Yield and Grain Quality Response of Winter Wheat to Different Nitrogen and Phosphorus Fertilization Doses

Abstract

Climate change increasingly challenges winter wheat production, making balanced nutrient management essential for maintaining crop productivity and grain quality. In this context, fertilization remains a key factor determining crop productivity and yield stability. This study was conducted during 2022–2025 within a long-term field experiment established in 1975 at the Agricultural Research and Development Station Secuieni, Romania, to evaluate the cumulative effects of nitrogen and phosphorus fertilization on winter wheat yield, yield components, grain quality, and soil chemical properties. The experiment was established on a medium-textured cambic chernozem using a two-factor split-plot design with five replications. Five phosphorus (P₀, P₄₀, P₈₀, P₁₂₀, and P₁₆₀ kg ha⁻¹ active ingredient) and nitrogen (N₀, N₄₀, N₈₀, N₁₂₀, and N₁₆₀ kg ha⁻¹ active ingredient) treatments were evaluated using the winter wheat cultivar Otilia grown in a bean–wheat–maize rotation. Fertilization significantly increased grain yield and improved grain quality. The highest winter wheat grain yield, 6897 kg ha⁻¹, was obtained with the N₈₀P₁₆₀ fertilization treatment, indicating that this nitrogen and phosphorus combination provided the most favorable response under the experimental conditions. Higher nitrogen rates significantly increased grain quality parameters, with protein content rising from 9.15% at N₀ to 14.20% at N₁₆₀, wet gluten content increasing from 17.29% to 28.97% and starch content decreasing from 70.95% at N₀ to 66.91% at N₁₆₀; however, the application of higher fertilizer rates did not result in further increases in grain yield. These findings emphasize the importance of balanced nitrogen and phosphorus management for improving winter wheat productivity and grain quality under changing climatic conditions.

1. Introduction

Winter wheat (Triticum aestivum L.) is one of the most widely cultivated cereal crops worldwide and ranks among the leading cereals in terms of global production [1]. Owing to its high nutritional value, it constitutes a major component of human diets by providing essential carbohydrates, dietary fiber, and protein, thereby contributing substantially to global food security [2,3,4]. As a major staple crop, winter wheat plays a crucial role in global food security by providing a substantial proportion of the calories and nutrients required for human consumption [5]. Ensuring stable and sustainable wheat production is therefore essential to meet the increasing nutritional demands of a growing global population [6].

Among the essential plant nutrients, nitrogen (N) and phosphorus (P) play a fundamental role in crop productivity and grain quality by regulating key physiological processes, including photosynthesis, root system development, biomass accumulation, and protein synthesis [7,8]. The efficient use of phosphorus (P) is a key factor in crop production, sustainable resource management, and food security [9]. Consequently, understanding the influence of soil properties, cultivar selection, and crop rotation on phosphorus use efficiency is essential for optimizing fertilizer management in wheat production [9]. Furthermore, the localized placement of nitrogen, particularly in the ammonium (NH₄⁺–N) form, together with phosphorus has been shown to improve phosphorus uptake, early plant growth, and grain yield compared with conventional broadcast fertilization. These beneficial effects have been confirmed in both controlled experiments and field studies, highlighting the importance of fertilizer placement for improving nutrient use efficiency [10,11]. Achieving high and stable grain yields and grain quality depends largely on adequate mineral nutrition, particularly nitrogen (N), which is the most yield-limiting nutrient in wheat production [12,13]. Appropriate nitrogen management strongly influences plant growth, photosynthetic activity, grain yield, grain protein content, and nitrogen use efficiency [13]. As a key macronutrient, nitrogen is essential for plant growth and development; therefore, a thorough understanding of the physiological processes governing nitrogen uptake and utilization is necessary to improve nitrogen use efficiency [14].

However, the efficiency of fertilizer use is influenced by numerous soil, climatic, and agronomic factors, while improper nutrient management may result in reduced crop productivity, lower nutrient use efficiency, and adverse environmental impacts due to nutrient losses [15,16,17,18]. Therefore, optimizing nitrogen and phosphorus fertilization strategies is essential for improving wheat yield, maintaining grain quality, and enhancing the sustainable use of agricultural inputs [12].

The application of mineral fertilizers to winter wheat improves crop nutrition, as its relatively poorly developed root system explores a limited volume of soil and has a low capacity to solubilize and absorb nutrients from soil reserves [19]. The maximum uptake of nitrogen and phosphorus by winter wheat occurs during the relatively short period from stem elongation to maturity, when plants absorb more than 80% of their total nitrogen and phosphorus requirements [20,21,22,23].

Worldwide, long-term fertilization and soil management experiments constitute essential research infrastructures for evaluating the cumulative effects of agricultural practices on crop productivity and soil fertility. Many of these experiments have been continuously maintained for periods ranging from 20 to more than 180 years, including the Rothamsted Experimental Station experiments in the United Kingdom, established in 1843, as well as numerous other long-term experimental platforms across Europe, North America, and Asia [24,25,26]. The results of these long-term experiments have demonstrated that long-term fertilization significantly influences nitrogen use efficiency, yield stability, and the dynamics of soil nutrient reserves [27].

In Romania, long-term fertilization experiments have been established within the network of agricultural research institutes and research stations, providing an important scientific basis for developing fertilizer recommendations adapted to different soil and climatic conditions. A representative example is the long-term experiment at the Agricultural Research and Development Station Secuieni, established in 1975, where the cumulative effects of different nitrogen and phosphorus application rates on crop yield and soil fertility have been investigated under a long-term crop rotation system [21,28,29].

Wheat grain yield and quality are the primary concerns of producers, as these parameters largely determine the economic efficiency of wheat production. To maximize grain yield and quality, producers can adopt and combine a range of agronomic practices, among which fertilizer application is one of the most effective measures [30].

Unlike short-term studies, long-term experiments enable the quantification of gradual changes in soil fertility, nutrient availability, and nitrogen and phosphorus use efficiency [31].

Although the effects of nitrogen and phosphorus fertilization on winter wheat are well documented, relatively few studies have simultaneously evaluated grain yield, technological grain quality, and the response of soil chemical properties across a wide N × P fertilization gradient within a long-term crop rotation under the specific pedoclimatic conditions of a cambic chernozem. Therefore, the present study provides an updated assessment of the long-term experiment at Secuieni under recent climatic conditions, with particular emphasis on identifying fertilization combinations that can achieve a balance among grain yield, grain quality, and soil fertility.

Therefore, the present study aimed to evaluate the response of the winter wheat cultivar Otilia to different N × P fertilization combinations within a long-term fertilization experiment conducted in Secuieni, Romania, during 2022–2025. The study investigated the effects of fertilization on grain yield, yield components, the main indicators of technological grain quality, and selected soil chemical properties under growing seasons characterized by contrasting weather conditions. The results contribute to the characterization of winter wheat responses to combined N and P fertilization and provide relevant information for assessing fertilization strategies under the specific pedoclimatic conditions of the study area.

2. Materials and Methods

2.1. Study Site and Environmental Conditions

The research was conducted at the Agricultural Research and Development Station (A.R.D.S.) Secuieni–Neamț, Romania, located at 26°51′00″ E longitude and 46°51′15″ N latitude (Figure 1) [32]. The experimental fields are located on both the first terrace of the Siret River and the Siret River floodplain.

The study area is characterized by a temperate continental climate, with short springs, hot summers, and mild winters [33].

Figure 1. Geographic location of the experimental site at the Agricultural Research and Development Station Secuieni–Neamț, Romania [34].

Figure 1. Geographic location of the experimental site at the Agricultural Research and Development Station Secuieni–Neamț, Romania [34].

2.2. Experimental Design and Crop Management

The field experiment was conducted from 2022 to 2025. A two-factor experiment was established using a split-plot design with five replications (Figure 2). The size of each experimental plot was 45.5 m² (3.5 m × 13 m).

Figure 2. Layout of the split-plot experimental design. Factor A—phosphorus (P₂O₅) fertilization rates: a1—P₀; a2—P₄₀; a3—P₈₀; a4—P₁₂₀; a5—P₁₆₀. Factor B—nitrogen (N) fertilization rates: b1—N₀; b2—N₄₀; b3—N₈₀; b4—N₁₂₀; b5—N₁₆₀.

Factor A included five phosphorus (P₂O₅) fertilization rates—0, 40, 80, 120, and 160 kg ha⁻¹ active ingredient—whereas Factor B included five nitrogen (N) fertilization rates—0, 40, 80, 120, and 160 kg ha⁻¹ active ingredient. For the preceding bean crop, lower nitrogen rates were applied, namely 0, 25, 50, 75, and 100 kg ha⁻¹ active ingredient.

The research was conducted on a typical cambic chernozem with a medium-textured soil profile. The physicochemical properties of each experimental treatment were determined and are presented in Section 3.4.

The biological material used in the study was the winter wheat cultivar Otilia, developed by the National Agricultural Research and Development Institute (NARDI) Fundulea. Officially registered in 2013, Otilia is characterized by good resistance to lodging, winter conditions, drought, and heat stress, as well as tolerance to most of the major pathogens affecting small-grain cereals [35]. As only one cultivar was included in the experiment, the responses observed in this study are specific to Otilia, and no comparisons among genotypes or genotype × fertilization interactions were assessed.

During the study period, fertilization was carried out using ammonium nitrate and single superphosphate, with application rates corresponding to the experimental treatments. The experiment was part of a three-year crop rotation system (bean–winter wheat–maize), with winter wheat grown after bean.

Ammonium nitrate is one of the most widely used mineral fertilizers in agriculture because of its high nitrogen content (33.5% active substance) (Azomureș, Târgu Mureș, Romania). Ammonium nitrate was applied in spring, at the BBCH 30 growth stage, in a single application, with the entire nitrogen dose for each experimental treatment applied.

Single superphosphate, containing 20% active ingredient as phosphorus, is commonly used as a phosphorus fertilizer in agricultural yield (Alcedo SRL, Roman, Romania). In this study, it was applied in autumn and incorporated into the soil during seedbed preparation.

Soil preparation included moldboard plowing to a depth of 15–20 cm using a Lemken reversible plow (Lemken, Romania), followed by soil loosening and leveling with a Unia Atlas 4 cultivator (Apan Agriculture Equipments, Brăila, Romania) to produce a well-prepared seedbed.

Sowing was carried out within the optimal planting period for the research station’s agroecological region, between 15 and 25 October of each experimental year, using an Amazone grain drill for small-grain cereals (Proinvest, Teleorman, Romania). The crop was sown at a density of 550 viable seeds m⁻², with a row spacing of 12 cm. Harvesting was performed at full crop maturity, between 1 and 20 July, depending on the climatic conditions of each experimental year and the progression of crop ripening. The experimental plots were harvested using a Wintersteiger Plot Combine (Wintersteiger Holding, Österreic, Austria).

Crop management practices focused on the control of monocotyledonous and dicotyledonous weeds. Throughout the entire five-year study period, the same systemic herbicide, based on amidosulfuron, iodosulfuron-methyl-sodium, and mefenpyr-diethyl, was applied at a rate of 0.15 L ha⁻¹ (Sekator Progress OD, BAYER, Bucharest, Romania). In addition, a fungicide based on prothioconazole, spiroxamine, and tebuconazole was applied in a tank mixture with the herbicide at a rate of 0.6 L ha⁻¹ (Impulse PRO 425 EC, BAYER, Bucharest, Romania) to control the complex of foliar diseases and prevent Fusarium head blight in wheat.

2.3. Data Collection and Measurements

During the growing season and at harvest, the following agronomic traits were evaluated: plant height (cm), number of grains per spike, grain weight per spike, thousand-kernel weight (TKW, g), test weight (TW, kg hL⁻¹), grain yield (t ha⁻¹), and the main grain quality parameters.

Plant height was determined after heading [36], at the BBCH 69 growth stage, to assess the influence of different nitrogen and phosphorus fertilization rates on plant growth and final plant height. Plant height was measured from the soil surface to the tip of the spike, excluding the awns. The number of grains per spike was determined by counting the grains from randomly selected spikes collected from each experimental treatment, after which the grain weight per spike was recorded. Thousand-kernel weight and test weight were determined according to standard laboratory procedures. Grain yield was determined by harvesting the net plot area of each experimental plot and expressing the results on a hectare basis (t ha⁻¹).

Grain quality was evaluated by determining protein content (%), wet gluten content (%), starch content (%), Zeleny sedimentation index (mL), and grain hardness (%). Quality analyses were performed on grain samples collected from each experimental treatment using a Perten IM9500 Near-Infrared (NIR) Analyzer (Perten Instruments, Stockholm, Sweden) [37].

2.4. Influence of N and P Fertilization on the Soil

To assess the impact of fertilization on soil fertility status, agrochemical analyses were performed for soil pH, nitrogen, and phosphorus content under increasing phosphorus (P₂O₅) and nitrogen application rates ranging from 0 to 160 kg ha⁻¹ active ingredient. The experiment was conducted on a cambic chernozem (typical chernozem) soil.

The first soil samples were collected in 2020 to assess the initial agrochemical characteristics of the experimental site. The results indicated a soil pH of 5.3, a total nitrogen content of 0.121%, and a phosphorus content of 34 mg/kg. In 2024, a new set of soil samples was collected from the same experimental field to enable a comparative assessment of changes in soil fertility over time. Soil sampling was performed before sowing the winter wheat crop. The soil samples were collected from the experimental field and analyzed at the National Research and Development Institute for Soil Science, Agrochemistry and Environmental Protection (ICPA Bucharest). Soil pH was determined using the potentiometric method [38], while nitrogen and phosphorus contents were determined by spectrophotometric analysis [39]. The analytical results obtained in 2020 and 2024 were used to characterize the agrochemical status of the soil and assess changes in soil fertility over the four-year period, providing a basis for the comparative evaluation of soil conditions before and after the preceding crop and prior to the establishment of the winter wheat experiment.

Given the descriptive and exploratory nature of the soil property assessment, these parameters were not subjected to statistical analysis. Soil samples were collected to monitor variations in soil nitrogen and phosphorus contents and to assess the potential relationship between these parameters and soil pH.

2.5. Statistical Analysis

Observations and measurements were performed on five replications for each experimental treatment. Grain yield data were subjected to factorial analysis of variance (ANOVA) to evaluate the effects of year (Y), phosphorus fertilization (P), nitrogen fertilization (N), and their interactions (Y × P, Y × N, P × N, and Y × P × N). The experimental data were statistically analyzed using the least significant difference (LSD) test. Differences among treatments were considered significant at p < 0.05, highly significant at p < 0.01, and very highly significant at p < 0.001. Pearson’s correlation analysis was performed using the Jamovi (Version 2.7) statistical software available online [40]. Second-order polynomial regression analysis was performed in PAST software to quantify the response of winter wheat grain yield to nitrogen and phosphorus application rates. The following model was fitted separately for each growing season:

Y = a₀; + a₁N + a₂P + a₃N² + a₄P² + a₅NP

where Y is grain yield (kg ha⁻¹); a₀, a₁, a₂, a₃, a₄ and a₅ are regression coefficients; N is the nitrogen application rate (kg N ha⁻¹); P is the phosphorus application rate (kg P₂O₅ ha⁻¹); and NP represents the interaction between nitrogen and phosphorus fertilization.

2.6. Calculation of Nitrogen and Phosphorus Use Efficiency

Nitrogen use efficiency (NUE) and phosphorus use efficiency (PUE) were calculated as the ratio of grain yield to the amount of nitrogen or phosphorus fertilizer applied, respectively.

$$\mathrm{NUE}={\displaystyle \frac{\mathrm{Y}}{\mathrm{N}}}$$

$$\mathrm{PUE}={\displaystyle \frac{\mathrm{Y}}{\mathrm{P}}}$$

where Y is grain yield (kg ha⁻¹), N is the nitrogen application rate (kg N ha⁻¹), and P is the phosphorus application rate (kg P₂O₅ ha⁻¹). NUE and PUE were expressed as kg grain kg⁻¹ nutrient applied. Values were not calculated for N₀ and P₀ treatments.

2.7. Weather Characteristics

Average monthly air temperature and precipitation recorded in central Moldova during the three growing seasons (October 2022–July 2025), together with the 60-year long-term averages, are presented in Figure 3 and Figure 4 [41].

Figure 3. Monthly temperature regime at ARDS Secuieni during the period October 2022–July 2025, compared with the 60-year average, including cumulative temperature for the BBCH 30–85 growth stages of winter wheat.

Figure 4. Monthly precipitation regime at ARDS Secuieni during the period October 2022–July 2025, compared with the 60-year average, including cumulative temperature for the BBCH 30–85 growth stages of winter wheat.

From a thermal perspective, the monthly mean temperatures recorded during the 2022–2025 growing seasons showed considerable variation relative to the 60-year long-term averages, indicating a generally warmer climatic regime in recent years. During the autumn months, temperatures were consistently higher than the long-term averages in all study years, suggesting an extended period of favorable conditions for crop growth and a delayed onset of the cold season. In October, for example, mean air temperatures ranged from 10.2 °C to 13.0 °C, compared with the long-term average of 9.2 °C.

During the winter period (December–February), even greater deviations from the 60-year long-term averages were observed. In the study years, mean monthly temperatures were generally above or close to 0 °C, indicating milder winters and a reduced risk of severe freezing conditions. From an agronomic perspective, this is particularly relevant because temperatures well above the seasonal average may influence the vernalization process and the overwintering capacity of winter crops.

From March to July, a general trend of higher-than-average temperatures was observed, with the most pronounced positive deviations occurring in March, April, June, and July compared with the long-term averages (Figure 3).

From a pluviometric perspective, the three growing seasons exhibited considerable variability in precipitation patterns compared with the 60-year long-term average. Overall, rainfall distribution was highly uneven, with frequent alternations between months characterized by precipitation deficits and those with above-average rainfall.

During the autumn and winter seasons, precipitation amounts were generally close to or below the long-term average, indicating limited soil moisture availability during the early stages of crop development. In contrast, spring and early summer were marked by substantial fluctuations in rainfall, including episodes of excessive precipitation. The highest monthly rainfall was recorded in May 2025 (116 mm), considerably exceeding the long-term average.

June and July showed pronounced interannual variability, characterized by alternating periods of substantial rainfall deficits and excessive precipitation, highlighting the increasing instability of the hydrological regime during the late stages of crop development (Figure 4).

3. Results

3.1. Effect of Fertilization on Growth and Yield Components

Nitrogen (N) and phosphorus (P) fertilization positively affected the main growth traits and yield components of winter wheat (Table 1). Plant height ranged from 70 to 91 cm, spike density from 490 to 576 spikes m⁻², spike length from 5.73 to 7.34 cm, the number of grains per spike from 27 to 42, and grain weight per spike from 1.03 to 1.25 g. The highest values for most of the measured traits were recorded under moderate-to-high fertilization rates, particularly in treatments receiving 80–160 kg N ha⁻¹ in combination with 80–160 kg P ha⁻¹.

Table 1. Effect of nitrogen and phosphorus fertilization on plant growth and yield components of winter wheat (mean values).

A Factor	B Factor	Plant Height (cm)	Spikes per Square Meter	Spike Length (cm)	Grains per Spike	Grain Weight per Spike (g)
P0	N0	70	490	6.11	31	1.03
	N40	72	512	5.86	30	1.06
	N80	73	576	6.06	34	1.24
	N120	72	527	6.80	39	1.24
	N160	79	544	6.53	36	1.08
P40	N0	70	546	6.18	33	1.07
	N40	79	533	6.50	33	1.16
	N80	84	499	7.06	40	1.06
	N120	82	532	6.27	31	1.08
	N160	78	527	6.81	36	1.17
P80	N0	75	518	5.91	30	1.04
	N40	78	533	6.53	36	1.19
	N80	84	567	6.61	38	1.10
	N120	86	557	7.07	39	1.11
	N160	83	509	7.12	39	1.17
P120	N0	78	542	5.73	27	1.13
	N40	77	527	6.32	32	1.15
	N80	84	535	6.44	33	1.19
	N120	88	535	7.10	37	1.14
	N160	91	550	7.35	40	1.13
P160	N0	69	528	5.97	29	1.15
	N40	81	566	6.32	32	1.09
	N80	74	526	6.67	32	1.13
	N120	87	571	6.95	39	1.25
	N160	86	552	7.34	42	1.13

No statistical comparisons were performed for plant height, spike density per m², spike length, number of grains per spike, and grain weight per spike, as these traits were presented as mean values, primarily to highlight the general trends in plant response to the application of different fertilizer rates. Consequently, the differences among the experimental treatments are interpreted descriptively and are not considered statistically significant. The results presented in Table 1 are expressed as averages calculated across all experimental years included in the study.

3.2. Effects of Year and Nitrogen and Phosphorus Fertilization on Yield

Factorial analysis of variance revealed highly significant effects of year (Y), phosphorus (P), and nitrogen (N) on winter wheat grain yield (p < 0.001). Significant interactions were also observed for Y × P, Y × N, P × N, and Y × P × N (p < 0.001), demonstrating that the response of grain yield to N and P fertilization varied among growing seasons (Table 2). The particularly strong year effect was also reflected in the mean grain yields, which were 5145 kg ha⁻¹ in 2023, 5175 kg ha⁻¹ in 2024, and 7771 kg ha⁻¹ in 2025.

Table 2. Analysis of variance for the effects of year and phosphorus and nitrogen fertilization on winter wheat grain yield.

Source of Variation	SS	df	MS	F	p-Value
Year (Y)	568,196,200	2	284,098,100	808.716	<0.001
Phosphorus (P)	35,765,510	4	8,941,378	75.716	<0.001
Y × P	26,594,820	8	3,324,353	28.151	<0.001
Nitrogen (N)	30,776,160	4	7,694,040	141.078	<0.001
Y × N	7,706,322	8	963,290.2	17.663	<0.001
P × N	6,675,561	16	417,222.6	7.650	<0.001
Y × P × N	19,320,730	32	603,772.7	11.071	<0.001
Error Y	2,810,363	8	351,295.3
Error P	5,668,405	48	118,091.8
Error N	13,088,990	240	54,537.46
Total	695,035,110	74

SS—sum of squares; df—degrees of freedom; MS—mean square; F—F-value; Y—year; P—phosphorus; N—nitrogen. All main effects and interactions were significant at p < 0.001.

The significant Y × P interaction showed distinct phosphorus responses among growing seasons (Table 3). In 2023, grain yield increased only moderately with increasing P rates, from 4933 kg ha⁻¹ at P₀ to 5276 kg ha⁻¹ at P₁₆₀, with no significant difference between P₁₂₀ and P₁₆₀. In 2024, the response to phosphorus was considerably stronger, with grain yield increasing from 4532 kg ha⁻¹ at P₀ to 6154 kg ha⁻¹ at P₁₆₀. In contrast, in 2025, the highest yields were obtained at P₈₀ (8101 kg ha⁻¹) and P₁₂₀ (7993 kg ha⁻¹), which did not differ significantly, whereas increasing the P rate to P₁₆₀ reduced yield to 7784 kg ha⁻¹. These results indicate that the yield response to phosphorus was strongly dependent on growing-season conditions.

Table 3. Interaction between growing season and phosphorus rate in winter wheat grain yield (kg ha⁻¹).

Year	P0	P40	P80	P120	P160
2023	4933 B	5112 BCD	5151 CD	5253 D	5276 D
2024	4532 A	4550 A	5032 BC	5605 E	6154 F
2025	7231 G	7744 H	8101 I	7993 I	7784 H

P₀, P₄₀, P₈₀, P₁₂₀, and P₁₆₀ represent phosphorus application rates of 0, 40, 80, 120, and 160 kg P₂O₅ ha⁻¹, respectively. Different letters indicate significant differences among means according to Duncan’s multiple-range test (p ≤ 0.05).

A significant Y × N interaction was also observed (Table 4). In 2023, grain yield increased from 4597 kg ha⁻¹ at N₀ to a numerical maximum of 5418 kg ha⁻¹ at N₁₂₀, with no further yield improvement at N₁₆₀. In 2024, yield increased progressively with N supply, reaching 5514 kg ha⁻¹ at N₁₆₀; however, N₁₂₀ and N₁₆₀ were not significantly different. A different response occurred in 2025, when N₈₀ produced the highest grain yield (8294 kg ha⁻¹) and was significantly superior to all other N rates. Increasing the nitrogen rate beyond N₈₀ did not improve productivity, with yields declining to 7869 kg ha⁻¹ at N₁₂₀ and 7732 kg ha⁻¹ at N₁₆₀. Thus, the nitrogen rate associated with maximum yield differed among growing seasons.

Table 4. Interaction between growing season and nitrogen rate in winter wheat grain yield (kg ha⁻¹).

Year	N0	N40	N80	N120	N160
2023	4597 A	5105 D	5270 EF	5418 GH	5335 EFG
2024	4792 B	4951 C	5236 DE	5380 FGH	5514 H
2025	7093 I	7865 K	8294 L	7869 K	7732 J

N₀, N₄₀, N₈₀, N₁₂₀, and N₁₆₀ represent nitrogen application rates of 0, 40, 80, 120, and 160 kg N ha⁻¹, respectively. Different letters indicate significant differences among means according to Duncan’s multiple-range test (p ≤ 0.05).

The P × N interaction significantly influenced winter wheat grain yield. The highest three-year mean yield was obtained with the P₁₆₀N₈₀ treatment (6897 kg ha⁻¹), which was significantly higher than all other fertilization combinations. High yields were also recorded for P₁₂₀N₁₂₀ (6555 kg ha⁻¹) and P₁₆₀N₁₂₀ (6540 kg ha⁻¹), followed by P₁₆₀N₁₆₀ (6436 kg ha⁻¹), P₁₆₀N₄₀ (6417 kg ha⁻¹), and P₁₂₀N₈₀ (6411 kg ha⁻¹). At lower phosphorus rates, increasing nitrogen generally increased grain yield, although the response was not consistently linear. At P₀, yield increased from 4703 kg ha⁻¹ at N₀ to 5971 kg ha⁻¹ at N₁₆₀, whereas at P₁₆₀, the highest yield was reached at N₈₀, followed by a decline at N₁₂₀ and N₁₆₀. These results demonstrate a clear interaction between nitrogen and phosphorus fertilization and indicate that the yield response to increasing nitrogen depended on the level of phosphorus supply (Table 5).

Table 5. Interaction between phosphorus and nitrogen fertilization (N × P) on three-year mean winter wheat grain yield (kg ha⁻¹).

	N0	N40	N80	N120	N160
P0	4703 A	5563 BC	5867 DEF	5722 CD	5971 FG
P40	5395 B	5681 CD	5853 DEF	6106 GH	5976 FG
P80	5722 CD	6040 FGH	6306 IJK	6190 HIJ	6217 HIJ
P120	5916 EF	6167 HI	6411 KL	6555 L	6370 JKL
P160	5734 CDE	6417 KL	6897 M	6540 L	6436 KL

P₀, P₄₀, P₈₀, P₁₂₀, and P₁₆₀ represent phosphorus application rates of 0, 40, 80, 120, and 160 kg P₂O₅ ha⁻¹, respectively. N₀, N₄₀, N₈₀, N₁₂₀, and N₁₆₀ represent nitrogen application rates of 0, 40, 80, 120, and 160 kg N ha⁻¹, respectively. Different letters indicate significant differences among means according to Duncan’s multiple-range test (p ≤ 0.05).

Pearson’s correlation analysis revealed significant positive relationships between grain yield and several yield-related traits in winter wheat (Table 6). Grain yield was strongly positively correlated with plant height (r = 0.722, p < 0.001), indicating that greater vegetative growth was associated with higher productivity. Significant positive correlations were also observed between grain yield and spike density (r = 0.479, p < 0.05), as well as between grain yield and spike length (r = 0.530, p < 0.01), highlighting the contribution of these traits to yield formation. Although positive, the correlations between grain yield and grain weight per spike (r = 0.372) and between grain yield and the number of grains per spike (r = 0.368) were not statistically significant. The strongest association was observed between spike length and the number of grains per spike (r = 0.919, p < 0.001), suggesting that spike development directly influenced grain set.

Table 6. Pearson correlation coefficients (r) between yield and yield-related traits of winter wheat.

	Yield	Plant Height (cm)	Spikes per Square Meter	Spike Length	Grain Weight per Spike	Grains per Spike
Yield	-
Plant Height (cm)	0.722 ***	-
Spikes per Square Meter	0.479 *	0.266	-
Spike Length	0.530 **	0.770 ***	0.126	-
Grain Weight per Spike	0.372	0.167	0.395	0.263	-
Grains per Spike	0.368	0.629 ***	0.230	0.919 ***	0.302	-

Symbols *, **, and *** indicate significant correlation coefficients at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001.

Quadratic Regression Analysis of Grain Yield Response to N and P Fertilization

To further evaluate the grain yield response to nitrogen and phosphorus fertilization, second-order polynomial regression models were developed separately for each growing season. The models included the linear effects of N and P, their quadratic terms (N² and P²), and the N × P interaction (Table 7). All models were highly significant (p < 0.001), although their ability to explain yield variation differed among years. The highest coefficient of determination was recorded in 2024 (R² = 0.799), followed by 2023 (R² = 0.687) and 2025 (R² = 0.606), showing that the relationship between grain yield and N and P fertilization varied considerably from one growing season to another.

Table 7. Second-order polynomial regression models describing winter wheat grain yield response to nitrogen and phosphorus fertilization during 2023–2025.

Year	Regression Equation	R2	Adjusted R2	F	p
2023	Yield = 4389.4 + 13.362N + 4.1618P − 0.053589N2 − 0.011132P2 − 0.0038988(N × P)	0.687	0.674	52.284	<0.001
2024	Yield = 4285.2 + 3.4435N − 0.075257P − 0.0084857N2 + 0.051443P2 + 0.032435(N × P)	0.799	0.791	94.839	<0.001
2025	Yield = 6090.9 + 28.515N + 23.257P − 0.11927N2 − 0.08529P2 − 0.077828(N × P)	0.606	0.589	36.602	<0.001

N—nitrogen application rate (kg N ha⁻¹); P—phosphorus application rate (kg P₂O₅ ha⁻¹); Yield—grain yield (kg ha⁻¹); R²—coefficient of determination; Adjusted R²—adjusted coefficient of determination; F—F-statistic of the regression model; p—probability value.

In 2023, the model showed that yield increased with nitrogen up to a certain level, with the highest predicted yield occurring at about 119 kg N ha⁻¹ and 160 kg P₂O₅ ha⁻¹. In 2024, no clear optimum N × P combination was identified within the tested range. In 2025, the model indicated a maximum yield at about 88 kg N ha⁻¹ and 96 kg P₂O₅ ha⁻¹. These results show that the N and P rates associated with maximum yield differed among growing seasons.

3.3. Nitrogen and Phosphorus Use Efficiency

Nitrogen use efficiency decreased consistently with increasing N application rates in all growing seasons. NUE ranged from 123.78 to 196.62 kg grain kg⁻¹ N at N40 and decreased to 33.34–48.33 kg grain kg⁻¹ N at N160. The highest NUE values were recorded in 2025 (Table 8). A similar decline with increasing fertilizer rate was observed for PUE, although the magnitude of the response differed among growing seasons (Table 9).

Table 8. Nitrogen use efficiency (NUE) of winter wheat as affected by nitrogen application rate and growing season.

Year	N Rate (kg N ha−1)	Yield (kg ha−1)	NUE (kg Grain kg−1 N)
2023	40	5105.12	127.63
	80	5270.24	65.88
	120	5418.16	45.15
	160	5335.12	33.34
2024	40	4951.08	123.78
	80	5235.60	65.45
	120	5380.28	44.84
	160	5513.88	34.46
2025	40	7864.90	196.62
	80	8294.30	103.68
	120	7869.00	65.58
	160	7732.20	48.33

Table 9. Phosphorus use efficiency (PUE) of winter wheat as affected by phosphorus application rate and growing season.

Year	P Rate (kg P2O5 ha−1)	Yield (kg ha−1)	PUE (kg Grain kg−1 P2O5)
2023	40	5112.36	127.81
	80	5151.04	64.39
	120	5252.84	43.77
	160	5276.24	32.98
2024	40	4549.96	113.75
	80	5032.12	62.90
	120	5605.24	46.71
	160	6154.16	38.46
2025	40	7744.30	193.61
	80	8101.30	101.27
	120	7993.20	66.61
	160	7783.60	48.65

3.4. Effects of Year and Nitrogen and Phosphorus Fertilization on Thousand-Kernel Weight and Test Weight

The combined application of nitrogen and phosphorus fertilizers significantly influenced grain physical characteristics and yield components. Thousand-kernel weight (TKW), an important yield component, ranged from 30.15 to 37.55 g, with the highest values observed in treatments receiving higher nitrogen and phosphorus application rates. Test weight (TW), an important indicator of grain physical quality and market value, showed less variation among treatments, ranging from 76.97 to 80.07 kg hL⁻¹ (Figure 5).

Figure 5. Effect of nitrogen and phosphorus fertilization on thousand-kernel weight (TKW) and hectoliter weight (HW) of winter wheat.

3.5. Effect of Fertilization on Grain Quality

Nitrogen fertilization had a pronounced effect on grain quality. Protein content increased progressively from 9.15% at N₀ to 14.20% at N₁₆₀, while wet gluten content increased from 17.29% to 28.97%. In contrast, starch content decreased progressively from 70.95% at N₀ to 66.91% at N₁₆₀. All nitrogen levels differed significantly according to Duncan’s multiple-range test. Phosphorus fertilization also significantly affected the three quality parameters, although the response was not progressive with increasing P rate. The highest protein and wet gluten contents were recorded at P₄₀, whereas starch content varied only slightly among phosphorus treatments. Mean values among years were very similar for all three parameters (Table 10).

Table 10. Effect of year and phosphorus and nitrogen fertilization on grain protein, wet gluten and starch content of winter wheat.

Variable	Protein (%)	Wet Gluten (%)	Starch (%)
2023	11.78 A	23.36 AB	69.09 A
2024	11.66 A	23.14 A	69.07 A
2025	11.84 A	23.42 B	69.14 A
P0	11.97 D	23.79 D	69.13 D
P40	12.02 E	23.93 E	68.92 A
P80	11.56 B	22.85 B	69.07 B
P120	11.71 C	23.19 C	69.08 C
P160	11.53 A	22.78 A	69.30 E
N0	9.15 A	17.29 A	70.95 E
N40	10.09 B	19.43 B	70.69 D
N80	11.70 C	23.17 C	69.55 C
N120	13.65 D	27.66 D	67.39 B
N160	14.20 E	28.97 E	66.91 A

P₀, P₄₀, P₈₀, P₁₂₀, and P₁₆₀ represent phosphorus application rates of 0, 40, 80, 120, and 160 kg P₂O₅ ha⁻¹, respectively. N₀, N₄₀, N₈₀, N₁₂₀, and N₁₆₀ represent nitrogen application rates of 0, 40, 80, 120, and 160 kg N ha⁻¹, respectively. Different letters indicate significant differences among means according to Duncan’s multiple-range test (p ≤ 0.05).

Correlation analysis revealed strong associations among the principal winter wheat quality parameters. Protein content, wet gluten content, the Zeleny sedimentation index, and grain hardness were all strongly and positively correlated with one another (r = 0.924–1.000; p < 0.001), demonstrating that these traits are closely associated and represent key determinants of wheat bread-making quality. In contrast, starch content was strongly and negatively correlated with all protein-related quality parameters, indicating that increased starch accumulation is associated with a decline in the technological quality of the grain (Table 11).

Table 11. Pearson correlation coefficients (r) between yield and wheat quality parameters.

	Yield	Protein (%)	Wet Gluten (%)	Starch (%)	Zeleny (mL)	Hardness (%)
Yield	-
Protein (%)	0.467	-
Wet gluten (%)	0.465 *	1.000 ***	-
Starch (%)	−0.419 0	−0.978 000	−0.978 000	-
Zeleny (mL)	0.481 *	0.965 ***	0.965 ***	−0.903 000	-
Hardness (%)	0.512 **	0.924 ***	0.924 ***	−0.852	0.961 ***	-

Symbols *, **, and *** indicate significant correlation coefficients at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, while ⁰ and ⁰⁰⁰ indicate significant negative correlations at p ≤ 0.05 and p ≤ 0.001.

3.6. Effect of Fertilization on Soil Properties

The results showed that increasing nitrogen application rates progressively reduced soil pH, indicating a gradual acidification of the soil. The highest pH values were observed in treatments that did not receive nitrogen fertilization, ranging from 5.49 to 5.75, whereas the application of the highest nitrogen rate (160 kg N ha⁻¹) reduced soil pH to between 5.33 and 5.51 (Table 12). Soil total nitrogen content was positively affected by nitrogen fertilization and generally increased with increasing nitrogen application rates. The measured values ranged from 0.123% in the unfertilized control treatment to 0.174%, with the highest concentration recorded in the P₁₂₀N₁₆₀ treatment.

Table 12. Effects of nitrogen and phosphorus fertilization on soil chemical properties.

A Factor	B Factor	pH	N %	P mg/kg as
P0	N0	5.75	0.123	41
	N40	5.53	0.152	57
	N80	5.42	0.157	41
	N120	5.42	0.154	37
	N160	5.33	0.154	37
P40	N0	5.49	0.133	75
	N40	5.33	0.155	89
	N80	5.47	0.160	92
	N120	5.53	0.155	78
	N160	5.39	0.156	84
P80	N0	5.71	0.151	177
	N40	5.52	0.154	156
	N80	5.6	0.17	168
	N120	5.61	0.149	170
	N160	5.47	0.149	180
P120	N0	5.67	0.123	165
	N40	5.61	0.135	157
	N80	5.53	0.157	183
	N120	5.44	0.158	174
	N160	5.51	0.174	184
P160	N0	5.6	0.137	242
	N40	5.56	0.143	234
	N80	5.54	0.140	219
	N120	5.40	0.163	253
	N160	5.33	0.145	251

Available phosphorus content responded primarily to phosphorus fertilization, increasing significantly with higher phosphorus application rates. Specifically, available phosphorus increased from 41 mg kg⁻¹ in the unfertilized control treatment (P₀N₀) to more than 200 mg kg⁻¹ in the treatment receiving the highest phosphorus rate (P₁₆₀) (Table 12).

4. Discussion

Throughout the winter wheat growing season, climatic factors play a fundamental role in determining crop productivity. They directly influence crop growth and yield by providing the temperature and moisture conditions required for plant development, while also exerting an indirect effect by promoting the absorption and utilization of soil nutrients.

According to Mahfoozi et al. [42], the influence of temperature varies across the different growth stages of wheat; however, it remains a key factor throughout the entire plant life cycle [43]. An optimum temperature range of 17–23 °C has been reported for the vegetative growth of wheat, whereas 0 °C and 37 °C are considered the lower and upper tolerance limits, respectively. In addition, previous studies have highlighted the remarkable ability of wheat to adapt to extreme temperature fluctuations [44].

The results of long-term experiments have demonstrated that the combined application of nitrogen and phosphorus significantly increases wheat yield and contributes to greater yield stability over time compared with the application of either nutrient alone [45,46,47]. Furthermore, the yield response of wheat to fertilization has been shown to depend on both soil fertility status and the climatic conditions of a given region [48,49].

Balanced N–P fertilization plays a crucial role in maximizing and maintaining stable wheat yields. When phosphorus availability is adequate, nitrogen application at rates of 80–160 kg ha⁻¹ can significantly enhance grain yield, even under unfavorable environmental conditions [50].

The decline in thousand-kernel weight (TKW) and test weight (TW) observed at nitrogen application rates above 120 kg N ha⁻¹ can be attributed to excessive vegetative growth, which promotes the allocation of assimilates to leaves and stems at the expense of grain filling. Furthermore, excessive nitrogen application may induce nutritional imbalances and reduce phosphorus use efficiency, thereby limiting dry matter accumulation in the grain and, consequently, decreasing grain weight and density [12,51].

Numerous studies have shown that nitrogen fertilization stimulates protein accumulation in the grain and increases wheat yield; however, nitrogen use efficiency is strongly influenced by phosphorus availability [8,52,53]. At the same time, long-term phosphorus fertilization contributes to maintaining plant-available phosphorus reserves in the soil and supports the productivity of intensive agricultural systems [54,55].

In the context of rising fertilizer costs and increasing demands to reduce environmental impacts, optimizing nitrogen and phosphorus application rates has become a key priority for the development of sustainable agricultural practices [17,56,57]. Recent studies have also demonstrated that the response of cereal crops to fertilization is closely related to environmental variability and to the ability of different genotypes to efficiently utilize available resources, thereby influencing both crop yield and grain quality parameters [58,59].

The marked decline in NUE with increasing N application rates indicates progressively lower grain yield per unit of applied nitrogen. This response was particularly evident at the highest N rates, where additional fertilizer produced only limited or no yield benefit. Together with the decrease in soil pH observed under higher N rates, these results indicate that increasing N input beyond the level required for maximum yield may reduce fertilizer-use efficiency and increase potential environmental risks.

Winter wheat yield is determined by the interaction among its principal yield components, namely spike density per unit area, the number of grains per spike, and thousand-kernel weight (TKW). The compensatory relationships among these components vary according to genotype and growing conditions, including nutrient availability [60].

In long-term experiments, the evaluation of the relationships between yield components and nitrogen and phosphorus application rates provides valuable insights into the mechanisms underlying yield formation and facilitates the identification of efficient fertilization strategies adapted to local conditions.

The statistical analysis showed that nitrogen fertilization had a pronounced effect on grain quality. Protein content increased progressively from 9.15% at N₀ to 14.20% at N₁₆₀, while wet gluten content increased from 17.29% to 28.97%. In contrast, starch content decreased from 70.95% at N₀ to 66.91% at N₁₆₀. Phosphorus fertilization also significantly influenced these quality parameters, although its effect was not progressive with increasing P rate. The highest protein and wet gluten contents were recorded at P₄₀, whereas starch content showed relatively small variations among phosphorus treatments.

The highest three-year mean grain yield recorded for the N₈₀P₁₆₀ treatment may reflect a favorable balance between nitrogen and phosphorus availability under the conditions of the experiment. The significant interactions with year indicate that this combination should not be considered universally optimal. At higher nitrogen rates (N₁₂₀–N₁₆₀), the additional yield response became progressively smaller, suggesting that nitrogen was no longer the main factor limiting grain production and that other environmental, nutritional, or cultivar-related factors became more important. This interpretation is also supported by the decline in nitrogen use efficiency with increasing N rates. In contrast, protein and wet gluten contents continued to increase at higher nitrogen rates, suggesting that additional nitrogen contributed increasingly to grain protein accumulation rather than to further increases in grain yield.

In other studies, a high nitrogen contribution has also been reported to increase protein content, gluten, and protein fractions [8]. Correlation analysis revealed a very strong relationship between nitrogen application and the protein and wet gluten contents of wheat grain, confirming the essential role of nitrogen in improving grain quality. In contrast, phosphorus exhibited a more moderate influence on the quality parameters evaluated [61].

An et al. [62] reported that nitrogen fertilization increased grain protein content by more than 25%. Similarly, Landolfi et al. [63] documented increases of 15–18% following the application of 80 and 160 kg N ha⁻¹, respectively, while Mor et al. [64] observed a 12% increase in grain protein content at nitrogen rates ranging from 120 to 160 kg N ha⁻¹. Phosphorus fertilization has also been reported to positively influence grain protein content, although to a lesser extent, with increases ranging from 4% to 11% [65].

The results indicate that increasing nitrogen application rates progressively reduced soil pH, reflecting a gradual trend toward soil acidification. The highest soil pH values were observed in treatments that did not receive nitrogen fertilization, whereas the application of the highest nitrogen rate resulted in a decrease in soil pH.

Previous long-term studies have shown that nitrogen (N) and phosphorus (P) fertilization did not exert a significant effect on soil pH, nitrate (NO₃⁻–N), ammonium (NH₄⁺–N), or available potassium (K) concentrations. Nevertheless, both grain yield and phosphorus uptake by crops increased significantly in response to the application of N and P fertilizers [66]. Therefore, identifying optimal combinations of nitrogen and phosphorus application rates is essential, as the most effective fertilization strategy varies according to site-specific conditions and the environmental characteristics of each growing season in long-term wheat production systems [67,68].

Sun et al. [69] reported that nitrogen fertilization significantly decreased soil pH, with higher N application rates generally associated with greater reductions in soil pH. These findings indicate that increasing nitrogen availability can contribute to soil acidification, particularly under higher fertilization rates.

5. Conclusions

Grain yield of the Otilia cultivar increased progressively with increasing fertilizer application rates until the optimum application range was reached. All fertilized treatments produced significantly higher yields than the unfertilized control (P₀N₀), highlighting the essential role of mineral nutrition in enhancing the productivity of this winter wheat cultivar under the experimental conditions.

The results also demonstrated that increasing fertilization intensity, particularly through higher nitrogen application rates, significantly improved the principal quality characteristics of the Otilia cultivar. Protein content and wet gluten content increased progressively with increasing nitrogen application rates, whereas starch content exhibited a declining trend. In addition, the Zeleny sedimentation index and grain hardness were positively influenced by higher nitrogen application rates, reaching values substantially greater than those observed in the control treatment, particularly when nitrogen was applied in combination with phosphorus.

Since only the Otilia cultivar was evaluated, the absence of genotype comparison represents a limitation of the present study. Therefore, the present findings should be considered specific to the soil, crop rotation, cultivar, and environmental conditions of the experimental site. Further multi-site and multi-cultivar studies are needed to determine whether similar responses occur under different soil and climatic conditions.