Soybean Response to Seed Inoculation with Bradyrhizobium japonicum and/or Nitrogen Fertilization

Abstract

Seed inoculation with symbiotic bacteria is a commonly employed practice in soybean cultivation. As a result, nodulation proceeds properly and plants self-supply atmospheric nitrogen, requiring either minimal or no additional nitrogen fertilization. The aim of the study was to investigate the response of soybeans to the application of the recommended or double dose of commercial inoculants (HiStick^® Soy or TURBOSOY^®) and/or mineral nitrogen fertilization compared to the untreated control. It was demonstrated that a double dose of the tested preparations had the most favorable effect on nodulation. However, the impact of weather conditions modified their effectiveness during the study years, which was especially visible in 2022. Sowing seeds without inoculation (control) resulted in the formation of sparse root nodules and consequently the lowest leaf area index (LAI) and soil plant analysis development (SPAD) measurements. In addition, the values of SPAD and LAI indices varied across the years of the study, indicating that weather conditions modified nitrogen uptake by plants. Overall, seed inoculation and/or nitrogen fertilization positively influenced the chemical composition of seeds compared to the control. The only decrease observed was in the oil content, while the double dose of HiStick^® Soy preparation reduced the polyphenol content. The double dose of the tested inoculants had the most favorable impact on yield components and seed yield. However, applying inoculation at the recommended dose or in combination with nitrogen fertilization yielded similar or slightly worse results, depending on the year. Therefore, soybean seed inoculation should be recommended, although the effectiveness of the procedure will depend on various factors, including the type of inoculant, dosage, nitrogen fertilization, and weather conditions.

1. Introduction

Climate change, particularly its warming, has a significant impact on crop production [1]. For example, in Poland, there is growing interest in soybean cultivation, driven by both habitat factors and economic considerations [2]. In the European Union, there is a significant demand for plant protein for feed and food production, primarily met by imported soybean [3]. Therefore, Prusiński [4] has argued that the cultivation of protein crops in Europe would increase because of the high demand and the availability of new cultivars that have stable yields and good-quality seeds. Studies by Ghani et al. [5] and Jarecki and Migut [6] have suggested that among leguminous crops, soybean possesses numerous valuable traits that are often underappreciated. However, Serafin-Andrzejewska et al. [7] described soybeans as a thermophilic and photophilic species highly sensitive to weather conditions; thus, cultivars for cultivation should be selected according to local agro-environmental conditions.

Prusiński et al. [8] argued that besides cultivar selection, it is important to improve agriculture practices, especially in new soybean cultivation regions. Fordoński et al. [9] demonstrated that in northeastern Poland, soybean should not be sown too early due to a high number of days with low temperatures and the risk of frost (April or May). On the other hand, Kulig and Klimek-Kopyra [10] showed in southern Poland that early sowing combined with higher nitrogen doses was an effective method to increase soybean yields. However, they have not recommended nitrogen fertilization when sowings are delayed, as it reduces the efficiency of nitrogen uptake. Prusiński et al. [8] have indicated that soybean cultivation in Poland encounters numerous challenges, such as low efficiency of symbiosis with Bradyrhizobium japonicum and/or nitrogen fertilization. Varying precipitation and temperatures in individual years of the study did not allow the authors to conclusively determine which option was most optimal: seed inoculation and/or mineral N fertilization. Salvagiotti et al. [11] have pointed out that the relationships between seed yield, biological N₂ fixation (BNF), and nitrogen fertilization are well described in the scientific literature. However, there is a lack of research in this area concerning new soybean cultivation regions and high-yielding cultivars. Hungria et al. [12] have reported concerns raised in Brazil about whether BNF is sufficient for increased N requirements in high-yielding cultivars, as well as doubts about the need for annual seed inoculation in fields where soybeans are commonly grown. In this aspect, Albareda et al. [13] demonstrated that the survival of root-nodule bacteria in subsequent years after application depended on soil type and bacterial strain.

Current research shows that about 50–65% [11,14] of nitrogen for soybeans is supplied by biological nitrogen fixation (BNF) and the rest from soil resources. However, in cases of high yields (>4.5 t ha⁻¹), it may be necessary to fertilize below the nodulation zone or apply nitrogen before flowering.

La Menza et al. [15] demonstrated that nitrogen fertilization allowed to obtain soybean yields above 6 t ha⁻¹ and an increase in seed protein content. As a result, protein and fat yields were higher after nitrogen fertilization compared to the control; however, this applied to cultivars with high yielding potential.

A study by Saito et al. [16] showed that improper nitrogen fertilization, especially with nitrate, inhibited nodule formation and nodulation processes, which was an undesirable phenomenon. Therefore, many authors [17,18,19,20] consider symbiosis with root-nodule bacteria to be crucial in soybean cultivation because it is an inexpensive and typically effective agronomic practice that provides adequate nitrogen supply for plants while simultaneously benefiting soil health. Krutylo et al. [21] advocated for the inoculation with various strains of root-nodule bacteria as a good agricultural practice compared to mono-inoculation. In a field experiment, after applying two strains for inoculation, these authors obtained a yield increase of 11.1–13.7% compared to mono-inoculation. Albareda et al. [13] demonstrated that each evaluated inoculant dose resulted in increased yield and seed quality compared to the control (without inoculation). However, additional nitrogen fertilization (50 N kg ha⁻¹) during soybean vegetative growth was unjustified. Hungria et al. [22] have confirmed that nodulation is improved when different species or strains of root-nodule bacteria are used for inoculation. Albareda et al. [23] and Flajśman et al. [24] have reported that some seed inoculants should be applied just before sowing, while others can be used 3 months before sowing or even earlier.

Panasiewicz et al. [25] demonstrated that the best results in soybean cultivation were achieved with the combined application of seed inoculation (Hi^®Stick Soy) and nitrogen fertilization (30 kg N ha⁻¹). Similarly, Ntambo et al. [26] recommend seed inoculation along with fertilization at a rate of 50 kg N ha⁻¹. On the other hand, Yokoyama et al. [27] demonstrated that nitrogen fertilization (30 kg ha⁻¹), while increasing initial plant growth, reduced nodulation and elevated neither yield nor seed quality. Therefore, they did not recommend nitrogen fertilization for soybeans. A study by Mourtzinis et al. [28] also showed that nitrogen fertilization did not result in the expected increases in soybean yields, and this applied to doses, timing, and foliar application compared to the control (without nitrogen).

Głowacka et al. [29] reported that the best effect on soybean yield and seed quality was observed with the application of 60 kg N ha⁻¹ in two doses: ½ or ¾, before sowing and the remainder during pod and seed formation, but in combination with sulfur application. Kubar et al. [30] demonstrated that the timing of nitrogen fertilizer (N) application modified the parameters of photosynthesis, dry biomass, and soybean seed yield. Therefore, they believed that nitrogen fertilization should be tailored to the developmental stage of the plants.

Many studies [8,31,32,33,34] demonstrated that the effectiveness of inoculation and/or nitrogen fertilization was dependent on various factors, including weather conditions. Low soil moisture significantly reduced nitrogen uptake (both atmospheric and soil) by plants. Zuffo et al. [31] confirmed that weather conditions had a significant impact on the quality of harvested soybean seeds, while inoculation and nitrogen fertilization exerted a smaller effect.

Also, soil conditions, especially acidity, salinity, and temperature, may negatively affect nodules and, consequently, reduce the ability of plants to fix nitrogen from the atmosphere [33,34]. In this aspect, Dolatabadian et al. [35] demonstrated that the adverse effects of salt stress on nodulation could be mitigated by the combined use of inoculant with genistein. Peoples et al. [36] confirmed that several factors, such as ineffective inoculant formulations, excessive tillage, or high nitrogen doses, could limit the symbiosis between plants and root-nodule bacteria. Furthermore, they have demonstrated that a significant portion of nitrogen is exported with the seed yield and thus does not return to the soil. Interesting findings were presented by Grossman et al. [37], indicating that organic soybean cultivation leads to an increase in root-nodule bacteria diversity in the soil compared to conventional cultivation. It should be added that certain chemical seed treatments could adversely affect the effectiveness of inoculation. Therefore, it is recommended using natural fungicides instead of synthetic (chemical) ones [38].

Vieira Neto et al. [39] advocated for the widespread adoption of inoculation to improve nodulation in soybean cultivation, considering the high costs of agricultural production, especially of nitrogen fertilizers.

An important aspect in soybean cultivation is the quality of seeds, which is influenced by genetics, environment, or agrotechnics. Soybean seeds contain many valuable ingredients such as protein and fat, including medicinal components such as polyphenols and others [40]. The phenolic compounds present in soybean seeds exerted a beneficial effect on the plants’ defense mechanism against environmental stresses (abiotic and biotic) [41]. On the other hand, Soedarjo et al. [42] and Choi et al. [43] considered the antioxidant properties of soybean seeds and research in this area to be particularly important.

The aim of this study was to evaluate the effect of seed inoculation with Bradyrhizobium japonicum and/or nitrogen fertilization on soybean yield and seed composition. The research hypothesis posited that the most favorable option would be the use of a double dose of inoculant, which would allow for the elimination of nitrogen fertilization.

2. Materials and Methods

2.1. Field Experiment

The soybean field experiment was conducted from 2021 to 2023 at the Agricultural Experimental Station in Boguchwała (21°57′ E, 49°59′ N), Poland. The studied factors included various seed inoculation treatments with Bradyrhizobium japonicum and nitrogen fertilization in comparison to the control group. A one-factor experiment was performed in four replicates in a randomized block design. Commercial formulations designed to inoculate soybean seeds were used in the following variants:

A—control (without inoculation and nitrogen fertilization),

B—nitrogen fertilization at a dose of 30 N kg ha⁻¹ (without inoculation)

C—inoculant—HiStick^® Soy

D—inoculant—HiStick^® Soy in a double dose

E—inoculant—HiStick^® Soy + nitrogen fertilization at a dose of 30 N kg ha⁻¹

F—inoculant—TURBOSOY^®

G—inoculant—TURBOSOY^® in a double dose

H—inoculant—TURBOSOY^® + nitrogen fertilization at a dose of 30 N kg ha⁻¹

The tested cultivar was Abelina (Saatbau Linz, Leonding, Austria), which belongs to cultivars with a medium-long growing season and high seed yield. The certified seed material was obtained from Saatbau Polska Sp. z o.o., Środa Śląska.

Ammonium nitrate (34% N) was used for fertilization at a rate of 30 N kg ha⁻¹. Nitrogen fertilization was applied before seed sowing, only in certain experimental variants. For seed inoculation, the following commercially available preparations were purchased: HiStick^® Soy (BASF SE, Ludwigshafen am Rhein, Germany) and TURBOSOY^® (Saatbau, Leonding, Austria), which were mixed with the seeds directly before sowing. Both preparations contained Bradyrhizobium japonicum at 2 × 10⁹ per gram and 2 × 10¹⁰ per milliliter, respectively. The inoculation procedure was carried out in “dry” (no water added to inoculants) conditions according to the information provided on the label of the preparations. Both inoculants were applied by hand either according to the recommendations (HiStick^® Soy—400 g na 100 kg nasion, TURBOSOY^®—250 mL na 100 kg nasion) or a double dose was used. Soybeans were cultivated for the first time in the field used for the experiment, and the preceding crop was maize grown for grain. Chemical seed treatment was not applied. The seeds were sown on the following dates: 21 April 2021, 19 April 2022, and 25 April 2023. The plot area was 18 m² (1.8 m × 10 m) with 1 m wide isolation strips. Row spacing was 45 cm, and the sowing depth was 3.5 cm. Sixty seeds were sown per square meter. Potassium and phosphorus fertilizers were applied in the fall at a rate of 60 kg ha⁻¹ K₂O (potassium salt 60%) and 40 kg ha⁻¹ P₂O₅ (superphosphate 19%). Subsequently, pre-winter plowing was carried out. In spring, harrowing was performed, followed by the use of a cultivator and subsequent sowing. Two herbicides containing metobromuron (Mandryl 500 S.C.) and bentazone + imazamox (Corum 502.4 SL) were used for chemical protection. Amistar Gold Max (azoxystrobin, difenoconazole) was applied for disease control. The doses and timings of the chemical products were applied according to the manufacturer’s recommendations.

2.2. Soil Conditions

The soil on which the experiment was conducted belonged to the Haplic Luvisol class, according to the WBR classification [44]. Soil samples for analysis were collected in the spring before nitrogen fertilization. The soil pH was slightly acidic (5.9–6.2 mol/L KCl), and the humus content (1.2–1.5%) and mineral nitrogen content were moderate. The content of assimilable phosphorus and potassium was very high or high (

Table 1. Chemical analysis of soil (30 cm).

Parameter	Unit	Year
		2021	2022	2023
pH in 1 mol/L KCl	-	6.2	6.0	5.9
Nmin	kg ha−1	78	72	65
Humus	%	1.5	1.3	1.2
K2O	mg kg−1 soil	209	204	197
P2O5	mg kg−1 soil	189	176	170

). Soil was analyzed in the Regional Chemical and Agricultural Station in Rzeszów, according to Polish standards.

2.3. Weather Conditions

Rainfall totals and average air temperatures during the soybean growing season are provided according to data from the Subcarpathian Agricultural Advisory Center in Boguchwała. The weather station was located next to the experimental field. Weather conditions varied during the study years. High precipitation was recorded in August 2021 compared to the long-term average (2001–2020). In contrast, low precipitation occurred in May, June, and August 2022, as well as August and September 2023. Air temperatures in April each year were below the long-term average. High temperatures were recorded in May, June, and August 2022, as well as in July 2021 and September 2023 (Figure 1).

2.4. Field and Laboratory Measurements

The developmental stages of the plants were provided according to the BBCH scale (Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie) [45] used in the European Union. At the flowering stage (BBCH 65), 10 roots were collected from each plot. Subsequently, the roots were washed on sieves, and the number of nodules was counted. The dry weight of the nodules was measured after their prior drying at a temperature of 22 °C.

Leaf area index (LAI—m²/m²) and chlorophyll content in SPAD value (SPAD—soil plant analysis development) were assessed twice in each plot, during the budding stage (BBCH 55) and at the flowering stage (BBCH 65). A SPAD 502P chlorophyll meter (Konica Minolta, Inc., Chiyoda, Japan) and an AccuPAR LP-80 apparatus (Meter Group, Inc., Pullman, WA, USA) were used for SPAD and LAI measurements, respectively. SPAD was measured on 10 upper leaves in the morning.

Before harvesting the soybeans, 10 plants were sampled from each plot for biometric measurements, including the number of pods per plant, number of seeds per pod, and thousand seed weight. Pre-harvest plant density was counted per 1 m². The harvest was conducted with combine (Wintersteiger) at full maturity stage in the second (2022) or third (2021 and 2023) week of September. Seed weight from each plot was converted to yield per hectare (moisture content of 14%).

2.5. Chemical Analysis of Seeds

The chemical composition, the bioactive compound content (total phenols and flavonoids), and the antioxidant capacity of soybean seeds were performed in the laboratory of the Faculty of Environmental Protection of the University of Oradea, Romania.

Soybeans had been ground with a coffee grinder. A total of 500 mg of dried soybean seeds were defatted using hexane extraction (3 × 5 mL) with 15 min of sonication followed by centrifugation at 5000 rpm. The defatted seeds were then extracted with 10 mL of 80% methanol for 12 h under agitation and centrifuged at 5000 rpm for 15 min. The supernatant was analyzed for total phenols, flavonoids, and antioxidant capacity using four different methods (DPPH, FRAP, CUPRAC, TEAC).

The total phenol content was assessed using the Folin-Ciocalteu method [46] with slight modifications [47]. Each methanol extract of soybean seeds (100 µL) was combined with 1700 µL of distilled water, 200 µL of freshly prepared Folin–Ciocalteu reagent (1:10 dilution, v/v), and 7.5% Na₂CO₃ solution. The absorbance was determined at 765 nm with a Shimadzu mini UV-Vis spectrophotometer. The findings were quantified as milligrams of gallic acid equivalent (GAE)/g dw (dry weight).

The total flavonoid content of soybean seeds was determined by the aluminum chloride colorimetric assay, where Al(III) is utilized as a complexing agent [48]. The absorbance was recorded at 510 nm versus blank. Quercetin was used as a standard for the quantification of total flavonoids, and the results were expressed as mg QE (quercetin equivalents/g dw).

Four methods with distinct principles were employed in this study to highlight the antioxidant capacity of soybean samples. DPPH is currently a highly used antioxidant assay, based on the scavenging of the purple DPPH radical in the presence of antioxidants. Instead, the TEAC assay involved the reduction of a blue/green ABTS radical cation by both lipophilic and hydrophilic compounds from samples. Two additional methods utilized in this study have been based on the mechanism through which the antioxidants in the samples are able to reduce a metal species, such as iron in the FRAP test and copper in the cupric CUPRAC test [49].

Radical scavenging capacity of soybean extract using the stable DPPH radical was determined according to the method of [50], with some modifications according to Vicas et al. [51]. The absorbance was measured at 517 nm, and the radical scavenging activity was calculated by equation 1, where A0 was the absorbance of DPPH free radical solution in methanol and A1 the absorbance of the seeds extract.

$$\%\mathrm{Radical}\mathrm{Scavenging}\mathrm{Activity}=\frac{\mathrm{A}0-\mathrm{A}1}{\mathrm{A}0}\times 100$$

(1)

The results were expressed as mmol Trolox equivalent (TE)/g dw.

The antioxidant capacity of soybean extract is evaluated based on the reduction of Fe3+ from the tripyridyltriazine Fe(TPTZ)3+ complex to the blue colored complex-Fe(TPTZ)2+ in an acidic medium [52]. Soybeen seed extract (100 µL) was combined with 2000 µL distilled water and 500 µL of freshly prepared FRAP working solution (containing 300 mM acetate buffer, pH 3.6, 20 mM FeCl3·6H₂O solution, and 10 mM TPTZ solution in a 10:1:1 ratio v/v/v) and kept in the dark at room temperature for 60 min. The absorbance was recorded at a wavelength of 595 nm, and the results were reported as µmol TE/g dw.

The assay was determined according with Apak et al. [53]. The method involves combining 1 mL of copper (II) chloride solution (1 × 10⁻² M), 1 mL of neocuproine (2,9-dimethyl-1,10-phenanthroline) alcoholic solution (7.5 × 10⁻³ M), 1 mL of ammonium acetate aqueous buffer (pH 7), and 100 μL of soybean seed extract. Water is then added to make the final volume 4.1 mL. The absorbance was measured at a wavelength of 450 nm after a duration of 30 min, in the dark. The results were reported in µmol TE/g dw.

The TEAC assay is based on the ability of antioxidants to reduce the life of a cation radical (ABTS. +), a green blue chromophore absorbing at 734 nm [51]. The cation radical was generated by combining a 7 mM solution of ABTS (2,2′-azinobis-(3 ethylbenzothiazoline-6-sulfonic acid)) with 2.45 mM of potassium persulfate and allowing the mixture to sit in darkness at room temperature for 12 h. The ABTS stock solution was diluted to achieve an absorbance of 0.70 ± 0.02 at 734 nm. A total of 100 μL of soybean seed extract was added to 2.9 mL of diluted ABTS stock solution, and the antioxidant capacity was measured at 734 nm. The results were reported in µmol TE/g dw.

The chemical composition of the seeds (fat, protein) was determined using the MPA FT-LSD spectrometer, near-infrared method (Bruker GMBH, Mannheim, Germany). The standard mass per hectoliter (MHL) was determined using Granolyser HL (Pfeuffer GMBH, Kitzingen, Germany).

2.6. Statistical Calculations

Statistical differences between the analyzed parameters were obtained using a two-way analysis of variance (ANOVA), followed by Tukey’s HSD test. All significant results of interactions between the studied factors and years were presented. If the interaction was insignificant, the results were described for the individual factors tested. Ward’s method was used to estimate the distance between clusters. The result of the test is a dendrogram, i.e., a graphic interpretation of the obtained results. In order to estimate the sources of variation, Pearson’s correlation (r). Statistical analysis was performed using TIBCO Statistica 13.3.0.

3. Results

As expected, seed inoculation significantly increased the number of root nodules compared to nitrogen fertilization or the control (Figure 2). The most favorable effect on the number of root nodules was observed with the use of a double dose of HiStick^® Soy or TURBOSOY^®. However, it should be noted that the differences between the recommended dose of inoculants and the 2-fold dose were dependent on the years of the study. In 2021, the differences in the number of nodules between these variants were insignificant. In 2022, the application of a double dose of the TURBOSOY^® preparation was significantly more effective than the recommended dose, whereas this was not statistically proven for HiStick^® Soy. The low number of nodules in 2022 is mainly the result of rainfall deficiency in May and June. In 2023, both preparations yielded the best results when a double dose was applied. In field experiments, the results obtained are often variable in the years of research, which is related to weather conditions.

The combined application of seed inoculation and soil nitrogen fertilization reduced the number of nodules compared to the double dose of the inoculant, but did not differ significantly from the recommended dose. Few nodules developed on the control roots and those after nitrogen fertilization.

The double dose of inoculants positively affected the dry weight of nodules, but the differences obtained compared to the recommended dose were not statistically significant. The combined application of inoculation and nitrogen fertilization reduced the dry weight of nodules, which was particularly visible in 2022, when low rainfall was noticed in May and June. After sowing seeds without inoculation (variants A and B), the dry nodule weight was at the limit of quantitation (Figure 3).

The first measurement of soil plant analysis development (SPAD) showed that inoculated and/or nitrogen-fertilized plants were better nourished compared to the control. The application of a double dose of the HiStick^® Soy or TURBOSOY^® preparation resulted in the high value of the second SPAD measurement. However, after applying the recommended dose, statistically significant differences were not observed. The application of inoculation combined with nitrogen fertilization or nitrogen fertilization alone yielded worse results, but still significantly better than in the control group.

The initial leaf area index (LAI) measurement indicated that the lowest green mass per 1 m² was observed in the control group. The second measurement demonstrated that the double dose of the HiStick^® Soy preparation had the most favorable effect on the LAI value. Significantly lower readings were obtained in variants A (control), B (nitrogen fertilization), and H (TURBOSOY^® + nitrogen fertilization at a dose of 30 N kg ha⁻¹).

The interaction between the studied factor and the years of research (IFxY) was not significant in both SPAD and LAI measurements. Differences in SPAD and LAI measurements were observed only between individual years. The lowest readings for both indices were recorded in 2022.

The plant density before harvest was not altered by the experimental treatments, but it varied across the study years. The lowest plant density per square meter was recorded in 2022 (

Table 2. Field measurements and plant density before harvest.

Factor	SPAD Value		Leaf Area Index (m2/m2)		Plant Density before Harvest (1 m2)
	BBCH 55	BBCH 65	BBCH 55	BBCH 65
Inoculation/Fertilization (IF)
A	30.29 ± 1.89 b	29.13 ± 2.13 e	2.49 ± 0.13 b	3.61 ± 0.14 c	51.38 ± 4.43 a
B	36.20 ± 2.16 a	34.52 ± 2.14 d	2.65 ± 0.14 a	3.77 ± 0.15 b	50.28 ± 2.71 a
C	36.65 ± 2.03 a	38.05 ± 1.97 abc	2.69 ± 0.14 a	3.85 ± 0.16 ab	49.92 ± 3.96 a
D	37.25 ± 2.15 a	38.78 ± 1.86 a	2.72 ± 0.13 a	3.90 ± 0.17 a	49.63 ± 5.05 a
E	37.39 ± 1.98 a	37.24 ± 2.07 bc	2.75 ± 0.15 a	3.81 ± 0.18 ab	51.20 ± 2.90 a
F	36.18 ± 1.96 a	37.89 ± 1.55 abc	2.63 ± 0.13 a	3.79 ± 0.16 ab	50.75 ± 5.74 a
G	36.69 ± 2.02 a	38.42 ± 1.33 ab	2.68 ± 0.14 a	3.86 ± 0.16 ab	50.74 ± 5.19 a
H	36.71 ± 1.89 a	36.80 ± 1.62 c	2.71 ± 0.15 a	3.76 ± 0.17 b	51.22 ± 4.47 a
Year (Y)
2021	37.64 ± 2.60 a	37.66 ± 3.24 a	2.67 ± 0.09 b	3.78 ± 0.11 b	52.93 ± 2.47 a
2022	33.79 ± 2.35 c	34.44 ± 3.47 b	2.52 ± 0.12 c	3.63 ± 0.12 c	45.62 ± 2.70 b
2023	36.32 ± 2.41 b	36.95 ± 3.02 a	2.80 ± 0.11 a	3.97 ± 0.12 a	53.36 ± 1.57 a
ANOVA p value
IF	***	***	***	***	n.s.
Y	***	***	***	***	***
IF × Y	n.s.	n.s.	n.s.	n.s.	n.s.

A–H—tested variants of inoculation and/or nitrogen fertilization, ***—indicate significant differences at p < 0.001, n.s.—non-significant. Mean values with different letters (a–e) in columns are statistically different. BBCH scale—Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie.

).

The highest protein content in the seeds was observed after the application of TURBOSOY^® in all variants (F, G, H), as well as after the double dose of HiStick^® Soy (variant D). Seeds from the control plot contained only 26.8% total protein but had the highest fat content, averaging 25.8%. Significantly lower fat content in seeds was observed after nitrogen fertilization and in each variant with seed inoculation (

Table 3. Labeled ingredients in soybeans.

Factor	Protein (% DM)	Fat (% DM)	Polyphenols (mg GAE/g dw)	Flavonoids (mg QE/g)
Inoculation/Fertilization (IF)
A	26.8 ± 1.01 d	25.8 ± 1.15 a	2.49 ± 0.01 a	2.40 ± 0.07 bc
B	29.0 ± 1.61 c	24.5 ± 1.16 b	2.41 ± 0.07 a	2.75 ± 0.47 a
C	36.5 ± 1.54 b	22.5 ± 1.25 cd	2.39 ± 0.05 a	2.17 ± 0.04 c
D	39.6 ± 1.56 a	21.8 ± 0.96 d	2.10 ± 0.01 b	2.50 ± 0.43 ab
E	34.1 ± 1.66 b	23.7 ± 1.02 c	2.42 ± 0.01 a	2.63 ± 0.56 ab
F	39.6 ± 2.06 a	21.8 ± 1.23 d	2.19 ± 0.09 ab	2.42 ± 0.27 bc
G	40.0 ± 0.96 a	22.0 ± 0.96 cd	2.20 ± 0.03 ab	2.53 ± 0.43 ab
H	38.1 ± 0.91 a	22.4 ± 1.41 cd	2.16 ± 0.01 ab	2.08 ± 0.12 c
Year (Y)
2021	36.41 ± 2.91 a	22.52 ± 1.78 b	2.32 ± 0.14 ab	2.44 ± 2.42 ab
2022	33.11 ± 2.56 b	23.92 ± 1.63 a	2.18 ± 0.23 b	2.38 ± 2.10 b
2023	34.92 ± 3.74 ab	22.74 ± 1.80 ab	2.41 ± 0.21 a	2.51 ± 2.81 a
ANOVA p value
IF	***	**	*	**
Y	***	*	***	***
IF × Y	n.s.	n.s.	n.s.	n.s.

A–H—tested variants of inoculation and/or nitrogen fertilization; ***, **, *—indicate significant differences at p < 0.001, p < 0.01 and p < 0.05; n.s.—non-significant. Mean values with different letters (a–d) in columns are statistically different.

).

The polyphenol content in the seeds decreased after applying a double dose of the HiStick^® Soy preparation. The seeds contained significantly higher levels of polyphenols after the recommended dose of the HiStick^® Soy preparation and/or nitrogen fertilization (B, C, E), as well as in the control (A). The content of flavonoids was high, especially after nitrogen fertilization alone. Significantly lower content of the discussed components was observed in the control seeds, as well as after the recommended dose of HiStick^® Soy or TURBOSOY^® application, and after combined administration of TURBOSOY^® with nitrogen fertilization (Table 3).

The content of the analyzed components varied across the years; however, statistically significant interactions between the experimental factors and the years of study were not confirmed.

In accordance with the results from the free radical removal tests (DPPH and TEAC), there are no significant differences between soybean seeds treated with HiStick^® Soy (sample C) and those treated with a combination of nitrogen fertilization and HiStick^® Soy. Results were consistent for treatments with TURBOSOY^®, except for sample H, which showed a significant increase in antioxidant value when using the TEAC method. Furthermore, regardless of the type of treatment used, there are notable enhancements in the antioxidant content when comparing these samples to the control samples (A) and those treated with nitrogen (B).

When using metal reduction methods (FRAP and CUPRAC), there are significant distinctions between control (A) and nitrogen fertilization (B), with the latter resulting in an enhanced antioxidant capacity. Increasing the dosage of HiStick^® Soy does not enhance antioxidant capacity. However, applying a double treatment of TURBOSOY^® significantly increases antioxidant capacity compared to samples F and H (

Table 4. Antioxidant capacity (mmol TE/g).

Factor	DPPH (2,2-Diphenyl-1-picryl-hydrazyl-hydrate) Assay	FRAP (Ferric Reducing Antioxidant Power) Assay	CUPRAC (Cupric Reducing Antioxidant Capacity) Assay	TEAC (Trolox Equivalent Antioxidant Capacity) Assay
Inoculation/Fertilization (IF)
A	5.27 ± 0.04 a	4.76 ± 0.32 c	10.39 ± 0.11 b	5.72 ± 0.34 a
B	5.78 ± 0.77 a	5.91 ± 0.95 a	13.50 ± 0.43 a	5.92 ± 0.25 a
C	4.18 ± 0.37 b	4.42 ± 0.04 cd	11.16 ± 0.43 b	4.74 ± 0.27 b
D	3.57 ± 0.31 c	3.77 ± 0.41 f	10.41 ± 0.58 b	4.16 ± 0.28 cd
E	4.38 ± 0.08 b	3.81 ± 0.04 ef	14.10 ± 0.53 a	4.71 ± 0.14 b
F	3.86 ± 0.12 bc	4.15 ± 0.08 de	10.47 ± 0.11 b	4.06 ± 0.33 d
G	3.62 ± 0.20 c	5.19 ± 0.07 b	12.94 ± 0.29 a	3.89 ± 0.33 d
H	3.62 ± 0.11 c	4.05 ± 0.24 de	10.42 ± 1.23 b	4.21 ± 0.02 c
Year (Y)
2021	4.28 ± 0.78 ab	4.38 ± 0.73 b	11.71 ± 1.84 a	4.58 ± 0.78 b
2022	4.21 ± 0.90 b	4.48 ± 0.93 b	11.50 ± 1.76 a	4.69 ± 0.70 ab
2023	4.38 ± 0.91 a	4.66 ± 0.94 a	11.81 ± 1.48 a	4.78 ± 1.04 a
ANOVA p value
IF	***	***	***	***
Y	***	***	***	***
IF × Y	n.s.	n.s.	n.s.	n.s.

A–H—tested variants of inoculation and/or nitrogen fertilization; ***—indicate significant differences at p < 0.001, n.s.—non-significant. Mean values with different letters (a–f) in columns are statistically different.

).

The components of yield and bulk density were dependent on the interaction between the experimental factor and the years of the study. In 2023, the double dose of HiStick^® Soy, combined use of HiStick^® Soy with nitrogen fertilization, or TURBOSOY^® with nitrogen fertilization, exerted the most favorable effects on the number of pods per plant. Similar results were obtained in 2021 with the application of a double dose of HiStick^® Soy. The highest number of seeds per pod was obtained in 2023 after applying the recommended or double dose of HiStick^® Soy. On the other hand, the highest thousand seed weight was obtained in 2021 and 2023 after applying a double dose of TURBOSOY^®. Bulk density (MHL) was significantly higher in the control seeds in 2023 compared to their counterparts from 2021 to 2022 (

Table 5. Yield component and bulk density.

Factor	Year	Number of Pods per Plant	Number of Seeds in the Pod	Mass of a Thousand Seeds (g)	MHL—Bulk Density (kg/hl)
A	2021	13.75 ± 0.38 ij	2.14 ± 0.10 de	136.00 ± 4.59 defgh	73.37 ± 0.80 b
	2022	12.77 ± 0.95 j	2.01 ± 0.17 e	126.25 ± 4.43 gh	62.85 ± 1.07 c
	2023	14.25 ± 0.85 ij	2.24 ± 0.12 cde	130.10 ± 5.76 gh	82.82 ± 0.95 a
B	2021	16.57 ± 0.84 hi	2.37 ± 0.08 abcd	136.27 ± 3.17 defgh	72.47 ± 0.94 b
	2022	14.17 ± 0.56 ij	2.19 ± 0.05 cde	123.62 ± 5.84 h	71.40 ± 0.90 b
	2023	16.82 ± 0.95 ghi	2.40 ± 0.04 abcd	122.37 ± 2.76 h	74.75 ± 1.01 b
C	2021	20.17 ± 0.88 cdef	2.46 ± 0.10 abc	148.85 ± 4.71 abcde	74.30 ± 1.47 b
	2022	19.97 ± 1.15 cdef	2.23 ± 0.07 cde	132.15 ± 5.97 fgh	73.45 ± 1.26 b
	2023	22.05 ± 0.97 abcd	2.64 ± 0.04 a	147.6 ± 3.00 abcde	75.43 ± 0.65 b
D	2021	23.77 ± 0.78 ab	2.32 ± 0.07 bcd	152.37 ± 8.43 abc	72.77 ± 1.55 b
	2022	21.95 ± 1.49 abcde	2.32 ± 0.10 bcd	141.00 ± 6.97 bcdefg	73.90 ± 1.63 b
	2023	24.3 ± 1.25 a	2.57 ± 0.10 ab	142.00 ± 2.16 bcdefg	74.70 ± 1.58 b
E	2021	21.30 ± 1.01 abcde	2.43 ± 0.09 abc	150.57 ± 7.92 abcd	73.30 ± 2.26 b
	2022	18.19 ± 1.99 efgh	2.36 ± 0.11 bcd	138.37 ± 8.98 cdefgh	72.45 ± 2.29 b
	2023	24.17 ± 1.37 a	2.47 ± 0.16 abc	143.92 ± 8.44 abcdef	74.35 ± 2.21 b
F	2021	19.17 ± 1.28 defgh	2.36 ± 0.10 bcd	148.85 ± 4.71 abcde	73.35 ± 2.02 b
	2022	17.90 ± 1.15 fgh	2.26 ± 0.07 cde	138.37 ± 5.97 cdefgh	72.38 ± 1.33 b
	2023	22.77 ± 0.97 abc	2.37 ± 0.08 abcd	141.00 ± 6.47 bcdefg	74.36 ± 2.11 b
G	2021	20.85 ± 1.49 bcdef	2.36 ± 0.06 bcd	159.15 ± 8.86 a	73.17 ± 2.32 b
	2022	19.77 ± 1.26 cdefg	2.30 ± 0.15 bcd	142.60 ± 6.73 bcdefg	72.22 ± 2.09 b
	2023	22.17 ± 1.10 abcd	2.44 ± 0.10 abc	154.32 ± 9.67 ab	74.15 ± 2.19 b
H	2021	21.30 ± 0.78 abcde	2.26 ± 0.08 cde	152.37 ± 7.92 abc	72.35 ± 2.05 b
	2022	20.95 ± 1.99 bcdef	2.22 ± 0.09 cde	134.65 ± 4.24 efgh	71.27 ± 1.46 b
	2023	23.30 ± 1.01 ab	2.37 ± 0.16 abcd	148.50 ± 8.98 abcde	73.40 ± 2.08 b
ANOVA p value
IF × Y		***	***	***	***

A–H—tested variants of inoculation and/or nitrogen fertilization; ***—indicate significant differences at p < 0.001, n.s.—non-significant. Mean values with different letters (a–j) in columns are statistically different for interaction.

).

Seed yield was most favorably affected by the double dose of HiStick^® Soy, as confirmed in each of the study years. Similar results were obtained after using a double dose of TURBOSOY^®. However, it should be noted that the recommended dose of inoculant was less effective compared to the double dose only in 2021 and when evaluating the effectiveness of the TURBOSOY^® preparation in 2023. It should be mentioned that the meteorological conditions in 2022 resulted in a decrease in seed yield in all treatments compared to 2021 and 2023 (Figure 4).

The combined application of seed inoculation with nitrogen fertilization yielded comparable results to a double dose of inoculant. Therefore, it has been demonstrated that nitrogen mineral fertilization can be substituted with an increased dose of inoculant. In the years 2021 and 2022, nitrogen fertilization (without seed inoculation) resulted in a significant increase in yield, but only compared to the control plants. Meanwhile, nitrogen fertilization was unfounded in 2023 (Figure 4).

Positive and negative correlations between the individual parameters were also evident for the traits analyzed (

Table 6. Correlation coefficients (r) between tested parameters.

A	1
B	0.43	1
C	0.88	−0.7	1
D	0.51	−0.3	−0.1	1
E	0.63	0.13	−0.2	0.18	1
F	0.27	0.37	0.17	0.21	0.12	1
G	0.87	0.15	0.83	0.36	0.62	0.09	1
H	0.77	0.21	0.68	0.34	0.60	0.11	0.91	1
I	0.81	0.33	0.75	0.45	0.48	0.23	0.78	0.75	1
J	0.68	0.65	0.62	0.21	0.31	0.44	0.51	0.53	0.61	1
K	0.32	−0.3	0.46	0.26	0.24	−0.1	0.56	0.51	0.33	0.12	1
L	−0.1	0.12	−0.2	−0.1	0.06	0.21	−0.3	−0.3	−0.2	−0.1	−0.6	1
M	0.18	0.52	0.11	−0.1	−0.1	0.35	−0.1	−0.1	−0.1	0.35	−0.5	0.12	1
N	0.19	0.36	0.12	−0.1	0.04	0.32	0.02	0.07	0.15	0.48	−0.3	0.08	0.43	1
O	−0.4	0.21	−0.5	−0.3	−0.5	0.18	−0.6	−0.6	−0.3	0.04	−0.7	0.28	0.44	0.48	1
U	−0.2	0.25	−0.3	−0.2	−0.3	0.19	−0.4	−0.2	−0.1	0.21	−0.4	−0.1	0.41	0.53	0.62	1
P	0.18	0.33	0.17	−0.1	−0.2	0.27	−0.1	−0.1	0.33	0.53	−0.1	0.02	0.25	0.47	0.41	0.43	1
R	−0.2	0.48	−0.3	−0.2	−0.3	0.38	−0.5	−0.4	−0.1	0.26	−0.7	0.28	0.54	0.48	0.76	0.53	0.38	1
	A	B	C	D	E	F	G	H	I	J	K	L	M	N	O	U	P	R

A—Seed yield, B—Plant density before harvest, C—Number of pods per plant, D—Number of seeds in the pod, E—Mass of a thousand seeds (g), F—MHL—bulk density (kg/hl), G—Number of root nodules, H—Dry weight of root nodules (g), I—SPAD, J—LAI, K—Protein, L—Fat, M—Polyphenols, N—Flavonoids, O—DPPH, U—FRAP, P—CUPRAC, R—TEAC.

). It was shown that seed yield was very strongly positively correlated with number of pods per plant (r = 0.88), number of root nodules (r = 0.87), and SPAD (r = 0.81). Number of root nodules was highly correlated with dry weight of root nodules (r = 0.91). Seed protein content was negatively correlated with fat (r = −0.6), DPPH (r = −0.7), and TEAC (r = −0.7).

The statistical calculations presented in Figure 5 show that a double dose of inoculation (HiStick^® Soy in a double dose or TURBOSOY^® in a double dose) or inoculation with nitrogen fertilization (30 N kg ha⁻¹) resulted in a similar effect. It should be noted that fertilizing only with nitrogen gives similar results to the control. The large differences were found between inoculation HiStick^® Soy and TURBOSOY^®.

4. Discussion

4.1. Nodule Number and Dry Weight as Affected by Treatment and Year

Biological preparations are increasingly being used in agriculture to stimulate plant growth and development. In soybean cultivation, these are typically inoculants containing specific symbiotic bacteria or other microorganisms [54]. As a result, the process of nodulation proceeds correctly, providing plants with approximately 60% of their nitrogen demand. The remaining portion of nitrogen originates from the soil or fertilization [55]. Previous research [12,56] has indicated that nitrogen in soybean cultivation is mainly derived from biological nitrogen fixation (BNF), and additional fertilization with this element is usually unnecessary, which is justified both economically and environmentally. Torres et al. [57] and Hungria et al. [58] have affirmed that soybeans, when properly nodulated, do not require supplementary nitrogen fertilization for achievement of high seed yield.

In the present experiment, it was confirmed that inoculation of soybean seeds significantly increased the number of nodules on the roots compared to nitrogen fertilization alone or the untreated control, where nodules were scarce. These results indicated that there were no symbiotic bacteria in the soil capable of establishing symbiosis with soybean plants. The double dose of inoculants positively affected the dry weight of nodules, but the differences obtained compared to the recommended dose were not statistically significant.

Zimmer et al. [59] have reported that symbiotic bacteria do not occur naturally in some soils; thus, they recommended inoculating seeds before sowing soybeans to ensure proper nodulation. In their study, non-inoculated soybeans did not form any nodules, and grain yield, thousand kernel weight, protein content, and protein yield were significantly increased by up to 57%, 20%, 26%, and 99%, respectively, after successful inoculation with Bradyrhizobium strains. Narożna et al. [60] reported that after introducing Bradyrhizobium japonicum bacteria into the soil, their presence and symbiotic capacity persisted in subsequent years. Albareda et al. [13] showed that the survival rate of root-nodule bacteria was dependent on the soil type and the bacterial strain. Vieira Neto et al. [39] also proved that inoculation was more effective when soybeans were cultivated for the first time and the soil did not contain symbiotic bacteria.

4.2. Influence of Treatments and Year on Seed Yield and Yield Components

In the present study, it was demonstrated that both inoculation preparations were effective in the nodulation and the quantity and quality of the yield. In the case of the first one, slightly better nodulation effects and seed yield were obtained, while the application of the second one resulted in better seed quality parameters. After applying a double dose of HiStick^® Soy, the number of nodules per root ranged from 17.3 to 26.2. Similarly, following a double dose of TURBOSOY^® preparation, the count ranged from 16.2 to 24.8 nodules.

Cigelske et al. [61] obtained between 24.4 and 30.3 nodules per soybean plant. They demonstrated that the number and size of nodules decreased with increasing nitrogen fertilizer dosage. It was confirmed by Lyu et al. [62] that the application of nitrogen before soybean sowing reduced nodulation and, consequently, nitrogen uptake from the air.

In the present study, the application of the recommended inoculant dose along with nitrogen fertilization yielded similar results for seed yield to using a double dose of inoculant without nitrogen fertilization. Therefore, twice the dose of inoculant can substitute nitrogen fertilizer, which has important implications for agricultural practice.

The double dose of HiStick^® Soy, combined use of HiStick^® Soy with nitrogen fertilization, or TURBOSOY^® with nitrogen fertilization, exerted the most favorable effects on the number of pods per plant. The highest number of seeds per pod was obtained after applying the recommended or double dose of HiStick^® Soy. On the other hand, the highest thousand seed weight was obtained after applying a double dose of TURBOSOY^®.

In 2021 and 2022, nitrogen fertilization alone (without seed inoculation) resulted in increased yields only compared to the control, while in 2023, it did not exert a significant effect. The yield components (number of pods, number of seeds per pod, and thousand seed weight) and bulk density were dependent on the interaction between the studied factor and the years of the study. The availability of nitrogen from symbiosis and fertilization was therefore dependent on weather conditions, making it difficult to recommend the best variant for soybean cultivation. The plant density before harvest was not altered by the experimental treatments, but it varied across the study years.

Kakabouki et al. [63] obtained the best results for seed yield with high nitrogen doses of 80 or 120 N kg ha⁻¹, but the higher dose was less effective. Capatana et al. [64] demonstrated that mineral fertilization with NPK and foliar fertilization resulted in a 30.2% increase in soybean yield compared to the control. On the other hand, seed inoculation with root-nodule bacteria further increased the yield by 3.76%. Latifnia and Eisvand [65] have stated that biological nitrogen fixation (BNF) is not sufficient for obtaining high yields and protein content in soybean seeds. They believe that this species requires supplementary nitrogen fertilization, as indicated by chlorophyll fluorescence measurements. On the other hand, Welch et al. [66] concluded that nitrogen fertilization of soybeans was not as effective as expected and was typically uneconomical. Prusiński et al. [8] did not observe a clear influence of inoculants and/or mineral nitrogen fertilization on the number of pods and thousand seed weights. However, they demonstrated significant varietal differences in the traits investigated. Khaledian et al. [67] obtained the highest number of pods and seeds per pod after the combined application of manure and chemical fertilizers. However, TSW was not modified by fertilization.

The present study demonstrated that a double dose of the HiStick^® Soy preparation increased soybean seed yield, which was confirmed in each year of the study. Similar results were obtained after using a double dose of TURBOSOY^®. However, it should be noted that the recommended dose of inoculant was also effective. The application of the recommended dose of inoculant compared to the double dose produced inferior results only in 2021 and when the effectiveness of TURBOSOY^® was tested in 2023.

Deaker et al. [68] demonstrated that using an inoculant at a dosage higher than recommended resulted in increased nodulation and seed yield. In some cases, they observed yield increases of up to 25%. Albareda et al. [13] confirmed that the application of higher inoculant doses positively influenced nodule dry weight, yield, and nitrogen content in seeds. However, additional nitrogen fertilization (50 kg N ha⁻¹) was unjustified, especially at the high dose of inoculant. Rahangdale et al. [69] have supported the notion that higher doses of inoculant are generally justified in soybean cultivation. In the aspect in question, an interesting experiment was presented by Cordeiro et al. [70], who demonstrated that nitrogen (N) fertilization was beneficial under unfavorable conditions such as high temperature and low soil fertility. However, among the variants tested, the highest soybean seed yields were obtained after applying increased doses of inoculant.

4.3. LAI and SPAD as Affected by Treatment and Year

The leaf area index (LAI) measurements performed in the current study showed that the lowest plant biomass per 1 m² was observed in the control (without inoculation and nitrogen fertilization). After the second measurement, it was demonstrated that the double dose of the HiStick^® Soy preparation exerted the most beneficial impact on LAI compared to variants A (control), B (nitrogen fertilization), and H (TURBOSOY^® + nitrogen fertilization at a dose of 30 N kg ha⁻¹).

Zerpa et al. [71] demonstrated that seed inoculation favorably affected nodulation, resulting in increased leaf area. It was shown by Prusiński et al. [8] that the leaf area index (LAI) increased after inoculation or inoculation combined with nitrogen fertilization. However, this effect varied across the years of study and depended on the cultivar. On the other hand, Kakabouki et al. [63] demonstrated that the LAI index and plant biomass significantly increased after the application of a high nitrogen dose (120 kg N ha⁻¹) compared to the control, resulting in increased seed yield.

In the present study, soil plant analysis development (SPAD) measurements indicated that plants under control conditions were less nourished compared to variants with inoculation and/or nitrogen fertilization. The second measurement demonstrated that the SPAD index was highest when inoculation with the HiStick^® Soy or TURBOSOY^® preparations was applied. The combined application of inoculation and nitrogen fertilization, or nitrogen fertilization alone, resulted in lower SPAD readings, yet still significantly better than those observed in the control group.

Vollmann et al. [72] considered nodulation and photosynthesis to be the two most important processes for the growth and development of leguminous plants, as they are closely interrelated. Additionally, measurements of the chlorophyll content in leaves can provide information about the nodulation process and nitrogen fixation by cultivated plants. Basal and Szabó [73] reported that conducting measurements of NDVI, SPAD, or LAI during the soybean growing season allowed for a preliminary estimation of seed yield.

4.4. Protein, Oil, Polyphenols, AOX Capacity as Affected by Year and Treatment

In the present study, the highest protein content in seeds was detected after the application of TURBOSOY^® in all variants (F, G, H) as well as after the double dose of HiStick^® Soy (variant D). Seeds from the control plot contained only 26.8% total protein but had the highest fat content, averaging 25.8%. The lowest fat content in seeds was observed after nitrogen fertilization combined with seed inoculation.

Jarecki [74] proved that sowing inoculated or coated seeds positively affected the protein content in the seeds compared to the control. However, the fat content was significantly higher in the control seeds compared to the variants with inoculants and coating. Bais et al. [55] showed significant negative relationships between oil and protein content, as well as yield and oil content. Higher doses of nitrogen (112 and 336 kg N ha⁻¹) significantly increased yield, protein content, and thousand seed weight. Szostak et al. [75] demonstrated that the applied fertilization significantly influenced the chemical composition of soybean seeds. They recorded the highest seed protein content after nitrogen fertilization at a dose of 30 kg ha⁻¹ at the BBCH 73–75 stage. However, considering both yield and the chemical composition of seeds, these authors recommended applying nitrogen fertilizers at a dose of 60 kg N ha⁻¹ (first before sowing, second during the pod and seed development stage). Wood et al. [76] showed that nitrogen fertilization had a beneficial effect on the yield and chemical composition of soybean seeds. However, this was dependent on the experimental location and was not always conclusive regarding the nitrogen dose, timing of fertilization, or interaction with the cultivar.

In the present study, the content of polyphenols in the seeds decreased after the application of a double dose of the HiStick^® Soy preparation. Significantly higher polyphenol content was found in seeds inoculated with the recommended dose of the HiStick^® Soy preparation and/or nitrogen fertilization (B, C, E) as well as in the control group (A). On the other hand, the content of flavonoids was high after nitrogen fertilization alone. Significantly lower levels of these components were observed in the control group as well as after the application of the recommended dose of inoculants or the combined use of TURBOSOY^® with nitrogen fertilization.

Malenčić et al. [77] have reported that soybeans contain many valuable nutrients, such as polyphenols and flavonoids. Therefore, they are a valuable resource for feed, food, and even pharmaceuticals. It has been demonstrated by Król-Grzymała and Amarowicz [41] that phenolic compounds found in soybeans have beneficial effects on human health as well as on plant defense mechanisms against environmental stresses (both abiotic and biotic).

According to our results concerning free radical removal tests (DPPH and TEAC), no significant differences were observed between soybean seeds inoculated with HiStick^® Soy and those inoculated with a combination of nitrogen fertilization and HiStick^® Soy. A significant increase in antioxidant value was observed in variant H when using the TEAC method. The application of a double treatment of TURBOSOY^® significantly increased antioxidant capacity compared to variants F and H.

Soedarjo et al. [42] and Choi et al. [43] particularly emphasized the antioxidant properties of soybean seeds. They proved that the antioxidant activity of DPPH, TEAC, and FRAP depended primarily on the cultivar and to a lesser extent on habitat conditions. Zilic et al. [78] also showed that the antioxidant properties of soybeans are mainly influenced by genetics. However, there is little information about the impact of agrotechnical treatments on these nutrients.

Atmospheric nitrogen fixation by leguminous plants is one of the key processes in agriculture, which requires further research to improve nodulation and biological nitrogen fixation from the air [79]. It should be noted that the results of agricultural field trials are often ambiguous and depend on genotype (G), environment (E), management practices (MP), and their interactions [80]. Therefore, it is important to conduct field experiments in various environmental conditions to achieve high yields of soybean with good seed quality [81].

4.5. Correlation and Statistical Dependencies

Our own research has shown that seed yield was very strongly positively correlated with number of pods per plant, number of root nodules, and SPAD. Number of root nodules was highly correlated with dry weight of root nodules. Seed protein content was negatively correlated with fat, DPPH, and TEAC. Hong et al. [82] also showed a negative correlation between protein and fat contents in soybean.

The Diagram Ward shows that a double dose of inoculation or inoculation with nitrogen fertilization resulted in a similar effect. It should be noted that fertilizing only with nitrogen gives similar results to the control. Jarecki and Migut [6] showed that a dendrogram is useful in comparing different species of legumes.

5. Conclusions

The results of the conducted research showed that inoculation of soybean seeds was necessary for the proper nodulation process to occur on soybean roots. The main reason for this was the absence of symbiotic bacteria in the soil, as indicated by measurements obtained in the control plot. Using a double dose of the inoculation formulation proved to be a beneficial treatment, confirming the research hypothesis. However, it should be noted that variable weather conditions during the years of the study modified the effectiveness of the applied inoculants and/or nitrogen fertilization. In 2022, low rainfall in May and June resulted in lower nodulation and, as a result, low seed yields compared to 2021 and 2023. Measurements using soil plant analysis development (SPAD) demonstrated that in the absence of available nitrogen (both symbiotic and soil-borne), plants were less nourished (low SPAD value) and had lower leaf area index (LAI) values, which was particularly noticeable in years with low rainfall. Seed inoculation generally had a beneficial effect on the chemical composition of soybean seeds, except for the fat content. Furthermore, a double dose of the HiStick^® Soy preparation reduced the polyphenol content, while nitrogen fertilization increased the flavonoid content. The double dose of inoculants had the most beneficial effect on yield components and seed yield, but inoculation at the recommended dose or the combination of inoculation with nitrogen fertilization resulted in similar outcomes depending on the year of the study. Nitrogen fertilization alone (without inoculation) yielded the least favorable results, but generally better than the control. Research on soybean seed inoculation should be continued to improve nodulation and atmospheric nitrogen fixation, which is of great importance for agricultural practice.