Effects of Different Rates of Nitrogen Fertilisation and Biological Preparations to Increase Nitrogen Use Efficiency on Yield Structure Elements in Maize

Abstract

The soil used for the field experiment was PLb-g4 Endohipogleyic Eutric Planasol. The research aimed to investigate the effects of different nitrogen fertilisation rates and biological preparations on yield structure elements and partial factor productivity of nitrogen in maize (Zea mays L.) grown for grain production. The factors studied were Factor A—nitrogen (N) fertiliser rates: (1) 100 kg ha⁻¹, (2) 140 kg ha⁻¹, and (3) 180 kg ha⁻¹, and Factor B—use of biofertilisers: (1) no biological preparations (BP) used, (2) biological preparation (AB)—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹), (3) biological preparations (AB + C)—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹) and cytokinin, and (4) biological preparations (AB + H)—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹) and humic acids. The research showed that the yield of maize grain was significantly increased not only by increasing the rates of nitrogen fertilisation but also by using biological preparations. The highest maize grain yield (11.5 t ha⁻¹) was obtained in 2020 using N₁₈₀ fertilisation, in combination with biological preparations AB + H. In all cases, the biological preparations and their combinations significantly increased the maize grain yield compared to the control field (no use of BP). The biological preparations in combination with N significantly increased the weight of 1000 grains and thus the grain yield per plant. The highest maize grain yield per plant (154.6 g) was obtained in 2020 using N₁₈₀ fertilisation, in combination with biological preparations AB + H. In most cases, positive, strong, very strong, and statistically significant correlations were observed between the different rates of nitrogen fertilisation and the indicators studied: r = 0.76–0.94 (p < 0.01, p < 0.05). No statistically significant correlation was found between nitrogen fertilisation rates and the number of grains per cob (p > 0.05). The highest partial factor productivity of nitrogen fertiliser (92.0 kg of maize kg⁻¹ of N) was obtained in 2020 using N₁₀₀ fertilisation, in combination with AB + H. Increasing the nitrogen fertiliser rates and not using biological preparations resulted in a decrease in the partial factor productivity of nitrogen fertiliser.

1. Introduction

Maize (Zea mays L.) is the second most widely grown crop after wheat, significantly contributing to global food security [1]. However, abiotic stresses, including nutrient imbalance, limit maize productivity, accounting for 50% of yield losses [2,3,4]. Climate change affects soil fertility by altering nutrient and moisture content, temperature, organic matter, and microbiota activity, necessitating sustainable agrotechnology to enhance soil productivity while protecting the environment and human health [5]. The use of chemical fertilisers contaminates soil and water, highlighting the importance of sustainable practices, like biostimulants [6,7].

Biostimulants, including algal extracts, humic substances, amino acids, and plant-growth-promoting bacteria, improve nutrient uptake, plant metabolism, and stress responses [8]. Szczepanek and Wilczewski [9] observed that humic acids enhance nutrient availability and mitigate abiotic stress, but their effectiveness depends on soil conditions and formulations. However, biostimulant efficacy varies under field conditions due to interactions with soil, plant physiology, microflora, and meteorological factors [10,11,12]. Canellas et al. [13] reported a 65% maize yield increase with combined humic substances and endophytic diazotrophic bacteria, compared to 20% with bacteria alone. These variations necessitate comprehensive evaluations across sites and conditions [14].

Nitrogen, a key nutrient in photosynthesis and biological processes, is critical for maize grain yields. Yet, excessive nitrogen application poses risks to groundwater and incurs significant environmental costs in Europe, estimated at USD 78–357 billion annually [15,16,17,18].

Emphasis is placed on growth regulators that enhance plant productivity and biological quality, particularly in challenging climatic conditions. Plants rely on the interaction of five primary growth hormones: auxins, cytokinins, gibberellins, ethylene, and abscisic acid. Auxins are the most critical, as they play a role in virtually all physiological activities, including root and bud development, cell division, and tropisms [19]. Cytokinins influence cell division, which supports overall plant growth—they also promote lateral bud development and slow down the aging of tissues and organs. Gibberellins help break plant dormancy, stimulate cell division, and encourage growth [20,21].

Scientists are investigating how cytokinins influence the anatomical structure of leaves. Microscopic analysis of plant leaves revealed that cytokinins cause mesophyll cell enlargement, significant lignification of tissues supporting the leaf, and an increased number of vascular bundles in the leaf [22]. Additionally, cytokinins promote the opening of stomata in mature and senescing leaves, facilitating CO₂ diffusion. These phytohormones also encourage chloroplast division and the development of chloroplast ultrastructure [23]. The beneficial effects of cytokinins on photosynthesis are linked to their ability to delay leaf senescence. This effect has been observed across various plant species treated with externally applied cytokinins and in genetically modified plants with heightened levels of endogenous cytokinins [24]. By slowing or preventing chlorophyll degradation and green pigment breakdown, cytokinins help maintain leaf vitality. Furthermore, cytokinins play a critical role in supporting essential plant functions and are regarded as an indispensable component [25,26].

Numerous attempts have been made to find ways to inhibit urease activity to slow down the hydrolysis process. One of the best ways to reduce nitrogen loss and slow down the rate of hydrolysis is to use urease inhibitors. Urease inhibitors have been shown to increase the efficiency of urea [27,28].

The ability of nitrogen-fixing microorganisms to fix nitrogen in a non-symbiotic manner has been further investigated. Scientific reports have shown the occurrence of these organisms in the rhizosphere of maize (Zea mays L.) and other plant roots [29].

Scientists argue that free-living nitrogen-fixing bacteria are important contributors to nitrogen availability and can replace or supplement chemical fertilisers and produce secondary metabolites, especially phytohormones and exopolysaccharides, which are not present in chemical fertilisers [30]. Nitrogen-fixing bacteria allow producers to reduce chemical fertiliser rates and indirectly reduce the spread of some pathogens [30]. Jadhav and Sayyed [31] suggest that this corresponds to the ability of nitrogen-fixing bacteria to degrade the cell wall of a fungal pathogen, which may be related to the synthesis of hydrolytic enzymes [32].

Nitrogen-fixing bacteria products are presented as biofertilisers and can increase the productivity of crops. The use of nitrogen-fixing bacteria represents the application of biotechnology to support the development of agricultural practices that reduce pollution and improve soil quality. Nitrogen-fixing bacterial products can play an important role in replenishing nutrient reserves available to plants [33].

Paenibacillus polymyxa is widely recognised as a plant-growth-promoting bacterium that directly benefits plants by improving atmospheric nitrogen fixation, phosphorus solubility, and plant iron uptake in the soil, as well as phytohormone production. This could reduce dependence on chemical fertilisers, which are currently the source of environmental conflicts and appear to be harmful to humans. Paenibacillus polymyxa is mainly used as a functional microbial species for the production of biofertilisers. Over the last couple of years, the use of Paenibacillus polymyxa has gained increasing momentum. The most recent discovery in the microbial industry related to this bacterium is the production of bioactive compounds, such as exopolysaccharides [34,35,36].

The integrated use of biological preparations in agricultural technology can contribute to more sustainable agriculture, reduced environmental pollution, and high agricultural productivity. This research aims to investigate the effects of different rates of nitrogen fertilisation and biological preparations on yield structure elements and nitrogen partial factor productivity in maize (Zea mays L.) grown for grain production.

We hypothesise that the use of nitrogen-fixing bacteria and other biological agents can significantly enhance nitrogen use efficiency in maize cultivation. These bacteria, such as Paenibacillus polymyxa, naturally convert atmospheric nitrogen into plant-available forms, thereby reducing the need for synthetic nitrogen fertilisers. Biological agents can also improve nutrient uptake and soil health. By integrating these biological solutions, farmers can reduce nitrogen fertiliser application rates while maintaining optimal maize yields. This approach is expected not only to lower costs but also to mitigate environmental impacts, such as soil degradation and water contamination caused by fertiliser leaching.

2. Materials and Methods

2.1. Research Location and Arrangement of the Experiment

The research object was a maize (Zea mays L.) crop fertilised using different rates of nitrogen fertiliser and treated with biological preparations.

The field experiment was carried out at Vytautas Magnus University Experimental Station (54°52′ N, 23°49′ E) from 2020 to 2022. The soil of the field experiment was Endohipogleyic Eutric Planasol (World Reference Base (WRB)), a moderate clay loam on sandy light loam covered with moraine clay [37].

The plow layer was 23–27 cm thick. The soil was neutral (pH~6.7), with a medium humus content of ~2.86%, medium potassium content of ~154 mg kg⁻¹, and high phosphorus content of ~266 mg kg⁻¹.

The field experiment was conducted with 36 plots, each with an initial (gross) area of 66 m² (width 5.5 m and length 12 m). The area of the reference (net) field was 45 m² (width 4.5 m and length 10 m). The field experiment was carried out in 3 replicates, with the plots arranged in a randomised manner in replicate blocks. Two factors were studied: Factor A—nitrogen (N) fertiliser rates: (1) 100 kg ha⁻¹, (2) 140 kg ha⁻¹, and (3) 180 kg ha⁻¹, and Factor B—use of biological preparations: (1) no biological preparations (BP) used, (2) biopreparation AB, (3) biopreparations AB + C, and (4) biopreparations AB + H.

Maize was sown on 27 April 2020, 11 May 2021, and 8 May 2022. The early maturing hybrid maize variety P7326 (breeding company DuPont Pioneer, Johnston, IA, USA) was used. The seed rate was 80,000 seeds ha⁻¹ and row spacing was 75 cm. PK fertilisers were applied in all fields before sowing of maize in all years of the study: double superphosphate, Ca(H₂PO₄)₂H₂O, at a rate of 60 kg ha⁻¹ P₂O₅, and potassium chloride, KCl, at a rate of 60 kg ha⁻¹ K₂O. In each year of the study, the maize crop was sprayed at stage BBCH 16 with the herbicide containing the active ingredients mesotrione 75 g L⁻¹ + nicosulfuron 30 g L⁻¹—at 1.0 L ha⁻¹. Maize was grown in monoculture.

Characteristics of the use of Factors A and B studied in the field experiment are listed below.

Factor A—different rates of nitrogen (N) fertiliser:

• (N₁₀₀) 238 L ha⁻¹ KAS-32 (solution of urea CO(NH₂)₂ and ammonium nitrate NH₄NO₃) applied to the soil surface immediately after sowing,
• (N₁₄₀) 333.2 L ha⁻¹ KAS-32 (solution of urea CO(NH₂)₂ and ammonium nitrate NH₄NO₃) applied to the soil surface immediately after sowing,
• (N₁₈₀) 428.4 L ha⁻¹ KAS-32 (solution of urea CO(NH₂)₂ and ammonium nitrate NH₄NO₃) applied to the soil surface immediately after sowing.

Factor B—use of biological preparations (BP):

• (Without BP) biological preparations were not used,
• (AB) biological preparation—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32,
• (AB + C) biological preparations—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32 and cytokinin (N—0.33%, P₂O₅—0.15%, K₂O—0.2%, moisture—97.6%, organic matter—1.7%, pH 6–7, phytohormones, cytokinin series 11 mg L⁻¹ (EMA), auxins 0.05 mg L⁻¹ (EMA), vitamins, trace elements, proteins, and carbohydrate; 0.7 L ha⁻¹), sprayed at the 6-leaf stage,
• (AB + H) biological preparations—nitrogen bacteria Paenibacillus polymyxa (1.0 L ha⁻¹) and humic acids (15% suspension of humic and fulvic acids, pH 4–5; 1.0 kg ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32.

When the maize had reached physiological maturity, i.e., when the black dot appeared on the kernel at the point of attachment to the cob, plant samples were taken from each field, randomly selecting 10 plants and sampling according to the methodology used in the field experiments to determine yield and yield structure elements of maize grain. The cobs were dried in a drying chamber at a temperature of 65 ± 5 °C to constant dry matter, de-husked, and threshed; then, the grains were weighed, and the following parameters were calculated and assessed: grain yield (t ha⁻¹), average grain yield per plant (g), number of grains in the cob (pcs.), and weight of 1000 grains (g). PFPN (partial factor productivity) = YN/FN, where YN is the yield with applied N (kg ha⁻¹), and FN is the amount of N applied (kg ha⁻¹) [38].

2.2. Statistical Analysis

Statistical analyses were conducted using the analysis of variance (ANOVA) and STAT modules within the SELEKCIJA software package [39]. A two-way ANOVA was employed to evaluate the research data. Treatment differences were assessed using the F-test and least significant difference (LSD) test. Significant interactions between the factors were identified; therefore, mean values are not presented in the data analysis. Differences between treatment means not sharing the same letters (a, b, c, etc.) were considered significant at p < 0.05. The reliability of the regression coefficient was determined using Fisher’s F-test, and correlations were assessed using Student’s t-test. At p ≤ 0.05, dependencies are statistically significant at the 95% confidence level; at p ≤ 0.01, they are significant at the 99% confidence level [39].

2.3. Weather Conditions

Temperatures in April 2020 were close to the long-term norm, with only 4.0 mm of precipitation (Figure 1). Temperatures in May were 2.7 °C below the long-term norm and precipitation was 32.7 mm above the long-term norm. The monthly HTK was 4.26 (excess humidity). In June, the temperature was 2.9 °C above the long-term norm and precipitation was 22.4 mm above the norm, with an HTK of 1.74 (excess humidity). In July, the temperature was 1.3 °C below the long-term norm and precipitation was 36.2 mm less than the norm, with an HTK of 1.12 (optimum humidity). The temperature in August was 1.4 °C above the long-term norm, with an HTK of 1.61 (excess humidity). The temperature in September was 2.3 °C above the long-term norm with only 13.3 mm of precipitation, with the HTK of 0.30 (very dry). October precipitation was close to the long-term norm and the temperature was 3.5 °C above the long-term norm.

In 2021, the temperature in April was 0.7 °C below the long-term norm. May was cool and rainy with temperatures 1.8 °C below the long-term norm and precipitation 59.9 mm above the long-term norm. The monthly HTK was 4.04 (excess humidity). June and July were hot and dry. The HTK for these months was 0.69 (arid). August was cooler than normal, with precipitation 33.3 mm above the long-term norm. The monthly HTK was 2.40 (excess humidity). September was cooler and drier than normal (HTK 1.05). The temperature in October was 1.3 °C above the long-term norm and the precipitation was 23.8 mm below the long-term norm.

April 2022 was 0.7 °C colder than normal. Monthly precipitation was close to the long-term norm. May was cold and wet, with temperatures 2.2 °C below the long-term norm, precipitation 22.3 mm below the long-term norm, and an HTK of 3.3 (excess humidity). The temperature in June was 1.6 °C above the long-term norm, and precipitation was close to the long-term norm, with an HTK of 1.46 (optimum humidity). In July, the temperature was 0.8 °C lower than the long-term norm and precipitation was close to the long-term norm, with an HTK of 1.80 (excess humidity).

August was unusually hot and dry, with monthly temperatures 3.6 °C above the long-term norm, and precipitation 50.2 mm below the long-term norm, with an HTK of 0.60 (arid). September was 1.5 °C colder, and October was 3.4 °C warmer than usual. During these months, precipitation was 34.0 and 33.3 mm below the long-term norm, with HTKs of 0.88 and 0.87 (insufficient humidity), respectively.

3. Results

3.1. Effects of the Factors Studied on Maize Grain Yield

During the years studied, increases in nitrogen fertiliser rates without the use of biological preparations significantly increased maize grain yields (Figure 2). The highest maize grain yield (8.8 t ha⁻¹) was obtained in 2020 with N₁₈₀ fertilisation. Biological preparations increased the efficiency of nitrogen fertilisers. During the experiment, a substantial increase in maize grain yield was observed when AB was applied in combination with N₁₀₀ compared to N₁₀₀ alone. Significantly higher efficiency of nitrogen-fixing bacteria was observed in all years of the research when maize was fertilised with N₁₀₀, N₁₄₀, and N₁₈₀ fertilisation rates. The highest maize grain yield (10.9 t ha⁻¹) was obtained in 2020 with the N₁₈₀ fertilisation rate, in combination with AB. However, the data from the experiment showed that there were no significant differences between the N₁₄₀ and N₁₈₀ fertilisation rates.

The combination of nitrogen-fixing bacteria and phytohormones (AB + C) showed significant differences compared to the control field; however, no significant differences were found between AB + C and AB, except in 2022 when used in combination with the N₁₄₀ fertilisation rate. The highest grain yield (11.1 t ha⁻¹) using AB + C was obtained in 2020 when used in combination with the N₁₈₀ fertilisation rate; however, there was no significant difference compared to N₁₄₀, and no significant difference was observed when compared to N₁₄₀ and using AB.

When nitrogen-fixing bacteria were used in combination with a humic acid preparation (AB + H) and fertilisation rate N₁₀₀, a substantial increase in grain yield was observed compared to fertilisation rate N₁₀₀ without the use of biological preparations (BP). The highest grain yield (11.5 t ha⁻¹) was obtained in 2020 when fertilisation rate N₁₈₀ was combined with AB + H, but no significant difference was found when comparing N₁₈₀ with N₁₄₀, with significant differences only in the years 2021 and 2022.

As regards the higher effectiveness of the tested biological products and combinations in 2020, it could be assumed that in 2020, during the active growth phase of maize, the rainfall in June was higher than the standard climatic norm and the air temperature was higher, which could have led to a higher activity of microorganisms in the soil. In 2021 and 2021, there was insufficient soil moisture in the critical growth stages of maize, which may have influenced the microorganism activity. Maize of the same variety under the same agro-techniques and under different meteorological conditions will produce different yields of grain and aboveground biomass.

The correlation and regression analysis revealed that positive, strong, very strong, and statistically reliable correlations were observed in all cases between the different nitrogen fertilisation rates (x—kg ha⁻¹) and maize grain yield: r = 0.85–0.93 (p < 0.01;

Table 1. Dependence of maize grain yield on nitrogen fertilisation rates (Factor A: x—kg ha⁻¹), 2020–2022.

Dependent Variables, y	Factor B	Regression Equation	Correlation Coefficient r	Coefficient of Determination r2	Probability Level
y(2020)— grain yield (t ha−1)	Without BP	y = 4.75 + 0.02x	0.93	0.86	p < 0.01
	AB	y = 5.88 + 0.03x	0.91	0.83	p < 0.01
	AB + C	y = 6.22 + 0.03x	0.87	0.76	p < 0.01
	AB + H	y = 6.61 + 0.03x	0.87	0.76	p < 0.01
y(2021)— grain yield (t ha−1)	Without BP	y = 4.52 + 0.02x	0.93	0.86	p < 0.01
	AB	y = 5.15 + 0.02x	0.91	0.83	p < 0.01
	AB + C	y = 4.90 + 0.03x	0.93	0.86	p < 0.01
	AB + H	y = 5.40 + 0.02x	0.92	0.85	p < 0.01
y(2022)— grain yield (t ha−1)	Without BP	y = 4.55 + 0.02x	0.91	0.83	p < 0.01
	AB	y = 5.69 + 0.02x	0.86	0.74	p < 0.01
	AB + C	y = 5.85 + 0.02x	0.85	0.72	p < 0.01
	AB + H	y = 6.11 + 0.02x	0.91	0.83	p < 0.01

Notes. Factor B: use of biological preparations (BP)—(without BP) no biological preparations were used; (AB) biological preparation—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32; (AB + C) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32 and cytokinin (0.7 L ha⁻¹), spread at the 6-leaf stage; (AB + H) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) and humic acids (1.0 kg ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32.

).

3.2. Effects of the Factors Studied on Maize Grain Yield per Plant

The experiment showed a significant increase in maize grain yield per plant in 2020 when AB was applied in combination with nitrogen fertiliser (N₁₀₀), compared to the maize crop not treated with biological preparations. When fertilised at the N₁₀₀ fertilisation rate, the highest maize grain yield per plant (131.8 g) was obtained using the nitrogen AB + H combination (Figure 3).

Increased rates of nitrogen fertilisation (N₁₄₀ and N₁₈₀) resulted in a significant increase in maize grain yield per plant compared to the N₁₀₀ fertilisation rate. The highest maize grain yield per plant in 2020 (154.6 g) was observed when the N₁₈₀ fertilisation rate was used in combination with AB + H. The data from the experiment showed no significant difference between N₁₄₀ and N₁₈₀ fertilisation rates when used in combination with AB + C or AB + H. A significant increase in maize grain yield per plant was observed when the N₁₀₀ fertilisation rate was used in combination with AB + H, as compared to the N₁₄₀ fertilisation rate without the use of biological preparations.

The experiment conducted in 2021 showed similar trends as in 2020. Increased nitrogen fertilisation rates increased maize grain yield per plant, but no significant difference was observed between N₁₀₀ and N₁₄₀ nor between N₁₄₀ and N₁₈₀ when no biological preparations were applied. Significant differences were observed in all cases where the N₁₀₀ fertilisation rate was used in combination with biological preparations. Fertilisation rate N₁₄₀ in combination with AB resulted in a significant increase in plant grain yield per plant, but no significant difference was observed between AB and AB + C.

AB + H significantly increased grain yield per plant, as compared to the N₁₄₀ fertilisation rate without the use of biological preparations. As in previous years, the highest grain yield per plant (136.7 g) was observed with the N₁₈₀ fertilisation rate in combination with AB + H. When using the N₁₈₀ fertilisation rate, no significant difference was detected between AB, AB + C, and AB + H.

The experiment conducted in 2022 showed similar trends as in 2020 and 2021. The highest (145.6 g) average plant grain yield was observed with the N₁₈₀ fertilisation rate in combination with AB + H. Plant productivity was significantly higher compared to the N₁₈₀ fertilisation rate without the use of biological preparations; however, no significant differences were found between AB + H, AB, and AB + C treatments. When using the N₁₄₀ fertilisation rate, no significant differences were detected between the combinations of biological preparations; however, in each studied case, there was a significant increase in the average grain yield per plant compared to the control field. When using the lowest nitrogen fertilisation rate (N₁₀₀), each of the cases studied showed a significant increase in average grain yield per plant, as compared to the control field. No significant differences were found between AB and AB + C.

The studied biological preparations increased the efficiency of nitrogen fertilisers during the years of the experiment.

The correlation and regression analysis revealed that positive, strong, very strong, and statistically reliable correlations were observed in all cases between the different nitrogen fertilisation rates (x—kg ha⁻¹) and maize grain yield per plant: r = 0.83–0.94 (p < 0.01;

Table 2. Dependence of maize grain yield per plant on nitrogen fertilisation rates (Factor A: x—kg ha⁻¹), 2020–2022.

Dependent Variables, y	Factor B	Regression Equation	Correlation Coefficient r	Coefficient of Determination r2	Probability Level
y(2020)— maize grain yield per plant (g)	Without BP	y = 77.3 + 0.28x	0.92	0.85	p < 0.01
	AB	y = 91.5 + 0.30x	0.91	0.83	p < 0.01
	AB + C	y = 87.5 + 0.35x	0.94	0.88	p < 0.01
	AB + H	y = 104.5 + 0.28x	0.85	0.72	p < 0.01
y(2021)— maize grain yield per plant (g)	Without BP	y = 71.4 + 0.27x	0.90	0.81	p < 0.01
	AB	y = 86.4 + 0.24x	0.89	0.79	p < 0.01
	AB + C	y = 79.4 + 0.28x	0.84	0.71	p < 0.01
	AB + H	y = 87.5 + 0.27x	0.94	0.88	p < 0.01
y(2022)— maize grain yield per plant (g)	Without BP	y = 91.0 + 0.18x	0.86	0.74	p < 0.01
	AB	y = 93.5 + 0.26x	0.86	0.74	p < 0.01
	AB + C	y = 92.2 + 0.29x	0.93	0.86	p < 0.01
	AB + H	y = 107.3 + 0.22x	0.83	0.69	p < 0.01

Notes. Factor B: use of biological preparations (BP)—(without BP) no biological preparations were used; (AB) biological preparation—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32; (AB + C) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32 and cytokinin (0.7 L ha⁻¹), spread at the 6-leaf stage; (AB + H) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) and humic acids (1.0 kg ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32.

).

3.3. Effects of the Factors Studied on the 1000-Grain Weight of Maize

The highest 1000-grain weight of maize (311 g) was recorded in 2020 when maize was treated with the N₁₈₀ fertilisation rate in combination with AB + H (Figure 4). In other cases, no significant differences were found between the different combinations of nitrogen fertiliser and biological preparations. A significant increase in the 1000-grain weight was observed in 2020 and 2022 when maize was treated with the N₁₀₀ fertilisation rate in combination with AB, as compared to nitrogen fertilisation without the use of BP. In all years of the study, there was a significant increase in the 1000-grain weight of maize using the N₁₄₀ fertilisation rate in combination with AB, as compared to nitrogen fertilisation without the use of BP.

The correlation and regression analysis revealed that positive, strong, very strong, and statistically reliable correlations were observed in all cases between the different nitrogen fertilisation rates (x—kg ha⁻¹) and 1000-grain weight of maize: r = 0.76–0.91 (p < 0.01, p < 0.05). Only in 2020 were statistically insignificant dependencies found when no biological preparations were used (p > 0.05;

Table 3. Dependence of the 1000-grain weight of maize on nitrogen fertilisation rates (Factor A: x—kg ha⁻¹), 2020–2022.

Dependent Variables, y	Factor B	Regression Equation	Correlation Coefficient r	Coefficient of Determination r2	Probability Level
y(2020)— 1000-grain weight of maize (g)	Without BP	-	-	-	p > 0.05
	AB	y = 208.5 + 0.48x	0.84	0.71	p < 0.01
	AB + C	y = 183.4 + 0.69x	0.91	0.83	p < 0.01
	AB + H	y = 220.2 + 0.51x	0.90	0.81	p < 0.01
y(2021)— 1000-grain weight of maize (g)	Without BP	y = 169.9 + 0.50x	0.87	0.76	p < 0.01
	AB	y = 190.3 + 0.46x	0.85	0.72	p < 0.01
	AB + C	y = 186.9 + 0.48x	0.76	0.58	p < 0.05
	AB + H	y = 202.1 + 0.44x	0.79	0.62	p < 0.05
y(2022)— 1000-grain weight of maize (g)	Without BP	y = 193.1 + 0.34x	0.88	0.77	p < 0.01
	AB	y = 196.3 + 0.50x	0.87	0.76	p < 0.01
	AB + C	y = 189.9 + 0.56x	0.89	0.79	p < 0.01
	AB + H	y = 224.1 + 0.39x	0.84	0.71	p < 0.01

Notes. Factor B: use of biological preparations (BP)—(without BP) no biological preparations were used; (AB) biological preparation—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32; (AB + C) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32 and cytokinin (0.7 L ha⁻¹), spread at the 6-leaf stage; (AB + H) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) and humic acids (1.0 kg ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32.

).

3.4. Effects of the Factors Studied on the Number of Maize Grains per Cob

The highest number of maize grains per cob (498) was found when maize was treated using the N₁₈₀ fertilisation rate in combination with AB + H.

The data in Figure 5 show that the nitrogen fertilisation rates and combinations of biological preparations studied did not have a significant effect on the number of grains per cob. An upward trend in the number of grains could only be seen with the use of biological preparations.

A correlation and regression analysis of nitrogen fertilisation rates (x—kg ha⁻¹) and y (2020, 2021, and 2022)—number of grains per cob—showed no statistically significant correlation (p > 0.05).

3.5. Effects of Factors Studied on Partial Factor Productivity of Nitrogen

Evaluation of the effects of fertilisation rates and biological preparations used in the experiment on the partial factor nitrogen efficiency showed that when maize was fertilised using the N₁₀₀ fertilisation rate in combination with AB, AB + C, and AB + H, the partial factor nitrogen productivity in 2020 was 86.0, 88.2, and 92.0 kg per maize grain kg⁻¹ N, respectively. Figure 6 shows a decrease in the partial factor productivity of nitrogen fertiliser when increasing the nitrogen fertilisation rates without the use of biological agents.

When nitrogen fertiliser was applied alone, the partial factor productivity of nitrogen varied considerably depending on the meteorological conditions of the year. The lowest nitrogen efficiency (46.1 kg maize grain per kg⁻¹ N) was observed in 2021 when using the N₁₈₀ fertilisation rate without the use of BP. With AB, the highest partial factor productivity of nitrogen (86.0 kg maize grain per kg⁻¹ N) was observed when using the N₁₀₀ fertilisation rate. Using the same fertilisation rate in combination with AB + C and AB + H, the partial factor productivity of nitrogen was 88.2 and 92.0 kg maize grain per kg⁻¹ N, respectively. The data showed that, depending on the meteorological conditions of the year, the partial factor productivity of nitrogen tended to decrease, but not significantly. Increasing the nitrogen fertilisation rate without the use of biological agents resulted in a decrease in nitrogen efficiency.

The correlation and regression analysis revealed that very strong and statistically reliable negative correlations were found between the different nitrogen fertilisation rates (x—kg ha⁻¹) and the partial factor productivity of nitrogen in terms of maize grain yield in all years of the study: r = −0.94–−0.98 (p < 0.01;

Table 4. Dependence of partial factor productivity of nitrogen on the nitrogen fertilisation rates (Factor A: x—kg ha⁻¹), 2020–2022.

Dependent Variables, y	Factor B	Regression Equation	Correlation Coefficient r	Coefficient of Determination r2	Probability Level
y(2020)— kg maize grain kg−1 N	Without BP	y = 95.4 − 0.26x	−0.96	0.92	p < 0.01
	AB	y = 117.6 − 0.32x	−0.97	0.94	p < 0.01
	AB + C	y = 121.8 − 0.33x	−0.97	0.94	p < 0.01
	AB + H	y = 127.8 − 0.35x	−0.97	0.94	p < 0.01
y(2021)— kg maize grain kg−1 N	Without BP	y = 90.2 − 0.25x	−0.97	0.94	p < 0.01
	AB	y = 100.6 − 0.28x	−0.97	0.94	p < 0.01
	AB + C	y = 100.9 − 0.28x	−0.94	0.88	p < 0.01
	AB + H	y = 107.8 − 0.30x	−0.95	0.90	p < 0.01
y(2022)— kg maize grain kg−1 N	Without BP	y = 92.3 − 0.25x	−0.94	0.88	p < 0.01
	AB	y = 106.8 − 0.30x	−0.98	0.96	p < 0.01
	AB + C	y = 110.6 − 0.31x	−0.97	0.94	p < 0.01
	AB + H	y = 116.8 − 0.34x	−0.97	0.94	p < 0.01

Abbreviations (for Factor B). Factor B: use of biological preparations (BP)—(without BP) no biological preparations were used; (AB) biological preparation—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32; (AB + C) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32 and cytokinin (0.7 L ha⁻¹), spread at the 6-leaf stage; (AB + H) biological preparations—nitrogen bacteria (1.0 L ha⁻¹) and humic acids (1.0 kg ha⁻¹) applied to the soil surface immediately after sowing in combination with KAS-32.

).

Summarising the results of the three-year experiment, it can be concluded that similar yield trends were observed as in the individual years of the experiment. Increasing the nitrogen rate from N₁₀₀ to N₁₈₀ without the use of biological agents resulted in an average increase in maize grain yield of 1.8 t ha⁻¹. The use of biological preparations and N₁₀₀ fertiliser resulted in an average increase in maize grain yield of 1.0–1.7 t ha⁻¹, with the highest increase (1.7 t ha⁻¹) being observed with AB + H. Increasing the nitrogen rate up to N₁₄₀ and the use of biological preparations resulted in an average increase in grain yield of 1.6–2.1 t ha⁻¹, in comparison with the control. The highest average increase (2.1 t ha⁻¹) in maize grain yield compared to the control (without BP) was obtained with N₁₄₀ and AB + H. It can be concluded that the tested biological preparations increased the efficiency of nitrogen fertilisers, even at higher nitrogen fertiliser rates.

4. Discussion

Higher rates of nitrogen fertiliser are a common tool for farmers to increase yields and manage risks. However, excessive and inefficient use of nitrogen fertiliser leads to increased production costs and degraded soil, water, and air quality [40]. Studies have shown that about 76% of anthropogenic nitrogen applied to the world’s land surface has been released to the environment through nitrification, denitrification, volatilisation, and leaching, likely leading to nitrogen losses and greenhouse gas emissions [41]. There is an increasing focus on the precise application of nitrogen fertiliser. Studies have attempted to determine the agronomically or economically optimal nitrogen rate as a guideline for nitrogen fertiliser rates, taking into account the objective of maize production [42].

The experiments we conducted demonstrated that achieving stable maize grain yields relies on selecting appropriate cultivation technologies and tailoring their application to specific soil and climate conditions. While intensive farming practices aim to maximise yields, they often negatively impact soil fertility, leading to environmental degradation, reduced plant productivity, and diminished crop quality. Supporting these findings, Niknam and Faraji [43] reported that nitrogen rates and their interaction with plant populations had no significant effect on key yield components, such as the number of seeds per cob, the number of cobs per unit area, and grain yield. They concluded that a nitrogen rate of 200 kg ha⁻¹ is optimal for a maize crop density of 130,000 plants ha⁻¹. As an alternative to conventional inputs, biological fertilisers offer a promising solution to enhance crop production sustainably.

Volatilisation, denitrification, and leaching cause significant losses of nitrogen. Therefore, the main goal of this research was to improve maize productivity by improving the efficiency of nitrogen fertilisers through the use of nitrogen-fixing bacteria, phytohormones, and humic acids. The data from the experiment suggested that, depending on the meteorological conditions of the year of production, a lower fertilisation rate can be chosen when using biological preparations.

Biofertilisers composed of nitrogen-fixing bacteria have been proposed as a substitute for (or in addition to) mineral nitrogen fertilisers in crop production to improve the availability of nutrients in the soil, to provide certain metabolites during the vegetative period of the plants, and to reduce the fertilisation rate [44]. This can be achieved by using soil microbes, in particular, the plant-growth-promoting nitrogen-fixing bacteria Paenibacillus polymyxa MVY–024 (AB).

According to the relevant literature, the genus Paenibacillus has more than 100 species with valid names. Approximately 20 members of the genus Paenibacillus have been reported to have the capacity of fixing nitrogen [45,46]. The plant rhizosphere provides a habitat for functional microorganisms, encompassing a complex and dynamic zone of interactions between networks of organisms and their plant hosts. A large part of strains isolated from the plant rhizosphere can directly or indirectly stimulate plant growth, development, and evolution. These strains are called plant-growth-promoting rhizobacteria. These bacteria can stimulate plant growth in a variety of ways, including fixing nitrogen from the atmosphere, solubilising phosphorus, synthesising siderophores, and synthesising antimicrobial agents (antibiotics, bacteriocins, and small peptides) and plant hormones, such as indole, cytokinins, or gibberellins [47,48].

Some Paenibacillus species may affect plant growth by one or more of these mechanisms. Environmentally friendly strains of Paenibacillus are the best option for replacing chemical fertilisers because of their wide host range and their ability to release plant-growth-promoting substances and produce a variety of antimicrobial substances. Paenibacillus strains can increase the productivity of agricultural plants, including aboveground and root biomass [49].

Recently, ways to increase plant productivity have gone beyond nitrogen fertilisers to include foliar fertilisation, which is commonly used to correct nutrient deficiencies as plants develop. In addition to micronutrient fertilisers, plant growth regulators (phytohormones) have become widely used.

Singh et al. [50] suggested that the effect of each phytohormone may depend on the concentration or activity of another phytohormone, a mechanism known as cross-signalling. A mixture of different hormones used as foliar fertilisers can have either a detrimental or a beneficial effect on the plant, and studies have shown that different plant hormones (auxins and cytokinins) induce responses, such as tillage improvement, although in most cases the responses are dependent on the varieties studied.

It is also difficult to predict how a plant may respond to phytohormones applied at early stages of vegetation, as tissue sensitivity may vary between plant species, and the thresholds of benefit or harm are strictly dependent on the rates of fertilisers and formulations applied, as well as on biotic and abiotic factors [51,52].

The experiment revealed that combinations of nitrogen-fixing bacteria (AB), phytohormones (C), and humic acids (H) significantly increased maize grain yield, while AB + C in combination with the N₁₄₀ fertilisation rate in 2021 showed no significant effect on grain yield. The data from the experiment suggested that, as reported in [51], the efficacy of biological preparations and their combinations (AB and AB + C) can be linked to fertilisation rates and environmental factors.

In terms of the partial factor productivity of nitrogen fertiliser, it was found that the studied biological preparations significantly increased the partial factor productivity of nitrogen fertiliser, as compared to the control field, when used in combination with the N₁₀₀ fertilisation rate. When increasing fertilisation rates to N₁₈₀ and using AB, no significant difference was found in 2021 and 2022 compared to the control field. Our findings are consistent with the biostimulatory effects of nitrogen-fixing bacteria, phytohormones, and humic acids on plant growth and development described in other studies [53,54]. During the experiment, in all cases, AB + H significantly increased the partial factor productivity of nitrogen fertiliser compared to the control field. As reported in [51], humic acids are active at relatively low concentrations, so the use of AB + H biological preparations resulted in an increase in the partial factor productivity of nitrogen fertiliser compared to the control field, where BP was not applied. Souza et al. [53] also pointed out that the positive effect on plant condition is more likely to be caused by the use of phytohormones or other biological preparations, or by the large-scale reprogramming of genes involved in various plant growth processes [55].

It can be concluded that biological preparations played an important role in improving the efficiency of nitrogen fertilisers; however, increasing the nitrogen fertiliser rates up to N₁₄₀ and N₁₈₀ resulted in a decrease in the efficiency of biological preparations. The use of biological preparations in combination with lower nitrogen fertilisation rates allowed a more efficient use of nitrogen fertiliser, leading to more sustainable and productive agricultural practices.

5. Conclusions

In all cases, the biological preparations and their combinations significantly increased maize grain yield compared to the control field (no use of biological preparations). The highest maize grain yield (11.5 t ha⁻¹) was obtained in 2020 using the N₁₈₀ fertilisation rate in combination with nitrogen-fixing bacteria and humic acids (AB + H). The biological preparations in combination with N significantly increased the 1000-grain weight and thus the grain yield per plant. Positive, strong, very strong, and statistically reliable correlations were found in most cases between the different N fertilisation rates and the parameters studied: r = 0.76–0.94 (p < 0.01, p < 0.05). No statistically significant correlation was found between nitrogen fertilisation rates and the number of grains per cob (p > 0.05).

The highest partial factor productivity of nitrogen fertiliser (92.0 kg maize grain per kg⁻¹ N) was observed in 2020 when maize was fertilised using the N₁₀₀ fertilisation rate in combination with AB + H. Increasing the nitrogen fertilisation rate without the use of biological preparations resulted in a decrease in the partial factor productivity of nitrogen fertiliser. The highest partial factor productivity of nitrogen in all years of the study was obtained using the N₁₀₀ fertilisation rate in combination with biological preparations. Very strong negative and statistically reliable correlations were found (r = −0.94–−0.98, p < 0.01).

In summary, biological preparations have a significant potential to increase the efficiency of nitrogen fertilisers by stimulating the activity of the soil microbiota, improving plant nitrogen uptake, and reducing fertiliser losses to the environment. Based on the results of the studies carried out and the experience gained, it would be worth further examining the combinations of different microorganisms and their interactions with nitrogen fertilisers and exploring combinations that bind nitrogen-fixing microorganisms with phosphorus mobilisers or mycorrhizae, which can further improve the efficiency of nitrogen fertiliser. Continued research and the integrated use of biological preparations can contribute to building a more sustainable farming system, reducing environmental pollution and maintaining high agricultural productivity.

The use of biological preparations can enhance the efficiency of nitrogen fertilisers, as these preparations may act as biostimulants and improve plants’ ability to absorb nutrients from the soil. It is likely that biological preparations can synergistically interact with nitrogen fertilisers, promoting plant metabolism and enhancing nutrient uptake. This may also help reduce nitrogen losses. Based on experimental data, it can be recommended to use combinations of nitrogen fertilisers with nitrogen-fixing bacteria and humic acids in farms located in temperate climate zones.