Improving phosphorus utilization and iron homeostasis in maize through nitrification inhibitors and plant growth-promoting bacterium

Introduction: This study aimed to investigate the effects of nitrification inhibitors (NIs), specifically DMPP (Dimethyl pyrazole phosphate) and DMPFA (Dimethyl pyrazole fulvic acid), and plant growth-promoting microorganisms (PGPM) on nutrient uptake, allocation, and plant growth in maize under low phosphorus (P) availability. The research questions explored whether NIs enhance P, manganese (Mn), and zinc (Zn) uptake through rhizosphere acidification, alter nutrient partitioning between roots and shoots, and whether DMPFA-PGPM combinations synergistically improve plant growth and nutrient acquisition.

Methods: Two rhizobox experiments were conducted using silt loam soil with low P content (8.7 mg kg₋₁ P-CAL, pH 6.4).

Results: In the first experiment, maize was subjected to ten treatments, including ammonium (NH_{4 +}) and nitrate (NO3 −) with or without DMPP, DMPFA, and rock phosphate (RP), compared to controls. The second experiment tested five treatments, including NH₄ + with DMPFA, fulvic acid, and Bacillus atrophaeus (ABi05) as PGPM. Measurements included rhizosphere pH, acid/alkaline phosphatase activity, root exudates, phytohormones, root morphology, plant biomass, and nutrient (P, Mn, Zn, Fe, Ca, Mg, K) concentrations in shoots and roots. Nutrient use efficiencies (PUE, PFPp, NRE) were calculated, and data were analyzed using one-way ANOVA with Fisher’s LSD test (p<0.05). In the first experiment, DMPP+RP and DMPFA+RP treatments increased biomass by 31.8% and 38.5%, respectively, compared to the negative control, with total root length rising by up to 169.5% in the positive control (NO₃ −+soluble P). Shoot Fe content was 60% higher in NI treatments, with Mn and Zn shoot concentrations increasing by up to 40.4% and 32.8%, respectively, in DMPFA treatments. The rhizosphere pH dropped by 0.5 units in NI treatments, thereby enhancing acid phosphatase activity. In the second experiment, DMPFA and DMPFA+ABi05 increased shoot biomass by 47.5% and 50.7%, respectively, and shoot P content by 45.1% and 62.7%. PUE was 56.2% higher with DMPFA+ABi05, and zeatin concentrations rose by 79.1% compared to controls.

Conclusion: DMP-based NIs significantly enhance P, Mn, and Zn uptake in maize by acidifying the rhizosphere and increasing nutrient solubility. NIs shift mainly Fe and Mn allocation toward shoots, improving nutrient mobilization. The synergistic effect of DMPFA and PGPM (ABi05) further boosts PUE and Zeatin.

Introduction

Plant growth and development heavily rely on P, a vital macronutrient element critical in physiological processes such as energy transfer, photosynthesis, and nutrient mobility. The availability of P in soil is one of the essential factors determining plant productivity. However, plants often struggle to access P because phosphate forms insoluble Fe and Al phosphates in acidic soils and Ca phosphates in alkaline soils. P limitation is projected to affect crop productivity on over 40% of arable land (Vance, 2001). Understanding, managing, and increasing P availability in soil is a key agricultural challenge.

Humic substances, a central component of soil organic matter, significantly influence soil properties and nutrient availability, including P (Pinton et al., 2009). These complex organic molecules stabilize the soil structure, increase soil water retention, and create ligands with Al and Fe, facilitating P release from soil minerals and organic matter (Senesi and Loffredo, 2005). The interaction of humic substances with P can increase its solubility and mobility, making it more available for plant uptake (Wang et al., 1995). Humic substances can stimulate root growth and enhance the root surface area, further aiding in the efficient absorption of P from the soil (Abbas et al., 2024). Fulvic acid (FA) could improve plant growth and nutrient uptake (Kumar Sootahar et al., 2020).

P availability is enhanced by plant growth-promoting microorganisms (PGPM) as well. These beneficial microbes, including certain bacteria and fungi, can solubilize inorganic P compounds and mineralize organic P sources, converting them into forms that plants can easily absorb (Rodríguez and Fraga, 1999; Khan et al., 2026). PGPM achieves this through various mechanisms, such as the secretion of organic acids, enzymes, and siderophores that chelate Fe and release bound P (Dhawi, 2023). PGPM contribute to stronger root development, increased nutrient uptake, and enhanced plant growth and yield. Soil microbial communities are significantly influenced by various agricultural practices, including fertilization (Francioli et al., 2016), microbial inoculation (Behr et al., 2023), and the use of nitrification inhibitors (Li et al., 2023). Nitrification inhibitors (NIs) temporarily retain nitrogen in the ammonium form, reducing nitrification losses and maintaining a more plant-available N pool in the rhizosphere (Zhang et al., 2023). Increased ammonium availability promotes proton release and localized acidification, which enhances microbial-mediated P solubilization and micronutrient mobilization (Marschner, 2012). In this improved chemical environment, PGPM can more effectively mobilize nutrients and facilitate plant uptake; therefore, the sustained ammonium supply provided by NIs is expected to enhance PGPM activity and result in synergistic improvements in nutrient-use efficiency.

DMP-based nitrification inhibitors have shown their effectiveness in previous research (Ruser and Schulz, 2015). DMPFA (Dimethyl pyrazole fulvic acid) is a double chelator with a non-covalent interaction between DMP and fulvic acid (Mazzei et al., 2022). By forming metal complexes, fulvic acids have the potential to inhibit nitrification, preventing Cu and Fe from serving as cofactors for microbial nitrifiers. DMPFA increased P availability in the soil and improved plant growth indicators (Malakshahi Kurdestani et al., 2024).

Despite increasing interest in the use of nitrification inhibitors to enhance nutrient use efficiency, limited information is available regarding the effects of DMPFA on soil phosphorus dynamics and crop performance. To date, only a few studies have reported on DMPFA, and its physiological and agronomic impacts, particularly in maize, remain largely unexplored. The potential synergistic effect of DMPFA with plant growth-promoting bacteria (PGPB) has not yet been examined, representing a significant knowledge gap in integrated soil fertility management. This study, therefore, aims to provide new insights into the role of DMPFA in improving soil phosphorus availability and maize growth, contributing to the limited body of knowledge on this novel nitrification inhibitor.

The combined application of NIs and PGPM can synergistically increase P availability in soils, leading to considerable agricultural benefits (Nkebiwe et al., 2016; Mpanga et al., 2019). Humic substances can enhance the colonization and activity of PGPM by providing a favorable environment and additional carbon sources, amplifying the positive effects on P solubilization and mineralization (Canellas et al., 2015). This holistic approach makes agriculture more sustainable by reducing reliance on chemical fertilizers, enhancing soil health, and increasing crop productivity.

Our hypotheses are: (1) DMP-based NIs increase plant nutrient uptake, notably P, Mn, and Zn uptake, in soils with low P availability by influencing rhizosphere processes and promoting soil acidification. (2) N form and NIs application alter nutrient allocation between root and shoot. (3) DMPFA-PGPM enhance plant growth indicators and nutrient uptake due to the synergistic effect between NIs and microorganisms. Two rhizobox experiments were conducted to address the hypotheses.

Materials and methods

We utilized maize (Zea mays var. Ronaldinio) and the same soil for both rhizobox experiments. A silt loam soil with low P content was employed for these experiments. The CAL extractable P content was 8.7 mg kg^-1, which indicates very low plant availability (VDLUFA, 2018). The near-neutral soil conditions are indicated by a pH of 6.4 in a 10–² M CaCl₂ suspension. The final substrate consisted of 2.2% organic carbon (C_org) and 0.02% total nitrogen (N_t), comprising 70% soil and 30% quartz by weight. For both experiments, NH₄⁺ and NO₃^- were added as ammonium sulphate and calcium nitrate, respectively. DMPP and DMPFA are used as nitrification inhibitors. N, including both NH₄⁺ and NH₄⁺ plus NIs, was also administered at 150 mg per kg substrate. Water-soluble P (calcium phosphate monohydrate) and rock phosphate were added as P sources at 150 mg P per kg substrate. K was added at 150 mg per kg of substrate. The rhizoboxes used had dimensions of 50 × 10 cm and were set up at an angle of 60 degrees to the vertical. The large side facing downward was covered with a removable plexiglass panel.

The rhizoboxes were filled with 2 kg of substrate, and the moisture content was carefully increased from 50% to 70% of the maximum water-holding capacity (WHC), coinciding with the onset of plant growth. Two maize seeds were planted in the substrate, close to the rhizobox glass window, at a depth of 2 cm. Following their emergence, one seedling was removed to ensure only one plant remained per pot. The rhizoboxes were positioned within a greenhouse at the University of Hohenheim, Stuttgart, Germany. The temperature during the growing period spanned from 12 to 35 °C. Six weeks after planting, the maize plants were ready for harvest.

First experiment (effect of NIs on nutrient solubilization)

The first rhizobox experiment was designed as a completely randomized block design with five replications. Treatments were: (1) without additional N and P (negative control), (2) without additional N but with RP, (3) with NH₄⁺ but without P, (4) with NH₄⁺ and RP, (5) with NO₃^- and water soluble P (positive control), (6) with NO₃^- but without P, (7) with NH₄⁺ and DMPP but without P, (8) with NH₄⁺ and DMPP and RP, (9) with NH₄⁺ and DMPFA but without P, (10) with NH₄⁺ and DMPFA and RP.

Second experiment (Synergistic effects of DMPFA-PGPM on plant growth)

The second rhizobox experiment was designed as a completely randomized block design with five treatments and five replications to investigate the synergistic effects of compound components on plant indicators. All the agronomic practices for the second experiment were identical to those of the first experiment.

In this experiment, treatments comprised: (1) with NO₃^- (positive control), (2) with NH₄⁺, (3) with NH₄⁺ and Fulvic acid, (4) with NH₄⁺ and DMPFA, (5) with NH₄⁺ and DMPFA and ABi05.

Microbial inoculation

Microbial inoculation was employed for the second experiment. RhizoVital^® C5, containing Bacillus atrophaeus (ABi05) at a concentration of 1 × 10^8 CFU kg^-1 substrate, served as the PGPM. This consortium contains spores of the bacterium in a water-based medium without another active compound. Maize seeds were immersed in RhizoVital^® C5 suspensions for one hour before sowing, ensuring initial microbial interaction. Following planting, 20 mL of the microbial suspension was carefully inserted into each seeding hole, fostering a localized and sustained association between the plant roots and the PGPM throughout the experiment. This method aimed to establish a beneficial relationship, enhancing nutrient uptake and overall plant health. Additional weekly applications of the PGPM suspension were made near the plant for two consecutive weeks.

Analysis of phytohormones

In the second experiment, 1 g of frozen maize shoot samples from each treatment was pulverized with liquid nitrogen and extracted twice using 2.5 ml of 80% methanol. The extracts were homogenized by ultrasonication at 10,000 rpm for 75 s to enhance homogenization. After transferring 2 ml of the methanol extracts to another tube, they were centrifuged for 5 minutes. Following that, 350 µL of the supernatant was combined with 700 µL of ultra-pure water and underwent centrifugation for 5 minutes. The supernatant was passed through a filtration membrane for purification and then transferred into HPLC vials (Almeida Trapp et al., 2014).

Biomass, root length, and rhizosphere pH measurements

For both experiments, growth indicators were measured. After six weeks, the plants were harvested from the rhizoboxes, and any bulk soil was gently removed from the roots by shaking. The last soil particles adhering to the roots were collected as rhizosphere soil. Following VDLUFA, 1991, soil pH was assessed. After drying in an oven at 60 °C, we measured the biomass of the shoot and root tissues. Root length was assessed by placing washed roots on clear Perspex trays covered with a thin layer of water, and images were captured using a scanner (Epson, model Expression 1000 XL). The WinRhizo Pro software (WinRhizo Pro V. 2009c, Regent Instruments Inc., Canada) was employed to compute the length of the digitized root samples. Root diameter was classified into three categories: fine roots (0–0.02 mm), medium roots (0.02–0.6 mm), and coarse roots (>0.6 mm).

Nutrient analysis

The same analysis methods were utilized in both experiments. The nutrient composition of the dried plant material (roots and shoots) was evaluated using a standard wet-chemical extraction technique after grinding the plant dry biomass (VDLUFA, 2011). P was determined at 436 nm using the molybdo-vandate technique through spectrophotometry (Gericke and Kurmis, 1952). Mn, Zn, Fe, and Mg were quantified via atomic absorption spectrometry, while K was measured using flame emission photometry.

Phosphorus use efficiency (PUE) and partial factor productivity of phosphorus (PFPp) were calculated using Equations 1 and 2, respectively (Dobermann, 2007). Nitrogen recovery efficiency (NRE), which measures the effectiveness of plants or systems in utilizing nitrogen from fertilizers, was determined using Equation 3.

$\begin{array}{ll}PUE(\%)=\frac{P content mg per plant}{Input P mg as fertilizer per plant}*100& (1)\end{array}$

$\begin{array}{ll}PFPp =\frac{Total biomass (g)}{Input P (g) as fertilizer per plant}& (2)\end{array}$

$\begin{array}{ll}NRE=\frac{Total mg N content in fertilized plant-Total mg N content in unfertilized plant }{Total mg Nmin in the soil}& (3)\end{array}$

Data analysis

For statistical analysis of significant differences between treatment groups, a one-way ANOVA followed by a Fisher’s LSD post hoc test (p<0.05) was performed using the R version 4.0.2 (R Core Team, 2020). The analysis employed the packages dplyr for data arranging, ggplot2, networkD3, plotly for plotting, and agricolae for RCBD analysis. These Python packages, including Plotly, Matplotlib, Holoviews, Semopy, and Statsmodels, are commonly used for creating various types of diagrams and visualizations.

Results

First rhizobox experiment

Plant biomass and root characterization

The biomass indicators for the positive control treatment (NO₃ with soluble P) were significantly higher than those of the other treatments (Figure 1). Compared to the negative control, the positive control showed increases of 59.3% in biomass, 157.4% in root length (0< root length< 0.2 mm), and 169.5% in total root length (Figure 2). Following the positive control, treatments containing NH₄⁺ and DMPFA plus RP, as well as NH4+ and DMPP plus RP, had the highest values. Conversely, the treatment with NO₃ without P showed reductions of 3.8%, 30.2%, and 23.3% in biomass, root length (0< root length< 0.2 mm), and total root length, respectively, compared to the negative control treatment (Figure 2). NH₄⁺ and DMPP plus RP and NH₄⁺ and DMPFA plus RP enhanced biomass at 31.8% and 38.5%, respectively, compared with the negative control. Total root length significantly increased using NIs compared with the negative control. RP application significantly increased all growth indicators compared to treatments without P, but not as much as soluble P. In addition, Figure 1 shows that shoot dry matter contributed the majority of total biomass across all treatments, whereas root dry matter represented a smaller but consistent fraction. The relative increase in shoot biomass was more pronounced in the NO₃ plus soluble P and NI-containing treatments. In contrast, differences among treatments were minor for root biomass, indicating a greater treatment effect on shoot growth than on root development. As shown in Figure 2, root architecture exhibited distinct patterns across diameter classes. Fine roots (0–0.2 mm) accounted for the most significant portion of total root length in all treatments, followed by medium roots (0.2–0.6 mm) and coarse roots (>0.6 mm). The NO₃ plus soluble P treatment produced the most significant fine and medium root lengths, while coarse roots were relatively less affected. Treatments with NH₄⁺ combined with DMPP or DMPFA plus RP also promoted higher root development across all diameter categories compared with the negative control, indicating consistent improvement in root system size and distribution.

Figure 1

Figure 1. Root and shoot dry weight (g) of Maize (cv. Ronaldinio) at 42 days after sowing across the treatments. Bars represent means of five replicates ± standard error (SE), analysed by one-way ANOVA with Fisher’s LSD test (α = 0.05). Lowercase letters indicate significant differences in root or shoot biomass, and uppercase letters denote significant differences in total biomass.

Figure 2

Figure 2. Treatment effects on maize (cv. Ronaldinio) on root architecture at 42 days after sowing. The means of five replicates were analysed by one-way ANOVA and Fisher’s LSD test (α = 0.05). Lowercase letters denote significant differences in root length by diameter (mm), and uppercase letters indicate total root length variations, revealing the impacts of nitrogen forms and inhibitors on root traits.

Nutritional status

Shoot P concentration in the treatment with NO₃^- without P is significantly (P ≤ 0.05) lower than in the negative control treatment (Supplementary Table S1). The positive control treatment was significantly higher than in the other treatments. A consistent trend was observed where ammonium-based treatments, mainly with NIs, enhanced shoot P concentrations when combined with RP compared to nitrate-based or no-N treatments, indicating improved P solubilization under acidified conditions. Root P concentration in the NH₄⁺ and DMPP plus RP was significantly higher than in other treatments (Supplementary Table S2). A trend of reduced P accumulation is observed in the absence of soluble P or under nitrate nutrition. Shoot P content was significantly lower in NO₃^- without P compared to the control (Figure 3). A positive correlation was observed between shoot P content and several variables, including soil P extracted by the calcium-acetate-lactate method (P-CAL), total root length, and NRE (Figures 4A–C). Conversely, a negative correlation was detected between shoot P content and shoot K concentration (Figure 4D). NRE was lowest in the nitrate without P treatment and highest in the NH₄⁺ and DMPFA plus RP treatment (Table 1). PUE in the NO₃- plus Sol P treatment was about 56.3% higher than without N plus RP treatment (Table 2).

Figure 3

Figure 3. Phosphorus content (mg per plant) in shoots and roots of maize (cv. Ronaldinio) at 42 days after sowing across the treatments. The means of five replicates are shown with standard error bars, analyzed by one-way ANOVA, Fisher’s LSD test (α = 0.05). Letters indicate significant differences in root or shoot P content.

Figure 4

Figure 4. Correlation of the shoot phosphorus content (mg per plant) with (A) total root length (mm), (B) nitrogen recovery efficiency (g/g), (C) soil P-CAL (mg/100 g), and (D) shoot Potassium concentration (mg/g) in Maize. The shaded regions depict 95% confidence intervals from the regression model, illustrating the correlated relationships.

Table 1

Table 1. Nitrogen recovery efficiency (NRE) and Phosphorus use efficiency (PUE) of maize (cv. Ronaldinio) at 42 days after sowing with different nitrogen and phosphorus sources.

Table 2

Table 2. Growth indicators and soil properties of maize (cv. Ronaldinio) 42 days after sowing with different nitrogen and phosphorus sources.

The shoot Ca concentrations were higher than the root concentrations (Supplementary Tables S1, S2). Except for NH₄⁺ and DMPFA plus RP, all treatments containing NIs demonstrated reduced shoot Ca concentration compared to the negative control. In all treatments containing RP, the root Ca concentration was lower than in the negative control. The shoot Ca concentration was highest in NO₃^- without P (+39% vs. negative control) and lowest in NO₃^- plus Sol P (-21% vs. control). Regarding the application of P, the positive control treatment had the highest root Ca concentration and the lowest shoot Ca concentration. In all treatments except those with NH₄⁺ and NH₄⁺ and DMPP without P, the shoot Ca content was higher than that of the control (Supplementary Figure S1).

The Fe concentration in the roots was significantly higher than in the shoots across all treatments, suggesting that iron tends to accumulate more in the roots (Supplementary Table S1). Except for the nitrate-containing treatments, the root Fe concentration was lower than that of the negative control in all treatments. Simultaneously, the shoot Fe concentration exceeded control levels in all treatments except the positive control and the NH₄⁺ treatment without P, with -21.4% and -9.7%, respectively. Regarding the use of P, there was no significant difference in shoot Fe concentration between treatments containing RP, without P, and sol P. Despite the higher shoot Fe contents observed in all treatments compared to the control, treatments containing NIs exhibited lower root Fe contents than the control (Supplementary Figure S2). In the treatment with nitrate, 31% of the iron accumulated in the shoots. This amount was 38% for NH₄⁺, while in treatments with NIs, 60% of the iron accumulated in the shoots and 40% in the roots. Shoot Fe content increased with root N (%) and decreased with rhizosphere pH. Compared to nitrate, NIs treatments result in higher micronutrient accumulation in the shoots.

K concentration was always greater in the shoot than in the root across all treatments. All treatments had lower root and shoot K concentrations than the negative control. Compared to the negative control, the most significant reductions in shoot K concentration were observed in the positive control (64.2% lower) and in root K concentration in the NH₄⁺ plus DMPP treatment without RP (77.6% lower). Treatments with RP had higher shoot K contents, but the Sol P treatment showed a higher root K content than the RP treatments (Supplementary Figure S3).

Shoots have higher Mg concentrations than roots, like Ca. The lowest root Mg concentration was observed in the treatment containing NIs. The two treatments without any additional N had the highest root Mg concentration. All treatments, excluding NO₃^- without P, contained more shoot Mg than the negative control (Supplementary Figure S4). Mn tended to accumulate more in the shoots, although there were noticeable differences between treatments, mainly with and without P. Treatments involving NH₄⁺ showed increased Mn uptake compared to those without. Roots had significantly lower Mn contents in the treatments containing NIs than in the others (Supplementary Figure S5). Zinc accumulated more in the roots compared to the shoots across all treatments (Supplementary Figure S6). Treatments with NH₄⁺ generally showed slightly higher zinc concentrations, especially with added P. Zn concentrations in shoots and roots, which were significantly reduced in the positive control compared to the negative control (56.4% and 45.7% reduction, respectively).

Treatments without N and then with nitrate had a higher rhizosphere pH than those with NH₄⁺. NIs decreased rhizosphere pH significantly, although adding RP did not create a significant difference through these treatments (Figure 5).

Figure 5

Figure 5. In the different treatments, rhizosphere pH (CaCl₂) in maize (cv. Ronaldinio) at 42 days after sowing. The means of five replicates are shown and analyzed using one-way ANOVA and Fisher’s LSD test (α = 0.05).

The most notable micronutrient was iron (Fe). When NH₄⁺ was used, NIs (average of NH₄⁺ and DMPP and DMPFA) altered Fe distribution between roots and shoots, while the distribution of other nutrients remained unchanged (Figure 6A). In treatments containing only NH₄⁺, 62% of Fe was accumulated in the roots and 38% in the shoots. However, using NIs shifted this distribution to 60% in the shoots and 40% in the roots. Nitrate did not cause a significant difference in N, P, K, Ca, and Mg distribution between roots and shoots compared to NH₄⁺. When using nitrate fertilizers, the shoots contained less Mn, Fe, and Mg but more N (Figure 6B). Acid phosphatase activity was lower than the control treatment, only in the treatment with NH₄⁺ without P. However, using NIs significantly increased acid phosphatase activity compared to the control (Supplementary Figure S7).

Figure 6

Figure 6. Sankey diagrams illustrating the nutrient distribution between Maize roots and shoots. Nutrient proportions (%) in shoots (top) and roots (bottom) beside each element are shown and analysed using the Kruskal-Wallis test with Dunn’s test and Bonferroni correction for multiple comparisons. (A) Ammonium alone vs. ammonium with nitrification inhibitors (NIs); (B) Nitrate vs. ammonium with NIs. Asterisks indicate significant differences in treatment means for each nitrogen form per element (*p< 0.05, **p< 0.01, ***p< 0.001).

Second rhizobox experiment

Plant biomass and root characterization

This experiment investigated how fulvic acid affects nutrient availability, comparing it with NH₄⁺ and DMPFA in the presence and absence of PGPM (ABi05). DMPFA significantly increased shoot biomass in this experiment compared to other treatments (Table 2). The increase was 47.5% for NH₄⁺ and DMPFA alone and 50.7% for NH₄⁺ and DMPFA combined with ABi05, compared to the control. No significant differences were observed among treatments with NH₄⁺ or NH₄⁺ plus fulvic acid. Total biomass was 41.9% and 46% higher in the treatments with NH4+ and DMPFA, and with NH₄⁺ and DMPFA plus ABi05, respectively, compared to the control (Figure 7). The rhizosphere pH (CaCl₂) was highest in the control treatment (5.8), while the lowest values were observed in treatments containing NH₄⁺ and DMPFA (5.3) and NH₄⁺ and DMPFA in combination with ABi05 (5.2). Acid phosphatase activity differed significantly between N application methods. Treatments with ammonium-based N exhibited higher acid phosphatase activity than those with nitrate-based N. Additionally, alkaline phosphatase activity was 22.8% greater in the treatment with NH₄⁺ and DMPFA+ABi05 than in the control.

Figure 7

Figure 7. Root and shoot dry biomass (g) across the treatments in maize (cv. Ronaldinio) at 42 days after sowing. The means of five replicates with standard error are shown and analyzed by one-way ANOVA and Fisher’s LSD test (α = 0.05). Different letters above the bars indicate significant differences.

Nutritional status and phytohormones

Shoot and root P content showed significant increases with the application of NH₄⁺ and DMPFA (Table 3). However, no significant differences were observed in P concentration between treatments for both shoots and roots. Shoot P content increased by 45.1% with NH₄⁺ and DMPFA and 62.7% with NH₄⁺ and DMPFA+ABi05 compared to the control. Similarly, root P content increased 48.8% with NH₄⁺ and DMPFA and 46.2% with NH₄⁺ and DMPFA+ABi05. Treatments containing NH₄⁺ and NH₄⁺ combined with fulvic acid exhibited decreased P-CAL compared to the control, with reductions of 8.6% and 4.2%, respectively. However, P-CAL levels increased by 9.7% with NH₄⁺ and DMPFA and by 14.6% with NH₄⁺ and DMPFA+ABi05.

Table 3

Table 3. Nutrient concentrations and contents in roots and shoots of maize (cv. Ronaldinio) at 42 days after sowing with various nitrogen and phosphorus sources.

The shoot and root K concentrations of all treatments were lower than those of the control. The lowest shoot K concentration was observed in treatments containing NIs, although the shoot K content was significantly higher. While some trends were noted in root K concentration and content, none were statistically significant. Shoot and root Ca concentrations were higher in the control group than in all treatments. The shoot Ca concentration was significantly lower in treatments containing NH₄⁺ and DMPFA than in other treatments. However, shoot Ca content was higher in the NH4+ and DMPFA treatments than in the other treatments. Root Ca concentration was lower in NH₄⁺ and NH₄⁺ with NH₄⁺ and DMPFA plus ABi05 treatments than in the others. There was no difference in shoot Mg concentration; however, shoot Mg content was higher in treatments containing NH₄⁺ and DMPFA than in the control, increasing by 38.2% in NH₄⁺ and DMPFA and 53.7% in NH₄⁺ and DMPFA+ABi05. Despite the higher root Mg concentration in the control, there was no difference in root Mg concentration and content between treatments.

There were no significant differences among treatments in shoot Fe concentration, although a trend was observed where the control exhibited higher levels than the other treatments. Root Fe concentration was significantly higher in the control than in the other treatments. No substantial differences in root Fe content were observed among the treatments; however, shoot Fe content was higher in the treatments with NH₄⁺ and DMPFA at 48.7% and NH₄⁺ and DMPFA+ABi05 at 50.2%. Treatments containing NH₄⁺ had higher shoot Mn concentrations and contents than those with nitrate; however, the root Mn contents did not differ significantly among treatments. The shoot Zn concentrations were higher at 40.4% with NH₄⁺ and DMPFA and 32.8% with NH₄⁺ and DMPFA+ABi05 than in the control. The shoot Zn content was significantly higher in treatments containing NH₄⁺ and DMPFA compared to other treatments, while no significant differences were observed among the remaining treatments. The root Zn concentrations did not differ significantly across treatments; however, higher concentrations were particularly noted in plants treated with NH₄⁺ and DMPFA. Additionally, the root Zn content was elevated in treatments containing NH₄⁺ and DMPFA+ABi05. DMPFA enhanced the shoot and root N concentrations (%) compared to the other treatments, whereas NH₄⁺ and NH₄⁺ combined with fulvic acid (FA+NH₄⁺) resulted in lower concentrations than the control.

In this study, shoot phytohormone concentrations were quantified. Jasmonic acid (JA) and zeatin (Z) were the only phytohormones detected across all treatments. While JA levels did not exhibit statistically significant differences among treatments, a trend towards increased JA concentration was observed in treatments containing DMPFA (Supplementary Figure S8). Specifically, treatments with NH₄⁺ and DMPFA alone and NH₄⁺ and DMPFA+ABi05 showed a 53.6% and 44.2% increase in JA levels, respectively, compared to the control. Zeatin concentrations varied significantly among treatments. The treatment combining RP and NH₄⁺ exhibited the lowest zeatin concentration, while the treatment with NH₄⁺ and DMPFA+ABi05 showed the highest. Relative to the control, zeatin concentrations were 46.6% higher in the NH₄⁺ and DMPFA treatment and 79.1% higher in the NH₄⁺ and DMPFA+ABi05 treatment.

The Ni treatments exhibited significantly greater PUE than all other treatments. PUE values for the NH₄⁺ and DMPFA and NH₄⁺ and DMPFA+ABi05 treatments were 46.6% and 56.2% higher than those of the control, respectively. Rhizosphere pH, shoot K and Ca concentration, and root Fe concentration presented a negative correlation with PUE in this experiment (Figure 8). PFPp was higher in the NH₄⁺ and DMPFA treatments than in all other treatments, which did not differ significantly from one another. PFPp was 46% greater in the treatment with NH₄⁺ and DMPFA+ABi05 and 41.9% larger in the NH₄⁺ and DMPFA treatment compared to the control.

Figure 8

Figure 8. Pearson correlation analysis of morphological and physiological indicators in maize. This graph displays positive (red) and negative (blue) correlations between key plant traits. Asterisks indicate significance levels: * (p< 0.05), ** (p< 0.01), *** (p< 0.001). B, Biomass; TRL, Total root length; Sh. Ca, Shoot ca concentration; Sh. K, Shoot K concentration; Sh. Zn, Shoot Zn concentration; TN, Total N content per plant; Rh. pH, Rhizosphere pH; Z, Zeatin; JA, Jasmonic acid; R. Fe, Root Fe concentration.

Discussion

The findings of this study highlight the critical role of N form, P availability, and microbial interactions in optimizing plant nutrient uptake, rhizosphere processes, and overall plant performance. Given that NH₄⁺ and DMPFA is a recently developed nitrification inhibitor with only two prior studies, the present findings contribute novel evidence regarding its potential to enhance soil P availability and crop performance. The integration of NIs, particularly NH₄⁺ with DMPFA and DMPP, demonstrated substantial effects on nutrient solubilization and rhizosphere acidification, offering valuable insights for sustainable agricultural practices.

Impact of nitrification inhibitors on nutrient availability and plant growth

In both experiments, higher nutrient concentrations were observed in the control treatment relative to the other treatments, indicating evidence of a “dilution effect” (Jarrell and Beverly, 1981; Wikström, 1994). Our data support previous findings (Vogel et al., 2020; Barth et al., 2001) that NH₄⁺ and DMPFA and DMPP significantly alter P availability and nutrient uptake. The presence of ammonium-based fertilization treatments led to enhanced P availability, likely due to their role in maintaining NH₄⁺ in the soil for longer durations (George et al., 2016). The effect of root exudation to enhance PUE has been explored by previous research (Menezes-Blackburn et al., 2016; Pantigoso et al., 2023). In the first experiment, the highest PUE was observed in the positive treatment, and no difference was found between the NH₄⁺ and DMPP and DMPFA treatments. The second experiment assessed PUE and PFPp as key indicators of fertilization efficiency. When N was applied as NH₄⁺, treatments with NH₄⁺ and DMPFA, both alone and in combination with PGPM, exhibited the highest PUE, demonstrating that this approach optimizes P utilization. The increases in PUE in these treatments suggest that the combination of nitrification inhibition and microbial activity enhances the efficiency of applied P fertilizers, potentially reducing the reliance on high-input chemical fertilization. These findings align with the previous work of Canellas et al. (2015), which highlights the role of humic substances in enhancing nutrient efficiency and plant productivity. In another study, Watson et al. (2019) demonstrated that applying NIs enhanced the uptake of P and Mg. However, in our experiment, no significant difference was observed in root Mg concentration, and the highest shoot Mg concentration was found in the treatment combining NH₄⁺ with FA. In a long-term experiment, the increase of total phosphorus in the soil was reported (Zhang et al., 2023). This effect supports the hypothesis that NIs can enhance nutrient efficiency by limiting rapid nitrification, thereby preventing nitrate leaching and sustaining ammonium-mediated rhizosphere acidification (Mpanga et al., 2019; Ruser and Schulz, 2015). The enhancement of NUE by NIs in our experiments supports previous studies and meta-analyses (Gilsanz et al., 2016; Wu et al., 2021; Yin et al., 2023). The reduced pH rhizosphere observed in treatments with NH₄⁺ and DMPFA and DMPP aligns with previous studies, which indicate that NH₄⁺ uptake lowers soil pH, thereby increasing the solubility of P and micronutrients such as Fe and Mn (Canellas et al., 2015).

The effect of NIs on root development and structure was illustrated by previous research (Xu et al., 2014; Pittaway et al., 2023). In our study, the increase in root and shoot biomass in treatments with the NIs indicates enhanced nutrient use efficiency beyond the simple N retention reported previously (Alonso-Ayuso et al., 2016). This observation aligns with previous studies (Kong et al., 2022; Wang et al., 2021), which reported enhanced nutrient efficiency and crop performance with the combined application of humic substances and nitrification inhibitors.

Path analysis from the first experiment revealed the direct and indirect effects of various factors on biomass through regression coefficients. Total root length exhibited a direct positive effect from NIs (Ravazzolo et al., 2020), significantly influencing several downstream variables, including plant-available P, shoot and root P content, and overall biomass. These findings indicate that increased root length enhances nutrient acquisition and promotes plant growth by facilitating greater soil exploration (Lynch et al., 2021). The analysis demonstrates a strong correlation between enhanced nutrient uptake (shoot and root P) and biomass accumulation (Figure 9).

Figure 9

Figure 9. Path analysis of direct and indirect effects on maize biomass under nitrification inhibitor treatments. This diagram illustrates the relationships between nitrification inhibitors, rhizosphere factors, nutrient uptake, and biomass in maize, with regression coefficients indicating the strength of the paths. Arrows represent hypothesized causal pathways with significant correlations (p< 0.05) denoted by asterisks (*). The green lines indicate positive effects, and the red lines denote negative effects, highlighting the role of inhibitors in enhancing biomass through nutrient dynamics. ** and *** indicate statistically significant differences at p < 0.01 and p < 0.001, respectively.

Role of microbial inoculation in enhancing plant nutrient uptake

The positive effect of Bacillus atrophaeus on P uptake was proven in previous research (Tanveer et al., 2024). The second rhizobox trial showed increased biomass and PFPp in the NH₄⁺ and DMPFA treatments. The increased activity of acid phosphatase in the treatment with NH₄⁺ was significantly higher than in the treatment with nitrate, indicating a different regulatory mechanism. Previous studies have shown a stronger effect of NH₄⁺ on acid phosphatase activity (Guo et al., 2022; Li et al., 2021; Min et al., 2011). The synergy between PGPM and humic substances facilitates microbial colonization, and root interactions increase PUE. Although the previous findings (Behr et al., 2023; Rodríguez and Fraga, 1999) highlight the important role of PGPM in enhancing soil nutrient bioavailability and plant performance, inoculation in our experiment increased zeatin, PUE, shoot Mg content, and shoot P content compared to no inoculation. Furthermore, improved root growth may enhance rhizosphere colonization of inoculated PGPM (Nkebiwe et al., 2017).

Effects on rhizosphere pH and nutrient mobilization

By maintaining NH₄⁺ concentrations, NIs induce root proton exudation, which lowers the rhizosphere pH by 0.5-1.1 units compared to the control and initial soil pH (Kravljanac et al., 2024). Our study confirms that ammonium-based treatments generally result in a lower rhizosphere pH (approximately 0.5 units) compared to nitrate treatments. Compared to the control and nitrate treatments, NIs acidified the rhizosphere. This acidification is crucial for mobilizing phosphorus and micronutrients, such as Fe and Mn (Rengel, 2015). Therefore, enhancing nutrient solubility in low-P soils requires a well-balanced N form and pH. This aligns with previous research indicating that soil acidification associated with NH₄⁺ uptake improves P solubility and micronutrient bioavailability (Senesi and Loffredo, 2005).

Nutrient partitioning between roots and shoots

An essential finding of this study is the differential accumulation of nutrients in roots and shoots under various fertilization treatments. According to Marschner (2012), we also found that nutrients like K, Ca, Mg, and P tended to accumulate more in shoots, whereas nutrients such as Fe and Zn exhibited higher concentrations in roots. Both are following Marschner (2012). This pattern suggests a correlation between the type of nutrient and its preferred site of accumulation. The application of DMP-based NIs altered Fe distribution (Figures 6A, B), shifting it from roots to shoots, which suggests a potential enhancement in Fe translocation mechanisms (Zou et al., 2001; Chen and Aviad, 1990).

In maize, mugineic acids, as phytosiderophores, chelate insoluble Fe³⁺ in soil, and these complexes are transported into root cells by YSL family transporters, such as ZmYS1 (Curie et al., 2009). While citrate plays a key role in internal Fe transport within the xylem, its exudation into the soil is less significant for Fe uptake in maize compared to phytosiderophores. NH₄⁺ improved shoot soluble Fe content by upregulating Fe translocation genes (FRD3, NAS1), thereby mitigating chlorosis and facilitating Fe redistribution from roots to shoots under Fe deficiency (Zhu et al., 2019). The higher Fe accumulation in shoots observed with NIs may be attributed to NH₄⁺ nutrition enhancing Fe solubility through rhizosphere acidification, promoting Fe³⁺ reduction (e.g., of ferric citrate) in plant tissues (Kosegarten et al., 1999), and facilitating Fe remobilization via apoplastic pH reduction (Marschner, 2012; Mengel, 1995).

Mn²⁺ is the sole plant-available form of manganese; it is efficiently taken up by root cells and subsequently translocated to the shoot. Mn oxides are reduced to soluble Mn²⁺ under acidic conditions, making Mn more available for root uptake; however, in highly acidic soils, this process can create toxicity (Santiago et al., 2020). The application of NH₄⁺ stimulated the exudation of protons, carboxylates, and enzymes, which enhanced micronutrient availability through increased solubilization and mobilization (Table 3). Root exudation of protons and organic acids enhances Mn solubility, while microbial reduction of Mn oxides further contributes to bioavailability. Within the plant, Mn²⁺ is primarily taken up through NRAMP and ZIP transporters at the root plasma membrane and subsequently allocated or sequestered by MTP, CAX, and BICAT transporters across vascular, Golgi, and chloroplast membranes (Schmidt and Husted, 2019; Meier et al., 2025). This effect is likely driven by low-molecular-weight carboxylate anions, amino acids, and other small bioactive compounds (Rengel, 2015).

Enhanced micronutrient mobility, driven by rhizosphere acidification, chelation, and microbial activity, has a profound impact on plant nutrient dynamics and physiology (Singh et al., 2024). It modulates nutrient interactions—promoting synergies, such as Fe–Mg cooperation in chlorophyll synthesis, while inducing antagonisms like Fe interference with Ca, K, and P uptake (Rietra et al., 2017)—and activates antioxidant enzymes (Mn-, Cu-, Zn-, and Fe-dependent SOD) to mitigate oxidative stress under drought or salinity conditions (Shahbaz et al., 2025). These integrated effects enhance photosynthesis, biomass accumulation, and hormonal signaling, underscoring the central role of micronutrient mobility in plant resilience and overall health.

Phytohormone regulation and stress response

The observed 54% increase in jasmonic acid (JA) and 79% increase in zeatin concentrations in the NH₄⁺ + DMPFA + ABi05 treatment compared to NH₄⁺ alone indicate a combined role of microbial activity and nitrification inhibition in promoting plant growth and stress adaptation. Fulvic acid alone had no significant effect on hormone levels, suggesting that its influence is primarily indirect, likely through enhanced nutrient solubility and root metabolism. Zeatin promoted cell division and root growth, aligning with the observed increases in biomass and root length (Scheres and Benfey, 1999). The increase in zeatin with DMPFA treatment suggests that prolonged NH₄⁺ availability and rhizosphere acidification affected hormone synthesis pathways associated with growth regulation (Wasternack and Hause, 2013). The additional increase under DMPFA + ABi05 highlights a synergistic interaction between microbial inoculation and DMPFA, where Bacillus atrophaeus (ABi05) likely amplified stress-related hormonal signaling through root–microbe communication. To further differentiate these effects, future experiments should include treatments such as ABi05 alone, ABi05 combined with fulvic acid, and DMP (without the fulvic acid component) under both NH₄⁺ and NO₃⁻ regimes. These comparisons would help to disentangle the specific contributions of microbial activity, organic matter interactions, and nitrogen form to hormone regulation and overall plant stress response. These hormonal shifts may be linked to the influence of the PGPM ABi05 on plant signaling pathways (Behr et al., 2023), which is amplified by fulvic acid’s role as a microbial carbon source (Canellas et al., 2015).

Conclusion and future perspectives

This study provides comprehensive evidence that DMP-based nitrification inhibitors, particularly DMPFA, significantly enhance iron homeostasis and phosphorus utilization in maize by altering nutrient partitioning and rhizosphere chemistry. Treatments that combined ammonium with DMPFA and DMPP significantly increased Phosphorus use efficiency (PUE), shoot and root biomass, total root length, and nutrient use efficiency in both experiments. These results indicate the strong potential of these nitrification inhibitors to maximize nutrient acquisition under low-P conditions. Significant rhizosphere acidification (up to 0.5 pH units) was caused by nitrification inhibition, which enhanced acid phosphatase activity and increased the solubility of micronutrients, particularly Fe, Mn, and Zn. Additionally, DMPFA enhanced nutrient mobilization and plant physiological balance by redistributing Fe and Mn from roots to shoots. These discoveries contribute to our current understanding of how DMP-based NIs interact with microbial and plant systems to enhance nutrient efficiency, providing a long-term approach to increasing crop yields and reducing reliance on fertilizers. To guide the development of next-generation bioactive fertilizers, future research should confirm these mechanisms in field settings and assess their long-term effects on microbial ecology and soil health.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://doi.org/10.7910/DVN/LAV7PJ.

Author contributions

AMK: Investigation, Funding acquisition, Conceptualization, Supervision, Methodology, Writing – original draft, Project administration, Formal Analysis, Writing – review & editing, Visualization, Resources, Validation, Data curation, Software. RR: Data curation, Formal Analysis, Validation, Project administration, Conceptualization, Methodology, Supervision, Writing – review & editing, Resources, Software, Investigation, Visualization, Funding acquisition. PN: Writing – review & editing, Validation, Conceptualization, Methodology. XC: Software, Investigation, Resources, Writing – review & editing, Funding acquisition, Visualization, Methodology, Formal Analysis, Validation, Project administration, Conceptualization, Supervision, Data curation. TM: Writing – review & editing, Investigation, Software, Supervision, Funding acquisition, Validation, Conceptualization, Visualization, Data curation, Resources, Methodology, Formal Analysis, Project administration.

Funding

The author(s) declared that financial support was received for this work and/or its publication. DFG (German Research Foundation) grant 328017493/GRK 2366 (Sino-German International Research Training Group AMAIZE-P) provided funding for this project.

Acknowledgments

We sincerely appreciate the invaluable support of Prof. Dr. Günter Neumann, Helene Ochott, Heidi Zimmermann, Sonja Kurz, Yufen Zhao, Sunah Yoon, Frazaneh Akbari, Nasim Gandomdoust, Atousa Eilgoli, and Afsaneh Yousefi in conducting the laboratory analyses. We are also profoundly grateful to Abitep GmbH and Dr. Kristin Dietel for providing ABi05 and their excellent collaboration. Thank you to Prof. Alessandro Piccolo for his outstanding collaboration and support.

Conflict of interest

PN was employed by company Yara GmbH & Co. KG.

The remaining authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer KSK declared a shared affiliation with the author(s) XC to the handling editor at the time of review.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fagro.2025.1720896/full#supplementary-material

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