Impact of Nitrogen Fertilisation and Inoculation on Soybean Nodulation, Nitrogen Status, and Yield in a Central European Climate

Abstract

Soybean (Glycine max [L.] Merr.) cultivation is expanding in Central Europe due to the development of early-maturing cultivars and growing demand for plant-based protein produced without the use of genetically modified organisms. However, nitrogen (N) management remains a major challenge in temperate climates, where variable weather conditions can significantly affect nodulation and yield. This study evaluated the effects of three nitrogen fertilisation doses (0, 30, and 60 kg N·ha⁻¹), applied in the form of ammonium nitrate (34% N) and two commercial rhizobial inoculants—HiStick Soy (containing Bradyrhizobium japonicum strain 532C) and Nitragina (including a Polish strain of B. japonicum)—on nodulation, nitrogen uptake, and seed yield. A three-year field experiment (2017–2019) was conducted in southwestern Poland using a two-factor randomized complete block design. Nodulation varied significantly across years, with the highest values recorded under favourable early-season moisture and reduced during drought. In the first year, inoculation with HiStick Soy significantly increased nodule number and seed yield compared to Nitragina and the uninoculated control. Nitrogen fertilisation consistently improved seed yield, although it had no significant effect on nodulation. The highest nitrogen use efficiency was observed with moderate nitrogen input (30 kg N·ha⁻¹) combined with inoculation. These findings highlight the importance of integrating effective rhizobial inoculants with optimized nitrogen fertilisation to improve soybean productivity and nitrogen efficiency under variable temperate climate conditions.

1. Introduction

Soybean (Glycine max [L.] Merr.) is a globally important legume crop, valued for its high protein content, balanced amino acid profile, and wide applicability in both animal feed and human nutrition [1]. From 1961 to 2023, global soybean cultivation expanded from 23.8 million to over 133.8 million hectares, accompanied by a significant increase in yield from 1.12 to 2.71 t·ha⁻¹ [2]. In recent years, interest in soybean cultivation has grown rapidly in Europe, including Central and Eastern countries such as Poland, driven by the development of early-maturing cultivars adapted to temperate climates [3]. In Poland, there has been an increase in soybean cropping from a negligible scale before 2000 to over 18,000 hectares in 2023 [2].

This expansion is also motivated by the need to reduce dependence on genetically modified (GMO) soybean imports, which cultivations remain restricted across many EU countries, yet dominate feed use (approximately 19 million tonnes imported annually in the EU) [4]. Therefore, expanding non-GMO soybean production in Europe aligns with broader goals of food security, sustainability, and climate resilience.

Plant available nitrogen (N) is a major determinant of soybean yield and quality. While soybean is capable of meeting much of its nitrogen requirement through symbiotic biological nitrogen fixation (BNF) with Bradyrhizobium spp., the efficiency of this process is sensitive to various agronomic and environmental factors [5,6]. Among these, soil temperature, water availability, and nitrogen fertilisation interact strongly with root nodulation and nitrogen acquisition [7,8,9,10]. In cooler climates, such as those in Central Europe, suboptimal root-zone temperatures and weather instability can limit nodulation and BNF, posing a major constraint to protein yield and nitrogen efficiency [11].

While numerous studies have assessed the response of soybean to nitrogen fertilisation and inoculation in warmer or more stable agroecological zones, relatively few have addressed the interactions between nitrogen inputs, root nodule development, and nitrogen partitioning in field-grown soybeans under temperate conditions in Central Europe. Therefore, there is still high need to investigate this problem. Moreover, the relationship between the number and mass of root nodules and nitrogen accumulation in reproductive (seeds) and vegetative (straw) tissues remains underexplored, despite its significance for nitrogen use efficiency (NUE) and sustainable yield optimization [5,12].

There is also ongoing debate about the optimal nitrogen strategy for soybean cultivation in moderate climates. While some studies report a clear inhibition of nodulation and BNF at high nitrogen levels [13,14], others have observed neutral or even transiently positive effects under specific conditions, such as early growth stages or limited nodulation capacity [15].

Optimal nitrogen fixation efficiency requires adequate numbers of compatible and effective rhizobia species, which are present in the rhizosphere. Rhizobia that colonize legume root nodules originate either from commercial inoculants applied to seeds at planting or from indigenous soil populations [16]. It is worth underlining that in Poland there is still not enough population of B. japonicum in arable soils; therefore, seed producers or farmers need to inoculate seed material themselves.

This study aimed to address current knowledge gaps regarding soybean cultivation under Central European conditions, where suboptimal temperatures, low indigenous rhizobial populations, and variable soil fertility often limit effective nodulation and biological nitrogen fixation (BNF). Although rhizobial inoculation and nitrogen fertilisation are widely recognized as agronomic practices, their interactive effects on nodulation efficiency, nitrogen uptake, and yield remain insufficiently explored in this region. To this end, a three-year field experiment was conducted in southwestern Poland to evaluate how varying levels of nitrogen fertilisation (0, 30, and 60 kg N·ha⁻¹) and seed inoculation with commercial B. japonicum preparations (HiStick Soy and Nitragina) influence soybean performance. We hypothesized that moderate nitrogen input, combined with effective rhizobial inoculation, would enhance nodule biomass and nitrogen use efficiency without substantially suppressing BNF, ultimately improving nitrogen accumulation and seed yield. By providing region-specific data, this study contributes to the optimization of sustainable nitrogen management strategies for soybean production in Central European agroecosystems. This experiment provides also the first multi-seasonal field-based comparison of the two commercial B. japonicum inoculants—HiStick Soy and Nitragina—with respect to both nodule biomass and nitrogen harvest index (NHI) in Central European conditions. Results demonstrate not only the distinct nitrogen partitioning efficiencies of each inoculant but also their variable seasonal performance.

2. Materials and Methods

2.1. Site Description, Experimental Design, and Weather and Soil Conditions

A three-year field experiment was established in 2017–2019 at the experimental fields of the Institute of Agroecology and Plant Production, Wrocław University of Environmental and Life Sciences, located in Pawłowice, Poland (51°11′ N, 17°85′ E). The research was designed as a two-factor randomized complete block design (RCBD) with four replications. The experimental treatments included nitrogen fertilisation (0, 30, and 60 kg N·ha⁻¹) and seed inoculation (no inoculation, HiStick Soy, and Nitragina), so it creates nine research combinations: 0 N + no inoculation, 0 N + Hi Stick Soy inoculation, 0 N + Nitragina inoculation, 30 N + no inoculation, 30 N + Hi Stick Soy inoculation, 30 N + Nitragina inoculation, 60 N + no inoculation, 60 N + Hi Stick Soy inoculation, 60 N + Nitragina inoculation.

HiStick Soy is widely used in soybean cultivation in many countries. It contains live cultures of Rhizobium group bacteria (B. japonicum). This product contains at least 2 × 10⁹ (at least 2 billion) live bacteria for use in soybean cultivation per gram of peat substrate. The original polymer was added to the peat substrate at a low concentration to ensure adhesion and safety.

Nitragina is a Polish inoculant containing live papillary bacteria of the genus Bradyrhizobium capable of fixing free atmospheric nitrogen in symbiosis with legumes. Perlite was used as a carrier for the bacteria. The standard content of live bacteria is 108 (one hundred million).

The inoculants certainly differed in composition, i.e., bacterial strains within B. japonicum. Both inoculants were typical commercial products commonly used in soybean cultivation in Poland during the years of field tests.

The field experiment was conducted on soil classified as a Luvisol according to the FAO/WRB classification. In the Polish national categorization system, the soil is described as autogenic, belonging to the brown earths order (Cambisols), lessivated type, typical subtype. The soil developed from light clay overlying medium clay and belongs to soil quality class IIIb, categorized as part of the “good wheat complex” in terms of agricultural suitability.

Soil samples were collected annually between 2017 and 2019 from the 0–20 cm topsoil layer. The samples were analysed for available macronutrient content and soil reaction. Available phosphorus (P), potassium (K), and magnesium (Mg) were determined using the Egner–Riehm method (DL version). Soil pH was measured potentiometrically in a 1 M KCl solution.

Based on the averaged results (Supplementary Materials Table S1), the soil at the experimental site was characterized by a high content of available phosphorus, very high levels of potassium and magnesium, and a slightly acidic reaction, according to the classification criteria of the Institute of Soil Science and Plant Cultivation (IUNG-PIB), Puławy, Poland [17].

Weather conditions during the years of the study (average monthly temperatures and monthly precipitation sums), due to their relation to soybean nodulation, are presented in the Results section in the form of graphs. The data come from a weather station belonging to the Wroclaw University of Life Sciences located exactly on the experimental fields. For the presentation of weather conditions, average daily temperatures and daily precipitation sums were used and converted to monthly values.

2.2. Agronomic Practice

Soybean was cultivated including recommended crop rotation, each year in different plot sites but always in the same field. Winter wheat was used as the previous crop in each year of the study and was harvested at the beginning of August. Following its harvest, the field was cultivated with a chisel plough and subsequently subjected to traditional winter ploughing. In spring, the soil was harrowed. Immediately prior to sowing, seedbed preparation was performed using a rotary harrow combined with a packer roller.

On the day of sowing, phosphorus and potassium fertilisation was applied at the following rates: 60 kg P₂O₅·ha⁻¹ in the form of triple superphosphate (40%) and 120 kg K₂O·ha⁻¹ in the form of potassium salt (60%).

Seeds were inoculated with HiStick Soy or Nitragina according to the field experiment design prior to sowing.

Soybean seeds of the cultivar Annushka were used at a sowing density of 90 seeds·m⁻². Annushka is an early cultivar, recommended for cultivation through Poland. Its growing season lasts 100–130 days. It is characterized by 36–40% of protein and 17.5–21% of fat content in seeds. The weight of 1000 seeds of this cultivar is 110–155 g. The quality of the seed material, as well as sowing and harvest dates for each growing season (2017–2019), are presented in Table S2. Each year new certified seed material was used.

Soybean seeds were sown in 10 rows spaced 15 cm apart, using a Tool Carrier 2700 plot seeder (Wintersteiger, Austria) at a sowing depth of 3–4 cm. Each plot had an area of 15 m² (10 × 1.5 m).

Immediately after sowing, pre-emergence weed control was conducted using Boxer 800 EC (prosulfocarb 800 g·L⁻¹) at a rate of 4.0 L·ha⁻¹. Post-emergence control of monocotyledonous weeds was performed using a foliar application of Targa Super 05 EC (quizalofop-P-ethyl 50 g·L⁻¹) at a dose of 2.5 L·ha⁻¹. To manage secondary weed infestation, Corum 502.4 SL (bentazon 480 g·L⁻¹ + imazamox 22.4 g·L⁻¹) was applied in combination with the oil adjuvant Dash HC at rates of 1.2 L·ha⁻¹ and 0.6 L·ha⁻¹, respectively.

For soybean growing stages determining, a BBCH (Biologische Bundesanstalt Bundessortenamt und Chemische Industrie) scale was applied, which is commonly used in Europe for many crops.

At the beginning of the flowering stage (BBCH 61) the mass of root nodules was determined on all plots by sampling five plants from the central row. Due to soil structure and drought, plants were carefully excavated to a depth of only 20 cm; then all collected roots were gently washed with running water using sieves and drained. The sieves were necessary to gather all nodules. Nodules were then separated manually from the roots, air-dried until the mass was not changeable. The air-dry mass of nodules was expressed on a per-plant basis.

Soybean field experiment, each plot separately (15 m²), was harvested using a Seedmaster Universal Hydrostatic plot combine harvester (Austria). Seeds and straw from a single plot were collected into separate bags and next weighed. Then a sample of 100 g was taken to measure the content of water.

2.3. Chemical Analyses

Samples (100 g) of seeds and straw from each plot were taken and analysed by gravimetric method immediately after harvest to determine their moisture content in accordance with the PN-EN ISO 18134 standard [18].

Total nitrogen content (as an indicator of crude protein) was determined in harvested seeds and straw using a modified Kjeldahl’s method, which is based on combustion or mineralization of nitrogen compounds, followed by distillation and titration.

Additionally, two nitrogen efficiency indices were calculated to assess nitrogen partitioning and utilization in the crop.

The Nitrogen Harvest Index (NHI) was calculated using the following equation:

NHI (%) = (N accumulated in seeds (kg·ha⁻¹)/Total above-ground N uptake (kg·ha⁻¹)) × 100

The Nitrogen Use Efficiency (NUE), expressed as the ratio of seed yield to nitrogen applied, was calculated as:

NUE = Seed yield (kg·ha⁻¹)/Nitrogen applied (kg·ha⁻¹)

In addition, the proportion of nitrogen remaining in straw was calculated to estimate the fraction of above-ground nitrogen that was not remobilized to seeds. It was computed as the complement of NHI:

Straw N Share (%) = 100 − NHI (%)

These indices were used to evaluate nitrogen remobilisation to seeds and the effectiveness of nitrogen fertilisation under different treatment conditions.

2.4. Statistical Analysis and Graphs Preparation

In accordance with the central limit theorem, the obtained results were assumed to follow a normal distribution. Levene’s test was used to verify the homogeneity of variances. Data collected during the field experiment were analysed using analysis of variance (ANOVA). Statistical significance was determined at p ≤ 0.05 using one-way and two-way ANOVA followed by Tukey’s HSD test (Statistica 13.1, StatSoft, Kraków, Poland). Homogeneous groups were determined using Tukey’s multiple comparison test, with letters assigned starting from ‘a’ to indicate the highest values. Additionally, standard deviation (SD) was calculated.

Graphs were prepared using both Statistica 13.1 and Microsoft Excel 2010 software.

Periods of water deficit were identified using a modified Bagnouls–Gaussen criterion. According to this method, a month was considered dry when the total monthly precipitation (P) was less than four times the mean monthly temperature (T), i.e., P < 4T [19]. In the ombrothermic diagram, dry periods are highlighted in yellow, indicating months with insufficient precipitation to meet general plant water demands.

3. Results

3.1. Environmental Conditions and Root Nodulation Response

Weather conditions during the growing season differed markedly across the study years. The three-year average was characterized by rainfall deficits that persisted throughout most of the vegetation period. In 2017, rainfall shortages between May and July might have affected early developmental stages and the onset of pod formation. In 2018, soil moisture remained insufficient during nearly the entire growing season. In contrast, 2019 experienced favourable conditions at the beginning of the season (Figure 1).

Environmental conditions (year), inoculation, and their interaction (Y × I) significantly affected both the number and weight of root nodules (p < 0.0001). Nitrogen fertilisation did not influence the number of nodules; however, it significantly increased nodule weight, indicating an impact on biomass rather than nodule quantity. A significant year × fertilisation (Y × F) interaction was also observed for nodule weight. Other interactions, including fertilisation × inoculation (F × I) and the three-way interaction (Y × F × I), were not statistically significant (p > 0.05) (Table S3).

Among the treatments, HiStick Soy resulted in the highest nodule number, with a tenfold increase compared to the non-inoculated control. Nitragina also significantly increased both the number and weight of nodules, though to a lesser extent. After averaging across years, the highest number of nodules and biomass were recorded in 2017. On the other hand, the lowest values were observed in 2019, which corresponds to a deficit in rainfall during the flowering and pod formation stage (BBCH 65-79) (

Table 1. Effect of year, nitrogen fertilisation (kg N·ha⁻¹), and rhizobial inoculation on root nodule number and weight (mean values for main factors).

Fertilisation (kg N·ha−1)	Inoculation	Year	Root Nodule per Plant
			Number [pcs]	Weight [g]
0			5.36 ± 0.94a	60.9 ± 9.0a
30			4.29 ± 0.90a	49.2 ± 8.0b
60			4.58 ± 0.92a	51.8 ± 10.2ab
	Control		0.98 ± 0.18c	14.3 ± 1.9c
	HiStick Soy		9.46 ± 1.16a	91.5 ± 11.6a
	Nitragina		3.79 ± 0.32b	56.2 ± 5.2b
		2017	6.78 ± 1.17a	87.5 ± 11.6a
		2018	5.44 ± 0.86a	54.3 ± 5.4b
		2019	2.00 ± 0.32b	20.1 ± 4.7c

± means SD. Different letters indicate significant differences (Tukey’s multiple range test).

, Figure 1).

In 2017, HiStick Soy produced the highest nodule count (~17 nodules per plant), significantly exceeding both Nitragina and the control. But in 2019, no significant differences among treatments were observed. Nitragina consistently resulted in moderate nodule numbers (4–5 per plant), outperforming the control (1–2 per plant), which showed the lowest counts across all years. Statistically significant differences were detected in 2017 and 2018 but not in 2019 (Figure 2).

In terms of mass, the highest nodule weight in 2017 was recorded at 60 kg N·ha⁻¹. In subsequent years, nodule biomass declined with increasing nitrogen rates. In 2018, both the control and the 30 kg N·ha⁻¹ treatment outperformed the 60 kg N·ha⁻¹ variant. By 2019, biomass decreased across all nitrogen levels, with no significant differences (Figure 3).

In 2017, the use of HiStick Soy caused the highest nodule biomass (>170 g per plant), significantly surpassing both Nitragina and the control. However, its performance declined in the following years, aligning with Nitragina by 2018. Nitragina maintained relatively stable biomass levels (60–80 g per plant), whereas the control consistently exhibited the lowest values (<50 g per plant). By 2019, differences between treatments remained statistically significant, despite further declines in HiStick Soy performance (Figure 4).

3.2. Nitrogen Content

Total nitrogen content in seed, straw, and above-ground biomass was significantly influenced by the growing season conditions and rhizobial inoculation but not by nitrogen fertilisation (Table S4). Significant two-way interactions were observed for year × fertilisation (straw), year × inoculation (above-ground parts), and fertilisation × inoculation (straw and above-ground parts). A significant three-way interaction was noted only for straw nitrogen content (Table S4).

The most pronounced variation in nitrogen content was recorded in the above-ground plant parts, ranging from 25.5 g·kg⁻¹ in 2017 to 51.9 g·kg⁻¹ in 2019. In the case of straw, the highest nitrogen content was observed in the first year, while the lowest was recorded in the second. Rhizobial inoculation increased nitrogen accumulation in all plant components. HiStick Soy resulted in the highest nitrogen content across all measured fractions, while Nitragina significantly increased nitrogen content only in seeds. The control treatment consistently showed the lowest values. Mineral nitrogen fertilisation rates had no significant effect on nitrogen content (

Table 2. Effect of year, nitrogen fertilisation, and rhizobial inoculation on nitrogen content in dry mass of seed, straw, and above-ground plant parts (mean values for main factors).

Fertilisation (kg N·ha−1)	Inoculation	Year	N Content [g·kg −1]
			Seed	Straw	Above-Ground Parts of Plant
0			52.3 ± 1.0a	6.61 ± 0.28a	35.0 ± 2.1a
30			51.7 ± 1.2a	6.61 ± 0.27a	35.8 ± 2.2a
60			52.8 ± 1.1a	6.66 ± 0.23a	36.3 ± 2.0a
	Control		50.6 ± 1.0b	6.53 ± 0.24b	33.6b ± 2.1a
	HiStick Soy		53.0 ± 1.1a	6.88 ± 0.31a	37.9 ± 2.1a
	Nitragina		53.1 ± 1.1a	6.47 ± 0.23b	35.6 ± 2.1b
		2017	46.7 ± 0.7c	8.13 ± 0.15a	25.5 ± 0.8c
		2018	51.0 ± 0.6b	4.78 ± 0.10c	29.8 ± 0.5b
		2019	58.9 ± 0.7a	6.97 ± 0.08b	51.9 ± 0.9a

± means SD. Different letters indicate significant differences (Tukey’s multiple range test).

).

In 2017, straw nitrogen concentration was the highest, with values ranging from 8.0 to 8.5 g·kg⁻¹, and did not differ significantly between fertilisation levels. In 2018, a marked decline in straw nitrogen content was observed across all fertilisation rates, with the lowest values recorded during the study period (~4.5 g·kg⁻¹). In 2019, straw nitrogen levels partially recovered, with reduced variability across both fertilisation and inoculation treatments (Figure 5).

In 2017, the highest straw nitrogen concentration was observed under the HiStick Soy treatment, while significantly lower values were recorded for the Nitragina and control variants. In 2018, nitrogen content in straw remained low regardless of the inoculant used, with no statistically significant differences between treatments. A partial recovery in straw nitrogen content was recorded in 2019; however, no significant differences between inoculant treatments were observed in that year either (Figure 6).

HiStick Soy inoculation at a nitrogen rate of 30 kg N·ha−1 resulted in the highest straw nitrogen concentration, significantly exceeding values obtained with Nitragina. However, no consistent relationship was observed between nitrogen fertilisation rate and inoculation effect (Figure 7).

The highest straw nitrogen content (9.10 ± 0.78 g·kg⁻¹) was recorded in 2017 with HiStick Soy at 30 kg N·ha⁻¹, while the lowest value (4.10 ± 0.21 g·kg⁻¹) occurred in 2018 under the same nitrogen rate with Nitragina inoculation.

In general, straw nitrogen levels in 2017 were higher across all treatments, particularly under inoculated conditions (

Table 3. Effect of year, nitrogen fertilisation, and rhizobial inoculation on straw nitrogen content in dry mass [g·kg⁻¹].

Fertilisation (kg N·ha−1)	Inoculation	2017	2018	2019
	Control	8.00 ± 0.77abcde	4.30 ± 0.24h	6.70 ± 0.21ef
0	HiStick Soy	9.00 ± 0.28ab	4.60 ± 0.63gh	7.30 ± 0.41cdef
	Nitragina	8.00 ± 0.68abcde	4.60 ± 0.39gh	7.00 ± 0.60def
	Control	7.60 ± 0.40abcde	5.10 ± 0.27gh	6.80 ± 0.92def
30	HiStick Soy	9.10 ± 0.78a	4.90 ± 0.47gh	7.20 ± 0.38cdef
	Nitragina	7.50 ± 0.72bcde	4.10 ± 0.21h	7.20 ± 0.38cdef
	Control	8.30 ± 0.46abcd	5.10 ± 0.16gh	6.90 ± 0.66def
60	HiStick Soy	8.60 ± 1.17abc	4.40 ± 0.25gh	6.80 ± 0.38def
	Nitragina	7.10 ± 0.96cdef	5.90 ± 0.19fg	6.80 ± 0.22def

± means SD. Different letters indicate significant differences (Tukey’s multiple range test).

).

3.3. Soybean Yield

Analysis of variance revealed that seed yield was significantly affected by all main factors: year, nitrogen fertilisation, and inoculation (Table S5). The year of cultivation exerted a significant influence on all yield components, with particularly strong effects on straw and total above-ground biomass (p < 0.0001).

Nitrogen fertilisation significantly affected seed yield and total biomass but had no effect on straw yield. Rhizobial inoculation significantly influenced seed yield only (p < 0.0001).

Among two-way interactions, both year × fertilisation and year × inoculation significantly influenced seed yield (p = 0.0137 and p = 0.0083), respectively (Table S5).

The highest seed yield was recorded in 2019 (3.69 ± 0.04 t·ha⁻¹), while the lowest was observed in 2017 (2.26 ± 0.07 t·ha⁻¹). Seed yield increased progressively with higher nitrogen rates, from 2.77 ± 0.12 t·ha⁻¹ at 0 kg N·ha⁻¹ to 2.97 ± 0.11 t·ha⁻¹ at 60 kg N·ha⁻¹. Straw yield remained stable regardless of fertilisation, while above-ground biomass increased, reaching a maximum of 5.86 ± 0.12 t·ha⁻¹ at the highest nitrogen dose. Rhizobial inoculation had no significant effect on straw or biomass yield; however, both Nitragina and HiStick Soy significantly improved seed yield compared to the control (

Table 4. Effect of year, nitrogen fertilisation, and rhizobial inoculation on seed, straw, and above-ground biomass yield.

Fertilisation (kg N·ha−1)	Inoculation	Year	Yield [t·ha−1]
			Seed	Straw	Total
0			2.77 ± 0.12b	2.71 ± 0.08a	5.48 ± 0.11b
30			2.89 ± 0.11ab	2.74 ± 0.09a	5.63 ± 0.12ab
60			2.97 ± 0.11a	2.89 ± 0.09a	5.86 ± 0.12a
	Control		2.74 ± 0.12b	2.72 ± 0.08a	5.45 ± 0.12a
	HiStick Soy		3.00 ± 0.10a	2.74 ± 0.09a	5.73 ± 0.10a
	Nitragina		2.89 ± 0.12a	2.88 ± 0.09a	5.78 ± 0.13a
		2017	2.26 ± 0.07c	3.00 ± 0.10a	5.27 ± 0.14c
		2018	2.68 ± 0.03b	2.94 ± 0.05a	5.62 ± 0.07b
		2019	3.69 ± 0.04a	2.40 ± 0.07a	6.08 ± 0.10a

± means SD. Different letters indicate significant differences (Tukey’s multiple range test).

).

In 2017, the lowest yields were recorded across all fertilisation treatments, ranging from 2.0 to 2.6 t·ha⁻¹. With more beneficial yields in 2018, and with the highest values obtained in 2019, yields exceeded 3.5 t·ha⁻¹ regardless of nitrogen application rate (Figure 8).

In 2017, HiStick Soy resulted in the highest seed yield, whereas lower values were recorded for Nitragina and the uninoculated control. In 2018, seed yield increased across all treatments, with no significant differences observed between inoculants. A similar lack of significant differences was found in 2019, the year with the highest yields overall (Figure 9).

3.4. Nitrogen Uptake and Allocation Efficiency

In 2017, total above-ground nitrogen accumulation was 110.7 kg N·ha⁻¹, of which 90.0 kg (81.1%) was allocated to seeds and 20.7 kg (18.9%) to straw. In 2018, total nitrogen accumulation increased to 128.3 kg N·ha⁻¹, with 116.4 kg (90.7%) in seeds and 11.9 kg (9.3%) in straw. The highest total nitrogen accumulation was recorded in 2019, reaching 199.2 kg N·ha⁻¹, with 185.0 kg (92.9%) in seeds and 14.2 kg (7.1%) in straw. Across the study years, nitrogen accumulation in above-ground biomass increased progressively, accompanied by a growing share of nitrogen in seeds and a decreasing share in straw (Figure 10).

Higher nitrogen uptake was consistently observed in inoculated plants (HiStick Soy and Nitragina) compared to the control. On average, seeds accounted for 87.6–88.8% of total nitrogen uptake, while straw accounted for 11.2–12.4%. In the control treatment, the proportion of nitrogen allocated to seeds was significantly lower than in inoculated treatments (Figure 11).

Across all treatments, seeds represented the primary site of nitrogen accumulation, increasing from 125.6 kg N·ha⁻¹ at 0 kg N·ha⁻¹ to 135.9 kg N·ha⁻¹ at 60 kg N·ha⁻¹. Nitrogen uptake by straw ranged from 15.2 to 16.4 kg N·ha⁻¹, showing no clear trend in response to increasing nitrogen doses. Overall, nitrogen fertilisation did not significantly affect the distribution of nitrogen between the above-ground plant components (Figure 12).

The highest nitrogen use efficiency (NUE) was recorded at 30 kg N·ha⁻¹, while a significant decrease in NUE was observed at 60 kg N·ha⁻¹. Inoculation with HiStick Soy resulted in the highest NUE values and the lowest nitrogen concentration in straw. Substantial year-to-year variation was evident, with 2019 showing the highest NUE and nitrogen harvest index (NHI), and 2017 the lowest (

Table 5. The effect of nitrogen fertilisation, inoculation, and year of cultivation on nitrogen use efficiency (NUE), nitrogen harvest index (NHI), and straw nitrogen share.

Fertilisation (kg N·ha−1)	Inoculation	Year	NUE (kg Seed per kg N)	NHI (%)	Straw N Share (%)
0			-	87.9 ± 6.4a	12.1 ± 6.4a
30			96.3 ± 22.1a	88.4 ± 5.2a	11.6 ± 5.2a
60			49.5 ± 10.8b	88.3 ± 5.1a	11.7 ± 5.1a
	Control		69.2 ± 27.6b	87.6 ± 5.9b	12.4 ± 5.9a
	HiStick Soy		76.6 ± 30.4a	88.8 ± 5.1a	11.2 ± 5.1c
	Nitragina		72.9 ± 30.2ab	88.2 ± 5.6ab	11.8 ± 5.6ab
		2017	59.0 ± 23.1c	81.1 ± 3.2c	18.9 ± 3.2a
		2018	67.1 ± 22.0b	90.7 ± 1.2b	9.3 ± 1.2b
		2019	92.7 ± 31.1a	92.9 ± 0.9a	7.1 ± 0.9c

± means SD. Different letters indicate significant differences (Tukey’s multiple range test).

).

4. Discussion

It is important to note that only one soybean cultivar was included in this study, which may limit the generalizability of the findings. As soybean nodulation, nitrogen fixation capacity, and responsiveness to rhizobial inoculation can vary significantly between genotypes, some of the observed differences may be partially attributed to genotype-specific traits. Future studies should incorporate multiple cultivars to account for genetic variation in nodulation efficiency and nitrogen use under temperate field conditions.

4.1. Environmental Conditions and Root Nodulation Response

In contrast to 2019, the 2017 growing season was characterized by a moderate precipitation deficit during the early developmental stages, while ensuring adequate water availability during the flowering period. These conditions were conducive to effective nodulation and nitrogen fixation, as confirmed by the highest recorded values of nodule number and biomass. Dayoub et al. (2017) [20] also demonstrated that early root biomass and intensive lateral root expansion are crucial for the establishment of root nodule symbiosis. Similarly, Mohammadi et al. (2012) [21] emphasized that reduced water availability can inhibit nitrogenase activity and lead to impaired nodule tissue permeability, ultimately decreasing symbiotic efficiency. This suggests that in 2017, nodulation was initiated before peak drought conditions and that nodules remained active during the reproductive phase, likely due to the absence of late-season water stress. The low nodule number and biomass recorded in 2019 were most likely the result of prolonged drought during the flowering period. According to the findings of Dhanushkodi et al. (2018) [22], water stress induces premature nodule senescence. Our results are consistent with this observation—both nodule number and biomass were the lowest in 2019, despite the use of inoculation, indicating not only limited initiation but also accelerated nodule degradation. In other studies, weather conditions influenced both the number and biomass of nodules on the plant [23]. Interestingly, although nitrogen fertilisation showed a statistically significant effect on nodule biomass, the actual variation between fertiliser rates was relatively small and non-linear. These findings partially align with previous studies, indicating variability in nodulation response to nitrogen inputs. Noh et al. (2006) [24] and Chaukiyal et al. (2013) [25] did not observe consistent differences in nodule biomass between nitrogen treatments in various legume species, suggesting that the effect of mineral nitrogen on nodule biomass may be limited. More defined results were received by Wysokiński et al. (2024) [26] and by Bais et al. (2023) [27], who stated that pre-sowing N application reduced the weight and number of root nodules. Importantly, inoculation itself was confirmed as a key factor influencing nodulation. Similar observations were reported by Szpunar-Krok et al. [6], who demonstrated a beneficial effect of seed inoculation on the number of nodules in soybean, further supporting the positive impact of rhizobial inoculants under various environmental conditions. In additional research [28] long-term co-application of rhizobium and PK promoted soybean nodule dry weight.

Specifically, nitrogen reduction increased the quantity and fresh weight of root nodules of peanuts in the early stage of fertility but decreased them in the harvest stage [10].

Additionally, the interaction between year and fertilisation significantly affected nodule weight but not number, pointing to a context-dependent response of nodule development to nitrogen levels across growing seasons. Such phenomenon is also reported by Jarecki et al. (2024) [23]. The authors found that changing weather during the studied growing seasons modified the effectiveness of fertilisation and also inoculation. In our study, similarly, the weight of root nodules varied significantly depending on the type of rhizobium inoculant and the year of cultivation. These results highlight a significant interaction between year and inoculant efficacy: HiStick Soy showed greater variability in effectiveness, depending on the growing season, while Nitragina had more consistent performance, during the three years of the study.

4.2. Nitrogen Content

Leguminous plants acquire nitrogen from two main sources: mineral nitrogen available in the environment and atmospheric nitrogen fixed via biological nitrogen fixation. The contribution of each source depends on environmental conditions such as temperature [29], precipitation, and moisture availability [30,31], as well as other climatic and agronomic factors [12]. Efficient nitrogen fixation requires the presence of compatible and effective rhizobial strains in the rhizosphere [32].

In the present study, inoculation with HiStick Soy and Nitragina significantly increased nitrogen content in soybean plants compared to the uninoculated control. Pronounced year-to-year differences also underscore the importance of meteorological conditions, in line with earlier findings [33,34]. According to Wysokiński et al. [26], supplemental nitrogen fertilisation is unnecessary when effective symbiosis is established. Authors tested large range of N doses (from 0 to 180 kg·ha⁻¹), and they did not influence the N content in seeds. Only the highest dose affected N content in straw. This is confirmed by our results, which showed no significant effect of mineral nitrogen application on nitrogen content in soybean seeds or straw. Similar conclusions were drawn by Wood et al. [35], who observed a fertilisation effect on protein content at only one of the experimental sites.

However, there are studies that show a beneficial effect of nitrogen fertilisation on the protein, or N, content of seeds. For example, applying mineral nitrogen at 30 or 60 kg·ha⁻¹ protein content increased by approximately 14% compared to the control in studies conducted by Księżak and Bojarszczuk [36]. Bais et al. [27] also found that nitrogen fertilisation increased the protein (and therefore nitrogen) content of the seeds, but very high doses had this effect, at 122 and 336 kg·ha⁻¹.

In our study, the highest nitrogen content in seeds was recorded in the 2019 season, during which rainfall supported vegetative development, while pod filling and seed maturation occurred under water-deficient conditions. The decisive role of weather variability in determining nitrogen content in soybean tissues is further supported by previous studies [33,34].

Overall, our results indicate that inoculation is a key factor determining nitrogen content in soybean, whereas mineral nitrogen fertilisation is unnecessary under conditions conducive to effective biological nitrogen fixation.

4.3. Soybean Yield

The presented results confirm the predominant role of environmental factors in shaping soybean yield—interannual variability had a significant impact on all yield components, particularly aboveground biomass. This finding aligns with the observations based on other research [37,38], which indicated that rainfall deficits can substantially limit soybean yield. Nitrogen fertilisation significantly increased seed yield and total aboveground biomass, especially up to the level of 60 kg N·ha⁻¹; however, this effect diminished in the most favourable year (2019). This outcome is consistent with earlier findings by Księżak and Bojarszczuk [36], who reported a yield increase of approximately 17% following the application of mineral nitrogen. Bais et al. [27] also found an increase in soybean yield after N fertilisation, but again, it should be borne in mind that this effect was produced by doses of 122 and 336 kg·ha⁻¹. According to Wysokiński et al. [26], pre-sowing nitrogen fertilisation is not crucial. They tested a wide range of N rates and showed no effect on soybean seed yield. This shows that it is important to remember that the applied fertilisation rate should be economically reasonable. Rhizobium inoculation significantly increased seed yield but had no effect on straw or total biomass. A significant interaction with the growing season suggests that inoculation efficacy is dependent on environmental conditions. Similar yield-enhancing effects of rhizobial inoculation were reported by other authors [39,40]. The beneficial effect of the HiStick Soy formulation, particularly in 2017, corresponds with the findings of Jarecki et al. (2024) [23], emphasizing its potential under less favourable growing conditions.

4.4. Nitrogen Uptake and Allocation Efficiency

The efficiency of nitrogen (N) uptake and its distribution among plant organs is a key factor determining soybean yield and the sustainable management of nutrient inputs. During the study period, there was a clear and consistent tendency for nitrogen to be directed towards the seeds, with the nitrogen harvest index (NHI) exceeding 88% in most treatment variants. This efficiency reflects the natural ability of soybean to remobilize nitrogen from vegetative tissues to generative organs—a trait particularly desirable in grain legume crops. Interannual variability in total nitrogen uptake was largely driven by climatic conditions, confirming previous findings by Bais et al. (2023) [27]. These authors demonstrated that in years with less favourable weather, the effects of fertilisation and inoculation were limited or statistically insignificant. In the present study, the effect of nitrogen fertilisation on nitrogen allocation was also limited. Although higher N doses slightly increased total N uptake, they did not significantly alter the distribution ratio between seeds and straw. NHI values remained relatively stable (89.2–89.5%), suggesting that nitrogen allocation efficiency had reached a physiological maximum, regardless of fertiliser dose. At the same time, nitrogen use efficiency (NUE) declined with increasing N doses—from 0.09 to 0.06 kg of seeds per 1 kg of applied N—indicating diminishing yield returns relative to nitrogen input. This aligns with the conclusions of Salvagiotti et al. (2008) [5], who found that once physiological needs are met, further nitrogen fertilisation offers no substantial yield benefits.

Biological nitrogen fixation plays a pivotal role in the total nitrogen supply for soybean. Inoculation with rhizobial strains—particularly with the commercial product HiStick Soy—resulted in a slight yet consistent increase in NHI, suggesting more efficient nitrogen allocation to seeds. The reduced nitrogen content in straw (down to 10.4% in the inoculated variant compared to 11.1% in the control) supports this observation. Similar results were reported by Allito et al. (2020) [41], who observed a greater contribution of symbiotically fixed nitrogen in inoculated plants and a more favourable nitrogen balance in legumes. It is worth noting, however, that the effects of inoculation may be more pronounced under marginal conditions—Cordeiro and Echer (2019) [42] demonstrated that soybean grown on degraded soils responded more favourably to a combination of inoculation and moderate fertilisation (50 kg N·ha⁻¹), resulting in significant yield increases.

The relationship between yield and nitrogen uptake was also confirmed in this study. Salvagiotti et al. (2008) [5] indicated that a coefficient of 12.7 kg of grain per 1 kg of N uptake signifies balanced growth, while lower values point to excess nitrogen or yield limitations due to other factors. The consistent NHI levels alongside declining NUE suggest that further increases in nitrogen fertilisation did not enhance nutrient use efficiency, potentially due to water limitations, soil structure issues, or insufficient seed sink capacity.

5. Conclusions

In conclusion, the obtained results show that inoculation and moderate nitrogen fertilisation can be effective strategies for enhancing soybean yield; however, their efficiency largely depends on prevailing weather conditions. Based on studied factors, inoculation plays a more important role, especially in fields without a long history of soybean cultivation. N fertilisation in soybean cultivation is not always justified and should only be implemented when it is economically beneficial, keeping in mind the environmental aspect in the definition of sustainable agriculture. Nitrogen allocation efficiency in soybean was more influenced by environmental conditions and inoculation than by the level of mineral fertilisation. High NHI values across all treatments confirm soybean’s inherent ability to efficiently direct nitrogen to the seeds. From the perspective of production optimization and sustainable development, a strategy combining inoculation with moderate fertilisation appears most effective. Future research should encompass a broader range of site conditions, including different soil types and climatic settings, and incorporate more precise indicators of nitrogen fixation efficiency. Such an approach would support the development of more stable and site-adapted inoculation and N fertilisation strategies for soybean cultivation in temperate climates.