Soil acidification refers to the increase in proton (H⁺) concentration within the soil, leading to a decrease in pH (Kochian et al., 2015; Ryan 2018). Acidic soil, typically defined by a pH of less than 7, usually range between 4.5 and 6. However, soils with pH below 5.5 often adversely affect plant growth and development (Munyaneza et al., 2024; Ryan 2018; von Uexküll 1995). These soils encompass lateritic red soil, lateritic brown soil, red soil, yellow soil, and dry red soil (Bao et al., 2000). Globally, acidic soil constitutes 40–50% of potentially arable lands (Kochian et al., 2015), with those below pH 5.5 covering approximately 39.5 million hm². In China, the red and yellow soil regions account for about 22.7% of the total land area and acidic soils below pH 6.5 spanning approximately 31.11 million km², representing 32.4% of the country’s land area (Zhao et al., 2023).

In acidic soils, Al exists in various forms, with their toxicity to plants following the order: Al³⁺ > Al(OH)²⁺ > Al(OH)₂⁺ > Al(OH)₃⁰ (Bojórquez et al., 2017; Nogueirol et al., 2015; Xia et al., 2023). At pH levels below 4.3, Al³⁺ becomes the dominant form, exerting the most significant toxic effects on plant growth (Nogueirol et al., 2015). When the soil pH is less than 5.5, crops often experience Al toxicity stress, characterized by inhibited root elongation, impaired nutrient absorption, disrupted physiological metabolism, and reduced yield (Kochian et al., 2015; Liu et al., 2023; Nerva et al., 2022). While Al toxicity is a major limiting factor in acidic soils, other constraints include deficiencies in essential nutrients such as phosphorus (P), nitrogen (N), calcium (Ca), and magnesium (Mg), as well as toxic levels of manganese (Mn) and iron (Fe) (Kopittke et al., 2016; Muhammad et al., 2019). To counteract Al toxicity, plants have developed several adaptive strategies, including internal chelation and external exclusion mechanisms (Kochian et al., 2015; Kopittke et al., 2016; Ma et al., 2014; Yan et al., 2021). These mechanisms involve the efflux of Al³⁺, secretion of organic acids and phenolic compounds, and production of catechol and flavonoids with high affinities for Al³⁺ efflux (Juan 2002; Kochian et al., 2015). Increasing the rhizosphere pH can alter the forms of Al in soil, shifting it from the highly toxic Al³⁺ to the less toxic forms Al(OH)²⁺ and Al(OH)₂⁺, and ultimately to the non- toxic Al(OH)₃⁰ as the pH rises from 4.5 to 6 (Bojórquez et al., 2017; Nogueirol et al., 2015). The role of mineral nutrition is also critical in mitigating Al toxicity. Nutrients such as boron (B), Ca, Mg, sulfur(S), P, N, and potassium (K) play an essential role in alleviating the stress associated with acidic soils (Muhammad et al., 2019; Xia et al., 2023). Additionally, plants secrete mucilage in response to Al toxicity, which is primarily composed of polysaccharides, glucose, and polysaccharides aldehydes (Yan et al., 2021; Yang et al., 2011).

P is a vital macronutrient indispensable for the growth and development of plants (Cordell et al., 2014; Herrera et al., 2016; Zou et al., 2022). It serves not only as a crucial structural component in many biological macromolecules but also plays a central role in various physiological and biochemical processes within plant tissues (Hammond 2008; Lambers et al., 2008). However, the tendency for P to bind with soil minerals leads to low efficiency in phosphate fertilizer utilization (Cordell et al., 2014; Kochian et al., 2004; Zou et al., 2022). As a result, crops exhibit minimal seasonal uptake of these fertilizers, with the unabsorbed fraction leaching into the environment, contributing to soil degradation and aquatic eutrophication, issues that pose significant ecological and environmental risks (Lambers et al., 2008; Wen et al., 2024). In acidic soil, the combined effects of low pH levels, elevated Al toxicity, and reduced bioavailable P severely hinder P uptake by plant roots, impairing crop growth and yield (Magalhaes et al., 2018; Wassen et al., 2005; Wen et al., 2024). As soil pH decreases, the interaction between P and Al oxides intensifies, further restricting the availability of inorganic phosphate (Pi) (Dai et al., 2020). In response to these challenges, plants have developed adaptive mechanisms to mitigate P deficiency (Liu et al., 2023). Microorganisms also play a pivotal role in regulating soil P availability and enhancing plant nutrient uptake (Liu et al., 2023). The application of chemical phosphate fertilizers raises soil P levels, thereby influencing the composition and activities of rhizosphere bacteria (Dai et al., 2020; Duan et al., 2020).

Considerable research has focused on the physiological, genetic, and molecular mechanisms underlying plant resistance to Al toxicity (Delhaize et al., 2012; Kochian et al., 2015). However, the potential of plant-microbe interactions in acidic soils to sustainably improve Al resistance and crop productivity in B. napus remains underexplored (Liu et al., 2023; Trivedi et al., 2020; Nerva et al., 2022; Schmitz et al., 2022; Sridhar et al., 2022). The rhizosphere microbiome plays a pivotal role in supporting plant growth and stress tolerance (Durán et al., 2018). Plant growth-promoting rhizobacteria (PGPR) can enhance nutrient acquisition, increase pathogen resistance, and improve overall stress tolerance (Durán et al., 2018; Nerva et al., 2022; Trivedi et al., 2020). Previous studies have highlighted the role of PGPR in mitigating the toxic effects of heavy metals, such as cadmium (Cd), copper (Cu), P and Mn, by modifying metal bioavailability through the secretion of organic acids and siderophores (Narayanan 2023; Rajkumar et al., 2012; Moudrikova et al., 2017). These findings inspired our exploration of microbial strategies to improve B. napus yield under acidic and Al stress conditions.

In this study, we address the issues of reduction in crop yield and the concurrent P deficiency induced by Al toxicity in acidic soils. To mitigate soil acidification and enhance Al resistance, we propose the use of P fertilizers, which can lower Al³⁺ concentrations and modify the soil microbiome. Our objectives of this study were: (1) to investigate the pH levels and concentrations of various Al forms within stratified soil profiles, (2) to analyze the effects of P fertilization (compared to no P fertilization) on the microbial community of acidic soils, and (3) to assess the impact of P fertilization on the growth, yield, and seed quality in B. napus. Our research represents an integrated approach to improving crop yield and promoting agricultural sustainability through the interactions of plants, soil, and the microbiome in acidic soils.

The pot experiment was performed at Huazhong Agriculture University, Wuhan, Hubei Province, China, from October to May of the following year. The pots, each filled with 7 kg collected acid soil, were arranged in a completely randomized design with six replicates per treatment. The experiment included the following treatments: (1) Original soil (OS), (2) Improved soil without P fertilization (OS + 0.2% Ca(OH)₂), (3) Original soil with P fertilization at 1.435 g/Kg KH₂PO₄ (OS + KH₂PO₄), and (4) Original soil with P fertilization at 2.87 g/Kg KH₂PO₄ (OS + + KH₂PO₄). Plants were sampled at three growth stages including seeding, flowering, and maturity.

At the flowering stage, rhizosphere soil was collected by gently removing bulk soil adhering to the roots, shaking the roots to dislodge loosely attached soil, and using a sterile brush to collect residual soil. The collected rhizosphere soil was quickly frozen and stored at − 80°C for high-throughput DNA sequencing analysis. Stratified non-rhizosphere soil samples (2 cm depth per layer, five layers per pot) were collected at seeding and flowering stages, air-dried, and used for pH determination and Al concentration measurements.

Soil pH was measured in a 1:2.5 soil-to-water solution using a pH meter (FE20, Mettler Toledo, Shanghai). To quantify different Al forms in the soil, 25 ml of air-dried soil was extracted using four different extractants (KCl, NH₄Ac, HCl, and NaOH). After shaking the mixtures for 30 minutes, 1 ml of the clear filtrate was mixed with 50 µl of ascorbic acid, followed by the addition of 0.5 ml of buffer solution and 0.25 ml of dye working solution. The absorbance of the resulting reaction was measured at 535 nm, and all colorimetric measurements were completed within two hours. The Al forms measured included Al³⁺, Al-HA, Al (OH)₃⁰ and Al(OH)₂⁺/ Al(OH)²⁺. Where Al³⁺, Al(OH)₂⁺, and Al(OH)^₂⁺ are highly toxic to plants, while Al (OH)₃⁰ and Al-HA have lower toxicity (Lin et al., 2018).

At the seeding stage, plants samples were harvested and their dry weights recorded. The samples were digested using HNO_3, and the concentrations of various mineral elements were quantified using inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent Technologies 5110, USA). The mineral elements included Al, Cadmium (Cd), Chromium (Cr), Lead (Pb), Nickel (Ni), Arsenic (As), Mg, P, boron (B), copper (Cu), iron (Fe), manganese (Mn), potassium (K), and Zinc (Zn). The concentration of each element was determined by comparing the absorbance values with standard calibration curves.

Rhizosphere soil samples were collected as described in Section 2.2. The samples were air-dried before analysis of available Cu, Fe, Mn, and Zn. To determine the availability of these elements in the rhizosphere, the air-dried soil samples were immersed in 50 ml of 0.1mol/L HCl and agitated at 180 r/min at 25°C for 1.5 hours. The analysis of Cu, Fe, Mn, and Zn were quantified using inductively coupled plasma-optical emission spectrometry (ICP-OES) (Agilent Technologies 5110, USA). The absorbance was proportional to the concentration within a specific range, quantified by comparison to a standard curve.

Total microbial genomic DNA was extracted from rhizosphere soil samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The quality and concentration of DNA were assessed by 1.0% agarose gel electrophoresis and with a NanoDrop2000 spectrophotometer (Thermo Scientific, United States). DNA was stored at -80 ℃ until further use. The hypervariable V3-V4 regions of the bacterial 16S rRNA gene were amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') on a T100 Thermal Cycler (BIO-RAD, USA). The PCR products were extracted from a 2% agarose gel, purified using the PCR Clean-Up Kit (YuHua, Shanghai, China), and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA). The purified amplicons were pooled in equimolar amounts and sequenced on an Illumina PE300/PE250 platform (Illumina, San Diego, USA) following standard protocols provided by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

At maturity, plants were harvested from each pot to record growth and yield parameters, including seed yield, thousand-seed weight, seed number per plant, pod number per plant, silique length, seed number per silique, silique weight, total dry weight, plant height, and straw weight. Seed were stored and dried until the water content fell below 10%. Seed quality traits, such as oil content, protein content, moisture content, unsaturated fatty acid, and saturated fatty acid, were measured using a near-infrared analyzer (NIR System3750, Sweden) at the National Rapeseed Engineering Center, Huazhong Agricultural University.

Soil acidification and Al toxicity are major challenges to crop production, often exacerbated by nutrient deficiencies and metal toxicity in acidic soils (Kochian et al., 2015; Kopittke et al., 2016; Lin et al., 2012; Muhammad et al., 2019). This study shows that inorganic P fertilizer significantly enhances B. napus growth and soil fertility under acidic conditions. P fertilizer treatments resulted in a remarkable 7.5-fold increase in shoot dry weight compared to the control as well as outperforming alkalized treatments. Soil P levels increased by up to 224%, accompanied by improved concentrations of B, Cu, and K, indicating enhanced fertility (Fig. 1,2). Moreover, P fertilizers effectively reduced Al toxicity, with a 39.8% decrease in Al concentration and a reduction in bioavailable Al³⁺ in surface layers (Fig. 2a). These changes facilitated the transformation of Al into less harmful forms, such as Al-HA, underscoring the critical role of P fertilizers in mitigating Al toxicity and improving soil conditions for plant growth.

Agricultural liming has been a traditional practice for centuries to counteract soil acidification, mitigate Al toxicity, and alleviate P deficiency in soil (Delhaize et al.,2012; Liu et al.,2023; Magalhaes et al.,2018). This study demonstrates that inorganic P fertilizers (OS + KH₂PO₄ and OS + + KH₂PO₄) significantly increase plant development as evidenced by substantial increases in plant height and biomass accumulation, particularly an increase in shoot dry weight compared to the control (OS). Notably, the P fertilizer treatments outperformed the alkalized treatment (OS + Ca(OH)₂), which showed a three-fold increase, underscoring that P application, rather than soil alkalinity alone, plays a pivotal role in promoting plant growth. Moreover, P fertilizers significantly reduced Al toxicity in P-fertilized treatments, fostering a more favorable soil environment for plant growth. Further analysis revealed that P fertilizers reduced bioavailable Al³⁺ concentrations, particularly in surface soil layers, however this effect diminished with soil depth. This suggests that the impact of P fertilizers on Al speciation is more pronounced near the surface (Fig. 1, 3, and Fig. S7). The transformation of Al into less harmful forms, such as Al-HA, highlights complex interactions between P fertilizers and soil components. However, the concentrations of Al-HA and Al(OH)₃⁰ remained relatively stable across P treatments, indicating their persistence and resistance to treatment-induced changes. The dominance of Al-HA across all treatments emphasizes the importance of organic matter in binding Al in soil (Bojórquez-Quintal et al., 2017; Nogueirol et al., 2015), further supporting the role of P fertilizers in alleviating Al toxicity and improving soil conditions for sustainable crop production.

Soil and plants microbiomes interact through intricate networks that are critical for maintaining ecosystem balance and plant health (Bai et al., 2022; Zhang et al., 2017; Zhu et al., 2022; Rösch et al., 2002). These interactions are particularly sensitive to environmental stress, which can lead to significant shifts in bacterial community structures (Pan et al., 2022; Zhang et al., 2015; Lundberg et al., 2012). Understanding soil-plant-microbe interactions under stress conditions, such as acidic soils with Al toxicity, is essential for developing sustainable agricultural practices (Miki et al., 2010; Nerva et al., 2022; Trivedi et al., 2020). Our study highlights the influence of rhizosphere microbiomes on plant health in such conditions, specifically through P fertilizer application. The application of P fertilizers (SP treatments) significantly altered the microbial community composition compared to the control (SAl), with notable increases in the relative abundances of Actinobacteriota and decreases in Proteobacteria, Myxococcota, Nitrospirota, and Bdellovibrionota. These shifts can be attributed to amendments modifying the soil environment, favoring microbial groups adapted to reduced Al toxicity and improved nutrient availability (Fig. 4). Consistent with prior findings, P amendments promoted microbial diversity, as evidenced by higher diversity indices in SCK and SP treatments compared to the SAl control (Fig. 6), reinforcing the critical role of soil chemistry in shaping microbial dynamics (Liu et al., 2023; Paries et al., 2023). A two-factor network analysis further revealed that Actinobacteriota positively correlated with soil pH and negatively correlated with Al³⁺ concentrations, while Proteobacteria, Chloroflexi, and Acidobacteriota exhibited opposite trends (Fig. 7). These correlations highlight the sensitivity of microbial communities to soil pH and Al³⁺ toxicity. Similar to previous studies, our results confirm that soil amendments not only mitigate Al toxicity but also promote beneficial microbial groups capable of enhancing soil health and plant growth (Simonin et al., 2020; Paries et al., 2023). The emergence of distinct biomarkers in SCK and SP treatments underscores the profound impact of P fertilizer on the microbial community, driving shifts in the dominant phyla and improving soil suitability for B. napus cultivation under acidic conditions.

Application of P fertilizer significantly improved yield components, oil content, and plant biomass, highlighting its importance in enhancing overall plant performance (Diepenbrock 2000; Fu et al., 2016; Gomez et al., 2011; Magalhaes et al., 2018; Wyatt et al., 2008). Notable increases in seed yield, 1000-grain weight, seed number per plant, and pod number per plant indicate a strong positive correlation between P availability and crop productivity (Fig. 8). These improvements are particularly significant for addressing food security challenges in regions where soil acidification and Al toxicity limit agricultural potential (Kochian et al., 2015). Interestingly, yield enhancements plateaued beyond certain application rates of P fertilizer, indicating a threshold effect that warrants further investigation to optimize application rates.

Beyond seed yield, parameters such as silique length, seed number per silique, silique weight, total dry weight, plant height, and straw weight were significantly enhanced by P application (Fig. 8), emphasizing its multifaceted benefits. P fertilizer also played a critical role in mitigating Al toxicity by reducing Al concentrations in roots, straw, and siliques, thereby promoting healthier root systems and facilitating nutrient uptake. This protective mechanism may involve the exclusion or sequestration of Al from sensitive tissues, contributing to improved plant health. Furthermore, the preferential accumulation of P in seeds enhances seed vigor and establishment, providing a strategic advantage for future generations and ensuring crop resilience (Zou et al., 2022). At the maturity stage, P fertilizer not only boosted B. napus yield and biomass but also significantly reduced Al concentrations in critical plant tissues. These results highlight its dual role as both a nutrient provider and a mitigator of Al toxicity. Comparatively, previous studies have demonstrated similar protective effects of P fertilizers against soil acidity and metal toxicity, aligning with findings that P improves soil pH and reduces bioavailable Al (Cordell 2014; Paries 2023).

This study highlights the critical role of P fertilization in mitigating Al toxicity and promoting the growth of B. napus in acidic soils. During the seedling stage, P fertilizer application reduced soil Al concentrations and improved soil pH, creating a more favorable environment for plant growth. By the flowering stage, P fertilizer significantly altered the soil's physicochemical properties, facilitating enhanced B. napus development. The application of P fertilizer also influenced microbial diversity, with differential bacterial communities emerging as potential contributors to the alleviation of Al toxicity. At the maturity stage, P fertilization markedly increased yield and biomass while reducing Al accumulation in roots, straw, and pods, thus protecting root health and promoting nutrient uptake. In summary, P fertilization serves as a dual-purpose strategy: providing essential P nutrition and alleviating Al toxicity in acidic soils. These findings provide valuable insights for developing sustainable agricultural practices in acidic soils. Future research should focus on determining the optimal P fertilizer rates and types to maximize productivity while minimizing environmental impacts, ensuring a balance between agricultural sustainability and ecosystem health.