Role and importance of cobalt in faba bean through rationalization of its nitrogen fertilization

Abstract

This study evaluates the combined effects of cobalt (Co) application and nitrogen (N) fertilization on faba bean (Vicia faba) growth, yield, and nutritional quality under field conditions during the 2021/2022 and 2022/2023 growing seasons. Field experiments were conducted at the Research and Production Station in El-Nubaryia, Egypt, using a split-plot design with three replicates. Nitrogen fertilizer levels (100%, 75%, 50%, and 25% (NH₄)₂SO₄) were applied to main plots, and cobalt concentrations (with and without 12 ppm) were applied to subplots. Cobalt treatment significantly enhanced root nodule formation, nitrogenase activity, and overall plant growth, especially under higher nitrogen levels. The highest positive effects on nodulation, yield components (such as number of pods and seed yield), and nutritional content (N, P, K, Mn, Zn, Cu) were observed at 100% nitrogen, followed by 75% and 50% nitrogen treatments. The results indicated that spraying with cobalt with 75% nitrogen fertilization was better or equal to 100% nitrogen fertilization without cobalt, which saves 25% of nitrogen fertilization. Cobalt improved faba bean productivity and nutrient content, particularly when combined with optimal nitrogen levels, suggesting its potential role in sustainable agriculture. The study underscores the beneficial impact of cobalt and nitrogen synergy in enhancing crop productivity and nutritional value.

Introduction

Faba bean (Vicia faba L.), one of the most important leguminous crops globally, plays a crucial role in human nutrition and sustainable agricultural systems due to its high protein content [1], ability to fix atmospheric nitrogen [2], and suitability in crop rotations [3]. In recent years, the role of micronutrients in enhancing plant growth, nitrogen fixation, and yield has gained significant attention [4–6]. Among these micronutrients, cobalt (Co) is not essential for all plants but plays a crucial role in legumes due to its involvement in nitrogenase enzyme activity, which is pivotal for biological nitrogen fixation [7, 8]. Nitrogen (N), as a primary macronutrient, is often a limiting factor in faba bean growth and productivity [2]. However, its efficient utilization in crops, particularly under varying environmental conditions, remains a challenge [9, 10].

Faba bean cultivation is commonly dependent on nitrogen fertilizers to meet its nutrient requirements, but over-reliance on synthetic nitrogen fertilizers can lead to environmental degradation, reduced soil fertility, and increased production costs [9, 10]. Recent studies have suggested that the addition of cobalt to nitrogen fertilization can improve nitrogen use efficiency, enhance growth parameters, and increase overall yield by facilitating better nitrogen fixation and reducing the dependency on high nitrogen fertilizer inputs [11, 12]. This potential for cobalt to optimize the growth and productivity of faba bean under varying nitrogen rates is an area of growing interest.

The present study investigates the effects of cobalt application on faba bean growth, yield, and nitrogen efficiency under different nitrogen fertilization regimes. The objective was to assess whether cobalt supplementation could enhance nitrogenase activity, improve nodulation, and ultimately increase the faba bean yield while reducing the required nitrogen input. Specifically, this research aims to evaluate cobalt’s impact under four nitrogen rates: 100% N, 75% N, 50% N, and 25% N, to better understand the interaction between cobalt and nitrogen on faba bean performance. By focusing on these aspects, the study seeks to identify an optimized cobalt-nitrogen strategy that could contribute to sustainable faba bean cultivation, improving yield and reducing environmental impact.

Materials and methods

Soil analysis

Composite soil samples were collected from a depth of 0–30 cm, air-dried, crushed, and sieved through a 2 mm mesh [13]. Soil texture was analyzed using the pipette method as described by Page et al. (1982). Calcium carbonate content (CaCO₃) was measured using the calcimeter method of Jackson (14). Soil organic matter content was determined through dichromate oxidation, while calcium carbonate content was measured using the calcimeter method of Jackson [14]. Soil salinity was assessed by measuring the electrical conductivity (EC) of a 1:1 soil-water extract using an EC meter (LOvibond 200 con, Germany), and soil pH was determined in a 1:2 soil-water suspension using a pH meter (Hanna Instruments pH 211, Romania) [14]. Available nitrogen (N) was extracted with 1% K₂SO₄ at a 1:5 soil-to-solution ratio, and 20 mL of the extract was distilled with 1 g of Devarda’s alloy using a micro-Kjeldahl apparatus [14]. Available phosphorus (P) was extracted using 0.5 M sodium bicarbonate (pH 8.5) and quantified using the stannous chloride method, with absorbance measured at 660 nm using a spectrophotometer (Unico 2000UV, Germany) [14]. Potassium (K) content was determined using a flame photometer (Jenway 7PFP, England) following the protocol outlined by Jackson (14). Micronutrients: iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were quantified using the procedures described by Page [15]. Additionally, the determination of soluble, available, and total cobalt concentrations in the soil was carried out in accordance with the method established by Ryan, Estefan [16]. The key physical and chemical properties of the investigated soils (mean of two seasons) are presented in Table 1. All reagents and chemicals used in the analysis were provided by El Gomhouria Company for Chemicals, Alexandria City, Egypt.

Table 1.

Physical and chemical properties of the investigated soil

Physical properties
Particle size distribution %								Soil moisture constant %
Sand	Silt		Clay			Soil texture		Saturation				FC				WP				AW
70.8	25.6		3.6			Sandy loam		32.0				19.2				6.1				13.1
Chemical properties
							Soluble cations (mmolcL− 1)							Soluble anions (mmolcL− 1)
pH 1:2.5	EC (dS m− 1)			CaCO3 g kg− 1		OM g kg− 1	Ca++			Mg++	K+		Na+	HCO3−			CO3		Cl−	SO4−−
8.49	1.74			34		2.0	3.5			1.9	0.8		11.2	0.2			-		12.4	3.8
Cobalt mg g − 1 soil							Available						Available micronutriments
							mg 100 g − 1 soil						mg g − 1 soil
Soluble		Available			Total		N		P		K		Fe		Mn			Zn		Cu
0.35		4.88			9.88		15.1		13.3		4.49		4.46		2.71			4.52		5.2

Where: FC = Field Capacity (%); WP = Wilting Point (%); AW = Available Water (%)

Experimental site and design

Field experiments were conducted at the Research and Production Station of the National Research Centre, located in El-Nubaryia, Behaira Governorate, Delta Egypt (30° 23’ 48” N, 30° 18’ 59” E) during the 2021/2022 and 2022/2023 growing seasons. To evaluate the role of cobalt in optimizing nitrogen fertilizer on faba bean yield (Vicia faba var. Giza 716). The faba bean seeds were obtained from Research and Production Station of the National Research Centre, located in ElNubaryia, Behaira Governorate, Delta Egypt. Seeds were sown on October 15 in both growing seasons. The field was plowed twice in perpendicular directions, marked with a 70–80 cm width. Seeds were planted on both sides of each row, with 25 cm spacing between holes. The experiment design was split-plot with three replicates. The amounts of nitrogen fertilizer were assigned to the main plots [Control (100% recommended (NH₄)₂SO₄; 75% (NH₄)₂SO₄; 50% (NH₄)₂SO₄; 25% (NH₄)₂SO₄] were provided by Salam International Fertilizers Co., Giza city, Egypt. While cobalt concentrations were devoted to the subplots [without cobalt and with cobalt (12ppm) were provided by El Gomhouria Company for Chemicals, Egypt]. Each plot had an area of 10.50 m². The study was carried out under a drip irrigation system to ensure efficient water utilization. Weeds were manually removed between rows to avoid competition with the crop. Preventive measures included two prophylactic pesticide sprays to minimize disease and insect infestation.

Fertilization and cobalt application

On preliminary findings in recent studies, the most effective cobalt concentration (12 ppm), those that exhibited the highest positive impact on growth and yield parameters were selected for use in these study [17–19]. Cobalt was applied in combination with varying nitrogen fertilizer levels at the third true leaf stage of faba bean plants.

All required agriculture managements for plants growth and production were carried out as recommended by the Ministry of Agriculture. A fertilizer injector was utilized to blend fertilizer with water and introduce it into the drip irrigation system. Nitrogen fertilizer was applied to plots [100% =475 kg ha^− 1; 75%=356 kg ha^− 1; 50%=237.5 kg ha^− 1; 25%=118.75 kg ha^− 1] in three equal doses: the first dose followed germination, the second occurred one month later, and the third was administered at the flowering stage. As per the guidelines of the Egyptian Ministry of Agriculture, 357 kg ha⁻¹ of mono superphosphate fertilizer (15.5% P ≈ 55.34 kg P₂O₅) was incorporated into the soil before preparation. In addition, 119 kg ha⁻¹ of potassium sulfate (K₂SO₄) was applied in two equal doses: the first dose 30 days after germination and the second dose at the onset of flowering. All fertilizers were provided by Salam International Fertilizers Co., Giza city, Egypt.

Nodule assessment and nitrogenase activity measurement

After 50 days from sowing, the number of nodules per plant, nodules fresh weight per plant, and nodules dry weight per plant were recorded for each faba bean plant, then transferred to the laboratory of the Plant Nutrition Department at the National Research Centre, Egypt. To determine nitrogenase activity, the acetylene reduction assay (ARA) was performed according to the method described by Hardy, Holsten [20]. Briefly, faba bean plants were gently uprooted to avoid damaging the root system, and the root nodules were carefully excised. The nodules were then placed in 500 mL serum bottles, which were sealed airtight using suba-seal rubber stoppers to ensure a gas-tight environment. To initiate the assay, 10% of the gas phase within the bottles was replaced with acetylene gas (C₂H₂) using a gas-tight syringe. The bottles were then incubated in the dark at room temperature (approximately 25 °C) for 2 h to allow nitrogenase enzyme activity to reduce acetylene to ethylene (C₂H₄). After the incubation period, a 1 mL gas sample was extracted from the headspace of each bottle using a gas-tight syringe and injected into a gas chromatograph (GC) equipped with a flame ionization detector (FID) for ethylene quantification. Nitrogenase activity was calculated based on the rate of ethylene production and expressed as micromoles of ethylene produced per gram of nodule fresh weight per hour (µmol C₂H₄ g⁻¹ h⁻¹).

Measurements of vegetative growth

After 80 days from sowing, various vegetative growth parameters of faba bean plants were measured to assess plant development and biomass accumulation. The measurements were conducted according to the guidelines provided by Gardner, Pearce [21]. The samples were transferred to the laboratory of the Plant Nutrition Department at the National Research Centre, Egypt. The following growth parameters were recorded: Plant height was measured from the base of the stem at soil level to the apex of the main shoot (cm), while root length was determined by excavating the root system carefully and measuring from the stem base to the longest root tip (cm). The number of branches (secondary stems ≥ 2 cm) and fully expanded leaves (lamina completely unfolded) per plant were counted. For biomass assessment, shoots and roots were separated, and fresh weights (g) were recorded immediately after harvesting. Dry weights (g) were obtained after oven-drying samples at 70 °C for 48 h (or until constant mass) and reweighing.

Measurements of plant yield

After 110–120 days from sowing, yield parameters of faba bean plants were measured to evaluate crop productivity. The samples were transferred to the laboratory of the Plant Nutrition Department at the National Research Centre, Egypt. The measurements were conducted according to the methods described by Steen, Peoples Mark [22]. The following yield parameters were recorded: The total number of fully developed pods (with visible seeds) per plant was counted, followed by immediate weighing of all pods to record fresh pod weight (g). Pods were manually threshed to separate seeds, which were then weighed to determine fresh seed weight (g). Seed size and uniformity were assessed by weighing a random 100-seed sample (g). Total pod yield was calculated per unit area, expressed in both kilograms per feddan (kg fed⁻¹) and metric tons per feddan (ton fed⁻¹) for standardized productivity comparison.

Determination of chemical constituents

The chemical composition of faba bean seeds, specifically the percentage of protein content, total carbohydrates, total soluble sugars, and vitamin A were determined. The samples were delivered to the Plant Nutrition Department’s laboratory at the National Research Centre in Egypt for further analysis. All reagents and chemicals used in the analysis were provided by El Gomhouria Company for Chemicals, Alexandria City, Egypt. The following procedures were used:

Total protein

The total protein content in faba bean seeds was determined using the Kjeldahl method [23], a widely accepted and reliable technique for protein quantification. Faba bean seeds were cleaned, dried, and ground into a fine powder using a mechanical grinder. A representative sample of the powdered seeds (approximately 0.5 g) was accurately weighed and transferred into a digestion flask. The sample was digested with concentrated sulfuric acid (H₂SO₄) in the presence of a catalyst (potassium sulfate (K₂SO₄)) to convert organic nitrogen into ammonium sulfate. The digestion process was carried out at a high temperature (approximately 400 °C) until the solution became clear, indicating complete digestion. The digested sample was then transferred to a distillation apparatus. Sodium hydroxide (NaOH) was added to the digested solution to release ammonia (NH₃) gas. The liberated ammonia was distilled and collected in a receiving flask containing a known volume of boric acid (H₃BO₃) solution. The ammonia absorbed in the boric acid solution was titrated with a standardized hydrochloric acid (HCl) solution (0.1 N) using a suitable indicator (methyl red). The endpoint of the titration was indicated by a color change from green to pink. The total nitrogen content was calculated using the volume of HCl consumed during titration and the normality of the HCl solution. The protein content was determined by multiplying the total nitrogen content by a conversion factor of 6.25, which is commonly used for plant materials.

where: 1.401 is the milliequivalent weight of nitrogen; 6.25 is the conversion factor for converting nitrogen to protein.

Total carbohydrates

The total carbohydrate content was determined using acid hydrolysis followed by the phenol-sulfuric acid method as described by DuBois, Gilles [24]. Briefly, 1.0 g of dried sample was hydrolyzed with 10 mL of 1 N H₂SO₄ in a water bath at 100 °C for 30 min. After cooling, the solution was neutralized with 0.1 g of BaCO₃, filtered through Whatman No. 1 filter paper, and diluted to 100 mL with distilled water. The total carbohydrate content was quantified using the phenol-sulfuric acid method, where an aliquot of the sample was mixed with 5% phenol and concentrated H₂SO₄, and the absorbance was measured at 490 nm. A standard curve was prepared using glucose solutions (10–100 µg mL^− 1) for quantification, and the results were expressed as a percentage of the dry sample weight. The following equation is used:

Total soluble sugars

The total soluble sugar content in faba bean seeds was quantified using the anthrone method, as described by Ebell [25], with slight modifications. A known weight (0.1 g) of finely ground, oven-dried seed sample was placed in a test tube and extracted with 10 mL of distilled hot water by incubating in a water bath at 90 °C for 30 min to solubilize the sugars. The mixture was then centrifuged at 4,000 rpm for 10 min, and the supernatant was collected. A 1 mL aliquot of the supernatant was transferred to a clean test tube and mixed with 5 mL of freshly prepared anthrone reagent (150 mg anthrone dissolved in 100 mL of concentrated sulfuric acid). The mixture was immediately placed in a boiling water bath for 10 min, then rapidly cooled in an ice bath to room temperature. The resulting green-colored complex, indicative of the sugar-anthrone reaction, was measured for absorbance at 620 nm using a UV-Visible spectrophotometer (model Unico UV-2100, Unico (Shanghai) Instrument Co., Ltd., China). The sugar concentration was calculated using a standard curve prepared with known concentrations of D-glucose, and the results were expressed as a percentage of total soluble sugars relative to the dry weight of the seed sample.

Determination of vitamin A content

Vitamin A content was determined using High-Performance Liquid Chromatography (HPLC), following standardized procedures outlined by Ball [26]. Briefly, approximately 2 g of finely ground seed sample was homogenized and subjected to saponification with 10 mL of alcoholic potassium hydroxide (KOH, 60% w/v in ethanol) at 60–70 °C for 30–45 min to hydrolyze vitamin A esters and release free retinol. The mixture was then cooled and extracted three times with 10 mL of hexane to isolate retinol into the organic phase. The combined hexane extracts were washed with distilled water until neutral pH was achieved, and the organic layer was collected. The solvent was evaporated under a stream of nitrogen to avoid oxidative degradation, and the dried residue was reconstituted in 1 mL of the mobile phase for chromatographic analysis. The analysis was conducted using an HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm particle size) and a UV-Vis detector set at 325 nm. The mobile phase consisted of methanol and water in a 95:5 (v/v) ratio, delivered at a flow rate of 1.0 mL/min under isocratic conditions. Quantification of vitamin A (retinol) was based on peak area comparison with a standard calibration curve generated from known concentrations of retinol. Results were expressed as milligrams of vitamin A per 100 g of dry sample weight (mg 100 g⁻¹).

Minerals compositions analysis

For chemical analysis, seeds were sampled from intact plants for each treatment. The collected samples were oven-dried at 70 °C for 48 h, then finely ground and stored for chemical determinations. For nutrient extraction, 0.2 g of finely powdered dry sample was digested using a mixture of sulfuric acid (H₂SO₄) and hydrogen peroxide (H₂O₂), following the method described by Cottenie, Verloo [27]. The analysis included the determination of nitrogen (N), phosphorus (P), potassium (K), manganese (Mn), zinc (Zn), copper (Cu), iron (Fe), and cobalt (Co).

Statistical analysis

All data were statistically analyzed using the SAS (1996) software package. Mean comparisons were conducted using the Least Significant Difference (LSD 5%) method, following the procedure outlined by Snedecor and Cochram [28].

Results and discussion

Nodulation and nitrogen fixation

The data presented in Table 2 indicate that cobalt had a significant positive effect on faba bean root nodule parameters under different nitrogen application rates compared to untreated plants. Cobalt treatment resulted in the highest number of nodules per plant, as well as the fresh and dry weights of nodules, when combined with 100% nitrogen (N). This was followed by treatments with 75% N and 50% N, while the lowest values were recorded at 25% N. These findings are consistent with Balai and Majumdar [29], who reported that cobalt significantly enhanced the number, fresh weight, and dry weight of cowpea nodules. Additional, recent researches reported that cobalt-treated plants showed a higher number and weight of root nodules, indicating improved nitrogen fixation plants compared to the control in chickpea plants [30, 31], cobalt-treated plants of pea [32], cobalt-treated plants of Lentil [8]. These researches reported that the presence of cobalamin (Co-enzyme) in legume nodules confirms cobalt’s essential role in nitrogen fixation. A deficiency in cobalt has been associated with reduced vitamin B12 production, leading to lower nitrogen fixation efficiency. The data presented in Table 2 further indicate that cobalt plays a vital role in enhancing nitrogenase enzyme activity in faba bean root nodules across all nitrogen levels compared to the control. The highest nitrogenase activity was observed at 100% N, followed by 75% N, while the lowest activity was recorded at 25% N. These results are consistent with Epstein and Bloom [33], who found that the Co-enzyme scopolamine contains cobalt (III) as a metal component, chelated to four nitrogen atoms at the centre of a porphyrin structure, similar to iron in hemin. In Rhizobium species, cobalt-dependent enzymes are primarily responsible for nodulation and nitrogen fixation in legumes.

Table 2.

Effects of cobalt application on nodulation parameters of faba bean under varying nitrogen levels at 50 days after sowing (mean of two growing seasons)

Nitrogen treatments %		Nodules No. plant− 1 (nodule)	Nodules fresh Weight (g)	Nodules dry Weight plant− 1 (g)	N-ase activity µmol C2H2 g− 1 h− 1
Without cobalt
100		136.4	14.32	3.97	18.8
75		112.2	12.49	3.60	17.2
50		102.0	11.44	3.28	16.0
25		95.6	10.56	2.88	14.8
With cobalt (12ppm)
100		154.5	16.65	5.43	22.0
75		148.	15.49	5.31	21.3
50		133.3	14.62	4.77	19.6
25		117.0	12.86	4.18	17.8
LSD 5%	N	6.6	1.3	0.16	0.36
	Co	5.3	0.9	0.14	0.24
	Co*N	3.9	0.8	0.11	0.21

Where: N = Nitrogen treatments %; Co = cobalt treatments; and Co*N = the interaction between Nitrogen treatments and cobalt treatments

These findings align with the work of Riley and Dilwarth [34], who established cobalt’s essential role in supporting microbial growth and proliferation during biological nitrogen fixation. While cobalt serves as a vital micronutrient in this symbiotic process, our results demonstrate significant interspecies variation in tolerance thresholds to cobalt excess. This variability likely stems from differences in plants’ physiological adaptation mechanisms and their capacity to regulate cobalt uptake and homeostasis. Finally, Manisha, Bhadoria (35) confirmed that root nodule parameters and nitrogenase enzyme activity in faba bean nodules were significantly influenced by cobalt. Similarly, Kandil, Gad [36] reported that cobalt significantly increased nitrogenase activity, which was directly related to nodule number and weight, particularly at 100% and 75% nitrogen fertilization levels.

Vegetative growth

Data presented in Table 3 indicate that applying cobalt at a concentration of 12 ppm significantly enhanced all growth parameters of faba bean, including plant height, number of branches and leaves, and the fresh and dry weights of both shoots and roots, compared to untreated controls. This positive effect was especially evident under 100% and 75% nitrogen levels. The consistent improvement in growth suggests that cobalt may play a regulatory role in key physiological processes. These findings align with those reported by Gad [37], who demonstrated that cobalt exerts a synergistic effect by stimulating the synthesis of endogenous hormones such as auxins and gibberellins, while simultaneously reducing the activity of oxidative enzymes like peroxidase and catalase. This hormonal and enzymatic shift favors anabolic over catabolic processes, thereby promoting enhanced plant development. However, excessive cobalt levels may reverse these benefits, promoting catabolic activity and inhibiting growth. These results are further corroborated by Banerjee, Sounda [38], who observed that cobalt application significantly improved leaf area index, dry matter accumulation in both shoots and roots, and pod yield in groundnut plants compared to untreated controls.

Table 3.

Effects of cobalt application on growth parameters of faba bean under varying nitrogen levels at 80 days after sowing (mean of two growing seasons)

Nitrogen treatments%		Plant hight (cm)	Number Plant− 1		Leaf area (cm2)	Fresh weight		Dry weight
			Branches	Leaves		Shoot	Root			Shoot		Root
Without cobalt
100		146	8.2	39.4	2182	121.0	19.7		24.5		4.76
75		128	7.1	32.2	2138	106.2	18.1		21.5		4.19
50		119	5.6	26.6	1790	87.2	17.5		17.6		3.79
25		108	4.7	21.3	1687	72.6	15.6		14.9		3.46
With cobalt (12ppm)
100		151	10.5	43.0	2391	131.8	21.7		27.1		5.21
75		146	9.9	36.5	2350	126.0	20.3		25.8		4.89
50		122	6.8	29.2	2214	104.9	18.4		21.9		4.31
25		112	5.7	24.1	1876	96.0	16.4		19.8		3.87
LSD 5%	N	11.7	0.75	3.1	41.1	4.6	Ns		2.3		Ns
	Co	6.3	0.66	2.7	34.6	3.9	Ns		1.9		Ns
	Co*N	5.5	0.51	2.3	29.5	3.2	Ns		1.6		Ns

Where: N = Nitrogen treatments %; Co = cobalt treatments; and Co*N = the interaction between Nitrogen treatments and cobalt treatments; Ns = no significance

As the nitrogen application rate decreased, the promotive effect of cobalt on faba bean growth also diminished. The lowest values for growth parameters, such as plant height, number of branches and leaves, and shoot and root biomass, were observed when cobalt was applied with 25% nitrogen. These results are in good agreement with the findings of Gad and Kandil [32], who reported that cobalt application resulted in the greatest improvement in groundnut growth parameters at 100% nitrogen, followed by 75% and 50% nitrogen. However, the lowest growth values were recorded when cobalt was combined with 25% nitrogen, compared to the control (100% nitrogen alone). This suggests that the beneficial effects of cobalt are highly dependent on the availability of nitrogen, with optimal growth promotion occurring under higher nitrogen levels. The application of cobalt (Co) at 20–40 mg kg^− 1 significantly enhanced plant height, branch number, fruit yield, and anthocyanin and flavonoid content in Hibiscus sabdariffa [39]. In maize, increasing Co levels (50–250 mg kg^− 1) initially improved root length, shoot height, cob number, and seed production, but these benefits declined at concentrations exceeding 100 mg kg^− 1 [40]. Similarly, in two onion cultivars, a Co dose of 10 mg kg^− 1 boosted growth, bulb yield, length, and quality, including nutritional and essential oil content, while also increasing bulb diameter and weight compared to the control [41]. However, Co concentrations above 10 mg kg^− 1 markedly reduced these positive effects. In Arachis hypogaea (peanut), Co concentrations of 100–200 mg kg^− 1 negatively impacted growth parameters, including shoot and root length, leaf area, nodule formation, and pigment content, while also reducing starch, sugar, amino acids, and protein levels in leaves. Additionally, antioxidant enzymes like catalase (CAT) were suppressed, and seedling growth and dry weight declined at higher Co levels [42]. Eleusine concana L. exhibited reduced seed germination, seedling growth, leaf area, and biomass when exposed to 5–100 mg/L of Co [43].

Yield characteristics

The results presented in Table 4 highlight the promotive effect of cobalt on faba bean yield parameters, including the number of pods per plant, pod weight per plant, seed weight per plant, and seed yield (ton feddan⁻¹). The most significant improvements were observed at 100% nitrogen, followed by 75% and 50% nitrogen. However, the lowest values for these yield parameters were recorded when cobalt was applied with 25% nitrogen. The addition of cobalt to the plant growth medium consistently enhanced seed yield, underscoring its beneficial role in faba bean productivity. These findings are consistent with the results of Balachandar, Nagarajan [44], who emphasized that cobalt is an essential element for legumes, particularly for nodule formation and nitrogen fixation. Furthermore, the results are consistent with those of Gad and Abdel-Moez [45], who reported that cobalt at 10 ppm, combined with 100% nitrogen, resulted in the greatest growth and yield of cowpea, followed by 75% and 50% nitrogen, while the lowest values were observed with 25% nitrogen. In Avena sativa L. (Oats), higher cobalt levels (20–320 mg kg⁻¹) significantly reduced grain yield, straw biomass, and root growth [46].

Table 4.

Effects of Cobalt application on yield parameters of faba bean under varying nitrogen levels at 120 days after sowing (mean of two growing seasons)

Nitrogen treatments %		Pods No. plant− 1 (pod)	Pods Weight plant− 1 (g)	100 seeds Weight plant− 1 (g)	Seeds yield ton fed− 1
Without cobalt
100		23.8	369	50.3	5.796
75		17.4	321	46.7	5.604
50		11.5	234	43.2	5.064
25		9.35	186	39.0	4.680
With cobalt (12ppm)
100		31.4	436	61.8	7.416
75		26.9	376	58.9	7.164
50		18.7	319	52.0	6.240
25		11.2	289	46.8	5.616
LSD 5%	N	3.2	2.7	3.1	0.215
	Co	2.9	2.4	2.7	0.207
	Co*N	2.2	2.1	2.2	0.193

Where: N = Nitrogen treatments %; Co = cobalt treatments; and Co*N = the interaction between Nitrogen treatments and cobalt treatments

Chemical compositions

The data presented in Table 5 demonstrate that cobalt application significantly enhanced the chemical constituents of faba bean, including total protein, total carbohydrates, total soluble sugars, and vitamin (A), across all nitrogen levels. Compared to untreated plants, cobalt had a pronounced positive effect on these chemical components, leading to marked increases in total protein, total carbohydrates, and total soluble sugars. These findings highlight the beneficial role of cobalt in improving the nutritional quality of faba bean under varying nitrogen conditions. Tomato plants showed concentration-dependent responses: at 6.2–1000 µM Co, symptoms included chlorosis of young leaves, decreased catalase (CAT) activity, and increased peroxidase, acid phosphatase and ribonuclease activities [47]. When treated with 0.05–0.5 mM Co, tomatoes exhibited reduced biomass, chlorophyll content, nucleic acids, nitrogen and carbohydrate levels, along with impaired photosynthesis and CAT activity [48]. In sunflower exposed to 5–50 mg kg^− 1 Co, researchers observed delayed seed germination, reduced shoot and root growth, and decreased enzyme activities [49].

Table 5.

Effects of Cobalt and nitrogen levels on the chemical composition of faba bean seeds (mean of two growing seasons)

Nitrogen treatments %		Protein %	Total Carbohydrates %	Total Soluble Sugars	Vitamine (A) (mg 100 g− 1)
Without cobalt
100		21.13	53.6	5.36	8.26
75		18.94	52.2	4.92	7.94
50		16.68	49.7	4.66	7.28
25		13.44	47.3	3.88	6.79
With cobalt (12ppm)
100		25.19	55.0	5.91	8.60
75		23.63	53.7	5.08	8.04
50		20.25	50.3	4.81	7.67
25		17.88	48.2	4.09	6.91
LSD 5%	N	0.24	1.41	0.15	0.34
	Co	0.19	2.11	0.40	0.11
	Co*N	0.11	0.88	0.23	0.12

Where: N = Nitrogen treatments %; Co = cobalt treatments; and Co*N = the interaction between Nitrogen treatments and cobalt treatments

Minerals compositions

The data presented in Table 6 indicate that cobalt significantly enhanced the content of nitrogen (N), phosphorus (P), and potassium (K) across all nitrogen application rates. The most pronounced increase was observed at 100% nitrogen, followed by 75% and 50% nitrogen levels. In contrast, the lowest values were recorded at the 25% nitrogen rate. These findings are consistent with the results reported by Gad, El-Moez [50], who demonstrated that cobalt significantly boosted N, P, and K concentrations in soybean seeds across various nitrogen levels compared to the control. Further support for these results comes from Jayakumar, Jaleel [51], who observed that cobalt application at 50 mg kg^− 1 soil positively influenced the mineral composition of Black gram. For Glycine max L. (soybean), Co levels of 4–16 mg kg^− 1 led to decreased concentrations of essential nutrients like nitrogen (N), phosphorus (P), and potassium (K) [36]. Together, these studies underscore the beneficial role of cobalt in enhancing nutrient uptake in plants under different nitrogen regimes.

Table 6.

Effects of Cobalt and nitrogen levels on the minerals’ composition of faba bean seeds (mean of two growing seasons)

Nitrogen treatments%		Macronutrients (%)			Micronutrients (ppm)
		N	P	K	Mn	Zn	Cu	Fe	Cobalt
Without cobalt
100		3.38	0.470	1.51	22.0	18.2	15.7	174	0.78
75		3.03	0.422	1.36	20.9	17.5	14.6	176	0.81
50		2.67	0.375	1.18	19.4	16.4	13.4	172	0.76
25		2.15	0.321	1.08	18.0	14.2	10.6	173	0.78
With cobalt (12ppm)
100		4.03	0.490	1.76	23.1	18.9	17.2	171	7.67
75		3.78	0.439	1.57	21.6	17.8	15.4	168	6.85
50		3.24	0.394	1.24	20.2	17.2	13.9	165	6.51
25		2.86	0.360	1.10	8.87	15.5	11.3	161	6.30
LSD 5%	N	0.15	Ns	Ns	0.34	0.22	0.46	0.32	0.21
	Co	0.13	Ns	Ns	0.29	0.18	0.39	0.61	0.32
	Co*N	0.10	Ns	Ns	0.25	0.15	0.35	0.11	0.22

Where: N = Nitrogen treatments %; Co = cobalt treatments; and Co*N = the interaction between Nitrogen treatments and cobalt treatments; Ns = no significance

Data presented in Table 6 further demonstrate that cobalt positively influenced the concentrations of manganese (Mn), zinc (Zn), and copper (Cu) in faba bean seeds across various nitrogen application rates. The most pronounced increases were observed at the 100% nitrogen level, followed by 75% and 50%, with the lowest micronutrient concentrations recorded at 25% nitrogen. These findings are consistent with those of Boureto, Castro [52], who reported that cobalt significantly enhanced the mineral composition of pea seeds under different nitrogen regimes. This consistency across studies underscores the beneficial role of cobalt in improving micronutrient uptake in legumes, particularly under optimal nitrogen conditions.

Additionally, Table 6 reveals that increasing cobalt levels in the plant medium resulted in a reduction in iron (Fe) content in faba bean seeds. This observation aligns with the findings of Bisht [53], who noted an antagonistic relationship between cobalt (Co) and iron (Fe), suggesting competition between the two elements during uptake. Further evidence is provided by Gad, Mohammed [54], who reported that cobalt enhanced nitrogen use efficiency, enabling a 25% reduction in the recommended nitrogen dose while still promoting improved growth and yield in cowpea. Collectively, these results highlight the complex interactions between cobalt and other micronutrients, and its potential role in optimizing nutrient management and improving the nutritional quality of leguminous crops.

Kobbia and Osman [55] emphasized that as plant roots absorb water, cobalt present in the soil migrates from the non-rhizosphere zone toward the root zone through mass flow. This process likely contributes to the enhanced uptake of cobalt by plants. Supporting this mechanism, data presented in Table 6 show a progressive increase in cobalt content within faba bean seeds in response to elevated cobalt concentrations in the growing medium. These results underscore the critical role of mass flow in facilitating cobalt uptake and its subsequent accumulation in plant tissues, particularly in leguminous crops such as faba beans. Similarly, Nadia Gad, I.M. El-Metwally [56] reported that cobalt application—especially when integrated with organic fertilizers—significantly improves chickpea growth, yield, and nutritional quality. Chickpea plants treated with cobalt and organic fertilizers exhibited higher concentrations of essential nutrients, including nitrogen (N), phosphorus (P), potassium (K), and cobalt (Co), compared to untreated controls [56]. These findings highlight the potential of cobalt, in synergy with organic amendments, as a viable strategy to enhance chickpea production and promote sustainable agricultural practices.

Correlation analysis with Mantel’s test

The correlation analysis presented in the figure reveals significant relationships among various measured parameters, as indicated by Mantel’s r and p-values (Fig. 1). The correlation coefficients range from 1.0 (perfect positive correlation) to -0.5 (moderate negative correlation), highlighting both strong and weak associations between the variables. Mantel’s r values, categorized as less than 0.1 or greater than or equal to 0.4, further elucidate the strength of these relationships, while the associated p-values (< 0.01, 0.01–0.05, and > = 0.05) indicate their statistical significance. The correlation analysis reveals significant relationships between Seeds Yield (SY) and various plant growth, nutrient, and yield parameters. SY shows strong positive correlations with Nodules Dry Weight per Plant (NDW/P), Plant Height (PH), Number of Branches per Plant (NB/P), Leaf Area (LA), Shoot and Root Fresh/Dry Weights (FSW, FRW, DSW, DRW), Pods Number and Weight per Plant (Pn/p, PW/p), and 100 Seeds Weight per Plant (100sW/p), indicating that enhanced growth and yield-related traits contribute to higher seed yield. Additionally, SY is positively correlated with essential nutrients like Nitrogen (N), Phosphorus (P), and Potassium (K), highlighting the importance of nutrient availability for maximizing yield. Weaker or negative correlations with parameters such as Total Soluble Sugars (TSSu) or Vitamin A (VA) suggest more complex interactions. These results emphasize the critical role of cobalt application in improving the utilization of nitrogen fertilization for plant growth, nutrient uptake and yield components to improve seed yield, providing valuable insights for agricultural practices and crop improvement strategies.

Fig. 1.

Correlation Matrix of Plant Growth, Nutrient, and Yield Parameters with Seed Yield (SY) of Faba bean plant. Where: Nodules No. Per plant = NN/P; Nodules fresh Weight = NFW/P; Nodules dry Weight/plant = NDW/P; N-ase activity = Nase; Plant hight = PH; Number of Branches/Plant = NB/P; Number of Leaves/Plant = NL/P; Leaf area = LA; Shoot Fresh weight = FSW; Root Fresh weight = FRW; Shoot Dry weight = DSW; Root Dry weight = DRW; Pods No. /plant = Pn/p; Pods Weight/plant = PW/p; 100 seeds Weight/plant = 100sW/p; Seeds yield = SY; Protein = Pro%; Total Carbohydrates = TC%; Total Soluble Sugars = TSSu; Vitamine (A) = VA; nitrogen = N; phosphorus = P; potassium = K; manganese = Mn; zinc = Zn; copper = Cu; iron = Fe; cobalt = Co

Conclusion

The results of this study demonstrate that the application of cobalt, particularly in combination with nitrogen fertilization, significantly enhances the growth, yield, and nutritional quality of faba bean (Vicia faba). Cobalt treatment positively influenced key parameters such as root nodulation, nitrogen fixation, and nutrient uptake, especially when applied alongside higher nitrogen levels (100% and 75%). The increase in nodulation and nitrogenase activity under cobalt supplementation suggests its vital role in optimizing nitrogen use efficiency in faba bean plants. Additionally, cobalt application improved the overall seed yield, as well as the content of essential nutrients such as nitrogen (N), phosphorus (P), potassium (K), and micronutrients like manganese (Mn), zinc (Zn), and copper (Cu). These findings highlight the potential of using cobalt and nitrogen synergistically to promote sustainable agricultural practices, enhancing both crop productivity and nutritional value. Future research could focus on further optimizing cobalt application rates and exploring its long-term impacts on soil health and plant performance in diverse environmental conditions.

Author contributions

All authors contributed significantly and equally to conceptualization, writing, editing and review of the current manuscript. All authors agreed to the submission of the current manuscript.

Funding

This research received no external funding.

Data availability

Data is provided within the manuscript.

Declarations

Compliance with ethical standards

This article does not contain any studies of human participants or animals performed by any of the authors.

Consent for publication

Not applicable

Competing interests

The authors declare no competing interests.