Ionomic Profile and Nutrient Use Efficiency in Sunflower Plants Treated with Plant-Derived Biostimulant Rich in Trigonelline

Abstract

In recent decades, the use of biostimulants has increased with the aim of creating an alternative to the use of chemical fertilizers and achieving sustainable agriculture. In this study, sunflower plants (Helianthus annuus L. cv. neoma) were grown under controlled conditions, and four trigonelline-rich extracts were applied as biostimulants through root and foliar applications. The plant growth parameters, nutrient concentrations, root metabolic activity, and nutrient use efficiency were evaluated. The results showed that the foliar application of extract 4 significantly improved the aboveground biomass and leaf area compared with the control treatments, with values of 44.30 g FW and 680.22 cm², respectively. Moreover, this extract enhanced nutrient accumulation. Meanwhile, foliar application of extract 3 improved macronutrient and micronutrient concentrations, as in the case of phosphorus, which increased by 74.2%, and iron, which increased by 107.3%. Root applications of extracts 3 and 4 increased apparent nutrient recovery, whereas foliar applications of extracts 3 and 4 improved internal nutrient use and productivity indices. Overall, the treatments showed no phytotoxicity and promoted growth, nutrient absorption, and nutrient use efficiency, with the best results observed in foliar treatments with high trigonelline content. These findings indicate that biostimulants with trigonelline-rich extracts could improve crop yield, reduce the use of fertilizer, and contribute to more sustainable agricultural systems.

1. Introduction

Currently, agriculture faces the challenge of developing sustainable and environmentally friendly systems that contribute to feeding the ever-increasing global population [1]. This population increase is expected to reach 9.6 billion people by 2050, putting pressure on horticultural production to increase its yield [2,3]. This significant population growth and the resulting global food demand have led traditional agriculture to overuse inorganic compounds to increase crop productivity. Consequently, this has caused degradation of terrestrial and aquatic ecosystems as well as soil quality. These inorganic compounds have also caused other harmful effects, such as the generation of nutritional imbalances in plants, water nitrification, and the development of pesticide-resistant pests [4,5]. Horticultural production is at risk due to environmental changes caused by climate change, the decrease in arable land, and the depletion of plant genetic potential, requiring the development of new methods to increase crop yields [1,3].

For these reasons, alternatives to fertilizers that do not negatively affect productivity or economic production are beginning to be used, such as biostimulants, which can be defined as “any product that stimulates plant nutritional processes, regardless of nutrient content, with the purpose of improving nutrient use efficiency (NUE), tolerance to abiotic stress, qualitative characteristics, and the availability of nutrients confined to the soil” [4,6,7]. Therefore, it would be possible to achieve a more sustainable agriculture. According to the Food and Agriculture Organization (FAO), sustainable agricultural systems can be defined as those designed to achieve three main objectives: (i) to maintain or enhance natural resources and ecosystem health, (ii) to ensure economic profitability, and (iii) to improve the well-being of farmers, agricultural workers, and rural communities [8].

Plant-derived biostimulants (PDBs) are one type of biostimulant that is currently of great interest in both research and industry. They can be defined as natural products derived from different parts of plants, such as seeds, roots, or leaves [3]. They contain antioxidant compounds, such as glutathione and ascorbate, essential nutrients, vitamins, essential amino acids and peptides, beneficial elements, and, in some cases, bioactive compounds. The main species from which extracts are obtained for use as PDBs are alfalfa (Medicago sativa L.), moringa (Moringa oleifera L.), aloe vera (Aloe barbadensis L.), and nettle (Urtica dioica L.) [9]. These extracts can be applied via foliar spraying, fertigation, or directly to the soil [3,10]. Biostimulants can be applied at all stages of agriculture, from seed treatment and plant growth to post-harvest [11]. In addition, they are considered environmentally safe and possess a wide range of biological activities, such as nitrogen metabolism activation or phosphorus release into the soil, microbial activity stimulation, and plant growth promotion [10].

Although further research is still needed, current evidence indicates that PDBs exhibit a wide range of beneficial functions on plant growth and development, as well as on resistance to adverse environmental conditions. These include: (i) enhanced nutrient absorption, (ii) resistance to abiotic and biotic stresses, (iii) increased photosynthesis, (iv) involvement in the regulation of hormonal balance, (v) stimulation of root growth, (vi) promotion of flowering and fruiting, (vii) stimulation of beneficial soil microbial activity, and (viii) reduction in the use of chemical fertilizers and pesticides, thereby supporting more ecological and sustainable agriculture [3,12]. All these functions make PDBs a sustainable and effective alternative to improve crop health and crop yield.

One of the plants proposed for use in agriculture as a source of biostimulant extracts is Trigonella foenum-graecum L., commonly known as fenugreek. Fenugreek is an annual legume crop belonging to the Fabaceae family that originates from the eastern Mediterranean. It is widely cultivated as a species in many parts of the world, and its leaves and seeds have been used for medicinal purposes, as a dietary supplement, and as raw material for various pharmaceuticals [13]. It is one of the oldest medicinal plants and has an exceptional nutritional profile. In addition to its numerous therapeutic applications—such as antioxidant, antibacterial, and antidiabetic activities—it shows moderate tolerance to drought, salinity, and heavy metals, as well as the ability to adapt to different climates [14].

Trigonella foenum-graecum L. has high nutritional value because its leaves contain calcium, phosphorus, iron, and vitamin C, whereas its seeds contain alkaloids, trigonelline, and saponins. Likewise, the use of fenugreek in human nutrition could help treat various disorders, such as cardiovascular diseases, liver conditions, and hyperglycemia [15]. Fenugreek owes much of its importance to one of these compounds—trigonelline. Trigonelline is synthesized through the methylation of nicotinic acid, a reaction catalyzed by nicotinate N-methyltransferase. Nicotinic acid, the precursor of trigonelline, is formed as a product of NAD degradation. The synthesis of trigonelline from nicotinamide is higher in the leaves than in the roots [16].

Although there are not many studies on the subject, the use of fenugreek extracts rich in trigonelline as plant biostimulants could be relevant due to its role influencing different physiological pathways in plants: (i) nitrogen metabolism, (ii) cell signaling and hormonal regulation, (iii) growth and development regulation, (iv) responses to abiotic stress, and (v) protection against pests and pathogens [17,18,19,20]. Trigonelline and PDBs rich in this compound may represent a promising alternative for the development of biostimulants because of these functions. Despite trigonelline-rich extracts showing great potential, research on their effect on nutrient use efficiency (NUE) is limited and constitutes a critical area with significant gaps in the literature. Most studies focus primarily on crop production [4], without offering a detailed physiological analysis of how these compounds influence plant nutrient absorption and utilization.

To address this gap, this study is focused on evaluating Trigonella foenum-graecum L. extracts as a biostimulant in sunflowers. Our research not only quantifies biomass production and compares different application methods. It also performs a comprehensive analysis of NUE, addressing different key parameters such as apparent recovery (RE). Furthermore, a relationship was established between the characterization of the extracts and the changes in the ionomic profile of the plants, offering a detailed vision of how it affects their nutritional status.

Therefore, this study proposes the use of trigonelline-rich extracts as plant biostimulants with two main objectives: (1) to determine the most beneficial dose and (2) to identify the most effective method of application for sunflower cultivation.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

In this study, sunflower (Helianthus annuus L. cv. neoma) was used as the plant material. The experiment was conducted from the 5 March 2025 to the 30 April 2025. The sunflower seeds were imbibed for one day and then sown in pots containing a 1:1 mixture of vermiculite and perlite. The pots were randomly distributed in a growth chamber under controlled environmental conditions: temperature of 25 °C/15 °C (day/night), a 16 h/8 h photoperiod, relative humidity of 60–80%, and 350 μmol m⁻² s⁻¹ of photosynthetically active radiation (PAR). The sunflower plants were irrigated with a complete nutrient solution based on the Hoagland model with some modifications: 10 mM NaNO₃ (PanReac AppliChem, Barcelona, Spain), 6 mM KCl (Carlo Erba Reagents, Val de Reuil Cedex, France), 4 mM CaCl₂ (Carlo Erba Reagents, Val de Reuil Cedex, France), 2 mM NaH₂PO₄·2H₂O (PanReac Montplet & Esteban SA, Barcelona, Spain), 2 mM MgSO₄·7H₂O (PanReac AppliChem, Barcelona, Spain), 5 ppm Fe·EDDHA (iron chelate) (Químicas Meristem S.L., Valencia, Spain), 1 mM H₃BO₃ (PanReac Química SA, Barcelona, Spain) 0.25 mM CuSO₄·5H₂O (PanReac Química SA, Barcelona, Spain), 0.1 μM Na₂MoO₄·2H₂O (Merck, Darmstadt, Germany), 1 μM ZnSO₄·7H₂O (Merck, Darmstadt, Germany), and 2 μM MnCl₂·4H₂O (PanReac Química SA, Barcelona, Spain). The pH of the nutrient solution was pH of 5.5–6 and was renewed every 3 days.

2.2. Treatment Description and Experimental Design

In this experiment, we used four different trigonelline extracts, which differed in terms of the solvent used for the extraction method and trigonelline concentration.

To extract trigonelline from each extract, the method described by Al-Khateeb (2019) [21] was followed with some modifications, employing methanolic extraction, chemical purification, and chromatographic isolation. The fenugreek seeds were crushed and defatted. For ETg1, ETg3, and ETg4 extracts, 500 g of fenugreek seeds were used, and for ETg2 extract, 1000 g. After filtration, the defatted seeds were dried and macerated with methanol. The mixture was then filtered, and methanol was added to the seeds. Methanol was used as the solvent in ETg3, water for ETg1 and ETg2 extracts, and ethanol for ETg4. Most of the solvent was evaporated under vacuum and subjected to alkaloid test, using Mayer’s and Dragendorff’s reagents. The gummy substances appeared to be precipitated by the addition of excess acetone in a 3:1 ratio to the solvent. Finally, once a clear solution was obtained, 1% of each ETg extract was diluted in 99% water.

Several parameters were analyzed to characterize the four trigonelline-rich extracts. First, the metabolite content, nutrient concentration, and other characteristics were determined (

Table 1. Characterization of the four trigonelline-rich extracts: metabolite and nutrient content, and other characteristics.

	ETg1	ETg2	ETg3	ETg4
Metabolites
Acetate (mM)	0.334	0.507	1.056	2.338
Citrate (mM)	n.d	2.368	0.497	0.928
Glucose (mM)	29.713	15.438	38.758	83.259
Sucrose (mM)	90.342	62.527	26.954	16.311
Trigonelline (mM)	0.311	1.576	2.481	5.317
Nutrients
C total (g 100 g−1)	0.485	0.490	0.365	0.685
N total (g 100 g−1)	0.010	0.020	0.025	0.025
P (mg L−1)	1.755	9.375	13.760	25.630
K (mg L−1)	21.110	79.415	74.600	141.34
Ca (mg L−1)	86.94	86.805	87.705	87.025
Mg (mg L−1)	42.960	44.925	46.365	48.320
S (mg L−1)	32.470	25.810	26.210	26.055
B (mg L−1)	0.115	0.155	0.175	0.205
Fe (mg L−1)	0.050	0.040	0.230	0.315
Mn (mg L−1)	0.295	0.320	0.325	0.350
Mo (mg L−1)	<0.01	<0.01	<0.01	<0.01
Zn (mg L−1)	0.040	0.060	0.160	0.370
Cu (mg L−1)	0.030	0.070	0.090	0.135
Other characterizations
Choline (mM)	0.068	0.373	0.899	1.751
Total phenols (mg gFW−1)	0.285	0.382	0.550	0.825
Antioxidant capacity (Abs gFW −1)	0.162	0.269	0.628	1.321

The metabolites have been expressed in mM; nutrients have been expressed in g 100 g⁻¹ and mg L⁻¹; total phenols have been expressed in mg gFW⁻¹; antioxidant capacity have been expressed in Abs gFW⁻¹. Antioxidant capacity analyzed through Reducing Power. Abbreviations: N: nitrogen; P: phosphorus; K: potassium; Ca: calcium; Mg: magnesium, S: sulfur; B: boron; Fe: iron; Mn: manganese; Mo: molybdenum; Zn: zinc, Cu: copper; ETg1: Extract Trigonelline 1; ETg2: Extract Trigonelline; ETg3: Extract Trigonelline; ETg4: Extract Trigonelline.

). The metabolite content was obtained using HMR (Agilent, Santa Cara, CA, USA); the concentration of macro- and micronutrients was determined using ICP-OES (Agilent, Santa Cara, CA, USA); and the remaining characteristics, such as antioxidant capacity, were determined by spectrophotometry (Tecan Trading AG, Männedorf, Switzerland).

The first application of trigonelline was made 1 month after sowing, and the following applications were performed every 7 days for a total of four applications. The trigonelline dose was 5 mL L⁻¹, and two application methods were used: root application, in which the trigonelline extract was added to the irrigation water, and foliar application, through spraying.

Therefore, the experimental design of this study consisted of a completely randomized block with 9 treatments, 3 replicates per treatment, and 8 plants per replicate: (1) control (water-sprayed), (2) extract 1 via root application (ETg1-R), (3) extract 2 via root application (ETg2-R), (4) extract 3 via root application (ETg3-R), (5) extract 4 via root application (ETg4-R), (6) extract 1 via foliar application (ETg1-F), (7) extract 2 via foliar application (ETg2-F), (8) extract 3 via foliar application (ETg3-F), and (9) extract 4 via foliar application (ETg4-F).

2.3. Plant Sampling and Biomass Measurement

Aerial parts of the sunflower plants were sampled 24 days after treatment initiation. All plants from each treatment were weighed to determine their fresh weight (FW). Half of the plants were frozen at −45 °C for biochemical analyses, while the other half were dried to obtain their dry weight (DW).

2.4. Plant Analysis

2.4.1. Leaf Area

To measure leaf area, the leaves were separated, and it was used a LI-COR optical reader, model LI-3000A (LI-COR, inc., Lincoln, NE, USA), where the results were expressed in cm².

2.4.2. Root Metabolic Activity

To measure root metabolic activity, segments of freshly sampled root tips were cut, and 0.8% TTC (Sigma Aldrich, St. Louis, MO, USA) was added for 30 min. Afterward, the roots were extracted, rinsed, and 95% ethanol was added to them. After 24 h, the absorbance was measured at 490 nm, and the results were expressed as ΔAbs at 490 nm [22].

2.4.3. Minerals and Nutrients Use Efficiency

Mineral nutrient concentrations were determined using ICP-OES. Sunflower leaves were mineralized according to Wolf’s method (1982) [23]. A 0.2 g weight of dry plant material was digested with 30% H₂O₂ (Carlo Erba Reagents, Val de Reuil Cedex, France) and HNO₃ (Carlo Erba Reagents, Val de Reuil Cedex, France) at 80 °C. The mineralized product obtained was used for ionic element analysis. To obtain the nitrogen concentration, 0.2 g of dried leaves was ground and mineralized with 30% H₂O₂ and H₂SO₄ (Carlo Erba Reagents, Val de Reuil Cedex, France) at 300 °C. The mineralized product was used for N analysis.

To determine the efficiency parameters for the use of various essential nutrients, such as RE (apparent crop recovery efficiency of the applied nutrient), IE (internal utilization efficiency of the applied nutrient), and PFP (partial factor productivity of the applied nutrient). The formulas defined by Dobermann (2007) [24] were used:

RE = (U − U₀)/F

IE = Y/U

PFP = Y/F

The U value is the plant’s total nutrient uptake in the aboveground biomass at maturity (mg). The U value can be measured for the control treatment and expressed as U₀. The F value is the amount of the nutrient applied (g). Finally, the Y value corresponds to the dry biomass of the aboveground part of the crop.

2.5. Statistical Analysis

All analyses were repeated in triplicate, and the results were evaluated statistically using a simple ANOVA with a 95% confidence interval. Fisher’s least significant difference test (LSD) was used at the 95% probability level to compare the different treatment means. Significance levels were expressed as: * p < 0.05; ** p < 0.01; *** p < 0.001; NS not significant. A principal component analysis (PCA) was conducted to assess the relationships between treatments and the analyzed parameters. All statistical analyses were performed using the Statgraphics Centurion software version 16.1.03.

3. Results

3.1. Plant Growth

As observed in

Table 2. Effect of trigonelline-rich extracts on aerial part biomass production, leaf area, and plant length.

Treatment	g FW Aerial Part	g DW Aerial Part	Leaf Area (cm2)	Plant Length (cm)
Control	25.24 ± 1.51 e	5.06 ± 0.47 b	534.26 ± 26.68 c	36.2 ± 0.7 c
ETg1-R	24.61 ± 3.74 e	5.15 ± 0.34 b	551.61 ± 45.94 c	34.5 ± 3.0 c
ETg2-R	29.24 ± 2.33 d	5.35 ± 0.32 b	585.31 ± 41.14 bc	34.6 ± 2.4 c
ETg3-R	28.51 ± 1.92 d	5.12 ± 0.17 b	530.48 ± 22.87 c	36.9 ± 0.6 bc
ETg4-R	30.89 ± 3.41 d	5.29 ± 0.50 b	574.71 ± 38.34 bc	36.7 ± 1.8 bc
ETg1-F	27.91 ± 2.10 d	5.20 ± 0.20 b	547.42 ± 27.55 c	36.2 ± 1.3 c
ETg2-F	36.38 ± 1.67 c	5.43 ± 0.31 b	654.70 ± 15.22 ab	40.8 ± 0.5 ab
ETg3-F	38.95 ± 1.18 b	5.99 ± 0.12 ab	662.30 ± 42.85 ab	40.6 ± 1.2 ab
ETg4-F	44.30 ± 3.10 a	6.48 ± 0.45 a	680.22 ± 19.96 a	42.8 ± 0.9 a
p-value	***	**	**	***

Values are means ± standard error (n = 8). The levels of significance are represented as ** (p < 0.01), and *** (p < 0.001). Values with different letters indicate significant differences. Abbreviations: ETg1-R: Extract Trigonelline 1–Root; ETg2-R: Extract 2 Trigonelline–Root; ETg3-R: Extract Trigonelline 3–Root; ETg4-R: Extract Trigonelline 4–Root; ETg1-F: Extract Trigonelline 1–Foliar; ETg2-F: Extract Trigonelline 2–Foliar; ETg3-F: Extract Trigonelline 3–Foliar; ETg4-F: Extract Trigonelline 4–Foliar.

and Figure 1, which present the data on fresh and dry biomass production, leaf area, and plant length, show that the application of trigonelline-rich extracts had a positive effect on the aerial part and biomass production, except for the ETg1-R treatment. The extracts that most promoted aerial growth with respect to the control treatment were ETg2-F, ETg3-F, and ETg4-F. In the case of fresh biomass, ETg2-F increased by 44.12%, ETg3-F by 54.33%, and ETg4-F by 75.55%. Among these treatments, ETg4-F showed the highest values of leaf area and plant length, with an increase of 27.35% and 18.23% in both parameters, respectively (Table 2 and Figure 1).

Regarding root growth, ETg3-R, ETg3-F, and ETg4-F treatments showed increased fresh and dry biomass values compared with the control treatment. Likewise, in root length, these treatments showed the highest levels, with increases of 25.84%, 15.73%, and 23.97%, respectively (

Table 3. Effect of trigonelline-rich extracts on root biomass production and root length.

Treatment	g FW Root	g DW Root	Root Length (cm)
Control	6.26 ± 0.46 b	0.60 ± 0.03 b	26.7 ± 2.5 bc
ETg1-R	6.07 ± 0.44 b	0.65 ± 0.03 b	27.8 ± 1.8 bc
ETg2-R	6.18 ± 0.35 a	0.63 ± 0.01 b	27.5 ± 1.8 bc
ETg3-R	6.80 ± 0.46 a	0.71 ± 0.02 a	33.6 ± 2.0 a
ETg4-R	6.20 ± 0.43 b	0.58 ± 0.03 b	25.9 ± 1.5 bc
ETg1-F	6.03 ± 0.34 b	0.61 ± 0.04 b	25.7 ± 1.8 bc
ETg2-F	6.11 ± 0.30 b	0.62 ± 0.03 b	24.8 ± 1.7 c
ETg3-F	6.98 ± 0.19 a	0.73 ± 0.02 a	30.9 ± 1.4 ab
ETg4-F	7.02 ± 0.63 a	0.72 ± 0.02 a	33.1 ± 1.4 a
p-value	**	**	**

Values are means ± standard error (n = 8). The levels of significance are represented as ** (p < 0.01). Values with different letters indicate significant differences. Abbreviations: ETg1-R: Extract Trigonelline 1–Root; ETg2-R: Extract 2 Trigonelline–Root; ETg3-R: Extract Trigonelline 3–Root; ETg4-R: Extract Trigonelline 4–Root; ETg1-F: Extract Trigonelline 1–Foliar; ETg2-F: Extract Trigonelline 2–Foliar; ETg3-F: Extract Trigonelline 3–Foliar; ETg4-F: Extract Trigonelline 4–Foliar.

and Figure 2).

3.2. Root Metabolic Activity

Treatments in which trigonelline extracts were applied via foliar application increased their root metabolic activity compared to the control treatment, with the ETg1-F treatment showing the highest values (Figure 3). However, in treatments in which trigonelline was applied via the root, the root metabolic activity decreased (Figure 3).

3.3. Minerals

This section addresses the effects of trigonelline-rich extracts on macronutrient concentrations in plants. First, nitrogen concentration in the treatments with the different trigonelline-rich extracts did not show significant differences compared to the control, except for the ETg3-R treatment, which exhibited the highest values (

Table 4. Effect of trigonelline-rich extracts on the foliar concentration of total macronutrients.

Treatment	N (mg g−1 DW)	P (mg g−1 DW)	K (mg g−1 DW)	Ca (mg g−1 DW)	Mg (mg g−1 DW)	S (mg g−1 DW)
Control	68.4 ± 0.1 abc	6.21 ± 0.02 d	59.06 ± 0.23 b	27.61 ± 0.01 d	12.84 ± 0.20 d	4.62 ± 0.01 c
ETg1-R	67.0 ± 0.8 bc	7.75 ± 0.16 c	50.73 ± 1.32 d	30.29 ± 0.92 c	19.53 ± 0.81 b	5.41 ± 0.10 b
ETg2-R	66.8 ± 1.5 cd	8.64 ± 0.04 b	49.57 ± 0.63 d	31.36 ± 0.34 c	18.28 ± 1.04 b	5.72 ± 0.02 b
ETg3-R	69.2 ± 0.1 a	8.30 ± 0.01 b	48.77 ± 0.57 d	34.43 ± 0.48 b	18.14 ± 0.86 b	5.46 ± 0.08 b
ETg4-R	67.0 ± 0.1 bc	10.43 ± 0.24 a	46.69 ± 0.10 d	31.25 ± 0.35 c	18.50 ± 0.91 b	5.97 ± 0.03 b
ETg1-F	68.6 ± 0.2 ab	6.71 ± 0.02 d	64.64 ± 1.11 a	26.96 ± 0.12 d	15.67 ± 0.22 c	4.77 ± 0.01 c
ETg2-F	67.4 ± 0.1 bc	7.92 ± 0.05 c	60.55 ± 0.76 b	25.95 ± 0.50 d	13.14 ± 0.11 d	4.85 ± 0.04 c
ETg3-F	65.1 ± 0.4 d	10.82 ± 0.14 a	53.52 ± 0.55 c	34.76 ± 0.52 a	23.07 ± 0.79 a	6.54 ± 0.07 a
ETg4-F	68.1 ± 0.1 abc	7.81 ± 0.03 c	59.10 ± 0.30 b	27.00 ± 0.21 d	12.75 ± 0.32 d	4.58 ± 0.01 c
p-value	**	***	***	***	***	***

Values are means ± standard error (n = 9). The levels of significance are represented as ** (p < 0.01), and *** (p < 0.001). Values with different letters indicate significant differences. Abbreviations: ETg1-R: Extract Trigonelline 1–Root; ETg2-R: Extract 2 Trigonelline–Root; ETg3-R: Extract Trigonelline 3–Root; ETg4-R: Extract Trigonelline 4–Root; ETg1-F: Extract Trigonelline 1–Foliar; ETg2-F: Extract Trigonelline 2–Foliar; ETg3-F: Extract Trigonelline 3–Foliar; ETg4-F: Extract Trigonelline 4–Foliar; N: nitrogen; P: phosphorus; K: potassium; Mg: magnesium; S: sulfur.

). The ETg3-F treatment showed a marked increase in phosphorus, producing the highest concentrations, with a 74.2% increase over the control plants (Table 4). Similarly, the potassium levels were higher in the ETg1-F and ETg2-F treatments than in the control. As shown in Table 4, the calcium concentration also increased in the treatments with trigonelline extract 3, both via root and foliar application (ETg3-R and ETg3-F). In the case of magnesium and sulfur, ETg3-F treatment increased the concentration of these nutrients by 79.6% and 41.6% compared with the control plants, respectively.

Table 5. Effect of trigonelline-rich extracts on the foliar concentration of total micronutrients.

Treatment	B (μg g−1 DW)	Fe (μg g−1 DW)	Mn (μg g−1 DW)	Mo (μg g−1 DW)	Zn (μg g−1 DW)	Cu (μg g−1 DW)
Control	101.06 ± 0.21 bc	89.77 ± 0.97 e	264.32 ± 0.81 d	4.53 ± 0.02 d	54.80 ± 0.55 d	16.98 ± 0.13 d
ETg1-R	92.36 ± 2.30 e	120.08 ± 3.81 d	277.43 ± 8.91 d	5.75 ± 0.16 c	55.12 ± 0.83 cd	15.89 ± 0.50 d
ETg2-R	95.32 ± 0.59 de	135.58 ± 0.90 c	295.53 ± 3.42 c	6.44 ± 0.04 b	62.18 ± 0.57 b	18.54 ± 0.10 c
ETg3-R	115.00 ± 0.65 a	139.53 ± 0.90 c	319.35 ± 5.54 b	5.85 ± 0.05 c	55.79 ± 0.48 cd	17.06 ± 0.08 c
ETg4-R	103.02 ± 1.25 b	164.35 ± 0.71 b	304.05 ± 2.63 b	7.05 ± 0.02 a	67.19 ± 0.26 a	20.36 ± 0.06 b
ETg1-F	99.91 ± 2.46 bc	131.96 ± 5.70 c	269.32 ± 1.08 d	4.32 ± 0.02 e	56.52 ± 0.02 c	16.15 ± 0.05 d
ETg2-F	93.43 ± 0.96 de	116.29 ± 2.74 d	255.50 ± 4.33 e	3.55 ± 0.04 g	51.23 ± 0.15 f	16.13 ± 0.05 d
ETg3-F	93.84 ± 1.21 de	186.11 ± 3.59 a	361.35 ± 5.54 a	7.01 ± 0.11 a	54.55 ± 0.95 de	21.57 ± 0.27 a
ETg4-F	97.10 ± 0.54 cd	93.93 ± 0.88 e	254.07 ± 0.86 e	3.79 ± 0.01 f	52.96 ± 0.43 e	15.57 ± 0.01 d
p-value	***	***	***	***	***	***

Values are means ± standard error (n = 9). The levels of significance are represented as *** (p < 0.001). Values with different letters indicate significant differences. Abbreviations: ETg1-R: Extract Trigonelline 1–Root; ETg2-R: Extract 2 Trigonelline–Root; ETg3-R: Extract Trigonelline 3–Root; ETg4-R: Extract Trigonelline 4–Root; ETg1-F: Extract Trigonelline 1–Foliar; ETg2-F: Extract Trigonelline 2–Foliar; ETg3-F: Extract Trigonelline 3–Foliar; ETg4-F: Extract Trigonelline 4–Foliar; B: boron; Fe: iron; Mn: manganese; Mo: molybdenum; Zn: zinc; Cu: copper.

shows the effects of trigonelline-rich extracts on micronutrient concentrations in plants. The boron (B) concentration increased in the ETg3-R and ETg4-R treatments compared with the control. Iron (Fe) levels were highest in the ETg4-R and ETg3-F treatments, showing increases of 83.1% and 107.3%, respectively. Likewise, the manganese (Mn) concentration also increased relative to the control by 20.8%, 15%, and 36.7% in ETg3-R, ETg4-R, and ETg3-F, respectively. The highest concentration of zinc (Zn) was observed in the ETg4-R treatment, while the highest copper (Cu) levels were observed in the ETg4-R and ETg3-F treatments (Table 5).

3.4. Principal Component Analysis

A principal component analysis (PCA) was conducted to identify general patterns in the data and to assess the relationships among the parameters. The first principal component (PC1) of the score plot clearly separated ETg3-R and ETg4-R from the other treatments, accounting for 64.58% of the total variance in the data. The second principal component (PC2) separated ETg3-F from the other treatments, explaining 17.38% of the variance (Figure 4). The PCA loading plot revealed the clustering of the nine treatments into three distinct zones (Figure 4). The first group, consisting of ETg3-R and ETg4-R, was associated with P, Ca, Mg, S, B, Fe, Mn, Mo, Zn, and Cu concentrations. The second group, consisting of control, ETg1-F, ETg2-F, and ETg4-F treatments, showed an association between root metabolic activity and N and K concentrations. Finally, in the third group, composed of ETg1-R, ETg2-R, and ETg3-F, different trends were observed. ETg2-R, located in the lower right part of the plot, was close to the variables representing macro- and micronutrient concentrations (except N and K). ETg1-R showed a moderate association with the micronutrients Zn, Mn, Mo, Cu, and Fe. In contrast, ETg3-F, which was positioned in the lower central area, exhibited a weaker association than the other treatments.

3.5. Parameters in Nutrient Use Efficiency

The results of the apparent crop recovery efficiency (RE) are shown in Figure 5 (see data in Tables S1 and S2). In general, RE values increased with the root application treatments, specifically with ETg3-R and ETg4-R, compared with the control treatment and the foliar application treatments. In the case of the ETg3-R treatment, the nutrients that showed the highest values were N, B, and Cu. In contrast, in the ETg4-R treatment, P, Ca, and Zn showed the highest values. Conversely, the lowest values of apparent crop recovery efficiency were observed in the ETg4-F treatment, particularly for Mg, S, Fe, and Mo.

According to the results obtained for the IE index, the ETg4-F treatment showed the most positive values (Figure 6) (see data in Tables S3 and S4). However, the ETg3-F treatment exhibited higher IE values for the nutrients N, K, Ca, and B. However, the ETg3-R and ETg4-R treatments showed the lowest levels across all nutrients. Moreover, the other foliar treatments and the control treatment showed values close to 0 or less negative compared to the treatments in which trigonelline was applied via the root (Figure 6).

Finally, Figure 7 represents the PFP parameter (partial factor productivity) (see data in Tables S5 and S6), where it is observed that the ETg1-R, ETg3-F, ETg4-R, and ETg1-F treatments presented negative values, while the ETg3-F and ETg4-F treatments showed positive ones. Once again, the ETg4-F treatment was the most effective, presenting the highest levels compared with the other treatments.

4. Discussion

4.1. Plant Growth

The use of plant biostimulants has become a common practice in agriculture because they offer multiple benefits for plant growth and protect plants against different types of stress [6]. According to the European Fertilizer Regulation, plant biostimulants can be defined as any product that stimulates the nutritional processes of plants to improve tolerance to abiotic stress, nutrient use efficiency, and plant growth [4]. The absence of phytotoxic effects and the stimulation of plant growth and productivity must be analyzed to verify that trigonelline-rich extracts can be used as biostimulants. To this end, several parameters were analyzed in this experiment, including the fresh and dry biomass of the aerial and root parts, leaf area, and the length of the aerial and root portions.

The application of trigonelline-rich extracts, specifically ETg2-F, ETg3-F, and ETg4-F, promoted the aerial biomass of sunflower plants. The ETg4-F treatment showed the highest values (Table 2). The increase in leaf area resulting from the application of trigonelline-rich extracts could be significant for plant development, as it may lead to a considerable increase in photosynthetic capacity by expanding the light-capturing area.

No phytotoxic effects on the root growth were observed with the application of the extracts. The treatments that significantly increased root growth were ETg3-R, ETg3-F, and ETg4-F (Table 3 and Figure 2). The increase in root length could benefit nutrient and water absorption by roots in areas with water deficit and nutrient limitation; however, further studies are needed.

4.2. Root Metabolic Activity

Biostimulants, in addition to enhancing plant growth, protect against stress and improve soil structure and root nutrition, such as the absorption of macro- and micronutrients, by increasing the cation exchange capacity of the soil [25]. In this sense, by increasing membrane H⁺-ATPase activity and membrane permeability, the root metabolic activity is stimulated, which could improve nutrient absorption [26]. It was observed that the foliar application of trigonelline-rich extracts generally increased root metabolic activity. In contrast, the root application decreased root metabolic activity (Figure 3). Based on these results, it could be deduced that the foliar application of trigonelline-rich extracts could improve nutrient absorption.

4.3. Minerals

For decades, the study of plant nutrition has been of scientific interest to understand how plants absorb, accumulate, transport, and use different chemical elements to complete their life cycle. In this way, knowledge about plant growth and composition in relation to different cultivation substrates, fertilization programs to be applied in various agricultural areas, and the appropriate concentrations of these elements in plant nutrient solutions has been expanded. A mineral nutrient is any chemical element that is essential for plants to grow and reproduce. For an element to be considered a nutrient, it must (i) be necessary for the plant to complete its life cycle, (ii) not be replaceable by another element, and (iii) be required by all plants for their development [27].

An increase in nutrients promotes the growth and development of several plant functions. Nitrogen (N) is considered one of the major essential nutrients; it is a component of the pyrimidines and purines that form nucleic acids (RNA and DNA), as well as being part of the amino group in amino acids in proteins, enzymes, and peptides [28]. Phosphorus (P) is also an essential macronutrient because it is a component of biomolecules involved in energy metabolism (ATP and NADPH), nucleic acids, and cellular membrane phospholipids. P owes its importance to its role in photosynthesis and the reactions of the Calvin cycle [28].

Along with N and P, potassium (K) is a major component of the most widely used fertilizers due to the importance of these three mineral elements. K is essential for maintaining correct osmotic regulation, stable cellular conditions, and photosynthetic CO₂ fixation [29]. Calcium (Ca) is an essential element in plants due to its role in cellular signaling; it also provides rigidity to the cell wall and is responsible for its stabilization [28,30]. Magnesium (Mg) is involved in the covalent bonding in chlorophyll, photon capture, and forms ionic bonds in cell walls [6,31]. Therefore, it is a key element in the transport of photosynthates and the distribution of carbohydrates in plant tissues.

However, a clear case of nutrient antagonism between K, Ca, and Mg has been observed. The concentrations of calcium and magnesium increase when the potassium concentration decreases. The ETg4-R and ETg3-F treatments have the highest values of Ca and Mg, corresponding to a decrease in K.

Sulfur (S) is another essential macronutrient for plants because it is a key component of various prosthetic groups in Fe-S proteins, which are involved in the photosynthetic electron transport chain, and it is a structural element of methionine [32].

In this case, there are synergistic relationships between nutrients, in which an increase in one nutrient’s concentration is accompanied by an increase in another. In the first place, synergism has been found between N and S. The N concentration remained high in all treatments, whereas the S concentration increased significantly, with a maximum in the ETg3-F treatment. Trigonelline-rich extracts could stimulate the metabolic machinery for biomass synthesis. This stimulation probably increases the plant’s demand for the synthesis of complex proteins and amino acids, enhancing the observed synergism between N and S to maintain the optimal N:S ratio necessary for the synthesis of proteins and compounds involved in plant defense [33]. This could explain the higher production results observed in this treatment (Table 2).

Furthermore, another case of synergism has been observed in the ETg3-F treatment. The ETg3-F treatment increased phosphorus concentration and markedly increased Ca, Mg, and S levels. This effect suggests that the extract is enhancing the solubilization of P and other ions in the soil or stimulates root development. The presence of soluble sugars (glucose and sucrose) and organic acids (acetate and citrate) in the ETg3 extract has a dual role: (i) acetate and citrate chelate or mobilize P and metals in the rhizosphere, and (ii) glucose and sucrose act as carbon sources for the soil microbiota. This microbial activation enhances the solubilization and uptake of P, Ca, Mg, and S ions, suggesting that the biostimulant action improves NUE [34].

In summary, regarding macronutrient concentrations, the foliar application of trigonelline extract 3 (ETg3-F) exhibited the highest values for P, Ca, Mg, and S, due to its ability to maximize the synergistic effects. This could explain the treatment’s effect in significantly increasing biomass production (Table 2). These results are also related to the characterization of the four trigonelline-rich extracts (Table 1). While potassium concentration increases linearly in the extracts from ETg1 to ETg4 (from 21.11 mg L⁻¹ to 141.34 mg L⁻¹), this nutrient decreases in plants. This confirms the effect of antagonism between K and Ca, and Mg, which reduces the mineral supply of the extract.

Regarding micronutrients, boron (B) is essential for stabilizing primary cell walls and is involved in cell division, cell elongation, and phytohormonal signaling [35]. The B content in the extracts increased significantly with the trigonelline concentration, from 0.115 mg L⁻¹ in ETg1 to 0.205 mg L⁻¹ in ETg4 (Table 1). This substantial supply of B is directly reflected in the highest foliar concentrations in ETg3-R and ETg4-R treatments (Table 5).

Iron (Fe) is a fundamental micronutrient that acts as a cofactor in proteins involved in catalysis, redox reactions, and electron transport. Fe cofactors can be found in three different forms: heme, non-heme, and Fe-S clusters for these proteins to carry out the previous functions [36,37]. Copper (Cu) is an essential microelement for all organisms. It functions as a catalytic cofactor for numerous proteins and enzymes and plays a key role in photosynthesis, cell wall lignification, and ROS elimination [35].

The Fe concentrations in ETg3 (0.230 mg L⁻¹) and ETg4 (0.315 mg L⁻¹) extracts were higher than those in ETg1 and ETg2 extracts. Similarly, the Cu concentrations in ETg3 (0.090 mg L⁻¹) and ETg4 (0.135 mg L⁻¹) are the highest. Likewise, ETg3-F treatment exhibited the highest concentrations of these micronutrients in plant tissues (Table 5). This synergism between Fe and Cu may be related to the increase in organic acids present in the extracts. Acetate and citrate act as external chelating agents, making the metal cations more available for root absorption [34].

Manganese (Mn) acts as a cofactor in Mn-dependent metalloenzymes, such as superoxide dismutase (Mn-SOD). It is also involved in lignin synthesis and serves as a cofactor in the oxygen-evolving complex (OEC) [35]. The ETg3 and ETg4 extracts showed the highest Mn content (Table 1), and the ETg3-F and ETg3-R treatments showed the highest foliar Mn levels. This increase could enhance the plant’s photosynthetic capacity because the extracts were used as biostimulants.

Molybdenum (Mo) is a cofactor in enzymes such as nitrate reductase and sulfite oxidase and participates in N and S metabolism, atmospheric N₂ fixation, phytohormone balance, and aldehyde detoxification [35]. However, the molybdenum content of the extracts is similar across all samples and has shown lower values. Despite this, ETg3-F treatment exhibited the highest foliar content of Mo, which may be related to the higher trigonelline content of this extract.

Zinc (Zn) acts as a protein cofactor and is involved in hormonal balance and plant growth [35]. A significant decrease has been shown in treatments with the highest P concentrations (specifically ETg3-F). This is a clear example of the antagonism between P and Zn [38]. High concentrations of P in plant tissues inhibit Zn translocation and assimilation, reducing the Zn content supplied by the extract (Table 1).

In summary, in the case of micronutrients, as was also observed for macronutrients, ETg3-F treatment notably improved the nutritional status of micronutrients, showing the highest concentrations for most of these elements. These results could explain the maximum biomass production values observed in plants treated with ETg3-F (Table 2). However, the ETg4-R treatment also showed higher values within the root application method, which was also reflected in the biomass results obtained through this application method.

One of the positive effects of applying biostimulants to plants is the improvement of their nutritional status, as there is a greater accumulation of essential mineral elements [39]. Plants exhibit a deficiency state when one of these nutrients is not present in sufficient quantities to perform its functions. Nutrient deficiencies have negative consequences for agricultural production on a global scale. Furthermore, inappropriate fertilizer use can cause pollution in terrestrial and aquatic ecosystems [28,35]. In the present study, the foliar concentration of macro- and micronutrients showed values considered normal, since no deficiency or excess of any nutrient was detected that could affect the growth of sunflower plants (Table 4 and Table 5) [25]. The application of trigonelline-rich extracts, both via root and foliar application, resulted in macro- and micronutrient concentrations similar to or higher than those of the control plants. Therefore, the use of these extracts improves the nutritional status of sunflower plants and, consequently, they could be used as a biofertilizer, particularly in the case of the ETg4-R and ETg3-F treatments, whose extracts presented the highest trigonelline concentrations.

4.4. Principal Component Analysis

The results obtained from PCA and NUE analysis crucially complement each other, offering a comprehensive view of nutrient assimilation and use strategies across treatments. PCA demonstrated that the main variance in the data is driven by mineral content, with treatments ETg3-R and ETg4-R accounting for 64.58% of the variance. These treatments resulted in strong and high accumulation of most nutrients (P, Ca, Mg, S, B, Fe, Mn, Mo, Zn, and Cu).

However, the ETg3-F treatment generally showed the highest nutrient concentration (Table 4 and Table 5), but the PCA analysis suggests a different profile than the ETg3-R and ETg4-R treatments. However, the IE and PFP heat maps (Figure 6 and Figure 7) showed that the ETg3-R and ETg4-R treatments were the least efficient in nutrient use, where high nutrient absorption did not effectively translate into productivity.

In contrast, the ETg4-F treatment, along with ETg3-F, was associated with higher root metabolic activity and N and K content (PC2) and was the most efficient in nutrient use (Figure 6 and Figure 7). This suggests that ETg4-F adopted an optimal nutrient balance strategy, avoiding excessive accumulation and using available nutrients more effectively for growth and biomass (Table 2 and Table 3).

4.5. Parameters in Nutrient Use Efficiency

Nutrient use efficiency expresses the relationship between the amount of each nutrient applied and various plant parameters, such as yield or biomass. This efficiency can be evaluated through different indicators, such as RE (apparent recovery efficiency of the applied nutrient by the crop), which is an index that considers the levels of the nutrient applied to the substrate, considering its absorption in untreated control plots. IE (internal utilization efficiency of the applied nutrient) is a parameter that relates biomass production to the nutrient concentration in the plant. From an economic perspective, it is interesting to determine how to maintain or increase crop yield while minimizing resources through PFP, which reflects the yield obtained per unit of nutrient applied. Therefore, greater efficiency in the use of nutrients could mean improved crop yield and quality, reduced use of fertilizers, lower environmental impacts, and decreased production costs [24,40].

In general, the RE values increased with root application treatments (Figure 5), which would explain the higher concentrations of nutrients such as N, P, Ca, B, Mo, Zn, and Cu obtained with the application of the two treatments (Table 4 and Table 5).

These results suggest that the application of trigonelline-rich extracts via root application may increase the ability of the plant to absorb nutrients, possibly by stimulating the root system [24]. This increase in RE not only reflects greater use of the applied nutrients but also represents an advantage in low-fertility soils, where absorption efficiency is important for sustaining crop yield (Figure 5).

The results obtained for the IE index suggest that the foliar application of extract 4, rich in trigonelline, produces an internal stimulation in the physiological use of nutrients, with higher values than those of control plants and the plants treated with the other trigonelline-rich extracts (Figure 6). These results can be related to the greater biomass production in plants with this treatment (Table 2), indicating that the nutrients available to the plant have contributed more effectively to plant growth and yield [24].

Regarding the PFP parameter, foliar treatments generally exhibited positive values, with the ETg4-F treatment showing the highest results (Figure 7). This suggests that the foliar application of this trigonelline-rich extract resulted in a higher yield per unit of nutrient applied, showing a productive advantage over the other treatments.

In conclusion, the application of trigonelline-rich extracts significantly improves NUE, with ETg4-F treatment being particularly notable, as it presented the highest values in the agronomic indices of IE and PFP, as well as in crop growth and production parameters (Table 2). On the other hand, ETg4-R treatment achieved the highest levels of RE, suggesting that a combination of foliar and root applications of extract 4 could jointly enhance nutrient absorption and physiological use. These results support the use of trigonelline-rich extracts as biostimulants under optimal growth conditions and open the possibility that the application of these extracts could be an effective strategy to reduce the use of fertilizers, improve crop yields in nutrient-poor soils, and promote more sustainable agriculture.

5. Conclusions

In conclusion, the findings of this study support the potential of trigonelline-rich extracts as biostimulants, particularly when the extracts are delivered through foliar application, which emerged as the agronomically superior strategy. Furthermore, a higher concentration of trigonelline in the extracts used as biostimulants translates into improved crop production and enhanced plant nutritional status. Our analysis demonstrated that while some root treatments accumulated high mineral concentrations (confirmed by PCA’s PC1, notably in ETg3-R and ETg4-R), the foliar-applied ETg3-F and ETg4-F achieved the highest NUE and biomass production. This critical distinction indicates that the biostimulant activity lies not in promoting excessive, non-functional accumulation of nutrients—where absorption exceeds metabolic needs—but rather in optimizing nutrient assimilation and metabolism, thereby improving biomass and nutritional status without requiring disproportionate absorption. This performance makes trigonelline-rich extracts effective biostimulants under optimal growth conditions and suggests their potential as a sustainable agricultural strategy to reduce fertilizer input and improve crop productivity, particularly in nutrient-deficient soils. Future studies should aim to validate these results under field conditions and explore the underlying biochemical and physiological mechanisms.