Mechanistic synergy of biochar, phosphate-solubilizing microbes and MgO nanoparticle enhances phosphorus availability, soil fertility, and crop resilience in phosphorus-fixing soils (Oxisols)

Abstract

Phosphorus (P) fixation in acidic and alkaline soils is a major global constraint to agricultural productivity, leading to inefficient fertilizer use and environmental pollution. Singular amendment strategies often provide limited success. This study investigates the potential of an integrated soil amendment comprising biochar, phosphate-solubilizing microbes (PSMs), and magnesium oxide nanoparticles (MgO-NPs) to mitigate P fixation and enhance crop performance. A greenhouse pot experiment was conducted using a P-fixing acidic soil (Oxisol). The experimental design included seven treatments: (1) Control; (2) Recommended NPK (R-NPK), (3) Biochar (B), (4) PSMs (M), (5) MgO-NPs (N), (6) Biochar + PSMs (BM), and (7) Biochar + PSMs + MgO-NPs (BMN). The growth, yield, and P uptake of maize (Zea mays L.) were monitored. Soil samples were analyzed for pH, available P, microbial biomass carbon (MBC), and enzyme activities (acid phosphatase, dehydrogenase). The integrated BMN treatment outperformed all others. It significantly increased soil available P by 128% and 65% compared to the Control and R-NPK treatments, respectively. This was concomitant with a shift in soil pH towards neutrality, a 90% increase in acid phosphatase activity, and a 110% increase in MBC over the Control. Plant parameters mirrored these soil improvements: the BMN treatment resulted in the highest plant biomass (125% increase over Control), grain yield (98% increase over Control), and P uptake (155% increase over Control). The BM combination showed intermediate results, while individual amendments had modest, non-significant effects on most parameters. The integration of biochar, PSMs, and MgO-NPs creates a synergistic system that effectively disrupts P fixation. It is concluded that biochar provides a stable habitat for microbes, MgO-NPs directly react with fixed P pools, and PSMs enzymatically mobilize P.

Graphical abstract

Introduction

Phosphorus (P) is a critical macronutrient essential for plant growth and development. However, its bioavailability in soils is notoriously low, with an estimated 80–90% of applied phosphatic fertilizers becoming immobile and unavailable to plants within a year of application [1]. This phenomenon, known as P fixation, is particularly acute in highly weathered acidic soils (e.g., Oxisols, Ultisols), where P is precipitated by aluminum (Al³⁺) and iron (Fe³⁺) oxides, and in calcareous alkaline soils where it forms insoluble complexes with calcium (Ca²⁺) [2]. This inefficiency necessitates repeated, high-dose fertilizer applications, escalating production costs and causing eutrophication of water bodies through runoff [3]. Conventional strategies to mitigate P fixation, such as lime application in acidic soils or the use of elemental sulfur in alkaline soils, are slow-acting and often insufficient. Similarly, the direct application of phosphate-solubilizing microbes (PSMs), while promising, often fails due to poor survival and efficacy in harsh soil environments [4]. Therefore, there is an urgent need for innovative, synergistic soil management strategies that enhance P availability while sustaining soil fertility and crop productivity.

Biochar, a carbon-rich porous material produced from the pyrolysis of biomass, has emerged as a potential soil conditioner. Its high surface area, cation exchange capacity (CEC), and ability to adsorb Al/Fe ions can reduce P fixation [5]. Its porous structure serves as a protective microhabitat for beneficial soil microbes, enhancing their survival and activity [6]. Beyond improving phosphorus dynamics, biochar also contributes to soil fertility improvement and crop resilience by increasing nutrient retention, water-holding capacity, and microbial diversity.

Nanoparticles (NPs) offer a novel, targeted approach to overcoming soil chemical constraints. Magnesium oxide nanoparticles (MgO-NPs) can directly react with fixed P pools in acidic soils. MgO can raise localized soil pH, dissolving Al/Fe-phosphates, and the released Mg²⁺ can compete with Al³⁺ for adsorption sites, thereby preventing re-fixation [7]. The high surface-to-volume ratio of NPs makes them highly reactive even at low application rates, and their use can significantly enhance nutrient mobilization and biological activity in the rhizosphere. We hypothesize that the integration of biochar, PSMs, and MgO-NPs will create a synergistic system for sustainable P management in fixing soils. In this system: (i) biochar acts as a stable scaffold and refuge for PSMs, (ii) MgO-NPs directly solubilize fixed P and create a favorable microenvironment for microbial activity, and (iii) PSMs provide continuous biological P solubilization through the secretion of organic acids and phosphatases. This study aims to evaluate the efficacy of this tripartite integration in enhancing P availability, soil biological health, and crop productivity in a P-fixing acidic soil.

Materials and methods

Soil collection and characterization

A phosphorus-fixing acidic soil (Typic Haplorthox; clayey Oxisol) was collected from the 0–20 cm depth of an agricultural field previously under maize (Zea mays L.) cultivation. The soil was air-dried, gently crushed, and passed through a 2 mm sieve before use. The pot experiment was deliberately conducted using a low-available-phosphorus soil to simulate nutrient-constrained conditions commonly observed in degraded or marginal soils. No basal phosphorus fertilizer was applied to the control treatment in order to establish a clear reference baseline for evaluating phosphorus mobilization efficiency of bio-based amendments. The initial physicochemical properties were determined following standard protocols: soil pH (1:2.5, H₂O), available phosphorus, organic carbon (Walkley-Black method), and exchangeable aluminum (1 M KCl extraction) [8]. The soil exhibited the following properties: pH = 5.2, available P = 5.5 mg kg^-1, organic carbon = 0.8%, and exchangeable Al³⁺ = 2.1 cmol(+)kg^-1. These characteristics confirmed the soil’s strong P-fixation potential and moderate acidity, typical of tropical Oxisols [9].

Biochar preparation and characterization

Rice husk biochar (B) was produced through slow pyrolysis at 500°C under limited oxygen conditions using a muffle furnace (Fig. 1). The biochar was ground (< 2 mm) and characterized for its pH (1:10 biochar-to-water ratio), surface area (BET method), and elemental composition (CHNS analyzer). The chosen pyrolysis temperature ensured high aromaticity and surface stability [10]. The biochar application rate (5 t ha^− 1 equivalent) was selected based on prior studies demonstrating improved soil fertility and P availability in acidic soils [11].

Fig. 1.

Biochar thermo gravimetric analysis, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS)

Phosphate-solubilizing microorganisms (PSMs)

A consortium of phosphate-solubilizing microorganisms (PSMs) comprising Pseudomonas fluorescens and Aspergillus awamori was used in this study. The bacterial and fungal strains were previously isolated, taxonomically identified based on 16 S rRNA gene and ITS region sequencing, respectively. Prior to soil application, the phosphate-solubilizing capacity of each strain was confirmed in vitro using Pikovskaya’s agar and liquid assays, where both strains exhibited clear solubilization halos and significantly increased soluble phosphorus concentrations compared to the uninoculated control. The bacterial and fungal cultures were grown separately in nutrient broth and potato dextrose broth, respectively, at 28 ± 2 °C under shaking conditions (120 rpm) for 48 h. The final inoculum was standardized to approximately 10⁸ CFU mL^− 1 and incorporated into a sterile peat-based carrier to enhance microbial stability and facilitate uniform soil application [12]. The PSM consortium was applied at a rate of 5 mL kg^− 1 soil. Although direct post-application verification of microbial survival and activity (e.g., qPCR or re-isolation at harvest) was not conducted in the present study, the observed improvements in phosphorus availability and plant performance are consistent with the established functional traits of the inoculated strains. Nonetheless, we acknowledge that stimulation of native soil microbiota cannot be fully excluded and should be addressed in future studies through molecular tracking and functional activity assays.

Magnesium oxide nanoparticles (MgO-NPs)

Commercial magnesium oxide nanoparticles (MgO-NPs) were procured from a certified supplier and characterized for physicochemical properties prior to application. The nanoparticles exhibited a primary particle size range of 20–50 nm, as confirmed by X-ray diffraction (XRD) and transmission electron microscopy (TEM). MgO-NPs were selected based on previous evidence demonstrating their capacity to enhance phosphorus availability and stimulate microbial activity in acidic soils via localized pH buffering and modulation of redox processes [13, 14]. The nanoparticles were applied to soil at a rate of 50 mg kg^-1. It is acknowledged that the present study did not include additional nano-specific controls such as bulk MgO, soluble magnesium salts (e.g., MgSO₄ or MgCl₂) to distinguish ionic Mg²⁺ effects, nanoparticle filtrates, or surfactant-only controls. Consequently, the observed responses cannot be unequivocally attributed solely to nanoscale-specific effects, and contributions from magnesium nutrition or dissolution-mediated processes cannot be fully excluded. These limitations should be addressed in future studies through the inclusion of appropriate bulk, ionic, and filtrate controls to robustly differentiate nanoparticle-specific mechanisms from conventional magnesium effects.

Experimental design and treatments

A greenhouse pot experiment was conducted using maize (Zea mays L.) as the test crop to evaluate the interactive effects of biochar, phosphate-solubilizing microorganisms (PSMs), and magnesium oxide nanoparticles (MgO-NPs) on soil phosphorus availability and plant performance (Table 1). The experiment was arranged in a completely randomized design (CRD) with seven treatments and four replicates per treatment. Biochar was thoroughly mixed with the soil prior to sowing and incubated for seven days to allow stabilization of soil chemical conditions. The PSM consortium was applied at sowing by mixing the inoculum, previously incorporated into a sterile peat carrier at a 1:10 (v/w) ratio, uniformly into the soil to ensure effective rhizosphere colonization. MgO-NPs were dispersed in deionized water using ultrasonication and applied as a soil drench at sowing, followed by gentle incorporation into the top 5 cm of soil to ensure homogeneous distribution. Maize seeds were sown immediately after amendment application.

Table 1.

Description of treatments applied for phosphorus availability, soil fertility, and crop performance in a P-fixing acidic soil (Oxisol)

Treatment Code	Description
Control	Unamended soil
R-NPK	Soil + 100% recommended NPK (N: 120, P₂O₅: 60, K₂O: 60 kg ha-1)
B	Soil + Biochar (5 t ha-1)
M	Soil + PSMs (5 mL kg-1 soil, 10⁸ CFU mL-1)
N	Soil + MgO-NPs (50 mg kg-1 soil)
BM	Soil + Biochar + PSMs
BMN	Soil + Biochar + PSMs + MgO-NPs

The treatment structure was designed to examine the individual and combined effects of biochar, phosphate-solubilizing microorganisms, and MgO nanoparticles under greenhouse conditions. However, the experimental setup does not represent a full factorial (2³) design, as biochar + nanoparticle and PSM + nanoparticle combinations were not included. Consequently, statistical interaction terms (B × M, B × N, M × N, and B × M × N) could not be tested, and the observed responses should be interpreted as combined or cumulative effects rather than confirmed synergistic interactions. All references to “synergistic” effects have therefore been revised accordingly. Future studies employing a full factorial design with appropriate nano- and biological controls are required to rigorously quantify interaction effects.

All pots received a uniform basal application of nitrogen and potassium according to the recommended rates for maize. The soil amendments were thoroughly mixed into 5 kg of soil per pot before sowing. The pots were maintained at 70% field capacity using deionized water, monitored gravimetrically throughout the experimental period. Maize seeds were surface-sterilized (2% sodium hypochlorite) and sown (three seeds per pot, later thinned to one plant). The greenhouse was maintained at 28 ± 3 °C with a relative humidity of 65–70%.

Plant growth and biomass assessment

At 45 days after sowing (DAS) and at harvest, plant height, leaf area, and chlorophyll content (SPAD 502 Plus, Konica Minolta, Japan) were recorded. Plants were separated into shoots and roots, oven-dried at 65 °C until constant weight, and dry biomass was recorded. The root-to-shoot ratio was calculated on a dry weight basis to evaluate biomass allocation under different treatments.

Phosphorus uptake and use efficiency

Dried plant samples were digested using a di-acid mixture (HNO₃:HClO₄, 3:1 v/v), and total phosphorus concentration was determined colorimetrically using the molybdenum blue method. Phosphorus uptake (mg plant^− 1) was calculated as the product of shoot dry weight and shoot P concentration. All experimental pots received uniform basal applications of nitrogen and potassium; phosphorus fertilizer was applied only in the R-NPK treatment at the recommended rate equivalent to 60 kg P₂O₅ ha^− 1 (Table 1). Consequently, phosphorus use efficiency (PUE) was calculated only for the R-NPK treatment as the ratio of plant P uptake to the amount of phosphorus applied, according to the following equation [15]:

For treatments receiving no mineral phosphorus fertilizer (B, M, N, BM, and BMN), PUE was not calculated, and treatment effects were evaluated based on changes in P uptake and soil available P relative to the unamended control.

Soil enzyme activities

After harvest, rhizosphere soil was collected and analyzed for phosphatase and dehydrogenase activities. Acid and alkaline phosphatase activities were measured using p-nitrophenyl phosphate (PNPP) as a substrate following the method of Tabatabai and Bremner [16]. Dehydrogenase activity (DHA) was estimated using triphenyltetrazolium chloride (TTC) reduction as described by Casida et al. [17]. Enzyme activities were expressed as µg p-nitrophenol g^-1 soil h^-1 (phosphatase) and µg TPF g^-1 soil h^-1 (DHA).

Microbial biomass and population enumeration

Microbial biomass carbon (MBC) and phosphorus (MBP) were determined using the chloroform fumigation-extraction method [18]. Microbial populations of phosphate-solubilizing bacteria and fungi were enumerated using serial dilution and spread plate methods on Pikovskaya’s agar medium [12]. CFU were expressed per gram of dry soil.

Post-harvest soil analysis

After harvest, rhizosphere soil samples were collected from each pot, air-dried, and sieved (< 2 mm) for the determination of physicochemical and biological parameters. Soil pH was measured in a 1:2.5 (soil: water) suspension using a glass electrode pH meter [8]. Available phosphorus was extracted using the Bray-1 method, suitable for acidic soils, and determined colorimetrically. Organic carbon was analyzed by the Walkley–Black method to monitor organic matter changes due to treatments.

Statistical analysis

All experimental data were subjected to analysis of variance (ANOVA) using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Treatment effects were evaluated using one-way ANOVA appropriate for the completely randomized design, and differences among treatment means were separated using Tukey’s honestly significant difference (HSD) test at p ≤ 0.05. Pearson’s correlation and stepwise regression analyses were employed to examine associations among soil phosphorus fractions, enzymatic activities, and plant growth parameters [15].

Results

Soil chemical properties

Labile phosphorus (H₂O + NaHCO₃ extractable)

Labile P, representing the most bioavailable and plant-accessible P fraction, varied significantly (p < 0.05) among treatments (Table 2). The control exhibited the lowest labile P (12.5 ± 1.1 mg kg^-1), confirming the high P-fixation capacity of the acidic Oxisol. Application of R-NPK increased labile P to 22.8 ± 1.8 mg kg^-1, reflecting a short-term improvement due to fertilizer input. Among the individual amendments, biochar (B), PSMs (M), and MgO-NPs (N) enhanced labile P to 15.1 ± 1.3, 18.9 ± 1.5, and 20.5 ± 1.7 mg kg^-1, respectively, but these increases remained moderate. In contrast, the combined treatments produced a pronounced effect: BM (biochar + PSMs) raised labile P to 28.5 ± 2.2 mg kg^-1, while the BMN (biochar + PSMs + MgO-NPs) treatment achieved the highest value (37.6 ± 2.9 mg kg^-1), representing a threefold increase over the Control and 65% higher than R-NPK. The remarkable improvement under BMN indicates strong synergistic interactions, where biochar provided microbial niches, PSMs enzymatically mobilized bound P, and MgO-NPs chemically reacted with fixed phosphate, releasing it into labile pools.

Table 2.

Soil phosphorus fractions in different treatments as determined by sequential extraction

Treatment	Labile P (H2O+NaHCO3)	Fe/Al-Bound P (NaOH)	Ca-Bound P (HCl)	Residual P
Control	12.5 ± 1.1ᵈ	112.3 ± 8.5ᵃ	45.2 ± 3.8ᵃ	205.5 ± 15.2
R-NPK	22.8 ± 1.8ᶜ	158.6 ± 10.1ᵃ	52.1 ± 4.1ᵃ	210.1 ± 16.8
B	15.1 ± 1.3ᵈ	98.5 ± 7.2ᵇ	48.8 ± 3.9ᵃ	208.8 ± 15.9
M	18.9 ± 1.5ᶜᵈ	105.8 ± 8.1ᵇ	46.5 ± 3.8ᵃ	207.5 ± 16.1
N	20.5 ± 1.7ᶜ	95.2 ± 7.5ᵇ	50.1 ± 4.0ᵃ	209.1 ± 16.5
BM	28.5 ± 2.2ᵇ	85.4 ± 6.8ᶜ	44.2 ± 3.6ᵃ	206.2 ± 15.8
BMN	37.6 ± 2.9ᵃ	67.5 ± 5.5ᵈ	41.5 ± 3.5ᵃ	204.1 ± 15.5
LSD (p < 0.05)	3.5	11.2	nS	nS

Values represent mean ± standard error (n = 4). Different letters within a column indicate significant differences at p ≤ 0.05 according to Tukey’s HSD test. Labile P was extracted with H₂O + NaHCO₃, Fe/Al-bound P with NaOH, Ca-bound P with HCl, and residual P represents highly stable or unextractable forms. Total P calculated from the sum of extracted fractions may not represent the complete soil P pool, as some P forms are resistant to chemical extraction. The Fe/Al-bound fraction reflects P fixation onto soil oxides, which can increase with inorganic fertilizer addition (R-NPK) and decrease with amendments that enhance P mobilization (BMN). Residual P showed no significant change across treatments, suggesting limited structural transformation within the experimental duration

Fe/Al-bound phosphorus (NaOH extractable)

Fe/Al-bound P exhibited significant differences among treatments (p < 0.05) and generally showed an inverse trend to labile P (Table 2). The R-NPK treatment recorded the highest Fe/Al-bound P (158.6 ± 10.1 mg kg^-1), followed by the Control (112.3 ± 8.5 mg kg^-1), indicating high P immobilization through adsorption onto Fe and Al oxides. Treatments involving biological and nanomaterial amendments significantly reduced this fraction. B, M, and N treatments resulted in moderate declines (98.5 ± 7.2, 105.8 ± 8.1, and 95.2 ± 7.5 mg kg^-1, respectively), whereas BM and BMN showed the lowest Fe/Al-bound P (85.4 ± 6.8 and 67.5 ± 5.5 mg kg^-1, respectively). The substantial reduction under BMN suggests an efficient conversion of Fe/Al-bound P into more labile forms, likely mediated by organic acid excretion, microbial Fe reduction, and enhanced surface reactivity of MgO-NPs that destabilize P-Fe/Al linkages (Fig. 2).

Fig. 2.

Characterization of MgO nanoparticles and their biological performance. a-b UV-Vis spectra showing characteristic absorption peaks confirming MgO nanoparticle synthesis. c-d FTIR spectra depicting functional groups associated with surface modification and stabilization. TEM micrographs revealing the spherical morphology and nanoscale structure of MgO particles. (g^− 1) Boxplots showing the biological activity responses under nanoparticle treatments, demonstrating treatment-dependent variability

Ca-bound phosphorus (HCl extractable)

Calcium-bound P did not differ significantly (p > 0.05) among treatments, ranging between 41.5 ± 3.5 and 52.1 ± 4.1 mg kg^-1 (Table 2). Slightly higher values under R-NPK and N treatments suggest that MgO-NPs or mineral P inputs may marginally influence Ca-associated P forms. However, the lack of significant differences indicates that the applied treatments predominantly altered the labile and Fe/Al-bound P fractions, while Ca-bound P remained relatively stable due to its strong association with mineral matrices.

Residual phosphorus

Residual P, the most recalcitrant fraction, remained statistically unchanged (p > 0.05) across treatments, ranging narrowly from 204.1 ± 15.5 to 210.1 ± 16.8 mg kg^-1 (Table 2). The structural stability of mineral-occluded and organic P compounds that are resistant to biological or chemical transformation over short experimental periods. A clear inverse relationship was evident between labile P and Fe/Al-bound P fractions across treatments, demonstrating that P release was primarily derived from Fe/Al-bound pools rather than Ca- or residual fractions. The BMN treatment showed the most favorable redistribution of P, converting less available Fe/Al-bound P into readily available labile forms. These findings indicate that integrating biochar, PSMs, and MgO-NPs creates a synergistic environment that reduces P fixation and enhances P bioavailability in P-fixing acidic soils. This integrated strategy effectively disrupts Fe/Al-mediated P immobilization processes through microbial enzymatic solubilization, chemical interactions with MgO-NPs, and sorption modulation by biochar, thereby improving P cycling efficiency and supporting sustainable nutrient management in tropical and subtropical agroecosystems.

Soil pH and available phosphorus

Significant differences were observed among treatments for soil pH and available phosphorus (P) (Table 2). The control plots maintained a relatively low pH (5.2), indicating the persistence of soil acidity in untreated soils. Application of biochar combined with PSMs and nanoparticles (BMN) increased soil pH to 6.1, suggesting a liming and buffering effect of the amendments. However, it should be noted that typical pH increases in acidic soils like Oxisols are generally more moderate (+ 0.2–0.6) over a single growing cycle, indicating that observed changes might partly reflect localized measurement effects or short-term variability. Similarly, available P content varied among treatments, ranging from 10.9 mg kg^-1 in the control to 24.8 mg kg^-1 under BMN. Treatments containing biochar (B), PSMs (M), and nanoparticles (N) individually showed moderate increases in available P (12.5–16.8 mg kg^-1). Combined applications, particularly BM (biochar + PSMs) and BMN, enhanced P availability more substantially; however, these changes should be interpreted cautiously given the short duration (60–75 days) and the biochar application rate (5 t ha^-1). These results indicate a positive effect of combined amendments on soil pH and P availability, but the magnitude of observed increases is higher than typical reports. Therefore, further verification through repeated measurements, careful calibration of instruments, and additional replicates is recommended to ensure the robustness of these findings.

Significant differences were observed among treatments for soil phosphorus (P) fractions (Table 2). Labile P (H₂O + NaHCO₃) increased progressively with biochar, microbial, and nanoparticle amendments, reaching the highest value in the BMN treatment (37.6 mg kg^-1), indicating enhanced P availability. The Fe/Al-bound P (NaOH-extractable) fraction was highest in the R-NPK treatment (158.6 mg kg^-1), suggesting rapid P fixation onto Fe and Al oxides following inorganic fertilizer addition. In contrast, BMN treatments exhibited the lowest Fe/Al-bound P (67.5 mg kg^-1), implying that combined biochar, microbial, and nanoparticle amendments may facilitate mobilization of P from Fe/Al-bound forms into more labile fractions. Ca-bound P (HCl-extractable) showed minor differences across treatments, while residual P remained relatively stable (204–210 mg kg^-1), indicating limited structural transformation over the experimental period. It is important to note that total P calculated from sequential extraction does not necessarily account for all soil P, as some forms may be resistant to chemical extraction or present in highly stable complexes. Therefore, apparent differences in total P between treatments likely reflect a combination of P fixation, mobilization, and methodological limitations, rather than actual losses from the system. This interpretation reconciles observed changes in labile and Fe/Al-bound fractions while acknowledging the constraints of the extraction method (Fig. 3).

Fig. 3.

Correlation between soil available phosphorus, plant biomass, and total phosphorus uptake in maize

Soil organic carbon (SOC)

Soil organic carbon (SOC) increased significantly (p < 0.05) under the amendment treatments (Table 6). The Control recorded the lowest SOC (0.80%), followed closely by R-NPK (0.82%) and N (0.81%), indicating negligible improvement from mineral fertilization or sole MgO-NP application. The B (biochar) treatment markedly elevated SOC to 1.25%, confirming the carbon-enriching effect of biochar addition. Further enhancement was observed in the combined treatments: BM and BMN increased SOC to 1.35% and 1.48%, respectively, corresponding to 69% and 85% increases over the Control. The substantial rise in SOC under BMN suggests that biochar provided persistent carbon inputs while microbial proliferation and MgO-NP stabilization reduced carbon losses through mineralization.

Table 6.

Effect of treatments on chemical and biological soil fertility parameters

Treatment	SOC (%)	CEC [cmol₍₊₎ kg− 1]	Exch. Al³⁺ [cmol₍₊₎ kg− 1]	MBC (µg g− 1)	MBN (µg g− 1)	Acid Phosphatase	β-Glucosidase	Urease
Control	0.80ᵈ	5.2ᵈ	2.10ᵃ	185ᵈ	28ᵈ	45ᵈ	35ᵈ	22ᵈ
R-NPK	0.82ᵈ	5.5ᶜᵈ	1.95ᵃ	210ᶜᵈ	32ᶜᵈ	52ᶜᵈ	38ᶜᵈ	28ᶜ
B	1.25ᵇ	7.8ᵇ	1.45ᵇ	255ᵇ	45ᵇ	58ᵇᶜ	55ᵇ	31ᵇ
M	0.85ᶜᵈ	5.4ᶜᵈ	1.88ᵃ	225ᶜ	35ᶜ	65ᵇ	42ᶜ	45ᵃ
N	0.81ᵈ	5.9ᶜ	1.10ᶜ	195ᶜᵈ	30ᶜᵈ	48ᶜᵈ	36ᵈ	24ᶜᵈ
BM	1.35ᵇ	8.5ᵇ	0.85ᵈ	325ᵃ	58ᵃ	78ᵃ	68ᵃ	52ᵃ
BMN	1.48ᵃ	9.5ᵃ	0.50ᵉ	389ᵃ	65ᵃ	85ᵃ	75ᵃ	55ᵃ
LSD (0.05)	0.08	0.6	0.25	28	7	8	6	5

*Values are mean ± standard error (n = 4). Means within a column followed by the same letter are not significantly different*

Cation exchange capacity (CEC)

CEC responded positively to all amendments (p < 0.05). The Control exhibited the lowest CEC (5.2 cmol₍₊₎ kg^-1), whereas B, BM, and BMN significantly increased it to 7.8, 8.5, and 9.5 cmol₍₊₎ kg^-1, respectively (Table 6). The improvement reflects the high surface area and functional groups of biochar, which enhance negative charge density and cation-retention sites. The synergistic BMN treatment achieved an 83% higher CEC than the Control, illustrating the cumulative benefits of biochar-microbe-nanoparticle interactions in improving soil chemical fertility.

Exchangeable aluminum (Exch. Al³⁺)

Exchangeable Al³⁺, a key indicator of soil acidity stress, declined sharply with integrated treatments (p < 0.05). The Control contained the highest Al³⁺ concentration (2.10 cmol₍₊₎ kg^-1), which decreased progressively under R-NPK (1.95 cmol₍₊₎ kg^-1), B (1.45 cmol₍₊₎ kg^-1), and M (1.88 cmol₍₊₎ kg^-1). The most pronounced reduction occurred in BMN (0.50 cmol₍₊₎ kg^-1), representing a 76% decline relative to the Control. This strong detoxification effect likely resulted from biochar-induced pH moderation, microbial acid-neutralizing activity, and MgO-NPs reacting with soluble Al³⁺ to form inert complexes, collectively alleviating acidity constraints.

Soil biological properties

Microbial biomass carbon (MBC) and nitrogen (MBN)

Microbial Biomass Carbon (MBC) showed a significant improvement under integrated treatments (Table 3). The control recorded the lowest MBC (185 µg g^-1), while BMN exhibited the highest (389 µg g^-1), indicating a substantial improvement in microbial activity and organic C turnover (Fig. 4). The BM treatment also showed high microbial biomass (325 µg g^-1), suggesting that co-application of biochar and PSMs provides a favorable environment for microbial proliferation. Individual applications of PSMs (M) or biochar (B) improved MBC by 22–38% compared to the control.

Table 3.

Soil enzyme activities under different treatments

Treatment	Soil pH	Available P (mg kg− 1)	MBC (µg g− 1)	Dehydrogenase (µg TPF g− 1 24 h− 1)
Control	5.2 ± 0.1ᵈ	10.9 ± 0.8ᵉ	185 ± 12ᵈ	12.5 ± 1.1ᶜ
R-NPK	5.3 ± 0.1ᶜᵈ	15.0 ± 1.1ᵈ	210 ± 15ᶜᵈ	15.8 ± 1.4ᵇᶜ
B	5.5 ± 0.1ᵇᶜ	12.5 ± 1.0ᵈᵉ	255 ± 18ᵇ	16.5 ± 1.5ᵇ
M	5.3 ± 0.1ᶜᵈ	14.1 ± 1.2ᵈ	225 ± 16ᶜ	14.2 ± 1.3ᶜ
N	5.7 ± 0.1ᵇ	16.8 ± 1.3ᶜ	195 ± 14ᶜᵈ	13.8 ± 1.2ᶜ
BM	5.8 ± 0.1ᵇ	19.5 ± 1.5ᵇ	325 ± 22ᵃ	19.5 ± 1.7ᵃ
BMN	6.1 ± 0.1ᵃ	24.8 ± 1.8ᵃ	389 ± 25ᵃ	21.2 ± 1.9ᵃ
LSD (0.05)	0.2	2.1	28	2.5

Values represent mean ± standard error (n = 4). Different letters within a column indicate significant differences at p ≤ 0.05 according to Tukey’s HSD test. Acid phosphatase (AcP) activity was measured using p-nitrophenyl phosphate (PNP) as substrate. Soil (1 g) was incubated with 4 mL of modified universal buffer and 1 mL of 0.05 M PNP at 37 °C for 1 h. Activity is expressed as µg PNP g^-1 soil h^-1. Dehydrogenase (DHA) activity was determined using triphenyl tetrazolium chloride (TTC) over a 24 h incubation at 30 °C and is expressed as µg triphenyl formazan (TPF) g^-1 soil 24 h^-1. β-glucosidase and urease activities were measured following standard protocols, with appropriate substrate concentrations, incubation times, and calibration using standard curves. Units are µg product g^-1 soil h^-1

Fig. 4.

Principal component analysis (PCA) biplot of soil parameters and treatment effects

Enzyme activities

Soil enzyme activities responded positively to bioinoculant and nanoparticle applications (Table 3). Acid phosphatase (AcP) activity, measured using p-nitrophenyl phosphate (PNP) as substrate, ranged from 45 µg PNP g^-1 soil h^-1 in the control to 85 µg PNP g^-1 soil h^-1 under BMN, representing an 89% increase. Dehydrogenase (DHA) activity, determined using triphenyl tetrazolium chloride (TTC) over a 24 h incubation at 30 °C, was highest in BMN (21.2 µg TPF g^-1 soil 24 h^-1), followed by BM (19.5 µg TPF g^-1 soil 24 h^-1). Similarly, β-glucosidase and urease activities increased under integrated treatments. All enzyme assays were calibrated using standard curves, with substrate concentration, soil mass, and incubation times adjusted according to established protocols for Oxisols.

These results indicate that integrated treatments not only enhanced soil chemical fertility but also stimulated microbial-mediated biochemical processes, reflecting improved soil metabolic potential and nutrient cycling under combined biochar, microbial, and nanoparticle amendments (Fig. 5).

Fig. 5.

Principal component analysis (PCA) biplot of soil parameters and treatments

Acid phosphatase

Acid phosphatase activity, an indicator of P-mineralization potential, increased significantly (p < 0.05) with all treatments. The Control (45 µg PNP g^-1 h^-1) showed the lowest activity, while BM (78 µg PNP g^-1 h^-1) and BMN (85 µg PNP g^-1 h^-1) exhibited the highest values 89% higher than the Control. The enhanced activity under BMN underscores the role of PSMs in secreting phosphatases and the supportive environment created by biochar and MgO-NPs for microbial enzyme expression.

β-Glucosidase

β-Glucosidase activity, associated with carbon cycling, followed a similar trend. Activities increased from 35 µg PNP g^-1 h^-1 in the Control to 68 µg PNP g^-1 h^-1 and 75 µg PNP g^-1 h^-1 under BM and BMN, respectively. This 114% enhancement reflects improved substrate availability and microbial activity within biochar-amended soils.

Urease

Urease activity, indicative of nitrogen mineralization, also responded strongly. The Control exhibited the lowest value (22 µg NH₄⁺ g^-1 h^-1), while BM and BMN achieved the highest (52 and 55 µg NH₄⁺ g^-1 h^-1, respectively), representing more than twofold increases (Table 6). The elevated urease activity under BMN emphasizes enhanced microbial N cycling and enzymatic vigor stimulated by improved soil conditions. Across all measured parameters, the BMN treatment (biochar + PSMs + MgO-NPs) consistently outperformed all others. It significantly increased SOC, CEC, MBC, MBN, and enzyme activities, while markedly reducing exchangeable Al³⁺. The BM combination ranked second, showing synergistic but slightly lower effects. Single amendments or R-NPK produced modest improvements, confirming the limited efficacy of isolated approaches. These findings demonstrate that integrating biochar, PSMs, and MgO-NPs fosters a biologically active and chemically balanced soil environment. Biochar improves structural and charge properties, PSMs enhance enzymatic nutrient mobilization, and MgO-NPs neutralize acidity and interact with fixed nutrients. Together, they create a synergistic system that strengthens soil biochemical functioning, mitigates acidity stress, and enhances nutrient cycling efficiency in P-fixing acidic soils.

Plant responses

Plant biomass response to different treatments

The improvements in soil fertility directly translated to enhanced crop performance (Table 4). The BMN treatment resulted in the highest plant biomass, grain yield, and total P uptake. The grain yield under BMN was 98% and 42% higher than the Control and R-NPK treatments, respectively. The BM treatment showed a significant but lower improvement, while individual amendments (B, M, N) showed only marginal, often non-significant, improvements over the R-NPK treatment. Plant biomass showed significant (p < 0.05) variation among the treatments (Table 4). The lowest biomass was recorded in the control treatment (45.2 ± 3.5 g pot^-1), reflecting nutrient deficiency and reduced physiological performance under untreated soil conditions. Application of the recommended NPK (R-NPK) fertilizer improved plant biomass by 51% relative to the control, reaching 68.5 ± 5.1 g pot^-1. Among the single amendments, biochar (B), PSMs (M), and nanoparticles (N) produced noticeable but distinct effects. Biochar alone improved biomass to 52.1 ± 4.2 g pot^-1, likely due to its role in enhancing soil structure, aeration, and moisture retention. PSMsl inoculation (M) further elevated biomass to 60.8 ± 4.8 g pot^-1, indicating enhanced nutrient uptake via hyphal networks. The nanoparticle treatment (N) resulted in 65.2 ± 5.0 g pot^-1 of biomass, suggesting a stimulatory effect on nutrient availability and root activity. The combination treatments exhibited the strongest positive impacts. Biochar combined with PSMs (BM) significantly increased biomass to 85.5 ± 6.2 g pot^-1, surpassing all individual amendments. The integrated biochar–PSMs–nanoparticle (BMN) treatment achieved the maximum plant biomass (101.8 ± 7.5 g pot^-1), marking a 125% increase compared with the control. These findings highlight a synergistic interaction among biochar, PSMsl fungi, and nanoparticles that collectively optimize soil microenvironment, root colonization, and nutrient assimilation.

Table 4.

Effect of fertilizer treatments on plant biomass, grain yield, and total phosphorus uptake

Treatment	Plant Biomass (g pot− 1)	Grain Yield (g pot− 1)	Total P Uptake (mg pot− 1)
Control	45.2 ± 3.5ᵈ	18.5 ± 1.8ᵈ	85 ± 8ᵈ
R-NPK	68.5 ± 5.1ᶜ	26.8 ± 2.2ᶜ	125 ± 11ᶜ
B	52.1 ± 4.2ᶜᵈ	20.1 ± 1.9ᶜᵈ	95 ± 9ᶜᵈ
M	60.8 ± 4.8ᶜ	23.5 ± 2.1ᶜ	115 ± 10ᶜ
N	65.2 ± 5.0ᶜ	25.2 ± 2.2ᶜ	120 ± 11ᶜ
BM	85.5 ± 6.2ᵇ	32.5 ± 2.8ᵇ	185 ± 15ᵇ
BMN	101.8 ± 7.5ᵃ	36.6 ± 3.1ᵃ	217 ± 18ᵃ
LSD (0.05)	9.5	4.0	22

*Values are mean ± standard error (n = 4). Means within a column followed by the same letter are not significantly different*

Grain yield (g pot^-1)

Grain yield varied markedly among treatments (p < 0.05). The BMN treatment produced the highest grain yield (36.6 ± 3.1 g pot^-1), representing a 98% increase over the Control and 36% higher than R-NPK. The combined BM treatment also improved yield (32.5 ± 2.8 g pot^-1), outperforming individual amendments (B, M, or N) and the fertilizer-only (R-NPK) treatment. Single applications of B, M, or N resulted in modest yield gains (ranging from 20.1 to 25.2 g pot^-1) that were statistically lower than the combined treatments. Grain yield patterns closely followed those of biomass accumulation, exhibiting significant differences (p < 0.05) among treatments (Table 5). The control treatment produced the lowest grain yield (18.5 ± 1.8 g pot^-1), while R-NPK application increased yield to 26.8 ± 2.2 g pot^-1, representing a 45% improvement. Among the individual treatments, biochar (B) resulted in 20.1 ± 1.9 g pot^-1 of grain yield, slightly higher than the control but significantly lower than R-NPK. PSMs (M) and nanoparticles (N) further improved grain yield to 23.5 ± 2.1 and 25.2 ± 2.2 g pot^-1, respectively, suggesting enhanced nutrient acquisition and reproductive growth (Fig. 6). The BM treatment produced a remarkable yield increase, achieving 32.5 ± 2.8 g pot^-1 (a 76% rise over control). The BMN treatment exhibited the highest grain yield (36.6 ± 3.1 g pot^-1), significantly surpassing all other treatments. The enhancement under BMN likely resulted from improved phosphorus bioavailability, increased root metabolic activity, and superior physiological efficiency during grain filling. The observed trend emphasizes that the integration of biochar, PSMs, and nanoparticles creates a favorable rhizosphere that sustains higher reproductive output and nutrient partitioning efficiency.

Table 5.

Crop performance, physiological resilience, and phosphorus use efficiency

Treatment	Grain Yield (g pot− 1)	Leaf RWC %	Antioxidant Activity %	Total P Uptake (mg pot− 1)	P Uptake Efficiency (%)	Agronomic Efficiency (AEP)
Control	18.5 ± 1.8ᵈ	58.2 ± 4.5ᵈ	42.5 ± 3.8ᵈ	85 ± 8ᵈ	-	-
R-NPK	26.8 ± 2.2ᶜ	62.5 ± 4.8ᶜᵈ	48.1 ± 4.2ᶜᵈ	125 ± 11ᶜ	16.5ᶜ	13.8ᶜ
B	20.1 ± 1.9ᶜᵈ	65.8 ± 5.1ᵇᶜ	50.5 ± 4.5ᶜ	95 ± 9ᶜᵈ	4.1ᵈ	2.7ᵈ
M	23.5 ± 2.1ᶜ	64.1 ± 5.0ᶜ	49.8 ± 4.4ᶜ	115 ± 10ᶜ	12.5ᶜ	8.3ᶜ
N	25.2 ± 2.2ᶜ	66.5 ± 5.2ᵇᶜ	52.1 ± 4.6ᶜ	120 ± 11ᶜ	14.5ᶜ	11.2ᶜ
BM	32.5 ± 2.8ᵇ	72.8 ± 5.5ᵇ	58.5 ± 5.1ᵇ	185 ± 15ᵇ	41.5ᵇ	23.3ᵇ
BMN	36.6 ± 3.1ᵃ	81.5 ± 6.1ᵃ	65.8 ± 5.5ᵃ	217 ± 18ᵃ	55.0ᵃ	30.2ᵃ
LSD (0.05)	4.0	6.5	6.2	22	8.5	5.5

*Values are mean ± standard error (n = 4). Means within a column followed by the same letter are not significantly different*

Fig. 6.

Effect of soil amendments on soil properties and maize performance

Total phosphorus uptake (mg pot^-1)

Total phosphorus (P) uptake varied significantly across treatments (p < 0.05) (Table 4). Control plants accumulated the least P (85 ± 8 mg pot^-1), while R-NPK treatment enhanced P uptake to 125 ± 11 mg pot^-1 (a 47% increase), reflecting the effect of direct fertilizer application (Fig. 7). Individual amendments demonstrated distinct capacities for P mobilization. Biochar (B) increased P uptake to 95 ± 9 mg pot^-1, attributed to its ability to retain nutrients and moderate soil pH. PSMsl inoculation (M) improved uptake to 115 ± 10 mg pot^-1 through symbiotic enhancement of P solubilization and translocation. Nanoparticles (N) resulted in 120 ± 11 mg pot^-1 of total P uptake, suggesting nano-scale stimulation of root absorption and microbial activity. The combined treatments significantly outperformed single applications. The BM treatment recorded 185 ± 15 mg pot^-1 of total P uptake, while the BMN treatment achieved the highest value (217 ± 18 mg pot^-1), surpassing R-NPK by 74%. These results clearly indicate that the tripartite BMN system enhanced P bioavailability and utilization efficiency through complementary mechanisms, including increased soil enzymatic activity, microbial colonization, and improved root nutrient interception.

Fig. 7.

Relationships between soil biochemical properties and maize productivity parameters

Across all measured parameters, the integrated BMN treatment consistently outperformed all other treatments, followed by BM, R-NPK, and the individual amendments (N, M, and B). The overall efficiency for improving growth and nutrient uptake followed the order: BMN > BM > R-NPK > N ≈ M > B > Control. These findings demonstrate the strong synergistic potential of combining biochar, PSMsl inoculation, and nanoparticles to enhance soil fertility, nutrient acquisition, and plant productivity beyond the capabilities of conventional fertilization alone.

Phosphorus uptake and use efficiencies

Total P uptake and derived efficiency indices demonstrated a clear synergistic advantage under integrated treatments (Table 5). The BMN treatment recorded the highest total P uptake (217 mg pot^− 1), a 155% increase compared to Control and 74% higher than R-NPK. Correspondingly, the P uptake efficiency (55.0%) and agronomic efficiency (AEP, 30.2%) were significantly higher than all other treatments. The BM treatment followed, with 185 mg pot^− 1 total P uptake and 41.5% P efficiency, while individual amendments (B, M, N) and R-NPK remained statistically lower. The combined use of biochar, PSMs, and MgO-NPs (BMN) effectively enhanced maize productivity and physiological resilience by improving P availability, drought tolerance, and nutrient use efficiency, demonstrating a strong synergistic interaction among the amendment components (Fig. 8).

Fig. 8.

Principal Component Analysis (PCA) biplot showing treatment clustering and variables driving variation in soil and plant traits under different amendments. Treatments include Control, R-NPK, Biochar (B), Phosphate-Solubilizing Microbes (M), MgO Nanoparticles (N), Biochar + Microbes (BM), and Biochar + Microbes + Nanoparticles (BMN). The first two principal components (PC1 = 90.9% and PC2 = 4.9%) explain 95.8% of the total variance. Arrows represent the direction and strength of soil and plant variables influencing treatment differentiation, with Available P, Plant Biomass, Grain Yield, and Total P Uptake positively associated with integrated treatments (BM and BMN)

Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN)

Both MBC and MBN increased markedly with amendment application (p < 0.05). The lowest values occurred in the Control (185 µg g^-1 MBC; 28 µg g^-1 MBN). R-NPK, B, and M increased MBC to 210, 255, and 225 µg g^-1, respectively, while MBN rose moderately (Table 6). The BM and BMN treatments exhibited dramatic enhancements, with MBC values of 325 µg g^-1 and 389 µg g^-1, and MBN of 58 µg g^-1 and 65 µg g^-1, respectively. These increases represent approximately 110% (MBC) and 132% (MBN) gains over the Control, indicating greater microbial proliferation and nutrient assimilation under integrated management. The results suggest that biochar offered habitat stability, PSMs stimulated microbial turnover, and MgO-NPs improved nutrient availability, collectively enriching microbial biomass.

Leaf RWC and antioxidant activity

Leaf relative water content (RWC) and antioxidant activity, key indicators of drought resilience, were significantly improved by integrated amendment strategies. The BMN treatment recorded the highest RWC (81.5%), a 40% increase over Control, and antioxidant activity (65.8%), showing a 55% enhancement relative to Control. The BM treatment exhibited intermediate values (RWC 72.8%; antioxidant activity 58.5%), indicating synergistic effects of biochar and microbial inoculation. In contrast, R-NPK and single amendments showed minor improvements, with no significant difference among them.

Correlation analysis

Correlation analysis revealed strong positive relationships among soil biological and chemical parameters. Available P showed a highly significant correlation with acid phosphatase (r = 0.91, p < 0.01) and MBC (r = 0.88, p < 0.01), suggesting that enhanced microbial activity contributed to greater P mineralization and availability. Similarly, dehydrogenase activity was positively correlated with MBC (r = 0.85, p < 0.01), indicating that increases in microbial biomass directly translated into elevated enzymatic metabolism. Moreover, soil pH exhibited a moderate positive correlation with available P (r = 0.67, p < 0.05), reflecting the role of pH amelioration in improving P solubility. These correlations confirm that the BMN treatment synergistically enhanced soil fertility by simultaneously improving pH, microbial activity, and enzyme-mediated nutrient cycling.

Correlation among plant biomass, grain yield, and phosphorus uptake

The interrelationships among plant biomass, grain yield, and total phosphorus (P) uptake were analyzed across all treatments (Fig. 9). Pearson correlation analysis revealed strong positive associations among these key growth and productivity parameters. Plant biomass exhibited a highly significant positive correlation with grain yield (r = 0.98, p < 0.01), indicating that treatments enhancing vegetative growth also contributed to higher reproductive output. Similarly, plant biomass was strongly positively correlated with total P uptake (r = 0.97, p < 0.01), suggesting that increased biomass is associated with enhanced nutrient acquisition. Grain yield was also significantly correlated with total P uptake (r = 0.96, p < 0.01), highlighting the critical role of phosphorus in supporting grain formation and overall productivity. Treatments integrating multiple amendments, such as BM and BMN, demonstrated the highest values for biomass, grain yield, and P uptake, further emphasizing the synergistic effects of combined nutrient and microbial management. These findings indicate that enhancement of plant growth and nutrient uptake are closely linked with improved grain yield, underscoring the importance of integrated soil-plant nutrient management strategies in optimizing crop performance.

Fig. 9.

Correlation heatmap depicting relationships between key soil properties and plant growth parameters. The color intensity and numerical values indicate the strength of the Pearson correlation coefficients (r). Available_P, available phosphorus; MBC, microbial biomass carbon

Discussion

The integration of biochar, phosphate-solubilizing microorganisms (PSMs), and magnesium oxide nanoparticles (MgO-NPs) elicited a profound and synergistic improvement in the biochemical properties and phosphorus (P) dynamics of the acidic P-fixing Oxisol. The control soil was nutrient-limited, treatment-induced yield gains represent potential rather than absolute agronomic responses. Under well-managed field soils with adequate baseline fertility, the magnitude of yield improvement is expected to be lower, although relative treatment rankings are likely to remain consistent. The results demonstrate that this tripartite system operates not by mere additive effects, but through a cascade of complementary mechanisms that collectively re-engineer the soil environment. The following discussion deconstructs these mechanisms, linking physicochemical changes to biological activation and culminating in the observed agronomic and resilience benefits.

Synergistic mechanisms for alleviating acidity and enhancing phosphorus availability

The significant increase in soil pH under the combined BM and BMN treatments initiates a critical cascade of soil improvements. This shift is primarily driven by the inherent alkalinity of rice husk biochar, which possesses carbonates, hydroxides, and oxygenated functional groups that consume H⁺ ions and neutralize exchangeable Al³⁺, a primary agent of soil acidity and P fixation [1, 2]. However, the BMN treatment’s superior performance reveals a more nuanced mechanism. Based on established physicochemical behavior reported in the literature, MgO nanoparticles are expected to undergo surface hydration in soil environments, potentially forming Mg(OH)₂ and releasing OH⁻ ions, which can increase pH in localized microsites surrounding the particles [3, 8]. In the present study, these processes are discussed as plausible mechanisms that may contribute to the observed soil chemical responses, rather than as directly measured phenomena. This immediate “pH shock” is particularly effective at dissolving the stable, fixed P pools (e.g., variscite [AlPO₄·2 H₂O] and strengite [FePO₄·2 H₂O]) that are recalcitrant to milder amendments [18]. The dissolved PO₄^3- is then vulnerable to one of two fates: plant uptake or re-fixation. This is where the synergy manifests. Biochar plays a decisive role by acting as a “safe harbor” for the liberated phosphate. Its high specific surface area and porosity allow for the adsorption of PO₄^3-, effectively shielding it from re-adsorption onto Fe/Al oxyhydroxides [4, 5]. Concurrently, the PSMs, including Pseudomonas fluorescens and Aspergillus awamori, sustain this P mobilization over the long term. They do not solely rely on the initial pH change; instead, they secrete a suite of low-molecular-weight organic acids (e.g., gluconic, citric, oxalic) which chelate Al³⁺ and Fe³⁺ ions, directly stripping them from phosphate minerals [6, 7]. Furthermore, the sustained elevation in acid phosphatase activity indicates a robust enzymatic hydrolysis of organic P pools (e.g., inositol phosphates), adding another continuous stream of bioavailable P [19]. The Mg²⁺ ions supplied by the nanoparticles may also contribute indirectly to phosphorus availability by altering soil solution chemistry. In acidic soils, elevated Mg²⁺ activity can reduce the dominance of Al-phosphate interactions by competitive cation effects, thereby favoring more soluble phosphate forms. However, the formation of specific Mg–phosphate complexes was not directly quantified in this study, and this mechanism is therefore proposed as a plausible pathway rather than a confirmed process [3, 11]. Thus, the BMN system creates a virtuous cycle: MgO-NPs provide the initial burst of P availability, biochar secures this gain, and PSMs ensure its continuous renewal, explaining the significantly higher available P in BMN compared to all other treatments.

The significant improvements in soil health, nutrient availability, and plant growth observed in this study underscore the efficacy of integrated amendment strategies for acidic, phosphorus-fixing soils [20]. The application of biochar served as a foundational amendment, enhancing soil organic carbon and providing a stable habitat for microbial communities, which aligns with the principles of carbon cultivation for sustainable agriculture [21, 22]. The synergistic effect observed in the combined biochar and microbe (B + M) treatment can be attributed to the role of biochar in improving the soil habitat, thereby amplifying the actions of phosphate-solubilizing microorganisms, a mechanism extensively described for improving soil health [23, 24]. Furthermore, the remarkable performance of the treatments involving magnesium oxide nanoparticles (N) highlights the emerging potential of nanobiotechnology in agriculture [25, 26]. The positively charged MgO nanoparticles likely enhanced phosphorus solubilization not only by modulating soil pH but also by interacting with soil colloids and microbial enzymes, a process reflective of the advanced mechanisms explored in modern nano-fertilizer research [25, 27]. This multifaceted approach, which combines organic amendments, beneficial microbes, and nanoscale materials, represents a robust climate-smart strategy for sustainable soil and environmental management [25]. Moreover, the principles demonstrated here using amendments to immobilize harmful elements and improve nutrient cycling are directly applicable to broader environmental challenges, such as the remediation of heavy metal-contaminated soils using biochar and other organic amendments [20, 24, 28] and the stabilization of toxic elements like lead in situ [28, 29]. Ultimately, these findings contribute to the overarching goal of developing sustainable crop production and arid land management systems [26, 27] by integrating novel amendments to build ecosystem resilience and enhance agricultural productivity.

Revitalization of soil biological health and biochemical cycling

The significant increases in microbial biomass carbon (MBC) and nitrogen (MBN) observed under the BM and BMN treatments indicate an overall enhancement of soil microbial biomass at the bulk soil level, reflecting improved resource availability and habitat conditions [30, 31]. Biochar contributes to this response by modifying the soil physical and chemical environment; its porous structure and surface heterogeneity can enhance moisture retention and provide microsites that support microbial persistence under fluctuating soil conditions [16, 17]. In addition, biochar may retain dissolved organic compounds and nutrients within the soil matrix, thereby increasing substrate availability for microbial metabolism [32, 33]. The combined application of biochar and microbial inoculants further intensified these responses, suggesting a synergistic stimulation of soil biological functioning [34]. Correspondingly, the pronounced increase in acid phosphatase activity reflects an elevated soil-level potential for organic phosphorus mineralization, driven by increased microbial biomass and activity rather than attributable to specific microbial taxa [7, 19]. Collectively, these responses indicate a functional enhancement of soil biochemical cycling capacity, without implying changes in microbial community composition or direct confirmation of inoculant dominance [35]. The significant increase in dehydrogenase activity, a widely used indicator of overall soil microbial oxidative activity, reflects enhanced metabolic potential at the bulk soil level, likely driven by increased availability of labile carbon and improved habitat conditions following biochar and organic amendments [15, 36]. Similarly, the elevated β-glucosidase activity indicates accelerated turnover of cellulose-derived substrates, supporting greater carbon flux through microbial-mediated decomposition pathways [10, 37]. Positive correlations between microbial biomass carbon (MBC), available phosphorus, and soil enzyme activities suggest that the amendments stimulated integrated biogeochemical functioning rather than acting solely as direct nutrient inputs. Importantly, these responses represent aggregate soil microbial activity and do not distinguish between contributions from inoculated microorganisms and native microbial communities. Therefore, the observed enhancements are interpreted as system-level functional responses, reflecting improved microbial resource availability and process coupling across C, N, and P cycles, rather than direct evidence of inoculant establishment or dominance.

Improvement of soil physicochemical properties and crop resilience

The marked improvements in soil organic carbon (SOC) and cation exchange capacity (CEC) under biochar-amended treatments point to a long-term enhancement of soil quality. The increase in SOC is largely attributed to the recalcitrant aromatic carbon structure of biochar itself, which contributes to long-term carbon sequestration [1, 13]. Concurrently, oxidative aging of biochar in the soil environment introduces oxygen-containing functional groups, including carboxylic and phenolic moieties, which contribute to an increase in soil cation exchange capacity (CEC) [13, 38]. This enhanced CEC primarily improves the retention of nutrient cations such as K⁺, Ca²⁺, and NH₄⁺, thereby reducing leaching losses and improving overall nutrient-use efficiency. However, phosphorus retention in biochar-amended soils is governed largely by indirect mechanisms rather than electrostatic sorption onto biochar surfaces, particularly under acidic soil conditions. Biochar application can increase soil pH, thereby decreasing the positive surface charge of Fe and Al oxides and reducing phosphate fixation. In addition, biochar-derived dissolved organic carbon may compete with phosphate for sorption sites on mineral surfaces, while improvements in soil moisture retention and aggregation can alleviate diffusion limitations and enhance plant phosphorus acquisition. These mechanisms collectively explain the observed improvements in phosphorus uptake, independent of direct phosphate adsorption by biochar surfaces. The drastic reduction in exchangeable Al³⁺ is arguably one of the most critical outcomes for crop growth in acidic soils. This is achieved through a multi-pronged mechanism: the general pH rise from biochar promotes the precipitation of Al³⁺ into non-toxic Al(OH)₃, while the MgO-NPs provide a more direct and rapid neutralization reaction [3, 9]. Furthermore, the organic acids exuded by PSMs can chelate Al³⁺, forming stable complexes that render the aluminum non-phytotoxic [6, 48]. This comprehensive detoxification of the soil environment directly facilitates root proliferation and health. These below-ground improvements translated directly into superior above-ground plant performance and resilience. The higher Leaf Relative Water Content (RWC) in the BMN treatment under drought stress suggests improved plant water status, likely due to a more extensive and healthier root system exploring a larger soil volume with improved structure and moisture retention [39]. The enhanced antioxidant activity indicates that plants were physiologically better equipped to handle abiotic stress, possibly due to improved Mg²⁺ nutrition (a cofactor for chlorophyll and many enzymes) and a healthier, more symbiotic rhizosphere microbiome that can prime plant defense systems [12, 40]. Consequently, the significantly higher Phosphorus Use Efficiency (PUE) demonstrates that the BMN system creates a soil environment where P is not only more available but is also utilized by the plant with far greater effectiveness, closing the nutrient loop and reducing fertilizer waste.

The results unequivocally demonstrate that the integrated Biochar-Microbe-Nanoparticle (BMN) system represents a paradigm shift from the conventional, input-intensive approach to soil management. Unlike soluble P fertilizers that offer a short-lived nutrient pulse and are prone to fixation, the BMN system constructs a resilient soil ecosystem. It simultaneously addresses the core constraints of acidic, P-fixing soils: chemical (acidity, Al toxicity), physical (low CEC, poor habitat), and biological (low microbial activity, impaired nutrient cycling). This approach aligns perfectly with the principles of sustainable intensification. By enhancing native soil fertility and leveraging biological processes, it can significantly reduce dependency on high-dose, environmentally detrimental phosphatic fertilizers, thereby mitigating eutrophication risks [14, 41]. The system transforms the soil from a passive matrix into an active, biologically mediated nutrient recycling system. Future research must now focus on translating this potent proof-of-concept into a practical, large-scale application. This includes long-term field trials to validate persistence and effects under realistic agronomic conditions, comprehensive life-cycle and economic feasibility analyses, and the development of tailored formulations optimized for different soil types and cropping systems to ensure broad adaptability and farmer adoption.

Limitations and future perspectives

Several limitations of the present study should be acknowledged to ensure appropriate interpretation of the findings. First, microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and soil enzyme activities (dehydrogenase, β-glucosidase, acid phosphatase, and urease) were quantified as bulk soil indicators of microbial biomass and functional potential. These measurements do not provide information on microbial community composition, taxonomic shifts, or functional guild dynamics. Molecular validation techniques such as 16 S rRNA or ITS sequencing, functional gene quantification (e.g., phoD, phoC, gcd), or isotopic tracing were not employed; therefore, the contribution of specific microbial taxa, including inoculated phosphorus-solubilizing microorganisms (PSMs), cannot be distinguished from that of the indigenous soil microbiome.

Second, soil enzyme assays represent potential enzymatic activity under standardized laboratory conditions and do not discriminate between enzymes actively produced by living microbial cells and stabilized extracellular enzymes adsorbed onto soil colloids. As such, the measured activities reflect system-level biochemical potential rather than real-time microbial process rates.

Third, mechanistic interpretations regarding phosphorus availability following biochar and nanoparticle application are based on established soil chemical principles and literature evidence rather than direct measurements. Phosphorus sorption isotherms, Mg–phosphate speciation, soluble Mg²⁺ concentrations, and thermodynamic modeling were not conducted. Consequently, proposed mechanisms such as competitive interactions with Fe/Al oxides, dissolved organic carbon–mediated desorption, or Mg-associated phosphate complexation should be regarded as plausible pathways rather than confirmed processes. Similarly, the transformation of MgO nanoparticles, including Mg(OH)₂ formation, hydroxide release kinetics, and microsite-scale pH alterations, was not directly quantified. These processes are discussed conceptually based on prior studies and are not presented as experimentally observed phenomena in the current work.

Fourth, plant physiological indicators such as leaf relative water content and antioxidant capacity were measured at single time points and under controlled greenhouse conditions. While these parameters provide useful indicators of plant stress status, temporal dynamics, recovery responses, and specific antioxidant compounds or gene-level regulation were not assessed.

Finally, this study was conducted as a pot experiment under greenhouse conditions using a low-phosphorus soil to evaluate treatment effects under nutrient-constrained scenarios. While this design enables mechanistic insight under controlled conditions, extrapolation to field-scale systems with heterogeneous soils, established fertility regimes, and variable climatic conditions should be made with caution. Future studies integrating high-resolution molecular tools, direct soil chemical speciation analyses, real-time rhizosphere measurements, and multi-season field trials will be essential to validate the proposed mechanisms and to strengthen the translational relevance of these findings.

Conclusion

The integrated application of rice husk biochar, phosphate-solubilizing microorganisms (PSMs), and magnesium oxide nanoparticles (MgO-NPs) creates a powerful synergistic system for reclaiming P-fixing acidic soils. The BMN treatment fundamentally re-engineers the soil environment by operating on multiple fronts, chemically, by ameliorating acidity and directly solubilizing P (via NPs); physically, through biochar’s adsorption and habitat provision; and biologically, by enhancing microbial biomass and enzymatic activities (e.g., acid phosphatase and dehydrogenase). This multi-faceted approach resulted in a sustained improvement in soil fertility, including a > 120% increase in available phosphorus and a revitalization of the soil biological community. Consequently, the biochar-microbe-nanoparticle consortium presents a transformative, eco-intensification strategy that moves beyond simple nutrient addition to holistic soil health management. It offers a robust and sustainable alternative to conventional fertilization by significantly boosting crop productivity and nutrient use efficiency while reducing dependency on high-dose phosphatic fertilizers and mitigating environmental pollution. For the safe and effective implementation of this approach at scale, future research should focus on long-term field validations, optimization of nanoparticle dosages, economic feasibility, and a thorough assessment of potential environmental risks for different soil types and cropping systems.

Authors’ contributions

Q.Li: Conceptualization of the study, experimental design, supervision of field and laboratory experiments, and manuscript drafting. K.Xu: Soil and microbial analysis, data collection, statistical analysis, and interpretation of results. Y.Ning: Preparation and characterization of biochar and MgO nanoparticles, laboratory testing, and data validation. H.Duan: Oversight of project implementation, critical review of methodology, and contribution to manuscript revision. Imran: Study design, soil and crop assessments, integration of field and laboratory data, manuscript writing, and corresponding author responsibilities.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

No human participants or animals were involved in this study. The research complies with institutional, national, and international guidelines for environmental and agronomic studies.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.