Effect of Nutrient Forms in Foliar Fertilizers on the Growth and Biofortification of Maize on Different Soil Types

Abstract

This research aimed to evaluate how different chemical forms of key nutrients, delivered through an advanced foliar product (PRO) and a standard formulation (TRA), influence maize performance when grown on contrasting soil types. Each fertilizer provided a set of macro- and micronutrients, including nitrogen, phosphorus, potassium, boron, copper, iron, manganese, molybdenum, and zinc, along with trace elements such as chromium, iodine, lithium, and selenium. In TRA, Fe and Zn were complexed with EDTA, and trace elements were present in mineral form. In PRO, Fe and Zn were chelated with amino acids, and trace elements were bound to plant extracts. The study examined increasing doses of PRO and their potential toxicity. Both fertilizers improved maize biomass: fresh weight increased by 5–8% and dry weight by 8–14%, depending on the dose. At the lowest dose, yields were similar. However, PRO was more effective in biofortifying maize with iron and zinc on sandy soil, increasing levels by 16% and 7% compared to TRA at the lowest dose and up to 29% at the highest dose. PRO was well tolerated at higher doses. No significant differences were observed between the second and third doses of PRO, suggesting reduced efficacy at the highest dose.

1. Introduction

Contemporary agriculture faces numerous emerging challenges, including climate change, water scarcity, limited arable land, environmental degradation, and the necessity to increase agricultural production while simultaneously improving its quality. Crop yield and nutritional value are primarily determined by soil condition and fertilization efficiency. Soil fertilization systems are still improved, and precision fertilization technologies are being introduced.

The importance of micronutrient fertilization in crop production is steadily increasing, primarily due to the introduction of high-yielding cultivars with elevated mineral nutrient requirements and intensive nutrient uptake from the soil. At the same time, many soils exhibit low levels of bioavailable micronutrients, which, combined with increasing crop yields, leads to their gradual depletion. Consequently, the reduced micronutrient content in plants adversely affects their nutritional value for both feed and food purposes. An adequate level of micronutrients is crucial for the proper functioning of the light phase of photosynthesis, nitrogen assimilation, carbohydrate metabolism, and protection against oxidative stress caused by various environmental stress factors. When soil conditions limit nutrient availability, foliar-applied fertilizers can correct nutrient deficiencies and improve plant growth and nutritional value [1].

Present-day farming places increasing emphasis on crop quality and safety, driven by growing consumer awareness, legal regulations, and efforts toward sustainable development. Researchers and farmers seek versatile fertilizer formulations that not only replenish deficiencies in essential macro- and micronutrients but also enhance crop tolerance to adverse environmental conditions, improve the nutritional value of agricultural produce, and are safe for the environment. The practice of foliar fertilization is perfectly in line with these trends. Hence, in response to this demand, various compound fertilizers containing not only micronutrients, i.e., copper (Cu), iron (Fe), manganese (Mn), boron (B), molybdenum (Mo), and zinc (Zn), but trace elements important for human and animal nutrition, such as chromium (Cr), iodine (I), lithium (Li), and selenium (Se), are also being tested [2].

Although trace elements such as Cr, Li, Se, and I are not classified as essential for plant nutrition, they can improve plant function, enhance resistance to environmental stresses, and contribute to increased yields. Se and I are essential for mammals and play a key role in metabolism and hormone production. In plants, these elements can provide protection against biotic and abiotic stresses by modulating the expression of defence-related genes. Li, used exogenously at low concentrations, acts as a mineral biostimulator to promote plant growth. Cr, in its trivalent form Cr(III), is essential for mammalian metabolism but has no important physiological functions in plants and is considered phytotoxic. The positive effect of these trace elements can be explained by the phenomenon of hormesis—a biological reaction in which minimal concentrations of substances usually considered harmful can stimulate growth and increase plant productivity. All these aspects were comprehensively discussed in our earlier study on foliar fertilization on triticale [3].

The effectiveness of foliar-applied mineral nutrients depends on multiple specific factors, with their chemical form and concentration playing a key role [1]. The formulation determines both the efficiency of nutrient transport into the leaf and their metabolic activity after uptake. Research is still ongoing to find the most effective foliar fertilizers.

It uses various organic compounds, including amino acids, peptides, and plant extracts, to increase the effectiveness of foliar fertilizers [4,5,6]. This amino acid complexation with metals potentially offers several physiological advantages. First, amino acid complexes typically have lower molecular weights than EDTA chelates (for example, glycine-metal complexes versus EDTA-metal chelates), which may facilitate more efficient penetration through cuticular pores and stomatal openings. Second, amino acids are naturally occurring compounds in plant tissues, potentially allowing their metal complexes to utilize existing amino acid transporters for active transport across cell membranes, rather than relying solely on passive diffusion as is often the case with synthetic chelates.

Furthermore, amino acid complexes generally maintain better stability and bioavailability across a wider pH range within leaf tissues than EDTA chelates, which can be particularly important given the varying pH conditions in different cellular compartments [7]. The amino acid ligands themselves may also serve as additional sources of nitrogen or as biostimulants, potentially enhancing overall plant metabolism beyond the direct effects of the micronutrients they deliver [8].

For trace elements combined with plant extracts, the bioactive compounds present in these extracts—such as polyphenols, flavonoids, and various secondary metabolites—may enhance membrane permeability, stimulate physiological processes, or activate defense mechanisms that indirectly improve nutrient utilization efficiency [9]. These plant-derived compounds may also facilitate better translocation of nutrients within plant tissues and potentially improve stress tolerance, which could be particularly beneficial under suboptimal growing conditions.

The assessment of foliar fertilizers’ effectiveness in enhancing crop yields should consider not only the form and concentration of their components but also environmental factors, particularly the physicochemical properties of the soil. The time and financial investments associated with their application may prove to be disproportionate to the achieved yield improvements. Recent studies on maize [10] have demonstrated that the primary factors influencing yield and biomass were the experimental location (soil) and genotype (maize cultivar), while the impact of foliar treatments was relatively minor. Additionally, a declining trend in yield quality was observed as the total yield increased.

Maize (Zea mays L.) is one of the most important crops worldwide and ranks first among cereals in terms of global production. One of their important applications is the production of high-quality silage for dairy cattle and sheep. Additionally, maize cultivation can play a significant role in soil protection, mitigating soil erosion and contributing to increased nitrogen content in the soil [11]. Maize grows well on a range of soils but does best on deep, well-drained, fertile soils that are slightly acid to neutral. The optimization of agronomic management includes foliar fertilization that makes it possible to supply key nutrients during periods of difficult uptake from the soil and during the phases of highest demand [2,3], reduces soil contamination, salinity, and acidification, and contributes to a reduction in the total amount of fertilizer applied, while maintaining high fertilizer efficiency [12]. Although it is native to tropical regions, it has adapted to cultivation in a variety of climates, including temperate climates.

The main aim of this study was to evaluate the effectiveness of two formulations of foliar fertilizers (TRA-standard and PRO-innovation) on maize grown in soils with diverse physicochemical properties and soil texture classes (loamy and sandy). The second objective was to evaluate the impact of increased doses of PRO fertilizer (two- and threefold the standard dose) on maize yield and its biofortification, as well as to verify whether they have a toxic effect on the plants.

The choice of chemical form for micronutrients in foliar fertilizers significantly influences their physiological behavior in plants. The innovative PRO formulation differs from standard TRA formulation primarily in that iron and zinc are complexed with amino acids rather than synthetic EDTA chelates, and trace elements are combined with plant extracts instead of being applied in simple mineral forms.

The introduction of new fertilizer formulations carries the risk of unpredictable plant reactions to various forms of nutrients used in foliar fertilizers [13]. For example, an excess of micronutrients such as copper (Cu) or manganese can cause toxicity and inhibit plant growth [14,15]. In addition, the timing and conditions of foliar fertilizer application are crucial, as improper use can lead to leaf burns, reducing plant health and yield [16]. Different chemical forms of nutrients, such as mineral salts, chelates, or organic compounds, have different availability and effects on plant metabolism. Therefore, assessing the potential negative effects of new foliar fertilizer formulations is essential to ensure their effectiveness and safety.

Building on these premises, we hypothesized that modern foliar formulations enriched with micronutrients such as iron and zinc bound to amino acids, and trace elements like chromium, iodine, lithium, and selenium delivered via plant-based complexes, would demonstrate enhanced effectiveness in increasing yield and biofortification of maize compared to standard foliar fertilizers containing the same elements in mineral or EDTA chelated form, with the efficacy depending on soil type. In particular, we would suggest that the use of foliar fertilizers with amino acid-complexed micronutrients contributes to more effective biofortification of maize plants with iron and zinc, especially in soils with lower availability of these elements (sandy soils). Furthermore, we expect that the effectiveness of foliar fertilization is strongly modified by soil type (sandy or clay soil), as reflected in differences in macro- and micronutrient uptake and final maize biomass yield.

2. Materials and Methods

2.1. Pot Experiment

The experiment was conducted in a controlled vegetation hall at the Department of Plant Nutrition of the Wrocław University of Life Sciences (Poland). Experiments were established on 18 May 2023 in four independent replicates using Wagner-type pots containing 5 kg of soil. Initially, 12 seeds were sown per pot, and after successful germination, plants were thinned to 4 per pot. Chemical analyses were performed on a representative sample comprising 4 plants from each pot.

The experiment was conducted on medium-late silage maize of the Legion variety, which is characterized by high yield potential, high nutritive value, and excellent digestibility.

The maize cultivation period lasted 100 days, with harvesting carried out at the kernel development phase. While temperature and lighting followed ambient environmental conditions, soil moisture was precisely regulated using distilled water to sustain approximately 60% of field capacity throughout the growth cycle.

2.1.1. Loamy Soil

Soil used in the pots was sourced from the top humus layer of an agricultural field located in Przeworno, Poland (50°68′ N, 17°18′ E), dominated by Haplic Luvisols (Episiltic, Endoloamic), characterized by silt loam texture (sand fraction 19%, silt 68%, clay 13%), a pH KCl of 5.2, and a total content of organic carbon (C_tot) of 1.25%. The levels of available nutrients in the soil, determined using the analytical methods described below, were as follows: phosphorus (P) 44 mg kg⁻¹ (low content), potassium (K) 187 mg kg⁻¹ (medium content), magnesium (Mg) 68 mg kg⁻¹ (medium content), manganese (Mn) 171 mg kg⁻¹ (medium content), iron (Fe) 1754 mg kg⁻¹ (medium content), copper (Cu) 5.9 mg kg⁻¹ (medium content), and zinc (Zn) 9.9 mg kg⁻¹ (low content). Before sowing, calcium carbonate was added to the soil (liming) at a rate of 8 g CaCO₃ per pot, calculated based on the hydrolytic acidity of the soil (Hh). In addition, prior to sowing, baseline levels of key macronutrients were ensured by supplementing the soil with the following rates per pot: nitrogen—0.8 g, phosphorus—1.0 g, potassium—1.5 g, and magnesium—0.3 g.

2.1.2. Sandy Soil

The pots were filled with topsoil obtained from the organic layer of an agricultural site in Miłoszyce, Poland (51°05′ N, 17°31′ E), dominated by Albic Luvisol (Epiarenic), characterized by sandy texture (sand fraction 90%, silt 7%, clay 7%), aa soil pH KCl of 5.9, and a C_tot of 0.95%. The levels of available nutrients in the soil were as follows: P—98 mg kg⁻¹ (high content), K—105 mg kg⁻¹ (high content), Mg—110 mg kg⁻¹ (high content), Mn—168 mg kg⁻¹ (medium content), Fe—973 mg kg⁻¹ (medium content), Cu—3.4 mg kg⁻¹ (high content), and Zn—13.6 mg kg⁻¹ (high content). Before sowing, calcium carbonate was added to the soil (liming) at a rate of 1.75 g CaCO₃ calculated based on the hydrolytic acidity of the soil. Prior to sowing, nitrogen was added at a rate of 0.5 g per pot to ensure the optimal baseline levels of this essential macronutrient.

2.2. Foliar Fertilization

The study focused on two distinct foliar compound fertilizers tested on maize grown in two contrasting soil types: loamy and sandy. Both the TRA and PRO formulations supplied essential macronutrients (N, P, K), micronutrients (Mn, Fe, Cu, Zn, B, Mo), and trace elements (Cr, Li, Se, and I); however, the nutrients were delivered in distinct chemical forms depending on the product. PRO represents a novel formulation, where iron and zinc—recognized as among the most critical deficiencies in human and animal diets—are chelated with amino acids, while trace elements are incorporated through complexes with plant-derived extracts. The chemical composition and nutrient rates are shown in

Table 1. Chemical forms of nutrients in foliar fertilizer compositions.

Elements		Nutrient Forms
		PRO	TRA
Macronutrients	N	Mineral	Mineral
	P	Mineral	Mineral
	K	Mineral	Mineral
Micronutrients	B	Mineral	Mineral
	Cu	EDTA chelate	EDTA
	Fe	Amino acid complexed	EDTA
	Mn	Manganese nitrate	Manganese nitrate
	Mo	Ammonium molybdate	Ammonium molybdate
	Zn	Amino acid complexed	EDTA
Trace nutrients	Cr	Complex with plant extracts	Mineral
	I	Complex with plant extracts	Mineral
	Li	Complex with plant extracts	Mineral
	Se	Complex with plant extracts	Mineral

and

Table 2. Elemental composition of foliar fertilizers at a standard application rate (1 L ha⁻¹).

Treatments	Macronutrients			Micronutrients						Trace Nutrients
	N	P	K	B	Cu	Fe	Mn	Mo	Zn	Cr	I	Li	Se
Nutrient dose g L−1
Control	0	0	0	0	0	0	0	0	0	0	0	0	0
TRA dose 1	200	45.8	36.4	0.28	1.68	36.0	12.0	12.0	7.00	0.40	0.98	6.60	0.40
PRO dose 1	200	45.8	36.4	0.28	1.68	36.0	12.0	12.0	7.00	0.40	0.98	6.60	0.40
PRO dose 2	400	91.6	72.8	0.56	3.36	72.0	24.0	24.0	14.0	0.80	1.96	13.2	0.80
PRO dose 3	600	137	109	0.84	5.04	108	36.0	36.0	21.0	1.20	2.94	19.8	1.20

[3].

The experimental design comprised five treatment variants: an untreated control, the reference fertilizer TRA applied at 2 L ha⁻¹ (dose 1), and the innovative PRO formulation applied at three increasing rates—2 L ha⁻¹ (dose 1), 4 L ha⁻¹ (dose 2), and 6 L ha⁻¹ (dose 3). For the PRO treatment, the objective was to assess the impact of increasing application rates. Based on a standard agricultural practice involving 200 L of water per hectare, these corresponded to working solution concentrations of 1.0%, 2.0%, and 3.0%, respectively. In the pot experiment, 10 mL of each solution was applied per pot at the corresponding concentrations for each dose level.

Foliar applications were carried out twice during the growing season, corresponding to key developmental stages: the first at the 3–4 leaf stage (BBCH 13–14) and the second at the 7–8 leaf stage (BBCH 17–18).

2.3. Chemical Analysis

Representative soil and plant samples were collected both before sowing and after harvest to conduct agronomic and chemical analyses. Soil samples were dried in the air at room temperature. The air-dried soils were gently crushed in an agate mortar and sieved through a 1 mm mesh sieve to remove stones and root fragments. The sieved samples were thoroughly mixed to ensure homogeneity. The soil pH was measured potentiometrically in 1 mol dm⁻³ KCl using a CP505 digital pH meter (Elemetron Co., Zabrze, Poland), while total carbon content was assessed with a carbon analyzer (LECO, Benton Harbor, MI, USA).

Plant-available phosphorus and potassium were determined using the Egner–Riehm method, while soluble magnesium content was analyzed following the Schachtschabel procedure. The contents of Mn, Fe, Cu, and Zn were determined by the Rinkis method using an AAS (Varian model SpectrAA 220FS, Varian Medical Systems, Inc., Charlottesville, VA, USA). Detailed descriptions of these analytical methods were provided in our previous study [3].

Plant samples were dried in an oven at 55 °C until a constant weight was achieved. The dried samples were ground in a laboratory mill and homogenized. The total nitrogen (organic N) in plant samples was quantified using the Kjeldahl method. For the determination of other elements, plant material was subjected to dry mineralization, and the resulting ash was dissolved in nitric acid. Phosphorus levels were measured using the vanadic–molybdate colorimetric method, while potassium and calcium were analyzed via flame photometry. Magnesium and selected micronutrients (Cu, Fe, Mn, Zn) were assessed by atomic absorption spectrophotometry. Trace elements including B, Cr, I, Li, Mo, and Se were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES; Thermo Scientific iCAP 7400, Waltham, MA, USA).

Nutrient uptake in the pot trial was calculated according to a standard formula, by multiplying the nutrient concentration in plant dry matter by the total dry biomass per pot: Nutrient uptake (mg pot⁻¹) = [Nutrient concentration (mg kg⁻¹ DM) × Plant dry mass (g pot⁻¹)]/1000.

2.4. Statistical Analysis

The experiment employed a two-factor randomized design with four replications. Data were analyzed using one-way or two-way analysis of variance (ANOVA), depending on the variable assessed. The effect size was estimated using generalized eta squared (η²G), which quantifies the proportion of variance explained by each factor. Effect sizes were interpreted as follows [17]: values <0.01 indicate small, 0.01–0.06 medium, and >0.06 large effects. Prior to conducting ANOVA, assumptions of homogeneity of variance and normality were verified using Levene’s test and the Shapiro–Wilk test, respectively. Differences between means were evaluated using Tukey’s post hoc test at a significance level of p < 0.05.

Regression Tree Analysis was performed using the rpart package (version 4.1.24.) in R [18], which enables recursive partitioning and allows for non-linear classification and prediction of the dependent variable through successive data segmentation. Model performance was evaluated based on the root mean square error (RMSE) and the coefficient of determination (R²). All statistical analyses were conducted using R software, version 4.4.1 (R Core Team, 2024).

3. Results and Discussion

3.1. Plant Growth—Biomass Yield

A significant impact of the applied foliar fertilization treatments on maize biomass yield was found (Figure 1 and Figure 2).

This is confirmed by the η²G values of 0.75 for fresh weight and 0.87 for dry weight. Although the increase in fresh weight after the application of the tested fertilizers did not exceed 10%, it was statistically significant (p < 0.001). The highest yield was in the variant with PRO fertilizer at dose 3 (539 g pot⁻¹), while the lowest value was recorded for control plants, not subjected to spraying (500 g pot⁻¹). A similar trend was observed for maize dry weight. However, the increases were slightly higher—from almost 9% for PRO and TRA fertilizers at dose 1 to almost 14% for PRO at dose 3. When comparing the efficacy of the two fertilizer formulations directly with each other rather than with the control, we observed some differences in yield between the standard PRO and TRA formulations. At dose 1, the PRO formulation gave a fresh biomass slightly higher compared to TRA at the same dose, with the difference not being statistically significant (p > 0.05). The innovative PRO formulation showed a dose–response relationship, with an increase in yield between doses 1 and 3 (from 526 to 539 g pot⁻¹, a 2.5% increase compared to PRO dose 1). For dry matter yield, PRO at dose 1 yielded 126 g pot⁻¹, identical to TRA at dose 1, but PRO at the higher doses showed improvement, with dose 2 yielding 129 g pot⁻¹ and dose 3 yielding 132 g pot⁻¹, a 4.8% increase from PRO dose 1 to PRO dose 3. For both the fresh and dry matter of maize, the increase in yield after applying PRO fertilizer at rate 3 compared to rate 2 was not statistically significant, suggesting the occurrence of a yield plateau. This is important for optimizing fertilization strategies to maximize fertilizer efficiency with minimal environmental impact and optimal cost.

Similar relationships were observed by other researchers who demonstrated the positive effect of foliar fertilizers, including nano-fertilizers and nano-fertilizers enriched with an amino acid complex, on plant growth and yield [19,20,21]. Kandil et al. [21] found that the presence of amino acids in fertilizers increases their effectiveness, leading to a 19% increase in wheat grain yield for mineral fertilizers and a 24% increase for nano-fertilizers. The researchers suggest that amino acids in fertilizers may participate in the biosynthesis of cellular components and stimulate plant growth. Rácz et al. [22] demonstrated the beneficial effect of a foliar fertilizer containing macro- and microelements (N, P, K, S, B, Cu, Fe, Mn, Zn) on physiological parameters and maize yield. They also found that foliar fertilization contributes to reducing the negative impact of adverse environmental conditions. Ramakrishna et al. [23] showed that foliar application of zinc at a concentration of 1% contributes to improving maize growth and increasing dry matter yield. The positive effect of zinc on growth and yield may result from stimulating plants to synthesize growth hormones and increasing the rate of photosynthesis. The yield increases recorded by the researchers ranged from 7 to 25%. The multi-component fertilizers used by us caused an increase in maize dry weight in a similar range, from 7 to 19%. Njoroge et al. [24] also showed a positive response of maize to the application of zinc and copper in its cultivation. In the case of NPK + Cu + Zn fertilization, the obtained yield was 20% higher compared to objects where only NPK was used. This experiment was conducted in two locations, and different results were obtained after applying higher doses of zinc and copper. In the first location, an increase in maize yield of 17% was observed, while in the second location, a decrease of 6% was observed. This shows that there is no single universal and effective cultivation and fertilization scheme that will ensure high yields in every location, every year, and under all conditions. The potential toxic effects of excessive doses of foliar fertilizers on plants should also be considered. In the conducted experiment, PRO fertilizer was applied in three different doses. The highest dose did not show a negative effect on plants. In this group, the highest values of fresh and dry maize weight were obtained, but the differences between the second and third doses were not statistically significant. The phosphorus contained in the analyzed fertilizer may play a key role in directly delivering this often deficient element to leaves. As indicated by Görlach et al. [25], foliar application of phosphorus is a justified and effective method of supplementing its deficiencies in situations of limited availability of this component in the soil. Studies of Leach and Hameleers [26] showed a positive effect of foliar fertilization with phosphorus and zinc on maize quality parameters, and no effect on dry matter yield.

Soil type also had a highly significant impact on maize yield (p < 0.001). Fresh mass was significantly higher in loamy soil (669 g pot⁻¹) compared to sandy soil (380 g pot⁻¹). Likewise, dry mass was greater in loamy soil (156 g pot⁻¹) than in sandy soil (95.5 g pot⁻¹). The soil type effect was dominant, as evidenced by very high η²G values (0.99 for both fresh and dry mass), indicating that soil properties were the primary determinant of yield variation. No significant interactions were detected between treatment and soil type (all values marked as “ns”—not significant). The p-values for interaction effects were 0.76 for fresh mass and 0.49 for dry mass, confirming the absence of a statistically significant combined effect. The η²G values for interaction were low (0.06 for fresh mass and 0.11 for dry mass), indicating that most of the variability in yield was attributed to the main effects (F1 and F2) rather than their interaction.

In summary, soil type was the dominant factor influencing maize yield, with significantly higher values observed in loamy soil compared to sandy soil. Fertilization treatments also significantly affected maize yield, with PRO dose 3 yielding the highest biomass. No significant interaction between treatment and soil type was observed, indicating that the effects of fertilization were consistent across both soil types.

3.2. Concentration and Uptake of Macronutrients

The results in

Table 3. Concentration of macronutrients in maize.

Effect	Nitrogen	Phosphorus	Potassium	Calcium	Magnesium
	Concentration g kg−1 DM
Treatment (F1)
Control	9.45 c	1.80 c	8.43 b	2.34 b	2.74 a
TRA dose 1	9.99 b	1.97 b	9.29 a	2.46 a	2.86 a
PRO dose 1	10.3 a	1.98 b	9.15 a	2.46 a	2.85 a
PRO dose 2	10.4 a	2.07 a	9.23 a	2.48 a	2.90 a
PRO dose 3	10.4 a	2.14 a	9.35 a	2.51 a	2.92 a
Soil (F2)
loamy	8.23 b	1.62 b	12.5 a	2.04 b	1.44 b
sandy	12.0 a	2.37 a	5.67 b	2.86 a	4.27 a
Interaction: (F1 × F2)
Control: loamy	8.02 d	1.39 f	11.5 b	1.93 d	1.33 b
TRA dose 1: loamy	8.25 d	1.61 e	12.9 a	2.04 cd	1.44 b
PRO dose 1: loamy	8.31 d	1.63 de	12.6 a	2.05 c	1.42 b
PRO dose 2: loamy	8.27 d	1.72 de	12.7 a	2.07 c	1.50 b
PRO dose 3: loamy	8.32 d	1.76 d	12.9 a	2.09 c	1.51 b
Control: sandy	10.9 c	2.22 c	5.34 c	2.74 b	4.16 a
TRA dose 1: sandy	11.7 b	2.33 bc	5.71 c	2.88 a	4.28 a
PRO dose 1: sandy	12.4 a	2.34 bc	5.69 c	2.87 a	4.27 a
PRO dose 2: sandy	12.6 a	2.42 ab	5.79 c	2.90 a	4.31 a
PRO dose 3: sandy	12.5 a	2.52 a	5.81 c	2.93 a	4.33 a
p-value F1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p = 0.414
p-value F2	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F1 × F2	p < 0.001	p = 0.181	p = 0.002	p = 0.961	p = 1
Effect size (η2G)—F1	0.80	0.84	0.74	0.68	0.12
Effect size (η2G)—F2	0.99	0.98	1.00	0.99	0.99
Effect size (η2G)—F1 × F2	0.67	0.18	0.42	0.02	0.00

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

indicate that the concentration of basic macronutrients (N, P, K, Mg, Ca), which influence the nutritional value of plants, increased under the influence of maize spraying with the tested fertilizers.

A comparative analysis of the fertilizer formulations revealed several notable differences in their effects.

At equivalent dose 1, the PRO formulation demonstrated superior performance for nitrogen, achieving 10.3 g kg⁻¹ DM compared to 9.99 g kg⁻¹ DM for TRA (3.1% increase, p < 0.05). This advantage of PRO over TRA was particularly pronounced on sandy soil, where nitrogen concentrations were 12.4 g kg⁻¹ and 11.7 g kg⁻¹, respectively (6.0% difference). The efficiency of increasing PRO doses was clearly demonstrated for phosphorus, with statistically significant concentration increases from 1.98 g kg⁻¹ at dose 1 to 2.14 g kg⁻¹ at dose 3 (8.1% increase, p < 0.05). Such a dose–response relationship was consistent across all analyzed macronutrients, with the highest values consistently observed at PRO dose 3.

Soil type significantly influenced the relative performance of both fertilizer formulations, with PRO showing its greatest advantage over TRA on sandy soil, where nutrient delivery efficiency appears to be enhanced by the amino acid complexation. This confirms our hypothesis that the innovative formulation would perform particularly well under conditions where nutrient availability is typically limited.

Magnesium was the only exception, showing no significant difference between foliar fertilization variants (p = 0.414).

Regarding nitrogen, a statistically significant difference was found between TRA and PRO fertilizers, with the latter showing higher effectiveness. Increasing PRO fertilizer doses in most cases did not lead to a significant increase in macronutrient concentration in maize. Therefore, it can be concluded that from the perspective of macronutrient content in maize yield, dose 1 is the most optimal.

The strongest plant response to foliar fertilization was observed for phosphorus. Given the need to reduce soil phosphorus fertilization, foliar fertilization is particularly important. Görlach et al. [25] indicate that foliar fertilization of crops is an effective solution to increase phosphorus utilization efficiency in agriculture. The increases observed in our experiment may be an effect of the applied TRA and PRO fertilizers, which contain this element. However, considering their relatively low content aimed at supplementing and securing potential deficiencies of key macronutrients in the early stages of plant growth and development, the positive reaction may result from synergistic mechanisms. As indicated by Zheng [27], foliar fertilization stimulates the uptake of nutrients available in the soil. Moreover, this positive effect may be a result of beneficial interactions between individual elements, leading to mutual enhancement of their uptake [28]. In contrast to the presented results, our earlier studies on triticale showed that macronutrient concentrations remained relatively constant, regardless of the applied foliar fertilization [3]. Chwil [29], in a three-year field experiment on wheat, showed that foliar feeding had a significantly smaller impact on plant mineral composition compared to soil fertilization. Nevertheless, the application of foliar micronutrient fertilizers led to a statistically significant increase in the content of certain macronutrients (N, Ca, Mg) in straw. Numerous studies confirm the positive effect of foliar fertilization on plant growth and yield quality parameters, including mineral composition [23,30]. Oprica et al. [31] showed that foliar application of a mixture of N, P, K, Fe, Cu, and Mn contributed to an increase in nutrient content in maize and sunflower and significantly increased plant yield.

Similar to biomass accumulation, soil type proved to be a key factor differentiating macronutrient content in maize aboveground parts. The effect of soil was statistically significant for all analyzed elements (p < 0.001). Maize grown on loamy soil was characterized by a significantly lower content of four macronutrients (N, P, Ca, and Mg) compared to plants grown on sandy soil. This effect can be explained by the previously described significant difference in plant yield. In this context, the so-called dilution effect, consisting of a reduction in mineral and other essential component concentrations due to increased biomass production, is particularly evident.

Similar results were obtained by Barczak et al. [32], who studied the effect of soil and fertilization type on macronutrient content in maize. Among other things, they showed a positive effect of foliar fertilization on the content of selected macronutrients and a higher nitrogen concentration in maize grown on sandy soil compared to loamy soil. These authors argue that this may result from the higher sulfur content in loamy soils, which affects nitrogen metabolism in the plant, as well as the presence of copper and molybdenum (also present in TRA and PRO fertilizers) in the foliar fertilizer, which are components of key plant nitrogen metabolism enzymes. Also, Jankowski et al. [33], studying the effect of various doses of mono- and multi-component foliar fertilizers, found that intensive foliar fertilization increased the nitrogen and potassium content in wheat straw.

In the case of macronutrient uptake (

Table 4. Uptake of macronutrients in maize.

Effect	Nitrogen	Phosphorus	Potassium	Calcium	Magnesium
	Uptake mg pot−1
Treatment (F1)
Control	1050 d	196 d	1070 c	258 d	275 b
TRA dose 1	1200 c	237 c	1280 ab	296 c	316 ab
PRO dose 1	1240 bc	240 c	1260 b	298 bc	316 ab
PRO dose 2	1280 ab	257 b	1300 ab	309 ab	333 a
PRO dose 3	1310 a	270 a	1330 a	318 a	344 a
Soil (F2)
loamy	1280 a	254 a	1950 a	318 a	225 b
sandy	1150 b	226 b	542 b	274 b	408 a
Interaction: (F1 × F2)
Control: loamy	1170 bc	203 g	1680 b	282 cd	194 c
TRA dose 1: loamy	1290 a	251 cd	2010 a	319 ab	224 c
PRO dose 1: loamy	1310 a	257 bcd	1990 a	324 a	225 c
PRO dose 2: loamy	1320 a	274 ab	2020 a	330 a	239 c
PRO dose 3: loamy	1340 a	282 a	2070 a	336 a	243 c
Control: sandy	931 d	190 g	457 d	235 e	356 b
TRA dose 1: sandy	1120 c	222 f	544 cd	274 d	407 ab
PRO dose 1: sandy	1170 bc	222 ef	541 cd	272 d	406 ab
PRO dose 2: sandy	1250 ab	240 de	574 c	288 cd	427 a
PRO dose 3: sandy	1280 a	259 bc	595 c	300 bc	444 a
p-value F1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F2	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F1 × F2	p < 0.001	p = 0.034	p = 0.034	p = 0.464	p = 0.731
Effect size (η2G)—F1	0.89	0.94	0.88	0.89	0.48
Effect size (η2G)—F2	0.81	0.82	1.00	0.91	0.93
Effect size (η2G)—F1 × F2	0.52	0.29	0.66	0.11	0.06

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

), the applied foliar fertilization variants and soil type significantly affected this parameter.

Statistically significant differences were found for all tested macronutrients (p-value F1 and p-value F2 < 0.001). It is worth noting that the term “uptake” refers to elements accumulated in the maize aboveground parts, in relation to a single pot. It includes elements absorbed by plant roots from the soil and supplied by foliar fertilization. In all cases, total uptake was higher after foliar fertilization. No significant differences were found between PRO and TRA applied at dose 1. The highest applied PRO dose resulted in the highest uptake, but this value usually did not differ significantly compared to dose 2 (except for phosphorus). Also, for this parameter, soil type was a very differentiating factor. The η²G values for the uptake of individual macronutrients ranged from 0.81 (for nitrogen) to 1.0 (for potassium).

3.3. Concentration and Uptake of Micronutrients

All foliar fertilization treatments led to an increase in the concentration of the analyzed micronutrients (Mn, Fe, Cu, Zn, B, Mo; p < 0.001) (

Table 5. Concentration of micronutrients in maize.

Effect	Manganese	Iron	Copper	Zinc	Boron	Molybdenum
	Concentration mg kg−1 DM
Treatment (F1)
Control	48.7 e	45.2 e	3.24 c	24.5 e	6.11 d	0.405 d
TRA dose 1	53.0 d	53.8 d	3.67 b	28.7 d	7.50 c	0.491 c
PRO dose 1	53.7 c	59.2 c	3.72 b	32.0 c	7.75 b	0.498 c
PRO dose 2	56.2 b	64.2 b	4.26 a	35.0 b	7.96 ab	0.564 b
PRO dose 3	57.2 a	69.4 a	4.50 a	37.4 a	8.18 a	0.589 a
Soil (F2)
loamy	37.6 b	65.4 a	3.78 b	16.2 b	9.25 a	0.382 b
sandy	69.9 a	51.4 b	3.98 a	46.8 a	5.75 b	0.637 a
Interaction: (F1 × F2)
Control: loamy	33.9 i	53.1 ef	3.28 ef	13.4 g	7.18 b	0.283 g
TRA dose 1: loamy	36.1 h	60.3 cd	3.55 def	14.7 g	9.66 a	0.356 f
PRO dose 1: loamy	37.0 g	63.9 c	3.68 cde	15.2 g	9.64 a	0.348 f
PRO dose 2: loamy	40.0 f	71.3 b	4.10 bc	17.8 f	9.85 a	0.452 e
PRO dose 3: loamy	40.9 e	78.2 a	4.30 ab	19.9 f	9.90 a	0.470 e
Control: sandy	63.4 d	37.3 g	3.19 f	35.5 e	5.04 e	0.528 d
TRA dose 1: sandy	70.0 c	47.2 f	3.79 cd	42.7 d	5.33 e	0.626 c
PRO dose 1: sandy	70.4 c	54.6 de	3.77 cd	48.8 c	5.85 d	0.650 bc
PRO dose 2: sandy	72.4 b	57 de	4.43 ab	52.2 b	6.06 cd	0.675 ab
PRO dose 3: sandy	73.5 a	60.7 cd	4.71 a	54.9 a	6.45 c	0.708 a
p-value F1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F2	p < 0.001	p < 0.001	p = 0.003	p < 0.001	p < 0.001	p < 0.001
p-value F1 × F2	p < 0.001	p = 0.057	p = 0.087	p < 0.001	p < 0.001	p < 0.001
Effect size (η2G)—F1	1.00	0.93	0.88	0.97	0.96	0.95
Effect size (η2G)—F2	1.00	0.90	0.26	1.00	0.99	0.99
Effect size (η2G)—F1 × F2	0.92	0.26	0.23	0.90	0.87	0.48

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

).

When directly comparing the effectiveness of fertilizer formulations on micronutrient acquisition, our data revealed distinct advantages of the PRO formulation over TRA, particularly for iron and zinc. At equivalent dose 1, PRO resulted in a significantly higher iron concentration (59.2 mg kg⁻¹ DM) compared to TRA (53.8 mg kg⁻¹ DM), representing a 10.0% improvement (p < 0.001). Similarly, for zinc, PRO at dose 1 achieved 32.0 mg kg⁻¹ DM versus 28.7 mg kg⁻¹ DM for TRA, an 11.5% increase (p < 0.001). This superior performance of PRO over TRA for these two nutritionally critical elements confirms the enhanced bioavailability provided by amino acid complexation compared to traditional EDTA chelation. The advantage of PRO was particularly evident on sandy soil, where iron concentration with PRO dose 1 was 15.7% higher than with TRA (54.6 vs. 47.2 mg kg⁻¹ DM), and the zinc concentration was 14.3% higher (48.8 vs 42.7 mg kg⁻¹ DM). These results directly support our research hypothesis regarding the efficacy of amino acid-complexed micronutrients in soil conditions with potentially limited nutrient availability.

Iron and zinc are particularly deficient elements in human and animal nutrition, emphasizing the significance of the observed biofortification effect as an important step toward improving the nutritional value of crops. Our previous study [3] on triticale demonstrated that this plant exhibited a stronger response to foliar fertilization following the application of the same multi-component fertilizer. Depending on the type of micronutrient and the applied spray dose, its concentration in straw was two to even five times higher compared to untreated plants. The weakest effect was observed for boron, whose levels were similar to those recorded in this study. It is also noteworthy that the innovative PRO formulation, in which Fe and Zn were complexed with amino acids, proved to be more effective than the TRA treatment. Similar conclusions were drawn by Ducatti and Tironi [34], who highlighted that the form of micronutrient metal complexation affects the efficiency of foliar fertilization. These authors suggest that natural compounds, such as amino acids, may be more effective than commonly used synthetic chelates (e.g., EDTA, DTPA, EDDHA) due to their lower molecular weight, which facilitates faster and easier penetration into plant tissues. Petković et al. [35] demonstrated that foliar micronutrient fertilizers (Zn, Se) are more effective when combined with urea. The increased effectiveness of such combination results from the physico-chemical properties of urea, which, when applied foliarly with other agrochemicals, serves as an efficient adjuvant [36]. Our experiments did not reveal significant differences in the effectiveness of TRA and PRO regarding their impact on the copper and molybdenum content in the above-ground parts of maize. As expected, increasing PRO doses resulted in higher concentrations of most micronutrients. An exception is copper with the application of the second and third fertilizer doses (no differences), as well as boron.

Soil type also significantly influences micronutrient concentrations. Higher values of Mn, Cu, Zn, and Mo are observed in maize grown on sandy soil, while loamy soil favours the accumulation of Fe, Cu, and B. However, it is worth mentioning that the loamy soil contained more iron and copper than the sandy soil. The effect of soil is statistically significant for all elements (p < 0.001) except Cu (p = 0.003). A recent study by Xue et al. [10] showed that foliar application of a solution containing Zn, Fe, Se, and N effectively increased the concentration of these elements in three maize varieties grown at different locations.

In most cases (Mn, Zn, B, Mo; p < 0.001), a significant interaction was observed between foliar fertilization and soil type, indicating that the effectiveness of treatment depends on soil conditions. Only for iron and copper was the fertilization effect similar across both soil types, as evidenced by the lack of significant interaction (p = 0.057 and p = 0.087). The highest manganese and zinc concentrations were recorded in maize grown on sandy soil and treated with the highest dose of the PRO formulation.

Analysis of differences in the uptake of the micronutrients (

Table 6. Uptake of micronutrients in maize.

Effect	Manganese	Iron	Copper	Zinc	Boron	Molybdenum
	Uptake mg pot−1
Treatment (F1)
Control	5.19 d	5.47 e	0.376 c	2.50 e	0.740 d	0.0432 d
TRA dose 1	6.15 c	6.95 d	0.457 b	3.18 d	1.01 c	0.0575 c
PRO dose 1	6.27 c	7.63 c	0.469 b	3.52 c	1.04 bc	0.0583 c
PRO dose 2	6.78 b	8.52 b	0.546 a	4.01 b	1.09 ab	0.0695 b
PRO dose 3	7.05 a	9.39 a	0.587 a	4.41 a	1.13 a	0.0740 a
Soil (F2)
loamy	5.88 b	10.2 a	0.591 a	2.54 b	1.45 a	0.0598 a
sandy	6.70 a	4.95 b	0.383 b	4.51 a	0.551 b	0.0612 a
Interaction: (F1 × F2)
Control: loamy	4.94 g	7.74 d	0.479 c	1.96 h	1.050 c	0.0412 e
TRA dose 1: loamy	5.64 ef	9.41 c	0.554 b	2.30 g	1.510 b0	0.0555 d
PRO dose 1: loamy	5.84 e	10.1 c	0.581 b	2.40 g	1.520 ab	0.0548 d
PRO dose 2: loamy	6.37 d	11.4 b	0.653 a	2.85 f	1.570 ab	0.0721 ab
PRO dose 3: loamy	6.58 cd	12.6 a	0.691 a	3.20 e	1.590 a	0.0755 a
Control: sandy	5.43 f	3.20 h	0.273 e	3.04 ef	0.432 g	0.0452 e
TRA dose 1: sandy	6.66 c	4.49 g	0.361 d	4.06 d	0.507 fg	0.0596 cd
PRO dose 1: sandy	6.69 c	5.19 fg	0.358 d	4.64 c	0.556 ef	0.0617 c
PRO dose 2: sandy	7.18 b	5.66 ef	0.439 c	5.17 b	0.601 de	0.0670 b
PRO dose 3: sandy	7.53 a	6.21 e	0.483 c	5.63 a	0.661 d	0.0725 a
p-value F1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F2	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p = 0.052
p-value F1 × F2	p = 0.001	p < 0.001	p = 0.884	p < 0.001	p < 0.001	p < 0.001
Effect size (η2G)—F1	0.98	0.95	0.90	0.97	0.96	0.97
Effect size (η2G)—F2	0.94	0.99	0.95	0.99	1.00	0.12
Effect size (η2G)—F1 × F2	0.45	0.54	0.04	0.84	0.85	0.61

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

) showed that the experimental factors, i.e., fertilization (F1), soil type (F2), and their interaction (F1 × F2), had a statistically significant effect (p < 0.001) on most of the microelements analyzed. The exceptions were the uptake of molybdenum (p = 0.052) and copper for the interaction of the factors tested (p = 0.884).

The application of PRO fertilizer at dose 1 was particularly effective in increasing iron and zinc uptake, confirming the effectiveness of the innovative method of complexing these micronutrients with amino acids. For the other microelements (Mn, Cu, B, and Mo), both formulations (PRO and TRA at dose 1) demonstrated similar efficacy.

All factors tested (treatments—F1, soil—F2, interaction (F1 × F2) are statistically significant (p < 0.001) for all micronutrients except Mo (p = 0.052) and for copper in the case of interaction (p = 0.884). PRO at dose 1 was more effective than TRA in enhancing iron and zinc uptake, confirming that the use of this innovative formulation is a well-founded choice. For the other micronutrients (Mn, Cu, B, and Mo), both formulations (PRO and TRA at dose 1) performed similarly. Increasing PRO doses resulted in increased uptake of individual micronutrients. Doses 2 and 3 of PRO fertilizer did not result in significant differences in copper and boron uptake.

Overall, the application of PRO fertilizer, especially at the highest dose, caused statistically significant increases in the concentration of micronutrients in maize, but they were lower than expected. It is possible that on soils with low micronutrient content, the effect would be significantly more pronounced. The results indicate that sandy soil favours Mn and Zn uptake by maize, leading to an increased concentration of these micronutrients in the aerial parts of the plant.

3.4. Concentration of Trace Elements in Maize

Foliar fertilization significantly increased the content of chromium and lithium in maize compared to the control, with a greater effect observed after applying PRO at higher doses (

Table 7. Concentration and uptake of trace elements in maize.

Effect	Chromium	Lithium	Chromium	Lithium
	Content mg kg−1 DM		Uptake µg pot−1
Treatment (F1)
Control	0.91 c	0.464 d	0.104 c	0.053 d
TRA dose 1	1.24 b	1.02 c	0.155 b	0.117 c
PRO dose 1	1.28 b	1.19 c	0.160 b	0.133 c
PRO dose 2	1.36 a	1.43 b	0.174 a	0.168 b
PRO dose 3	1.41 a	1.78 a	0.182 a	0.213 a
Soil (F2)
loamy	1.17 b	0.725 b	0.183 a	0.114 b
sandy	1.32 a	1.630 a	0.127 b	0.160 a
Interaction: (F1 × F2)
Control: loamy	0.85 e	0.442 f	0.125 d	0.0645 ef
TRA dose 1: loamy	1.23 d	0.634 ef	0.191 a	0.0989 de
PRO dose 1: loamy	1.20 d	0.631 ef	0.190 a	0.0997 de
PRO dose 2: loamy	1.27 cd	0.878 de	0.202 a	0.1400 c
PRO dose 3: loamy	1.29 cd	1.040 d	0.207 a	0.1670 bc
Control: sandy	0.97 e	0.486 f	0.083 e	0.0417 f
TRA dose 1: sandy	1.25 cd	1.410 c	0.119 d	0.1340 cd
PRO dose 1: sandy	1.36 bc	1.750 b	0.130 cd	0.1660 bc
PRO dose 2: sandy	1.46 ab	1.980 b	0.145 bc	0.1970 b
PRO dose 3: sandy	1.54 a	2.530 a	0.157 b	0.2590 a
p-value F1	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F2	p < 0.001	p < 0.001	p < 0.001	p < 0.001
p-value F1 × F2	p = 0.004	p < 0.001	p = 0.005	p < 0.001
Effect size (η2G)—F1	0.94	0.94	0.95	0.93
Effect size (η2G)—F2	0.72	0.94	0.95	0.72
Effect size (η2G)—F1 × F2	0.40	0.82	0.38	0.64

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

).

No statistically significant differences were observed between the TRA and PRO treatments, or between the second and third fertilizer doses, with regard to chromium levels. However, increasing fertilizer doses led to a significant increase in lithium concentrations in the aboveground parts of the maize, both compared to the control and between successive dose levels.

The highest lithium content was recorded after applying PRO fertilizer at dose 3 (an increase of 284% compared to the control and 24% in relation to dose 2). A similar trend occurred in lithium uptake. Differences noted for chromium content and uptake were also significant, but not as large as in the case of lithium.

Analyzing the effect of soil on the element level in maize showed that plants grown on sandy soil had higher concentrations of chromium and lithium than on loamy soil. The soil effect was statistically significant (p < 0.001), which means that soil type had a strong influence on the analyzed parameters. On sandy soil, the effect was stronger with higher PRO fertilizer doses. Significantly greater differences were observed in the case of lithium. Lithium uptake on sandy soil was 40% higher than on loamy soil. In the case of lithium, the interaction of soil and spraying was particularly evident (p < 0.001; η²G—F1 × F2 = 0.82), and fertilization yielded better results. The highest concentration and uptake of elements were found for PRO 3 on sandy soil. For lithium, the concentration was 2.53 mg kg⁻¹ DM, and the uptake was 0.259 µg pot⁻¹.

In summary, we can conclude that foliar application of chromium and lithium is an efficient tool in plant biofortification and thus an effective alternative to soil fertilization, which requires significantly higher doses and therefore carries the risk of soil contamination. The accumulation of heavy metals in soils, including chromium, is one of the most serious abiotic stresses. When plants are grown under high concentrations of chromium and lithium, these elements can cause oxidative stress and other dysfunctions [37,38]. Usually, the uptake and translocation of elements increase with increasing availability. Most studies focus on the toxic effects of chromium on plants and finding effective ways to mitigate its adverse effects [38,39]. The main goal of using lithium and chromium in the fertilizer we studied was to biofortify maize with these metals. On the other hand, thanks to the phenomenon of hormesis, their presence was intended to improve maize health and productivity [37,40]. The positive effect of foliar lithium application on plant biofortification and their functioning, including increasing photosynthesis intensity and water use efficiency, has been demonstrated in studies by various authors [37,41,42]. ACM dos Santos et al. [41], comparing the effectiveness of two forms of lithium, Li₂SO₄ and LiO, found that LiOH is more effective in the context of biofortification. They also showed that, regardless of the form, foliar application of lithium significantly affects the content of macronutrients (N, P, K) in plants. These studies also indicate the need to adjust the Li dose to its chemical form—the maximum safe concentrations were 46.8 mg kg⁻¹ for LiOH and 91.5 mg kg⁻¹ for Li₂SO₄. Exceeding these values, especially in an inappropriate form, can lead to the toxic effects of lithium and yield reduction. Buendía-Valverde et al. [37] indicated that excess lithium can cause oxidative stress, protein degradation, damage to the photosynthetic apparatus, photosynthesis and gas exchange disorders, and phytotoxicity symptoms such as necrosis, leaf curling, and a reduction in root and shoot mass. However, in our experiment, no harmful effect of lithium or chromium on plants was observed. Selenium, another important trace element in human and animal nutrition, is not essential in plant nutrition but inappropriate doses can have a stimulating effect [10,43]. Our results show a significant increase in the Se content in maize (

Table 8. Selenium concentration and uptake in maize on loamy soil.

Effect	Selenium
	Content mg kg−1 DM	Uptake µg pot−1
Loamy soil
Control	0.0455 c	0.0066 c
TRA dose 1	0.162 b	0.0253 b
PRO dose 1	0.163 b	0.0257 b
PRO dose 2	0.175 ab	0.0279 a
PRO dose 3	0.178 a	0.0286 a
p-value	p < 0.001	p < 0.001
Effect size (η2G)	0.99	0.99

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

).

Foliar fertilization contributed to an almost threefold increase in content. However, no significant differences were noted between TRA and PRO fertilizer at dose 1. Increasing the PRO dose contributed to an increase in concentration in relation to dose 1, 7% for dose 2, and 9% for dose 3. The selenium content recorded in maize dry matter is within safe, non-toxic levels from 0.1 to 1 mg kg⁻¹ [44]. The effect of increasing selenium doses on its accumulation in aboveground parts and roots of various plants was studied by Borowska and Koper [45], who showed, in an experiment, that increasing selenium dose contributes to an increase in its accumulation only up to a certain point (different for individual species), after which, despite increasing doses, its content decreases. It is also worth noting that selenium was detected only in maize grown on loamy soil. On sandy soil, the Se concentration in maize dry matter was below the detection level and was not determined. This may be an effect of its low content in sandy soil, as the decrease in selenium content in soil is positively correlated with a decrease in silt and clay content [46].

In soils, selenium exhibits a wide range of oxidation states from +6 in selenates to −2 in selenides. Its availability decreases with decreasing soil pH, and its transformation from available to unavailable forms occurs after addition to acidic or neutral soils [45]. Therefore, in the case of unregulated, i.e., low or neutral soil pH, the best way to provide it to plants is foliar application. The effect of various delivery methods on growth, yield, and macro- and microelement content was studied by Boldrin et al. [47]. They showed the positive effect of selenate and selenite application on yield and selenium content, as well as on the macro- and microelement content in rice. Also, Xue et al. [10] showed a positive effect on the increase in iron, zinc, and manganese content, with a simultaneous decrease in heavy metal content, such as chromium and cadmium, as a result of foliar selenium application. The effect of various doses and application methods (soil fertilization, foliar fertilization, and seed treatment) and various selenium applying dates in spring wheat cultivation was studied by Radawiec et al. [48]. They showed that a single seed treatment before sowing increases the selenium content in the yield; however, it is the least effective method. In contrast, the combined application of soil and foliar treatments proved to be the most efficient. In no case was a significant effect of Se on grain yield observed.

3.5. Nutrient Interactions and Their Effects on Maize Yield Depending on Soil Type

The tree in Figure 3 shows the critical threshold values for the concentration of micronutrients affecting maize dry matter yields.

Molybdenum (Mo) emerged as the main separating variable (threshold: 0.576), followed by iron (Fe) and copper (Cu) with thresholds of 57.2 and 4.62, respectively. Numbers at the nodes indicate average dry matter yield (g), sample size (n), and percentage of all observations. While the tree structure prioritises Mo > Fe > Cu as threshold-dependent determinants, global variable importance analysis identified a hierarchy of B > Mn > Fe > Cu > Zn > Mo. This discrepancy between tree structure and the importance of variables indicates that, while molybdenum is the first separating factor in tree structure, global yield variability is most strongly related to boron and manganese content. The model parameters (RMSE = 2.48, R² = 0.89) indicate high predictive accuracy, with a potential yield increase of 21% due to micronutrient optimization.

The tree in Figure 4 shows the critical threshold values for the concentration of micronutrients affecting maize dry matter yields in loamy soils.

Molybdenum (Mo) emerged as the main separating variable (threshold: 0.34), followed by copper (Cu) and iron (Fe) with thresholds of 3.54 and 77.6, respectively. The numbers at the nodes indicate the average dry matter yield (g), sample size (n), and percentage of all observations. The tree structure indicates a hierarchy of Mo > Cu > Fe as threshold-dependent determinants, which is consistent with the global variable importance analysis that identified Mo (326.46) as the most important variable, followed by Fe (273.32), Mn (268.47), Zn (250.91), B (246.06), and Cu (205.62). This consistency between tree structure and variable significance suggests that molybdenum plays a dominant role in determining yield variability in loamy soils. The model shows high predictive accuracy (RMSE = 5.45, R² = 0.90), with a potential yield increase of about 9% due to micronutrient optimization (comparing node 2: 148 g with node 15: 161 g).

3.6. Soil Properties After Experiment

In the loamy soil, PRO doses 2 and 3 significantly reduced the available concentrations of P, K, Mg, Mn, and Fe, with a decrease in Cu and Zn levels, while maintaining a stable soil pH (

Table 9. Post-experimental physicochemical characteristics of the soil.

Effect	pH KCl	Phosphorus	Potassium	Magnesium	Manganese	Iron	Copper	Zinc
		Soluble Forms mg kg−1 Soil
Loamy soil
Control	6.06 a	126 a	97.5 a	80.6 a	155 a	1560 a	5.57 a	9.49 a
TRA dose 1	6.07 a	121 b	94.8 ab	80.2 a	158 a	1550 a	5.36 a	9.10 abc
PRO dose 1	6.09 a	120 b	94 ab	78.9 a	157 a	1550 a	5.29 a	9.17 ab
PRO dose 2	6.04 a	112 c	88.5 bc	69.3 b	137 b	1520 a	4.97 b	8.28 c
PRO dose 3	6.05 a	112 c	87.0 c	68.9 b	136 b	1520 a	4.89 b	8.32 bc
p-value	p = 0.985	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.021	p < 0.001	p < 0.001
Effect size (η2G)	0.023	0.949	0.684	0.817	0.89	0.517	0.835	0.674
Sandy soil
Control	5.46 a	95.4 a	63.2 a	43.9 a	150 a	759 a	3.20 a	13.3 a
TRA dose 1	5.39 a	84.3 b	53.0 bc	40.7 ab	147 ab	758 a	3.17 a	12.6 a
PRO dose 1	5.39 a	83.4 b	55.0 b	43.3 a	148 a	745 a	3.18 a	12.7 a
PRO dose 2	5.31 a	75.5 c	48.2 cd	36.9 b	140 b	724 a	3.07 a	10.1 b
PRO dose 3	5.32 a	75.9 c	43.9 d	36.4 b	140 b	728 a	3.08 a	10.0 b
p-value	p = 0.758	p < 0.001	p < 0.001	p < 0.001	p < 0.002	p = 0.017	p = 0.125	p < 0.001
Effect size (η2G)	0.111	0.927	0.878	0.697	0.645	0.53	0.364	0.943

Averages followed by the same letter do not differ significantly (p > 0.05, Tukey’s HSD test).

).

The sandy soil showed similar trends, with PRO 2 and 3 doses causing significant reductions in nutrients, especially P, K, Mg, and Mn, and a marked reduction in Zn concentration. More pronounced changes in nutrient content in loamy soil compared to sandy soil suggest that fertilization strategies differently influence their distribution, transformations, and plant uptake depending on soil type.

4. Conclusions

The results of the experiment show the varying effectiveness of the foliar fertilizer formulations tested. At equivalent doses (dose 1), both PRO and TRA formulations achieved similar yield results. However, the innovative PRO formulation showed better biofortification abilities, especially for iron and zinc, on sandy soil. This confirms our hypothesis that amino acid-complexed micronutrients provide better bioavailability than traditional EDTA chelates, especially in soils with less fertile conditions. Increasing PRO doses (doses 2 and 3) further enhance both yield and nutrient concentrations, with dose 3 resulting in the highest values for most parameters. However, in many analyses, the differences between dose 3 and dose 2 were not statistically significant, suggesting that the optimal solution for PRO fertilizer is dose 2.

Soil type proved to be a dominant factor influencing the comparative effectiveness of both fertilizers, with PRO showing its greatest advantage over TRA on sandy soil. These findings support our research objective to evaluate the impact of both formulation type and increased application rates on maize yield and biofortification.