis for evaluation. The software facilitated feature peak detection and library matching by correlating MS/MS and MS spectrometer data with metabolic data sets, with the MS error threshold established at around 10 ppm. Metabolites were discovered with secondary mass spectrometry, and identical results. PCA and PLS-DA were conducted on the metabolites utilizing ropls program in R, with model firmness assessed through seven-cycle interaction corroboration. Furthermore, Student's t test and fold-shift assessment were conducted. Differential metabolites identified by variable weights and the top thirty diverse metabolites from all groups were compared.

Statistical analysis

GraphPad Prism and Microsoft Excel 365 were employed to arrange and graph the experimental data. Statistical evaluation was conducted utilizing Duncan’s multiple range test and one-way ANOVA to assess the disparities in indicators, including Fusarium wilt incidence, root morphological characteristics, growth characteristics, soil enzymatic activities, Al level, and soil physicochemical properties, among the MSAl, SSAl, and SSAl + Se, with statistical significance established at p < 0.05. Bacterial community's alpha diversity (Simpson, Chao, and Shannon indices) was examined with Rogers's approach, and within-group variances were analyzed with Wilcoxon rank sum analysis. Beta diversity assessment utilizing Bray‒Curtis distances was employed to examine the microbial community structure among the treatments, which was confirmed and visualized via PCoA. The data in the figures and tables are expressed as the mean ± SD of three repetitions. Correlation analysis among attributes was performed utilizing Spearman's technique. The metabolomics of the microbiota and rhizosphere soil, along with their relationship maps with divergent metabolites were evaluated and produced utilizing a free online bioinformatics cloud platform.

Results

Exogenous SeNPs and intercropping synergy mitigate aluminum stress and Fusarium wilt in soybean

In comparison to monoculture of soybean (MSAl) subjected to Al stress, each intercropping (SSAl) and intercropping with SeNPs (SSAl + Se) enhanced soybean growth. The SSAl and SSAl + Se treatments markedly enhanced the dry weights (both underground and aboveground) by 79.3% and 130.1%, and increased the crop height by 30.2% and 60.1%. Additionally, these treatments greatly diminished Fusarium wilt incidence by 10.9% and 34.4% and reduced aluminum accumulation in crop leaves by 16.9% and 57.4% (Figure 1). Nonetheless, SSAl sole did not greatly diminish Fusarium wilt incidence in comparison to SSAl + Se. The results demonstrated that intercropping with SeNPs greatly diminished Al buildup in crops and reduced soil-borne diseases prevalence. Subsequent examination of soybean root morphology under various treatments (Table 1) revealed that both the SSAl and SSAl + Se treatments enhanced the surface area diameter, perimeter, root length diameter, and root length to differing extents while diminishing the root surface area and average root diameter. The rises in the root length of 7.5% and 7.9%, as well as the surface area diameter of 5.5%, 25.9%, 24.1% and 20.1%, were statistically significant. In comparison to MSAl alone, SSAl + Se did not greatly augment the root length, surface area, circumference, and average diameter; nevertheless, it considerably improved the surface area and root length diameter of the root components. This demonstrated that intercropping with SeNPs could significantly enhance the total root length and diameter of root components, hence augmenting the resistance to Al toxicity and pathogen stress. Furthermore, SeNPs can enhance soybean development to a certain degree.

Figure 1.

Impact of intercropping (MSAl) and the application of SeNPs (SSAl + Se) on the growth of soybean and incidence of Fusarium wilt. Panels (a-d) illustrate the condition of crop growth, (a) dry weights of both belowground and aboveground tissues, (b) plant height, (c) incidence of Fusarium wilt, and (d) Al level in crop leaves. The values are expressed as mean ± SD. The distinct lowercase letters signify substantial changes among the treatments (p < 0.05).

Table 1.

Morphological characteristics of soybean roots under various treatment conditions.

Treatments	Root length (cm)	Average diameter (cm)	Perimeter (m)	Surface area (m2)
MSAl	149.60 ± 29.12b	0.80 ± 0.30a	169.03 ± 37.14a	3.90 ± 0.90a
SSAl	170.30 ± 24.10a	0.60 ± 0.06b	190.10 ± 26.54ab	3.29 ± 0.60b
SSAl + Se	171.12 ± 16.34a	0.62 ± 0.10ab	204.54 ± 20.14a	3.59 ± 0.69ab

Various lowercase letters in the same column designate significant variances between treatments (p < 0.05). Data are exhibited as mean ± standard deviation (n = 3).

Exogenous SeNPs and intercropping synergistically augment antioxidant activities and systemic defense in soybean

The concentrations of O₂⁻, MDA, and H₂O₂ in crop leaves indicate oxidative damage and the extent of membrane lipid peroxidation, serving as critical markers of crop stress resilience. In comparison to MSAl treatment, SSAl and SSAl + Se treatments showed a tendency to diminish the levels of O₂⁻, MDA, and H₂O₂, with the SSAl + Se treatment greatly reducing these values by 36.9, 73.9, and 89.8% (Figure 2a–c). The defensive enzymatic activities in leaves indicated that both SSAl and SSAl + Se treatments markedly enhanced the levels of POD (79.9 and 210.1%), APX (16.1 and 39.9%), and SOD (17.2 and 33.1%) enzymes. Furthermore, treatment with SSAl + Se led to substantial enhancement in the activities of all the enzymes in comparison to SSAl alone (Figure 2d–f). The results demonstrated that intercropping with SeNPs, both separately and in conjunction (SSAl + Se), may significantly improve the antioxidant and systemic defense processes of soybean under aluminum and soil-borne pathogenic stress.

Figure 2.

Impact of intercropping (MSAl) and the application of SeNPs (SSAl + Se) on the antioxidant and defensive capabilities of soybean. (a–c) Illustrates concentrations of O₂⁻, MDA, and H₂O₂ in the leaves of soybean. (d–f) Illustrates enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase (POD). The values are expressed as mean ± standard deviation (n = 3). The distinct lowercase letters denote statistically significant differences among the treatments (p < 0.05).

Impact of exogenous SeNPs and intercropping synergy on physicochemical parameters of rhizosphere soil in soybean cultivation

When evaluating the efficacy of planting strategies and exogenous treatments for Al-polluted soil, the amount of accessible Al in the rhizosphere of soybean may be a crucial indicator. The SSAl and SSAl + Se treatments reduced the accessible Al content while increasing the total Al content to varied degrees when compared to the MSAI treatment. When compared to MSAl alone, SSAl + Se treatment dramatically decreased the accessible Al content (31.9%) and its percentage of total Al (Figure 3a,b). When compared to MSAl treatment, the SSAl and SSAl + Se treatments greatly boosted soil enzyme activity, an essential marker of soil system function and health. 23.9% and 26.8% for CAT activity, 48.1% and 99.7% for dehydrogenase level, 103.7% and 77.7% for URE level, 14.1% and 41.8% for cell levels, and 22.9% and 43.1% for SUC enzymatic levels were among the notable increases (Figure 3). While SSAl + Se treatment did not substantially boost URE and CAT activities, it did increase SUC, cell, dehydrogenase, and NPase enzymatic activities as compared to MSAl.

Figure 3.

Impact of intercropping (MSAl) and the application of SeNPs (SSAl + Se) on the antioxidant and defensive capabilities of soybean. (a,b) Illustrates total Al and available Al in the rhizosphere. (c–h) Illustrates soil CAT, NPase, urease, cellulase, and sucrase. The values are expressed as mean ± standard deviation (n = 3). The distinct lowercase letters denote statistically significant differences among the treatments (p < 0.05).

In contrast to the MSAl approach, both the SSAI and SSAl + Se treatments showed an upward trend in AK, NH₄⁺–N, OM and pH levels, while Olsen-P and NO₃⁻–N contents decreased, according to the physicochemical parameter results of the soybean rhizosphere soil. In the SSAl + Se treatment, the NH₄⁺–N content dramatically increased by 22.9%, while NO₃⁻–N level greatly decreased by 71.9%. While the increase across all physicochemical variables was insignificant, SSAl + Se treatment decreased the NO₃⁻–N level by 65.1% when compared to SSAl (Table 2). Nutrient cycling enzymes such as URE had a strong positive correlation with NO₃⁻–N, according to a Spearman correlation study (|R| > 0.6, while Al had a significant negative correlation with URE, hydrogen peroxide, AK, and OM (Figure 4). This implies that the transformation of nitrate nitrogen and ammonium was largely influenced by intercropping and its further treatment (SSAl + Se), which improved the physicochemical characteristics of the soil.

Table 2.

Alterations in physicochemical properties of soybean rhizosphere soil after various treatments.

Treatments	pH	OM (g kg−1)	NH4+–N (mg kg−1)	NO3−N (mg kg−1)	Total P (g kg−1)	Olsen P (mg kg−1)	TK (g kg−1)	TK (mg kg−1)
MSAl	7.11 ± 0.08a	28.39 ± 1.29a	28.10 ± 3.10b	2.70 ± 0.30a	0.54 ± 0.10a	64.13 ± 3.60a	0.18 ± 0.01a	180.11 ± 1.10b
SSAl	7.30 ± 0.20a	31.10 ± 0.70a	30.12 ± 1.70ab	2.10 ± 0.30a	0.71 ± 0.03a	48.12 ± 3.60b	0.20 ± 0.02a	210.49 ± 4.20a
SSAl + Se	7.19 ± 0.30a	34.25 ± 1.09a	34.78 ± 1.80a	0.75 ± 0.03b	0.46 ± 0.10a	54.14 ± 2.61b	0.18 ± 0.01a	210.12 ± 4.53a

Different lowercase letters in the same column indicate significant differences between treatments (p < 0.05). Data are presented as mean ± SD (n = 3).

Figure 4.

Spearman correlation study of rhizosphere soil physicochemical parameters (pH, organic matter, ammonium nitrogen, nitrate nitrogen, total phosphorus, available phosphorus, total potassium, available potassium, aluminum, and selenium) and soil enzyme activity (hydrogen peroxide, sucrase, cellulase, urease, dehydrogenase, and neutral phosphatase). ***p < 0.001, **0.001 < p < 0.01, and *0.01 < p < 0.05.

Impact of intercropping and SeNPs synergy on rhizosphere microbial community of soybean

Rhizosphere soil 16S rRNA sequence results indicated that, in comparison to MSAl treatment, both the SSAl and SSAl + Se treatments exhibited an upward trend in diversity (Shannon value) and richness (Chao value) indices. In particular, the SSAl treatment greatly decreased the Simpson value, but the SSAl + Se treatment considerably elevated the Shannon and Chao indices (Figure 5a), signifying an enhancement in microbial community richness and diversity. The SSAl and SSAl + Se treatments diminished both the quantity and percentage of distinct OTUs within the bacterial population in comparison to MSAl treatment. Community β diversity data indicated a distinct distinction between the SSAl and SSAl + Se and MSAl treatments, but insignificant separation was observed between the SSAl and SSAl + Se treatments (Figure 5c), implying that the incorporation of SeNPs did not affect the rhizosphere microbial community.

Figure 5.

Impact of intercropping (MSAl) and the application of SeNPs (SSAl + Se) on the rhizosphere microbial community of soybean. α diversity indices (Simpson, Shannon and Chao) of the bacterial population across several treatments (MSAl, SSAl and SSAl + Se); (b) bacterial OTU overlapping across various regimens; (c) β diversity assessment of the community of bacteria; (d) microbial communities at the genus and phylum tiers compositional study; and (e) predominant bacterial genus intergroup analysis, Spearman correlation study of predominant bacterial species in connection to environmental parameters. ***p ≤ 0.001, **0.001 < p ≤ 0.01, and *0.01 < p ≤ 0.05.

Rhizosphere microbial composition analysis indicated that, in comparison to the MSAl treatment, the SSAl and SSAl + Se treatments enhanced Actinobacteria, Chloroflexi, and Proteobacteria relative abundance at the phylum scale while diminishing the Acidobacteriota number. At genus, Streptomyces, Marmoricola, Intrasporangium, and Sphingomonas relative abundances surged while Gemmatimonas abundance diminished. Subsequent investigation of intergroup variances indicated notable disparities among Nocardioids, Intrasporangium, and Sphingomonas. Nocardioids had a significant positive correlation with urease, NO₃⁻–N concentrations and hydrogen peroxide level, while Sphingomonas showed a positive correlation with cellulase, NO₃⁻–N, pH, hydrogen peroxide, sucrase, Se content and available phosphorus. Intrasporangium had a significant positive correlation with urease. The data indicated that SeNPs and intercropping treatment predominantly attracted beneficial microbes, thereby enhancing the rhizosphere microbial community by affecting soil physicochemical parameters and specific enzymes.

Impact of intercropping and SeNPs synergy on soybean rhizosphere metabolites

A metabolomic study was performed on soybean rhizosphere soil specimens affected by intercropping and SeNPs. The PLS-DA findings exhibited a distinct distinction across all of the treatments and robust grouping among the repeated specimens (Figure 6a). The model permutation parameters Q² and R²Y were −0.5529 and 0.9879, showing that the PLS-DA model exhibited strong performance and facilitated subsequent data processing. In comparison to MSAl treatment, the SSAl treatment led to notable alterations in 64 differential metabolites (32 downregulated and 32 upregulated), while SSAl + Se treatment exhibited significant alterations in 26 metabolites (16 downregulated and 10 upregulated) relative to SSAl. A Venn study of all the metabolites identified 17 distinct metabolites for SSAl and 6 for SSAl + Se in comparison to MSAl. The KEGG chemical classification of such metabolites categorized them into 7 principal groups (Figure 6-f), with flavonoids as the most predominant, succeeded by phenylpropanoids and terpenoids. Subsequent examination of metabolites enrichment indicated that, in contrast to MSAl and SSAl, which augmented the secretion of compounds, including scymnol, 4-hydroxy-5-phenyltetrahydro-1, 3-oxazin-2-one, and 5-deoxyribose-1-phosphate, while diminishing the secretion of compounds such as cis-p-coumaric acid and dehydrotropingosine.

Figure 6.

Metabolites classification and study in the soybean rhizosphere by intercropping (SSAl) and SeNPs superposition (SSAl + Se). (a) PLS-DA of metabolites among the MSAl, SSAl, and SSAl + Se treatment groups; (b) PLS-DA model permutation assessment; (c) Venn diagram illustrating metabolites among the treatments; metabolite volcano plot; (d) SSAl-vs-MSAl; (e) SSAl + Se-vs-SSAl analyses; and (f) KEGG compound grouping and statistical diagram.

In comparison to SSAl, SSAl + Se treatment elevated metabolite concentrations, including ectoine, gemfibrozil, and isopentanol, while diminishing the concentrations of substances such as phthalic acid and isorhamnetin (Figure 7a-b). The metabolites affected by SSAl treatment are predominantly enriched in specific pathways, including Fc gamma R-triggered phagocytosis, carotenoid biosynthesis, and GnRH signaling. The differential metabolites impacted via SSAl + Se were predominantly improved in cell cycle-associated metabolic pathways and ABC transporters (Figure 7c-d).

Figure 7.

Rhizosphere metabolite cluster study and KEGG enrichment research of differential metabolites in soybean utilizing inter-cropping (SSAl) and SeNPs combination (SSAl + Se). Cluster heatmap study of the thirty most differential metabolites in the SSAl_vs_MSAl (a); SSAl + Se_vs_ SSAl metabolome (b); KEGG channel enrichment research of differential metabolites in the SSAl_vs_MSAl (c); and SSAl + Se_vs_ SSAl (d).

Joint examination of exogenous SeNPs and cumulative effects of intercropping on the microbiome interactions and rhizosphere metabolome of soybean.

Comprehending the correlation between rhizosphere microbes and metabolites is essential for enhancing crop growth strategies via SeNPs and intercropping application. Therefore, we initially examined the comprehensive associations between metabolite expression and the rhizosphere microbial community. The findings revealed a considerable consistency in patterns between metabolite expression rates and microbial community arrangement across several groups, indicating a robust association between the two datasets (Figure 8-a). Subsequent examination of association between the main differential metabolites and predominant bacteria indicated that Intrasporangium, Sphingomonas, and Nocardioids displayed differing levels of negative relationship with metabolites such as cinobufagin and aflatoxin B2, while demonstrating positive associations with antibacterial substances, including sordarin, 15-methylpalmate and lactucin (Figure 8-b). These correlations underscore essential factors driving alterations in soil physicochemical parameters and the promotion of crop development resulting from the use of intercropping and SeNPs.

Figure 8.

Joint examination of microbiome interactions and metabolome inside the rhizosphere of soybean. Correlation study of microbial populations and rhizosphere metabolites with the Procrustes test (a); Spearman correlation heatmap depicting the associations between the principal differential metabolites and the predominant top 15 bacterial genera. ***p < 0.001, **0.001 < p < 0.01, and *0.01 < p < 0.05.

Discussion

Aluminum and Fusarium represent significant biotic and abiotic stressors that impact contemporary soybean cultivation. The collaborative impact of intercropping and exogenous SeNPs enhances the advantages of Se nanoparticles and interspecific root contacts. The combined effects augment rhizosphere soil enzymatic activity and soil physicochemical property alterations, mitigate the detrimental impacts of soil-borne pathogens and aluminum on host crops, and boost plants' resilience to stress. This study's findings endorse application of SeNPs to enhance capacity of intercropped soybean in alleviating Fusarium wilt and Al toxicity, while also clarifying the interaction processes between metabolites and root microbiota.

Intercropping and exogenous SeNPs significantly mitigate Al toxicity in soybean and manage Fusarium wilt

Heavy metals such as Al toxicity can impair agricultural development and product security, posing a risk to health via food system.⁶ The present study demonstrated that, in contrast to monoculture soybean under Al stress (MSAl), sorghum-soybean intercropping (SSAl) greatly diminished Al accumulation in soybean, enhanced crop growth, and mitigated soil-borne infections, aligning with findings from analogous research on spiny sowthistle intercropped with bell bean.²³ Nevertheless, some research has suggested that intercropping soybean with maize, millet, and peanut can enhance metal accumulation in the aerial portions of soybean.^24,25 The disparities may be ascribed to types of intercropped plants, physicochemical characteristics of soil, and experimental circumstances such as hydroponics, field studies, and soil culture.²⁶ Nitrogen promotes the cysteine and various compounds synthesis, mitigates metal stress, stimulates nutrient absorption, and diminishes disease in fodder alfalfa.²⁷ The present research determined that intercropping elevated effective Al concentration in soybean rhizosphere, likely due to improved root interactions between sorghum and soybean, which stimulated organic acids secretion, potentially increasing effective Al levels in rhizosphere.²⁸ Contemporary intercropping methods primarily emphasize crop intercropping and hyperaccumulating plants utilization to investigate the sorption and toxicology of Al.²⁹ Nevertheless, limited research has examined the detrimental impacts of crop-crop inter-cropping on certain targeted crops. The present research demonstrated that intercropping soybean with sorghum can alleviate the negative impact of Al on soybean.

This work demonstrated that intercropping with SeNPs (SSAl + Se) considerably diminished Al buildup in soybean, lowered rhizosphere Al availability, reduced Fusarium wilt incidence, and enhanced plant development circumstances compared to SSAl treatment (Figure 1). Se is crucial for alleviating Al toxicity in crops by sequestering toxic metals and preventing their translocation to aerial tissues. It fortifies crop's cuticle and establishes defense system within cell wall, so impeding pathogen infiltration and diminishing disease prevalence.³⁰ SeNPs, as a novel category of nanomaterial, provide improved stress resilience in plants facing abiotic and biotic stressors.³¹ SeNPs exhibit superior efficacy in enhancing plant development and stress resilience compared to conventional Se compounds, as they may be assimilated by crops without reliance on Se-specific transport proteins.³² The superior adsorption ability of SeNPs and their capability to diminish Al flow in crops are essential mechanisms for mitigating Al toxicity.³³ SeNPs combined with intercropping therapy showed more efficacy in mitigating Al toxicity and managing soybean Fusarium wilt compared to MSAl. This may be due to the deposition of SeNPs in crop epidermal tissues, which could impede the penetration of Fusarium bugs, hence increasing their ability to resist Fusarium wilt.^34,35 Furthermore, we initially observed that SSAl + Se treatment markedly increased the total length of roots, surface area diameter, and root diameter, which may establish a vital basis for N fixation, nutrient uptake, and plant growth facilitation in soybean root nodules.

Improvement of defensive tolerance and soil enzymatic activity in soybean via intercropping with SeNPs

The negative impacts of Al on crops manifested not only as stunted development but also as disturbances in biochemical and physiological processes, including heightened oxidative damage and modified resilience enzyme activity.^36,37 This investigation indicated that, in comparison to MSAl regimen, both the SSAl and SSAl + Se treatments diminished O₂⁻ and MDA levels while greatly enhancing antioxidant enzymatic activities. The reduction in oxidation degrees and the Al concentration may be associated with interspecific root relationships that foster Al-tolerant endophytic bacteria, thereby enhancing oxidoreductase activities in crops.³⁸ Plants react to stress by excessively generating ROS, reactive N species, and reactive S species. If not eliminated promptly, they can induce oxidative impairment of cells and could result in plant mortality.³⁹ Our research results demonstrate that intercropping and incorporation of SeNPs can augment antioxidant activity and inhibit Fusarium wilt in host crops, which is identical to the results in cucumber.^40,41 Significantly, the combination of SSAl + Se treatment surpassed the efficacy of SSAl alone in diminishing oxidative damage and augmenting resilience enzyme activity (Figure 2), suggesting that soybean-sorghum intercropping, when paired with SeNPs, synergistically bolstered crop antioxidant ability and defense mechanisms, thereby presenting an effective approach for mitigating soil-borne organisms and Al stress. It is essential to recognize that the impact of SeNPs reported in this work was under Al stress, and caution must be taken in assessing whether SeNPs may enhance antioxidant ability and stimulate soybean growth.

The rhizosphere soil is intimately linked to plants and is essential for evaluating heavy metal bioavailability and crop health by monitoring alterations in enzymatic activities and physicochemical characteristics.⁴² This work demonstrated that SSAl and SSAl + Se treatments markedly enhanced the CAT, SUC, cell, URE, dehydrogenase, and NPase enzymatic activities in soil, which are essential for nitrogen cycling, crop development, and soil-borne disorder mitigation.⁴³ Furthermore, SSAl + Se treatment markedly elevated the available potassium and NH₄⁺–N concentrations while diminishing Olsen-P and NO₃⁻–N levels, in contrast to SSAl treatment only. This may result from the increased function of the NH₄⁺–N bacteria, which are augmented by SeNPs and possess substantial SSA and significant adsorption ability, thus facilitating P adsorption. This results in a reduction of Olsen-P and NO₃⁻–N levels in the soil. The pronounced negative association between Se and Al, coupled with their positive relationship with the majority of enzymes, underscored advantageous impacts of SeNPs in modulating soil characteristics (Figure 4). These results demonstrate that SeNPs and intercropping collaboratively improved soil enzymatic activities and facilitated nitrogen cycling. Moreover, URE functions as an enzyme responsive to heavy metal pollution and serves as a crucial indicator for assessing Al toxicity.⁴⁴ When implementing SeNPs and intercropping in agricultural practices, it is crucial to consider the possible detrimental impacts of various nutrients, particularly in regulating soil total potassium concentration, to alleviate Al toxicity.

Intercropping and SeNPs interactions modulate rhizosphere microbiota

Rhizosphere soil microbes, also known as the 'second genome', can act as crucial indicators of plant health.⁴⁵ Under the influence of heavy metals, organisms generally undergo specific alterations, including the homogeneity of the species makeup and a decline in functional diversity.^46,47 This study found that intercropping, particularly when paired with SeNPs, enhanced bacterial community variety and richness relative to MSAl treatment. While the SSAl treatment demonstrated a trend of improvement, SSAl + Se treatment markedly augmented these microbiological metrics (Figures 5a–c), which aligns with prior intercropping research.^48,49 Current studies indicate that silicon can increase nutrient availability and plant diversity while mitigating Al toxicity. Both the SSAl and SSAl + Se treatments elevated the accessible Al concentration in the rhizosphere; however, the SSAl + Se treatment diminished this concentration relative to the SSAl treatment only, suggesting that SeNPs decreased soil Al bioavailability.⁵⁰ These alterations are intricately associated with heightened rhizosphere microbes' diversity. This study initially showed that, under Al stress, intercropping has a minimal impact on improving rhizosphere microbial variety and abundance. Conversely, intercropping integrated with SeNPs showed superior efficacy in enhancing microbial diversity inside the rhizosphere of soybean.

Proteobacteria, Chloroflexi, and Actinobacteria were the predominant bacterial groups in the intercropping system and SeNP regimens. They are recognized for their functions in digesting organic materials, enhancing soil enzymatic function, and facilitating soil nitrogen cycling.⁵¹ Actinobacteria are known to synthesize catechol and IAA, which serve as Fe chelators, and to form compounds with Al to diminish its concentration. The presence of Al can stimulate Pseudomonas aeruginosa to synthesize pyocyanin, therefore diminishing the population of pathogenic fungus, including Fusarium, thus combating soil-borne disorder and enhancing crop growth.⁵² Subsequent study of bacterial genus makeup across various regimens indicated microbial species relative abundance in the SSAl and SSAl + Se treatments augmented alongside enhancements in the soil chemical characteristics and crop growth metrics, in contrast to those in the SSAl treatment. The genera Nocardioids, Streptomyces, Marmoricola, Intrasporangium, and Sphingomonas exhibited a notable rise in the SSAl and SSAl + Se regimens. Sphingomonas not only enhances plant development, suppresses pathogens, and engages in antioxidation and N cycling but also possesses the ability to sequester soil toxic metals.⁵³ Beneficial bacteria, including Nocardioids and Streptomyces, are essential for enhancing crop growth and managing soil-borne illnesses in the rhizosphere.⁵⁴ These results elucidate the beneficial impacts of intercropping under Al stress, particularly when integrated with Se nanoparticles, on restructuring rhizosphere microbial populations and enhancing crop performance. In this investigation, SeNP incorporation under Al toxicity did not result in a notable increase in beneficial microbes, including P. aeruginosa and Paenibacillus. The presence of pathogenic bacteria and Al stress in continuous soybean cropping may lead to the recruitment of an increased array of functional microbes in the rhizosphere rather than depending solely on individual stress-tolerant and growth-enhancing bacteria, indicating crop approach to equilibrate growth and combat stresses.

Rhizosphere metabolites regulation by intercropping superposition and SeNPs, together with their relationship processes with microbes

Alterations in crop rhizosphere metabolites can act as critical markers of crop phenotypic reactions to ecological stress.⁵⁵ Rhizosphere metabolites can profoundly affect disease resilience and crop growth.⁵⁶ The present research analyzed monoculture root metabolites, intercropping, and intercropping augmented with SeNPs, revealing a significant effect on soybean rhizosphere metabolites. Flavonoids are the predominant metabolites (Figure 6-f), essential for modulating ecosystem‒plant interactions, attracting beneficial microbes, mitigating free radical damage, easing the toxic effects of Al, and combating disease invasion.⁵⁷ Terpenoids facilitate plant adaptability to stress, suppress pathogens, and develop resistance, yielding beneficial impacts.⁵⁸ Differential metabolite analysis indicated an elevation in ribonucleotides associated with DNA biosynthesis, alongside metabolites such as 4-hydroxy-5-phenyltetrahydro-1, 3-oxazin-2-one and 5-deoxyribose-1-phosphate, which facilitated plant development and defense mechanisms. These compounds were concentrated in the EGG pathways associated with hormonal signal transduction and cell phagocytosis. SeNPs augment antioxidant secondary compounds release, including ectoine, which assists in regulating plant cell integrity.⁵⁹ The primary enhanced KEGG pathways, encompassing cell cycle and ABC transporters, facilitate plant tolerance to salinity and other stresses.⁶⁰ The crop rhizosphere is a vital region for microbe‒plant‒soil interactions, where the metabolome and microbiome are essential for comprehending the mechanisms of crop oxidative stress reactions, disease resilience and optimal growth.⁶¹

The connection analysis of differential metabolites and dominant bacteria demonstrated a robust link between metabolomic and microbial datasets. Microbes associated with crop stress, such as Nocardioides, Intrasporangium, and Sphingomonas, demonstrated significant positive correlations with 15-methylpalmate, lactucin, and sordarin (Figure 8). These antibacterial substances are linked to crop defensive capabilities and the rhizosphere defense system.⁶² Prior research has demonstrated that the uptake of SeNPs by crops can augment rhizosphere amino acids, organic acids and sugars metabolism, enhance bacterial nutrition, and promote the colonization of Al-resistant bacteria, consequently mitigating the toxic effects of Al in the rhizosphere.⁶³ The present study indicated a robust link between metabolites and the recruited microbiota in response to Al stress, regardless of SeNP treatment and intercropping. Compounds including 15-methylpalmate, lactucin, and sordarin, were markedly increased.

In conclusion, our results demonstrate that SeNP treatment and intercropping substantially enhanced differential metabolites and essential microbes associated with soybean root zone protection, providing initial and crucial insights into improving soybean resistance against stressful conditions. The results presented here illustrate the crucial functions of rhizosphere compounds in influencing microbe populations and enhancing crop resilience to soil-borne pathogens. Correlation between metabolites and microbes elucidates how intercropping and Se nanoparticles can mitigate the combined stress of Al and soil-borne pathogens via improving crops antioxidant's ability, increasing rhizosphere-soil enzymatic activities, altering bacterial community composition, and modulating essential rhizosphere metabolites, thereby fostering robust plant growth.

Conclusion

Our findings demonstrated that, in contrast to monoculture soybean subjected to Al stress, SSAl and SSAl + Se diminished Al buildup in crops, reduced soybean Fusarium wilt disease prevalence, and enhanced the total length and diameter of roots. These treatments augmented crops' systemic defense and antioxidant mechanisms, elevated root enzyme activities including SUC, dehydrogenase, and NPase enhanced soil physicochemical attributes, modulated flavonoid secretion, and facilitated functional bacteria (Intraporangium, Sphingomonas, and Nocardiides) proliferation and secondary metabolites (15-methylpalmate, lactucin, and sordarin) in the rhizosphere. The intimate relationship between microbes and their metabolites disclosed rhizosphere microecological processes through which SSAl and SSAl + Se mitigated Al toxicity and conferred resistance to Fusarium wilt. While SSAl alone enhanced these parameters to degree, the regulatory impact of intercropping in conjunction with SeNPs was more significant. This research revealed substantial benefits of employing SeNPs and intercropping to mitigate pathogen and Al stress in soybean, indicating great possibilities for extensive agricultural applications in future years.

Funding Statement

This work was supported by Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (KFU261646).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the raw data in this research can be obtained from the corresponding authors upon reasonable request.

Consent for publication

Not applicable.

Ethics approval statement

This study does not include human or animal subjects.

Clinical trial number

Not applicable.

Statement on guidelines

All experimental studies and experimental materials involved in this research are in full compliance with relevant institutional, national and international guidelines and legislation.