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Article

Synergy of Arbuscular Mycorrhizal Fungi and Biochar-Based Fertilizer Reshapes Soybean Nutrient Acquisition and Drives Yield Enhancement

by
Lingbo Meng
1,†,
Huawei Yang
2,†,
Yue Fan
2,
Jiang Li
2,
Diwei Song
2,
Xiaozhe Ma
2 and
Shumin Li
2,*
1
School of Geography and Tourism, Harbin University, Harbin 150086, China
2
Resource and Environmental College, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2025, 17(22), 10355; https://doi.org/10.3390/su172210355
Submission received: 20 October 2025 / Revised: 14 November 2025 / Accepted: 16 November 2025 / Published: 19 November 2025
(This article belongs to the Section Sustainable Agriculture)
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Abstract

Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with most crops. They function as promising sustainable agricultural amendments by synergizing with biochar to enhance plant nutrient uptake. However, the effects of AMF and biochar interactions on the yield and nutrient uptake of leguminous crops and the underlying mechanisms remain insufficiently understood. This study employed a two-factor experimental design. Under the baseline conditions of no fertilization (CK), chemical fertilizer application (CF), and biochar-based fertilizer application (BF), treatments with and without AMF inoculation were established, resulting in a total of six experimental treatments. Compared to BF treatment alone, the combined application of AMF and BF (AM + BF) synergistically increased soybean biomass (12.81%) and grain yield (19.45%). This synergistic effect was accompanied by increased plant nitrogen (14.04%) and potassium (21.82%) accumulation. Notably, despite the highest yield, the AM + BF treatment showed a 22.22% reduction in nodule formation rate. This reveals that plant nitrogen acquisition strategies have shifted from relying on biological nitrogen fixation to efficient mycorrhizal pathways, reflecting an inherent optimization of carbon economy. The PLS-SEM model revealed that AMF inoculation altered yield-driving mechanisms: in the absence of AMF, yield could be directly predicted by soil nutrient levels; however, this relationship was disrupted after AMF inoculation. The soil nutrient pathway became non-significant, indicating a transition from a soil chemistry-dependent model to a biologically driven one, where AMF–plant symbiosis became the primary regulator of nutrient uptake. These findings highlight that AMF-BF synergy creates a novel soil–plant feedback mechanism that enhances nutrient acquisition efficiency and optimizes carbon allocation, providing a sustainable approach to boost legume crop yields and reduce environmental footprints.

1. Introduction

Soybean is the world’s leading high-protein, high-fat grain and oil crop, with significant social, political, and economic implications [1]. The yield of soybeans directly affects food and oil security, as well as the future development of agricultural production [2]. However, traditional soybean cultivation practices have not been suitable for meeting current sustainable development requirements. Long-term monoculture and unsustainable farming practices have led to a continuous decline in soil productivity, becoming a key issue constraining the industry’s efforts to enhance quality and efficiency [3]. Among the various factors affecting soybean yield and quality, soil nutrient imbalance and non-standardized production techniques are particularly prominent. Due to soybeans’ specific preferences for nutrient absorption, long-term monoculture easily leads to severe deficiencies in key elements such as phosphorus and potassium. This makes it difficult to provide sufficient nutrition for the whole growth period, ultimately leading to poor plant development [4]. At the same time, continuous cropping has exacerbated the spread of soil-borne diseases, such as root rot [5]. This also causes an imbalance in the soil microecosystem, leading to a decline in beneficial microorganisms while pathogenic fungal populations proliferate extensively [6]. In actual production, the improper use of inputs such as pesticides and chemical fertilizers poses further potential risks to soybean quality and the ecological environment of farmland [7,8]. These factors have severely impacted the development of the soybean industry, causing significant declines in both yield and quality [9]. Therefore, there is an urgent need to strengthen the introduction of soybean cultivation technology and to develop sustainable cultivation techniques and methods that take into account the local environment and growing conditions, in order to improve soybean yield and quality.
Arbuscular mycorrhizal fungi (AMF) are prevalent in agroecosystems and represent the most common plant symbiotic fungi. They form mutualistic relationships with over 80% of terrestrial plants, facilitating bidirectional nutrient exchange [10]. AMF connect with soil and plants primarily through mycelial bridges, which regulate plant metabolism, competition, and soil physicochemical properties. This interaction enhances plant nutrient uptake and energy flow, thereby influencing plant growth, development, and vegetation restoration [11]. AMF play an important role in enhancing crop nutrient uptake that could significantly improve the absorption of nitrogen (N), phosphorus (P), potassium (K), and micronutrients [12]. In addition, biochar-based fertilizer, a granular compound derived from biochar and chemical fertilizers, combines the properties of both components [13]. As a slow-release compound fertilizer, biochar-based fertilizer utilizes its porous structure to adsorb and retain nutrients such as N, P, and K, thereby effectively slowing nutrient release rates. This characteristic significantly reduces leaching and volatilization losses of easily leached nutrients like N, extending the duration of sustained nutrient supply [14]. In recent years, the synergistic effects between biochar and AMF have become a hot topic in agroecological research due to their important functions in improving crop nutrient use efficiency. Studies indicate that AMF and biochar could enhance soil fertility and permeability, address soil compaction and acidification, and create favorable conditions for soil microorganisms, thereby facilitating plant–microbe–soil interactions [15,16]. The synergistic effect of biochar and AMF can also promote plant root growth, alter soil microbe structure, and enhance crop nitrogen uptake efficiency [17,18]. The analysis of plant carbon and nitrogen metabolic pathways revealed that the combined application of AMF and biochar exhibits a synergistic effect, promoting plant growth, photosynthesis, and carbon and nitrogen metabolic activity [19]. The combination of biochar and wood ash elevated soil pH and nutrient levels, regulated rhizosphere metabolism to mitigate continuous cropping disorders by enriching beneficial AMF communities, enhanced phenylalanine metabolism, and suppressed soil-borne pathogens such as Fusarium [20]. The synergistic effect of AMF and biochar can also alter the rhizosphere microbial community, establishing functional synthetic communities. The selected synthetic microbial communities significantly increase plant biomass [21].
Despite these findings, the coupling mechanisms between AMF and biochar, particularly their effects on soil quality, plant growth, and soybean yield, remain insufficiently explored. In this experiment, soybeans served as the tested crop to evaluate the effects of AMF inoculation combined with biochar based fertilizer on growth indices, nutrient accumulation, number of nodules, mycorrhizal colonization rate, and changes in soil nutrients, enzyme activities, and yields at different soybean growth stages. The objectives were to determine (1) the combined effects of AMF and biochar-based fertilizers on nutrient uptake, soil physicochemical properties, and crop yields in a soybean filed, and (2) the key drivers influencing yields, including the direct and indirect pathways through which AMF and biochar-based interactions impact yields. This research aims to provide a theoretical and practical foundation for applying AMF and biochar-based fertilizers in sustainable agricultural production.

2. Materials and Methods

2.1. Experimental Site

The experimental site is located at the experimental station of Northeast Agricultural University in Acheng District, Harbin City, Heilongjiang Province (45°50′ N, 126°39′ E), China. The terrain is flat, with an elevation of 150 m, and the climate is cold-temperate continental monsoon. The local soil is classified as mollisol, with the following fertility parameters: soil organic matter 32.5 g·kg−1, total nitrogen 1.41 g·kg−1, available phosphorus 34.1 mg·kg−1, available potassium 121.24 mg·kg−1, and soil pH 6.16.

2.2. Experimental Design

A two-factor randomized split-plot design was used in this experiment, with six treatments: (1) no mycorrhizal fungi inoculation and no fertilization (CK); (2) no mycorrhizal fungi inoculation with chemical fertilizer application (CF); (3) no mycorrhizal fungi inoculation with carbon-based fertilizer application (BF); (4) mycorrhizal fungi inoculation with no fertilization (AM + CK); (5) mycorrhizal fungi inoculation with chemical fertilizer application (AM + CF); and (6) mycorrhizal fungi inoculation with carbon-based fertilizer application (AM + BF). Each treatment was replicated three times, resulting in 18 experimental blocks. Each block had a planting area of 24.7 m2 (5.2 m × 4.75 m), with eight rows of soybeans planted in each block. Soybean row spacing is 8.5 cm, with a seed density of 196,000 per hectare. In the biochar-based fertilizer treatment, the application rate of biochar compound fertilizer was 674.7 kg·ha−1. In the fungal inoculant treatment, the AMF inoculant was applied at a rate of 450 kg·ha−1. The chemical fertilizer treatment was formulated to match the nitrogen (N), phosphorus (P), and potassium (K) content of the biochar-based fertilizer, with the following composition: N 54 kg·ha−1, P2O5 128.2 kg·ha−1, and K2SO4 87.7 kg·ha−1, derived from urea, triple superphosphate, and potassium sulfate. All fertilizers and microbial agents were uniformly applied by hand. The biochar-based fertilizer was sourced from Nanjing Sanju Biomaterial Co., Ltd., located in Nanjing, Jiangsu Province, China. The biochar was pyrolyzed at a temperature between 500 and 600 °C. Its key properties were as follows: total carbon content of 636.7 g·kg−1, total nitrogen content of 4.21 g·kg−1, total phosphorus content of 0.55 g·kg−1, and a pH of 7.38 (1:1 H2O). Soybean seeds (Glycine max L., Dongnong-252) were sown in April and harvested in October in 2022. Cultivation and field management practices were consistent across all plots. Weed control was conducted manually throughout the growing season. The AMF used in this experiment was Glomus etunicatum (BGC NM03F) purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, China. The inoculum was grown through pot cultivation using a sand substrate in a controlled indoor environment.

2.3. Sample Collection and Determination

2.3.1. Plant Sample Collection and Analysis

Samples were collected during the soybean seedling stage, flowering stage, pod-setting stage, grain-filling stage, and maturity stage through random sampling within each plot. Five soybean plants were selected from each plot, and scissors were used to separate the aboveground and belowground parts. The aboveground plant material was brought back to the laboratory for blanching at 105 °C, followed by drying at 75 °C to constant weight to determine aboveground biomass. At the maturity stage, soybean stems and seeds were dried separately, ground, and thoroughly mixed. For the underground part, a 20 cm × 20 cm × 25 cm soil sample was excavated around the plant, placed in a bag, and transported to the laboratory. Soil was washed off using a mesh sieve, and root nodules were picked from the root system with tweezers and counted. Fresh root segments (1.0 cm long) were stained with Taiwan blue to assess mycorrhizal colonization rates under a microscope. Nitrogen, phosphorus, and potassium contents were measured using a continuous flow analyzer, vanadium-molybdenum yellow colorimetric method, and flame photometry, respectively.

2.3.2. Soil Sample Collection and Analysis

During the soybean seedling stage, flowering stage, pod formation stage, grain filling stage, and maturity stage, select an additional 5 soybean plants. Remove the surface soil around each plant, uproot the soybeans, shake off loose soil, and brush any soil adhering to the roots into sterile resealable bags. Store these bags in a refrigerator and transport them to the laboratory for processing as soon as possible. A portion of the soybean soil samples is air-dried at room temperature. After passing through a 2 mm sieve to remove impurities, these samples are stored in plastic resealable bags for soil nutrient analysis. Another portion of fresh samples is sieved through a 0.5 mm sieve and immediately used for soil enzyme activity assays.
Soil pH was assessed using a glass electrode meter in a 1:2.5 soil:water (w/v) suspension. Available phosphorus was extracted with NaHCO3 (0.5 M) and measured using the molybdenum antimony colorimetric technique. Available potassium was extracted with NH4OAc (1 M) and quantified using a flame photometer. Soil ammonium (NH4+) and nitrate (NO3−) N were extracted with 2 mol·L−1 KCl and analyzed using an FIAstar 5000 Analyzer. Soil urease activity was determined using the indophenol colorimetric method. Neutral phosphatase activity was determined using the sodium phosphate colorimetric method.

2.3.3. Determination of Crop Yield and Yield Components

In each block of the yield measurement zone, 4 m of double-row soybeans were selected. All soybean plants were collected, packed, and transported to the laboratory. After thorough drying, 10 soybean plants were randomly selected for seed testing. Parameters measured included plant height, pod height, stem diameter, number of main stem nodes, number of effective branches, number of pods per plant, number of seeds per plant, seed weight per plant, and hundred-seed weight.

2.4. Statistical Analysis

Experimental data were summarized, organized, and calculated using Microsoft Office Excel 2019. One-way and two-way analysis of variance (p < 0.05) was performed using SPSS Statistics 23.0 to assess differences between treatments and interactions between the fungicide and fertilizer factors. A random forest model was constructed using the ‘RandomForest’ package in R version 4.3.2 to predict key factors influencing yield. The model was constructed using 18 observed samples and validated through 10-fold cross-validation. Importance values represent the average across 500 decision trees. Correlation heatmap analysis was performed using the ‘psych’ and ‘corrplot’ packages. Based on the results of the correlation analysis, examine the variance inflation factors for each measurement indicator. Remove any indicators with a factor greater than 10. Construct a database for remaining indicators. A partial least squares structural equation model (PLS-SEM) built with the ‘plsm’ package identified potential indirect and direct effects on soybean yield. Graphs were generated using Prism 10.

3. Results

3.1. Effects of AMF and Biochar-Based Fertilizer Interactions on Soybean Plant Growth

Both AMF inoculation and fertilization promote soybean plant growth and development as well as biomass accumulation (Figure 1a). As the growth stage progresses, the dry weight of soybean plants gradually increases, exhibiting a slow-fast-slow trend. Prior to flowering, dry matter accumulation occurs at a relatively slow pace. The period from flowering to the grain-filling stage represents the fastest rate of dry matter accumulation. Following the grain-filling stage, the accumulation rate slows down, with the maximum dry matter accumulation occurring at maturity. During the pod-setting stage, grain-filling stage, and maturity stage of soybeans, the treatment sequence across all treatments was consistently: AM + BF > AM + CF > BF > CF > AM + CK > CK. The most significant biomass differences in soybeans occurred during the pod-setting stage. Compared to the CF, BF, AM + CK, and AM + CF treatments, the AM + BF treatment showed increases of 25.44%, 22.68%, 45.25%, and 15.86%, respectively. Furthermore, an interaction existed between different inoculation levels and different fertilization types (p < 0.05). At maturity, the AM + BF treatment increased soybean dry matter accumulation by 18.16%, 12.83%, 23.82%, and 1.61% compared to the CF, BF, AM + CK, and AM + CF treatments, respectively. Under identical fertilization treatments, the inoculated AMF treatment significantly outperformed the non-inoculated AMF treatment (p < 0.05). Furthermore, results from the two-way ANOVA revealed a significant interaction between different inoculum levels and different fertilization treatments (p < 0.05).
The number of root nodules at different growth stages of soybeans under various treatments showed an initial increase followed by a decrease as the growth stage progressed (Figure 1b). The number of root nodules reached its maximum during the flowering period and showed a gradual decline thereafter. During the soybean flowering period, all treatments reached maximum nodule counts. The flowering stage of soybeans is a period when both vegetative and reproductive growth coexist. During this phase, soybeans transition from vegetative to reproductive growth, experiencing high nutrient demand while root nodule activity reaches its peak. Consequently, under the regulation of the soybean-rhizobium symbiotic nitrogen fixation system, the number of root nodules reaches its maximum, and rhizobium activity also peaks. At the same fertilization level, the AM + CK treatment was significantly higher than the CK treatment; the CF treatment was significantly higher than the AM + CF treatment; and the BF treatment was significantly higher than the AM + BF treatment. AM + BF treatment reduced levels by 31.42%, 22.22%, 25.20%, and 6.78% compared to CF, BF, AM + CK, and AM + CF treatments, respectively. During the pod-setting stage, the number of root nodules gradually decreased. During this period, reproductive growth dominates, and the nutrients absorbed by the soybean plant must support the development of both the stems, leaves, and pods. Nutrients from the plant’s roots, stems, and leaves start to be redirected toward the pod growth points to supply the nutrients required for pod development. Compared to CF, BF, AM + CK, and AM + CF treatments, AM + BF reduced the number of root nodules during the soybean pod-setting stage by 42.50%, 40.19%, 36.91%, and 13.65%, respectively.
The soybean mycorrhizal colonization rate showed a gradual upward trend with increasing growth stage. From the seedling stage to the podding stage, the colonization rate increased rapidly, and after the podding stage, it exhibited a slow upward trend (Figure 1c). Mycorrhizal colonization rates across treatments showed consistent patterns throughout all stages, with inoculated treatments exhibiting significantly higher infection rates than non-inoculated treatments. The overall ranking among treatments was: AM + BF > AM + CF > AM + CK > BF > CF > CK. At identical inoculation levels, fertilized treatments consistently outperformed non-fertilized treatments, though no significant difference was observed between equal-rate fertilization and charcoal-based fertilizer treatments. At the soybean pod formation stage, mycorrhizal colonization rates reached their maximum values across all treatments. Mycorrhizal colonization rates ranged from 30.82% to 52.63% among the treatments.
During the soybean maturation stage, nitrogen and phosphorus accumulation in soybean plants across treatments showed consistent patterns: AM + BF > AM + CF > BF > CF > AM + CK > CK (Figure 1d,f). Compared with CF, BF, AM + CK, and AM + CF treatments, AM + BF treatment increased plant nitrogen accumulation by 24.72%, 14.04%, 40.13%, and 9.73%, respectively. Under identical inoculation levels of AMF inoculants, plants treated with carbon-based fertilizers exhibited significantly higher nitrogen accumulation than those treated with equivalent amounts of chemical fertilizers. For treatments with the same fertilization type, plants with AMF inoculants consistently accumulated more nitrogen than those without inoculation. In addition, potassium accumulation in plants treated with AM + BF increased by 35.29%, 21.82%, 62.60%, and 19.68% compared to CF, BF, AM + CK, and AM + CF, respectively. But during the soybean maturation stage, phosphorus accumulation varied among treatments (Figure 1e). Phosphorus accumulation in plants across treatment groups showed the following pattern: BF > AM + CF > AM + BF > CF > AM + CK > CK. The AM + BF treatment increased by 8.48% and 28.71% compared to the CF and AM + CK treatments, respectively, while it was 3.27% and 2.84% lower than the BF and AM + CF treatments, respectively.

3.2. Effects of AMF and Biochar-Based Fertilizer Interactions on Soil Nutrients Content and Enzyme Activities

The soil nitrate nitrogen content across treatments followed the pattern: AM + CF > BF > CF > AM + CK > AM + BF > CK (Figure 2a). The AM + BF treatment reduced emissions by 14.34%, 5.83%, 13.88%, and 8.15% compared to the AM + CF, AM + CK, BF, and CF treatments, respectively. The two-way ANOVA results indicate that the AMF inoculation did not significantly affect soil nitrate nitrogen content, while fertilizer type significantly influenced soil nitrate nitrogen content. However, a significant interaction existed between the two factors. The application of AMF and fertilizers increases soil nitrate nitrogen content. However, the interaction between AMF and carbon-based fertilizers reduces soil nitrate nitrogen content compared to the application of equal amounts of chemical fertilizers alone, carbon-based fertilizers alone, or the interaction between AMF and equal amounts of chemical fertilizers. The ammonium nitrogen content in the soil generally followed the pattern AM + BF > CF > AM + CF > BF > CK > AM + CK across all treatments (Figure 2b). The ammonium nitrogen content in the AM + BF treatment increased by 7.43%, 27.91%, 12.66%, and 3.45% compared to the AM + CF, AM + CK, BF, and CF treatments, respectively. The inoculation level of AMF did not significantly affect soil ammonium nitrogen content, while fertilizer type significantly influenced it. However, a significant interaction was observed between the two factors. This indicated that the interaction between AMF and carbon-based fertilizer exhibits a synergistic effect on soil ammonium nitrogen content, enhancing soil nutrients and promoting crop growth. Significant differences in available phosphorus content were observed among treatments, generally following the order CF > BF > AM + CF > AM + BF > CK > AM + CK (Figure 2c). AM + BF treatment reduced levels by 34.40%, 27.48%, and 8.58% compared to CF, BF, and AM + CF, respectively, and increased levels by 42.09% compared to AM + CK. AMF inoculation significantly enhanced phosphorus uptake and utilization by soybean plants, thereby reducing soil available phosphorus content. Results from two-way ANOVA indicated that both AMF inoculation and fertilizer type exerted extremely significant effects (p < 0.01) on soil available phosphorus content, with an extremely significant correlation (p < 0.01) between the two factors. The results indicate that inoculation with AMF combined with fertilization significantly alters soil available phosphorus content and enhances phosphorus utilization efficiency. The addition of AMF inoculum significantly increased soil available potassium content (Figure 2d). AM + BF treatment increased the available potassium content by 17.98%, 0.45%, 15.33%, and 16.93% compared to CK, CF, BF, and AM + CK treatments, respectively. However, the effect on soil pH was not significant (Figure 2e). In each treatment, soil urease activity exhibited the following pattern: AM + CF > AM + BF > BF > CF > AM + CK > CK (Figure 2f). The AM + BF treatment showed increases by 9.57%, 7.47%, and 43.05% compared to the CF, BF, and AM + CK treatments, respectively, while it decreased by 6.79% compared to the AM + CF treatment. Fertilization treatments all increased soil neutral phosphatase activity, while inoculation with AMF alone significantly reduced soil phosphatase activity (Figure 2g). Neutral phosphatase activity across treatments exhibited the following pattern: AM + BF > AM + CF > BF > CF > CK > AM + CK.

3.3. Effects of AMF and Biochar-Based Fertilizer Interactions on Soybean Yield

Both AMF inoculation and fertilizer application enhanced soybean yield, with the synergistic effect of AMF and charcoal-based fertilizer significantly surpassing (p < 0.05) the effects of AMF inoculation or fertilizer application alone (Table 1). The AM + BF treatment achieved a soybean yield of 3869.28 kg·ha−1, representing increases of 56.74%, 22.29%, 19.45%, 33.83%, and 6.81% compared to the CK, CF, BF, AM + CK, and AM + CF treatments, respectively. The yield hierarchy was AM + CF > BF > CF > AM + CK > CK, positively influencing all yield components. Plant height followed the pattern AM + BF > AM + CF > BF > CF > AM + CK > CK, with AM + BF showing increases of 3.59% (CK), 3.68% (CF), 5.00% (BF), 7.25% (AM + CK), and 10.54% (AM + CF) over other treatments. AM + BF treatment elevated bottom pod height by 18.38% and 6.88% compared to CK and AM + CK, respectively, but decreased by 11.08%, 2.08%, and 2.66% in CF, BF, and AM + BF treatments. The number of pods per soybean plant across treatments followed the pattern: AM + BF > AM + CF > BF > CF > AM + CK = CK. The AM + BF treatment increased pod numbers by 28.13% compared to CF, 20.59% compared to BF, 38.98% compared to AM + CK, and 13.89% compared to AM + CF. The number of seeds per soybean plant across treatments followed the pattern: AM + BF > AM + CF > BF > CF > AM + CK > CK. Compared to the CF, BF, AM + CK, and AM + CF treatments, the AM + BF treatment increased seed numbers by 17.31%, 14.02%, 26.10%, and 3.39%, respectively. Except for the significant difference between AM + BF and CK, other treatment differences were not significant.

3.4. Comprehensive Analysis of Plant Nutrient Uptake, Soil Nutrient and Yield

Neutral phosphatase activity (p < 0.01) and soil pH (p < 0.05) were significantly correlated with yield (Figure 3a). Urease emerged as a crucial factor influencing yield (Figure 3b). Partial least squares models were employed to determine the primary drivers of AMF and biochar effects on yield (Figure 4 and Figure 5). Yields without AMF inoculation can be predicted based on soil nutrients and enzyme activity. Soil nutrients, in turn, significantly influence soil enzyme activity(Figure 4a). In contrast, the yield trajectory of crops treated with AMF inoculants can be predicted through plant nutrient uptake (Figure 4b). The addition of AMF inoculant significantly altered the direction of plant nutrient influence (Figure 5a,b). Fertilizer addition modified the influence pathways of enzyme activity on yield (Figure 5c–e). At the same time, it has significantly altered the predictive effectiveness of soil nutrients on yield (Figure 4c–e). Although biochar basal fertilizer altered its effect on crop nutrients, this change was not significant in CF treatment (Figure 5e).

4. Discussion

4.1. Effects of AMF and Biochar Basal Fertilizer Interaction on Crop Growth

Our study demonstrated that AMF inoculation and fertilization treatments enhanced dry matter accumulation in soybean plants at different growth stages. The AM + BF treatment resulted in the highest dry matter accumulation across all stages, followed by the AM + CF treatment (Figure 1a). These findings suggest that the interaction between AMF and fertilizers can stimulate soybean growth potential and increase dry matter accumulation during the cultivation period. Additionally, the combined effect of AMF and carbon-based fertilizer was significantly more effective in promoting soybean growth than AMF alone, chemical fertilizers, or carbon-based fertilizers. Many studies have reported that AMF inoculation, biochar basal fertilizer application, and the combined use of AMF and biochar can substantially enhance crop growth, development, and dry matter accumulation [22,23,24]. The AM + BF treatment resulted in the highest biomass, likely due to differences in nutrient availability and efficiency for soybean growth.
The dry matter accumulation trend exhibited a slow-fast-slow pattern, with nodule numbers initially increasing and then decreasing as the growth stage progressed (Figure 1b). As the plants developed, the number of nodules in the AMF + CF and AMF + BF treatments was significantly lower than in the CK treatment, indicating a shift in plant nutrient strategy. In the presence of AMF, plants reduced nutrient allocation to nodules and instead adopted a more efficient root-based nutrient uptake system. This variation in nutrient uptake reflects the synergistic effect of AMF and different fertilizers on soybean nutrient acquisition (Figure 1d–f). Nitrogen and potassium, being mobile nutrients in the soil, exhibited accumulation patterns that were consistent with biomass accumulation, highlighting the beneficial impact of the treatments on plant growth and nutrient absorption. The BF treatment showed superior performance in phosphorus accumulation (Figure 1e). AMF and biochar promote corn growth and nutrient uptake through different mechanisms [25]. The mechanisms by which AMF promotes crop growth primarily involve two aspects: (I) Enhancing plant absorption of nutrients and water while improving stress tolerance, thereby stimulating plant growth [26]. (II) Promote photosynthesis in plants and facilitate carbon accumulation within them [27]. AMF can explore a larger soil volume than roots by generating extensive mycelial networks [28]. Therefore, it can enhance the efficiency of plant nutrient uptake. In addition to mycelial expansion, the external hyphae of AMF can also secrete protons to promote the dissolution of insoluble nutrients in acidic soils. This is the primary reason why AMF inoculation promotes soybean growth in this study [29]. Additionally, biochar possesses a porous structure and high specific surface area, which can increase soil porosity and specific surface area [30]. Enhance soil water retention capacity and aeration, improving soil physical properties [31]. It also has a certain impact on the ecological environment of soil microbe [32]. These properties enhance the contact area between crop roots and soil, promoting nutrient uptake. Biochar increases crop root geotropism and root length, boosting root surface area and root volume. This, in turn, expands the root system’s contact area with the soil and enhances its nutrient absorption capacity [33]. Additionally, biochar can enhance the activity and availability of various ions in the soil through adsorption and exchange processes, thereby promoting nutrient uptake by crops [34]. Therefore, the application of biochar can significantly enhance the mycorrhizal colonization rate of AMF and jointly increase nutrient uptake in soybeans. Simultaneously, AMF also promotes the efficient utilization of biochar. AMF can penetrate into biochar through minute pores inaccessible to other plant roots, thereby aiding plants in acquiring nutrients. Consequently, the interaction between AMF and biochar exerts a synergistic effect on soybean growth and nutrient uptake.

4.2. Effects of AMF and Biochar Base Fertilizer Interaction on Soil Properties

Soil plays a crucial role in material cycling, energy flow, biological succession, and information transfer within ecosystems [35]. The quality of soil directly impacts its ecological functions. Following AMF inoculation, soil nutrient levels decreased (Figure 2b,c). This decrease reflects the nutrient retention by AMF hyphae, where the nutrients absorbed by the hyphae are not immediately transferred to plants but are partly used to build AMF biomass [36]. However, the AMF + BF treatment enhanced plant growth (Figure 1a) and increased the levels of ammonium nitrogen and available phosphorus in the soil (Figure 2b,d). The addition of biochar can also supply nitrogen nutrients to the soil. Meanwhile, due to its excellent adsorption properties, biochar can absorb and immobilize nitrogen present in fertilizers and soil, thereby reducing nitrogen loss and increasing the content of alkaline hydrolyzable nitrogen and total nitrogen in the soil. This may explain why adding biochar leads to increased soil nitrogen content [37]. Biochar regulates soil pH [38], enhances soil water retention [39], and creates favorable microenvironments that support AMF reproduction and activity. At the same time, biochar improves soil available phosphorus content by increasing soil pH, thereby altering the activity of cations (Al, Fe, and Ca) in the soil, reducing phosphorus adsorption or increasing phosphorus desorption [40]. Biochar can also serve as an external nutrient source to increase soil available phosphorus content. Since the pyrolysis process of organic materials breaks down organic phosphate chemical bonds, biochar contains substantial soluble phosphate, which can directly enhance available phosphorus levels in soil [41,42]. This is attributable to biochar serving as a direct phosphorus source. During high-temperature carbonization, the breaking of chemical bonds significantly increases soluble inorganic orthophosphate and polyphosphate, leading to the enrichment of total phosphorus in biochar. Its content is markedly higher than before carbonization [43]. Thus, biochar-based fertilizer improved the growth conditions for AMF, promoted AMF colonization of soybean roots, and enabled AMF to absorb additional nutrients from the soil through its extensive hyphal network, enriching the soil near the soybean roots and enhancing nutrient content. The addition of biochar alters soil physicochemical properties, which in turn affects AMF. This interaction between AMF and biochar may be the reason for the changes in soil physicochemical properties.

4.3. Comprehensive Analysis of Plant Nutrient Uptake, Soil Nutrient and Yield

The AM + BF treatment achieved a soybean yield of 3869.28 kg·ha−1, representing increases of 56.74%, 22.29%, 19.45%, 33.83%, and 6.81% compared to the CK, CF, BF, AM + CK, and AM + CF treatments, respectively. Soybean yield could be enhanced by the application of microbial agents or fertilizers; however, the most significant increase was observed when AMF and biochar were used as the basal fertilizer, resulting in a 56.74% increase in yield (Table 1). Correlation analysis between soybean yield and environmental factors revealed that neutral phosphatase activity (p < 0.01) and soil pH (p < 0.05) were significantly correlated with yield, while urease activity was also a key factor influencing soybean yield (Figure 3). Neutral phosphatase is a biocatalyst secreted by plant roots and soil microorganisms [44]. Elevated phosphatase activity indicates a faster transformation rate of organic phosphorus to available phosphorus, thus enhancing the phosphorus supply capacity of the soil [45]. Urease regulates the release of nitrogen sources, converting organic nitrogen into inorganic nitrogen for plant and microbial uptake [46]. Urease and phosphatase activities were enhanced by the AMF + BF treatment (Figure 2f,g). These results suggest that AMF and biochar can stabilize soil pH and promote a healthy and balanced soil enzyme activity system.
PLS-SEM analysis revealed that soybean yield was influenced by the soil nutrient path in the absence of AMF addition (Figure 4a). However, this relationship became non-significant upon the addition of AMF (Figure 4b). The direction of the total effect for each variable changed (Figure 5a,b). This shift suggests that AMF application altered the original nutrient supply pattern. Without AMF, soybean primarily relied on its roots for nutrient uptake [47]. After AMF inoculation, the passive absorption mechanism, driven by soil chemical properties, transitioned to a biologically driven mode dominated by AMF [48]. AMF became the primary regulator of nutrient distribution, diminishing the straightforward linear relationship between the soil’s direct nutrient pool and yield. Even when soil nutrient levels are low, plants can still access sufficient nutrients through AMF functioning [49]. As a result, predicting yield based on soil nutrients alone becomes challenging, leading to the insignificance of this path. Thus, the determinant of yield shifts from soil to the efficiency of the AMF–plant symbiosis.
PLS-SEM analysis revealed that the direction of effects of plant nutrients, soil nutrients, and soil enzyme activity on yield shifted with fertilizer addition (Figure 5c–e). This change may be attributed to nutrient limitation in the system when no fertilizer is applied, with nutrient increases having a positive, linear effect on yield. However, as fertilizer is added, it addresses nutrient limitations, resulting in more complex relationships. Both CF and BF treatments influenced yield pathways, but biochar-based fertilizer more significantly altered the direction of impact on crop nutrients, which was not observed under CF treatment (Figure 4c–e). Under CF treatment, yield and plant nutrient concentrations are primarily determined by fertilizer input, and they remain strongly correlated. Chemical fertilizers provide readily available nutrients, and plant nutrient content remains positively correlated with yield. BF treatment optimizes the entire production system. Application of biochar-based fertilizer can change soil physical, chemical, and biological properties to indirectly affect plant nutrition [50]. Furthermore, the combination of biochar and AMF significantly enhances AMF colonization and promotes the development of hyphal networks, thereby facilitating the establishment of a symbiotic system [39]. This explains why the AMF + BF treatment exhibited the most effective synergistic effect, as it combines the strongest bioabsorption network with the most favorable soil conditions, maximizing biointeraction potential.

5. Conclusions

The interaction between AMF and biochar-based fertilizer significantly enhanced the growth and development of soybean plants. Dry matter accumulation in soybean plants was enhanced throughout each growth stage because the uptake and accumulation of nitrogen, phosphorus, and potassium were notably increased during each growth period. Mycorrhizal infection rate was significantly enhanced in AM + BF compared with the other treatments. But it reduced the nodule number at the flowering, pod-setting, and seed-filling stages separately. Furthermore, the combination of AMF and conventional fertilizer (CF) markedly stimulated soil urease and neutral phosphatase activities. Urease activity decreased by 6.79% compared to AM + CF, but increased by 7.47–47.43% relative to the other five treatments. Neutral phosphatase activity increased by 7.71–19.06% compared to the other five treatments. Additionally, the AM + BF treatment positively influenced soybean yield components, resulting in soybean yield elevated by 6.81–56.74% compared to other treatments. The interaction between AMF and biochar-based fertilizers offers a sustainable management strategy with potential applications for maintaining grain yields. Future research should expand to include the exploration of soil microbial communities to better understand the mechanisms driving improvements in soybean yields and soil properties.

Author Contributions

Conceptualization, L.M., H.Y. and S.L.; Methodology, L.M., H.Y., J.L., D.W. and S.L.; Software, H.Y., Y.F. and D.W.; Validation, H.Y. and Y.F.; Formal analysis, H.Y., Y.F., D.W. and X.M.; Investigation, H.Y., J.L. and X.M.; Resources, L.M.; Data curation, H.Y., J.L. and X.M.; Writing—original draft, H.Y.; Writing—review & editing, L.M. and S.L.; Visualization, H.Y. and D.W.; Supervision, H.Y. and S.L.; Project administration, L.M. and S.L.; Funding acquisition, L.M. and S.L. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the National Natural Science Foundation of China (32271657): Study on mechanisms of carbon and nitrogen distribution between host and hypha mediated by arbuscular mycorrhizal fungi and their regulation of soil organic carbon in maize/soybean intercropping.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of AMF addition and biochar interaction on soybean plant biomass (a), number of soybean root nodules (b), AMF colonization rate (c), soybean nitrogen accumulations (d), soybean phosphorus accumulations (e), and soybean potassium accumulations (f). The data in the figures were mean values ± standard deviation of three repeated. A: AMF; F: Fertilization type; A × F: Interaction between AMF and fertilization type. * means significant difference at p < 0.05, ** means significant difference at p < 0.01, *** means significant difference at p < 0.001, ns means no significant difference. Different letters indicate significant differences between treatments (p < 0.05).
Figure 1. Effects of AMF addition and biochar interaction on soybean plant biomass (a), number of soybean root nodules (b), AMF colonization rate (c), soybean nitrogen accumulations (d), soybean phosphorus accumulations (e), and soybean potassium accumulations (f). The data in the figures were mean values ± standard deviation of three repeated. A: AMF; F: Fertilization type; A × F: Interaction between AMF and fertilization type. * means significant difference at p < 0.05, ** means significant difference at p < 0.01, *** means significant difference at p < 0.001, ns means no significant difference. Different letters indicate significant differences between treatments (p < 0.05).
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Figure 2. Effects of AMF addition and biochar interaction on soil nitrate nitrogen (a), soil ammonium nitrogen (b), soil available phosphorus (c), soil available potassium (d), soil pH (e), soil urease activity (f), and soil neutral phosphatase activity (g). The data in the figures were mean values ± standard deviation of three repeated. A: AMF; F: Fertilization type; A × F: Interaction between AMF and fertilization type. * means significant difference at p < 0.05, ** means significant difference at p < 0.01, *** means significant difference at p < 0.001, ns means no significant difference. Different letters indicate significant differences between treatments (p < 0.05).
Figure 2. Effects of AMF addition and biochar interaction on soil nitrate nitrogen (a), soil ammonium nitrogen (b), soil available phosphorus (c), soil available potassium (d), soil pH (e), soil urease activity (f), and soil neutral phosphatase activity (g). The data in the figures were mean values ± standard deviation of three repeated. A: AMF; F: Fertilization type; A × F: Interaction between AMF and fertilization type. * means significant difference at p < 0.05, ** means significant difference at p < 0.01, *** means significant difference at p < 0.001, ns means no significant difference. Different letters indicate significant differences between treatments (p < 0.05).
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Figure 3. Correlation analysis between soybean yield and soil environmental factors (a). Random forest model predicting key factors influencing soybean yield (b). Abbreviations: AK, soil available potassium; AP, soil available phosphorus; NH4-H, soil ammonium nitrogen; NO3-H, soil nitrate nitrogen; NA, nitrogen accumulations in plants; PA, phosphorus accumulations in plants; KA, potassium accumulations in plants; Biomass, soybean plant biomass; pH, soil pH; Urease, soil urease activity; NP, soil neutral phosphatase activity. *, **, and *** indicate the significant level at p < 0.05, p < 0.01, and p < 0.001, ns indicate no significant difference.
Figure 3. Correlation analysis between soybean yield and soil environmental factors (a). Random forest model predicting key factors influencing soybean yield (b). Abbreviations: AK, soil available potassium; AP, soil available phosphorus; NH4-H, soil ammonium nitrogen; NO3-H, soil nitrate nitrogen; NA, nitrogen accumulations in plants; PA, phosphorus accumulations in plants; KA, potassium accumulations in plants; Biomass, soybean plant biomass; pH, soil pH; Urease, soil urease activity; NP, soil neutral phosphatase activity. *, **, and *** indicate the significant level at p < 0.05, p < 0.01, and p < 0.001, ns indicate no significant difference.
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Figure 4. The directed graph of the Partial Least Squares Structural Equation Model (PLS-SEM) shows the effects of soil properties, soil enzyme activity, and plant nutrient uptake under different treatments on soybean yield (a–e). Line transparency correlates significantly with path strength. Red lines indicate positive paths, while blue lines denote negative paths. Numbers beside arrows represent standardized path coefficients, and line widths indicate effect magnitude. Abbreviations: AK, soil available potassium; NH4-H, soil ammonium nitrogen; PA, phosphorus accumulations in plants; KA, potassium accumulations in plants; pH, soil pH; Urease, soil urease activity; NP, soil neutral phosphatase activity. *, **, and *** indicate the significant level at p < 0.05, p < 0.01, and p < 0.001.
Figure 4. The directed graph of the Partial Least Squares Structural Equation Model (PLS-SEM) shows the effects of soil properties, soil enzyme activity, and plant nutrient uptake under different treatments on soybean yield (a–e). Line transparency correlates significantly with path strength. Red lines indicate positive paths, while blue lines denote negative paths. Numbers beside arrows represent standardized path coefficients, and line widths indicate effect magnitude. Abbreviations: AK, soil available potassium; NH4-H, soil ammonium nitrogen; PA, phosphorus accumulations in plants; KA, potassium accumulations in plants; pH, soil pH; Urease, soil urease activity; NP, soil neutral phosphatase activity. *, **, and *** indicate the significant level at p < 0.05, p < 0.01, and p < 0.001.
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Figure 5. Standardized total effect analysis of soil properties, soil enzyme activity, and plant nutrient uptake on soybean yield under the treatments of no AMF inoculation (a), AMF inoculation (b), CK (c), CF (d), and BF (e).
Figure 5. Standardized total effect analysis of soil properties, soil enzyme activity, and plant nutrient uptake on soybean yield under the treatments of no AMF inoculation (a), AMF inoculation (b), CK (c), CF (d), and BF (e).
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Table 1. Effect of AMF interaction with carbon-based fertilizer on soybean yield composition factors.
Table 1. Effect of AMF interaction with carbon-based fertilizer on soybean yield composition factors.
TreatmentsPlant Height (cm)Bottom Pod Height (cm)Pod Number of per PlantGrain Number of per PlantSeed Weight per Plant (g)100-Seed Weight (g)Yield (kg·ha−1)
CK116.00 ± 1.9 c15.43 ± 1.40 c59.20 ± 1.5 c121.00 ± 6.24 d24.60 ± 1.45 c19.54 ± 0.71 b2468.58 ± 184.98 d
CF122.11 ± 2.7 b20.55 ± 0.84 a64.47 ± 5.7 bc138.67 ± 9.45 c27.56 ± 1.41 bc20.63 ± 0.95 ab3164.06 ± 206.03 bc
BF123.67 ± 3.5 ab18.66 ± 1.05 ab68.20 ± 4.3 b142.67 ± 10.12 bc29.04 ± 1.71 b20.41 ± 0.75 ab3239.35 ± 106.64 b
AM + CK119.56 ± 2.1 bc17.10 ± 0.51 bc59.13 ± 3.2 c129.00 ± 9.00 cd26.42 ± 2.56 bc20.21 ± 0.56 ab2891.16 ± 146.62 cd
AM + CF123.78 ± 4.0 ab18.77 ± 1.84 ab72.53 ± 4.1 b156.33 ± 9.02 ab32.95 ± 0.95 a20.86 ± 0.56 ab3622.58 ± 172.81 a
AM + BF128.22 ± 2.3 a18.27 ± 1.13 ab82.07 ± 4.4 a162.67 ± 11.59 a35.14 ± 8.24 a21.52 ± 0.63 a3869.28 ± 55.76 a
ANOVA
A**ns******ns***
F**ns**********
A × Fnsns*ns*nsns
Notes: The values are means ± standard deviation (n = 3). A: AMF; F: Fertilization type; A × F: Interaction between AMF and fertilization type. * means significant difference at p < 0.05, ** means significant difference at p < 0.01, *** means significant difference at p < 0.001, ns means no significant difference. Different letters indicate significant differences between treatments (p < 0.05).
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Meng, L.; Yang, H.; Fan, Y.; Li, J.; Song, D.; Ma, X.; Li, S. Synergy of Arbuscular Mycorrhizal Fungi and Biochar-Based Fertilizer Reshapes Soybean Nutrient Acquisition and Drives Yield Enhancement. Sustainability 2025, 17, 10355. https://doi.org/10.3390/su172210355

AMA Style

Meng L, Yang H, Fan Y, Li J, Song D, Ma X, Li S. Synergy of Arbuscular Mycorrhizal Fungi and Biochar-Based Fertilizer Reshapes Soybean Nutrient Acquisition and Drives Yield Enhancement. Sustainability. 2025; 17(22):10355. https://doi.org/10.3390/su172210355

Chicago/Turabian Style

Meng, Lingbo, Huawei Yang, Yue Fan, Jiang Li, Diwei Song, Xiaozhe Ma, and Shumin Li. 2025. "Synergy of Arbuscular Mycorrhizal Fungi and Biochar-Based Fertilizer Reshapes Soybean Nutrient Acquisition and Drives Yield Enhancement" Sustainability 17, no. 22: 10355. https://doi.org/10.3390/su172210355

APA Style

Meng, L., Yang, H., Fan, Y., Li, J., Song, D., Ma, X., & Li, S. (2025). Synergy of Arbuscular Mycorrhizal Fungi and Biochar-Based Fertilizer Reshapes Soybean Nutrient Acquisition and Drives Yield Enhancement. Sustainability, 17(22), 10355. https://doi.org/10.3390/su172210355

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