Foliar Application of Aqueous Extracts from the Algal Biomass of Laminaria digitata and Phaeodactylum tricornutum as Strategy to Mitigate Boron Toxicity in Tomato (Solanum lycopersicum L.)

Abstract

Boron (B) toxicity is a relevant problem in Mediterranean regions, where irrigation water may present high concentrations of this micronutrient. In this study, the potential of aqueous extracts from the brown macroalga Laminaria digitata and the diatom Phaeodactylum tricornutum, applied alone or in combination with metalloids (Se, Si) and micronutrients (Mn, Fe, Zn), was evaluated to improve the tolerance of tomato plants (Solanum lycopersicum L.) grown under excess B (15 mg L⁻¹). The extracts were applied foliarly, and growth parameters, gas exchange, chlorophyll content, mineral composition, B accumulation, oxidative stress, and metabolic profile were analyzed. Excess B significantly reduced root development, net photosynthesis, and metabolic balance, evidencing a strong physiological impact. The application of algal extracts partially mitigated these adverse effects, mainly through improvements in photosynthesis, water use efficiency, and the accumulation of osmoprotective metabolites (proline, tryptophan, glucose). In particular, L. digitata promoted a significant increase in total biomass and greater physiological recovery compared with P. tricornutum. Conversely, formulations enriched with metalloids and micronutrients did not provide consistent additional benefits and even induced metabolic imbalances. Multivariate analysis (PCA) confirmed that relative tolerance was associated with physiological and metabolic variables rather than nutritional changes. Overall, these results highlight the potential of algal extracts, especially L. digitata, as effective biostimulants to mitigate boron toxicity in tomato.

1. Introduction

In recent years, agriculture has faced numerous challenges, including the need to increase productivity and to develop strategies aimed at improving crop tolerance to various abiotic stresses. Within this context, biostimulants have emerged as a sustainable alternative that promotes plant development and enhances physiological efficiency [1,2]. Among the resources most commonly used in the formulation of biostimulant products, algae stand out due to their diverse biochemical and nutritional profiles.

Algae are photosynthetic organisms, either unicellular or multicellular, that mainly inhabit aquatic environments. They are broadly classified into two groups: macroalgae, represented in this study by brown algae, and microalgae, which include diatoms. These species have evolved defense mechanisms that enable them to withstand adverse conditions such as high salinity or temperature fluctuations, largely through the synthesis of bioactive compounds [3,4]. Nevertheless, their composition and activity may vary depending on the species, environmental conditions, and extraction methods [5].

In agriculture, algal extracts are applied due to their beneficial effects on crop performance, as they enhance processes such as germination, root development, and stress tolerance [6]. Recent studies have also demonstrated the potential of microalgae and cyanobacteria as sustainable biofertilizers that can stimulate plant growth and enhance physiological and metabolic processes. For example, Romanowska-Duda et al. [7] reported that foliar biofertilization with Chlorella sp. and cyanobacteria (Microcystis aeruginosa and Anabaena sp.) significantly increased growth, photosynthetic activity, and biomass production of willow (Salix viminalis L.), achieving over 25% higher yield compared to untreated plants. These microorganisms improved nutrient assimilation and enzymatic activity, demonstrating their capacity to enhance plant performance while contributing to sustainable biomass production for bioenergy purposes. Species proposed as biostimulants include the brown alga Laminaria digitata and the diatom Phaeodactylum tricornutum. Shibaeva et al. [8] reported that extracts of L. digitata stimulated growth in wheat and cucumber plants, increasing vegetative development, biomass accumulation, and chlorophyll content. Similarly, a commercial product based on Seaweed (Ascophyllum nodosum and Laminaria digitata) and Yeast Extracts was shown to mitigate drought stress in tomato plants by improving water potential, reducing oxidative stress, and enhancing photosynthetic pigment levels [9]. Regarding P. tricornutum, Rachidi et al. [10] found that extracts from this diatom strengthened the defense capacity of tomato plants, enhancing their resistance against both abiotic and biotic stresses. Likewise, Guzmán-Murillo et al. [11] demonstrated that applying P. tricornutum extract to pepper seeds alleviated the negative effects of salt stress, promoting root growth and germination rate.

In addition to algae, biostimulant formulations often incorporate certain metalloids and essential micronutrients. Among metalloids, selenium (Se) and silicon (Si) are particularly relevant, while manganese (Mn), iron (Fe), and zinc (Zn) are among the most widely used micronutrients. These elements, in either conventional or nanostructured forms, have been shown to improve plant metabolism, photosynthetic efficiency, and tolerance to various abiotic stresses [12,13]. For instance, Si contributes to cell wall synthesis, regulates water-use efficiency, and modulates antioxidant enzyme activity [14]. Se promotes growth, activates antioxidant defense systems, and enhances nutrient uptake [15]. Meanwhile, Mn, Fe, and Zn are involved in photosynthesis, antioxidant enzyme activation, chlorophyll biosynthesis, carbohydrate metabolism, and overall plant development [13].

In some agricultural regions, the scarcity of high-quality water has driven the use of non-conventional water resources, which often contain elevated concentrations of boron and other potentially toxic substances [16]. Although boron (B) is an essential micronutrient involved in processes such as cell wall integrity, nitrogen and carbohydrate metabolism [17], its optimal concentration range is extremely narrow, and slightly higher levels may result in toxicity, particularly in alkaline and saline soils [18]. Boron toxicity is one of the major abiotic stresses limiting crop productivity, and is associated with symptoms such as leaf chlorosis and necrosis, accumulation of reactive oxygen species (ROS), inhibition of photosynthesis, and impaired nutrient assimilation [19,20,21]. In the Mediterranean region, B toxicity is a widespread problem affecting several crops, including tomato.

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated climacteric fruits worldwide, valued for its nutritional quality and antioxidant compounds [21]. Although it is considered moderately tolerant to B, concentrations above 6.0 mg·L⁻¹ in irrigation water can negatively affect its growth and physiology [22]. For example, Sirajuddin et al. [23] reported that tomato plants exposed to increasing concentrations of B (10, 20, and 50 mg·L⁻¹) for 10 weeks showed reduced vegetative growth, decreased chlorophyll content, increased oxidative stress, and higher proline accumulation in leaves. However, despite its importance, relatively little information is available on how tomato responds to excess B in irrigation water. Based on these considerations, we hypothesize that extracts of L. digitata and P. tricornutum, applied individually or in combination with metalloids and micronutrients, may enhance tomato tolerance to boron toxicity. The specific objectives of this study were (i) to evaluate the effects of B toxicity (15 mg·L⁻¹) on tomato plants; (ii) to determine whether the application of algal extracts, alone or in combination with metalloids and micronutrients, can mitigate the adverse effects of excess B in the nutrient solution; and (iii) to identify the treatment that provides the most effective response. To this end, physiological, biochemical, and metabolic parameters were assessed.

2. Materials and Methods

Tomato (Solanum lycopersicum L.) plants of the cultivar ‘Óptima’ were obtained from a commercial nursery (BabyPlant, Santomera, Murcia, Spain). When seedlings reached an average height of 10–20 cm, homogeneous plants without nutritional deficiencies or disease symptoms were selected and transplanted into 1.5 L pots filled with silica sand as an inert substrate.

2.1. Greenhouse Conditions and Crop Management

The experiment was conducted between April and June 2022 in a multi-span greenhouse at the experimental farm of the Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC, Santomera, Murcia, Spain). Temperature was controlled using a cooling system that maintained values below 35 °C, supplemented with a 30% aluminum shading net. Yellow sticky traps were installed, and ecological foliar treatments were periodically applied to control pests and diseases.

Irrigation was applied according to crop demand using 2 L h⁻¹ self-compensating drippers, adjusting the volume to ensure drainage at each event. Fertigation was performed with a 100% Hoagland nutrient solution (NS) consisting of KNO₃ (54 g 100 L⁻¹), Ca(NO₃)₂ (84 g 100 L⁻¹), KH₂PO₄ (14 g 100 L⁻¹), MgSO₄ (26 g 100 L⁻¹), Fe-EDTA (2 g 100 L⁻¹), and micronutrients (2 g 100 L⁻¹). An automated system regulated both the frequency and volume of irrigation, maintaining 10–15% leaching to prevent salt accumulation in the substrate.

After one week of acclimation, plants were divided into two groups: (i) control, irrigated with Hoagland NS containing 0.25 mg L⁻¹ B; and (ii) boron treatment, irrigated with Hoagland NS containing 15 mg L⁻¹ B, supplied as boric acid (H₃BO₃). The experiment was conducted over a total period of five weeks following transplanting. During the first week, plants were acclimated to pot conditions under optimal irrigation. Beginning in the second week, irrigation with a boron-enriched nutrient solution was initiated, and foliar applications of the algal extracts were carried out simultaneously at weekly intervals for three consecutive weeks. Irrigation with the boron solution was maintained throughout the entire experimental period to ensure continuous exposure to B stress.

2.2. Preparation and Application of Algal Extracts

Aqueous extracts were prepared from freeze-dried biomass of the brown alga Laminaria digitata and the diatom Phaeodactylum tricornutum. Extracts were obtained by heating the dry biomass in distilled water (4 g 100 mL⁻¹) in a water bath (JP Selecta, Barcelona, Spain) for 1 h at 90 °C, followed by filtration through Whatman No. 42 filter paper (Whatman plc, Maidstone, UK).

For each algal species, four formulations were prepared:

• Mix 1 (alga): 500 mL L⁻¹ of the algal extract.
• Mix 2 (alga + mtd): 500 mL L⁻¹ algal extract supplemented with Se (1 mg L⁻¹) and Si (400 mg L⁻¹).
• Mix 3 (alga + nut): 500 mL L⁻¹ algal extract supplemented with Mn (100 mg L⁻¹), Fe (28 mg L⁻¹), and Zn (190 mg L⁻¹).
• Mix 4 (alga + mtd + nut): 500 mL L⁻¹ algal extract enriched with both the metalloids and micronutrients described above.

Tween-80 was added as a surfactant to improve foliar coverage and absorption. Treatments were applied weekly by foliar spraying for three weeks, ensuring full leaf coverage. Thus, for each algal extract, six treatments were evaluated: (1) Control -B, (2) Control + B, (3) Alga + B, (4) (Alga + mtd) + B, (5) (Alga + nut) + B, and (6) (Alga + mtd + nut) + B.

2.3. Growth Parameters

At the end of the experiment, tomato plants were harvested and separated into shoots and roots. Fresh weight (FW) was recorded using a precision balance (PS 600.R2, Radwag, Radom, Poland). Samples were washed with distilled water, dried in an oven at 60 °C for at least 48 h, and then weighed to obtain dry weight (DW). Total biomass per plant and shoot-to-root (S:R) ratio were calculated.

2.4. Gas Exchange Parameters

Net photosynthetic rate (PN; µmol CO₂ m⁻² s⁻¹), stomatal conductance (gs; mmol H₂O m⁻² s⁻¹), transpiration rate (E; mmol H₂O m⁻² s⁻¹), intercellular CO₂ concentration (Ci; µmol mol⁻¹), and water-use efficiency (WUE; µmol CO₂ mmol⁻¹ H₂O) were measured at the end of the experiment using a portable gas-exchange analyzer (CIRAS-2, PP Systems, Amesbury, UK) between 8:30 and 10:00 h. During measurements, photosynthetically active radiation (PAR) was set to 1200 µmol m⁻² s⁻¹ and CO₂ concentration to 400 ppm.

2.5. Chlorophyll Content

Relative chlorophyll content was measured in fully expanded leaves from the middle (Chl-M) and upper (Chl-U) canopy using a portable chlorophyll meter (CL-01, Hansatech Instruments Ltd., King’s Lynn, UK). The device calculates relative chlorophyll values based on absorbance at 620 and 940 nm, with automatic calibration and temperature compensation. Results are expressed as SPAD units.

2.6. Mineral Analysis

Three leaf samples per treatment were collected at the end of the experiment. Samples were washed, oven-dried (48 h, 60 °C), and ground to a fine powder. Nutrient analysis was performed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Iris Intrepid II, Thermo Electron Corporation, Waltham, MA, USA) after digestion with HNO₃:H₂O₂ (5:3, v/v) in a microwave digester (CERM Mars Xpress, Apex, NC, USA) at up to 200 °C. Concentrations of Mg, K, Ca, and P were determined (g 100 g⁻¹ DW), along with B, Mn, Fe, and Zn.

2.7. Oxidative Stress

Lipid peroxidation was determined by quantifying malondialdehyde (MDA) in three leaf samples per treatment. Samples were washed, frozen at −80 °C, and lyophilized (LyoQuest, Telstar, Terrassa, Spain). MDA was measured following the colorimetric method of [24], with minor modifications. Briefly, 50 mg DW was homogenized with 1 mL of 0.1% trichloroacetic acid (TCA), shaken for 10 min, and centrifuged (5000 rpm, 10 min). Two aliquots of 125 µL were taken: one mixed with 20% TCA and the other with 20% TCA + 0.5% thiobarbituric acid (TBA). Samples were incubated for 1 h at 90 °C, cooled on ice, and absorbance was measured at 440, 532, and 600 nm (BIOTEK Powerwave XS2, Marshall Scientific, Hampton, Virginia, USA). MDA equivalents (nmol mL⁻¹) were calculated as:

• A = [(A₅₃₂ + TBA − A₆₀₀ + TBA) − (A₅₃₂ − TBA − A₆₀₀ − TBA)]
• B = [(A₄₄₀ + TBA − A₆₀₀ + TBA) × 0.0571]
• MDA (nmol mL⁻¹) = [(A − B)/157,000] × 10⁶

Results were expressed as nmol g⁻¹ DW.

2.8. Metabolic Analysis

Metabolic profiling was conducted in leaves using four samples per treatment. Samples were washed, frozen (−80 °C), and lyophilized (LyoQuest, Telstar). Spectra were acquired using a 500 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5 mm CryoProbe Prodigy BBO. Proton NMR (¹H-NMR) spectra were processed with Chenomx NMR Suite v.9 (Chenomx Inc., Edmonton, AB, Canada). Detected metabolites included alanine (Ala), glutamine (Gln), proline (Pro), citrate (Cit), glucose (Glc), and trigonelline (Tri).

2.9. Statistical Analysis

For each alga, a unifactorial experimental design with six treatments was used: (1) Control -B, (2) Control + B, (3) Alga + B, (4) (Alga + mtd) + B, (5) (Alga + nut) + B, and (6) (Alga + mtd + nut) + B. In total, 36 plants per algal extract were analyzed (six treatments × six plants each). Data were analyzed by one-way ANOVA using SPSS v.29 (IBM Corp., Armonk, NY, USA). Growth, physiological parameters and boron concentration were evaluated using six biological replicates (n = 6), metabolite analyses were performed on four replicates (n = 4), and oxidative stress indicators (MDA) and mineral nutrients were determined using three replicates (n = 3). When significant differences were detected (p < 0.05), means were separated by Duncan’s multiple range test.

A principal component analysis (PCA) was also performed using physiological, mineral, metabolic, chlorophyll, boron, and MDA data (growth variables excluded to avoid circularity). The PCA was based on a standardized correlation matrix (z-scores), reducing dimensionality to two principal components. Relative tolerance was calculated as the ratio between total biomass under algal treatments and that of the +B control, expressed as a percentage. Pearson’s correlation analysis was used to evaluate relationships between relative tolerance and PC1/PC2 scores.

3. Results

3.1. Vegetative Growth Results

In plants irrigated with excess boron, root growth was markedly reduced, with root dry weight decreasing by nearly 60% compared with plants grown without boron. This limitation also affected total biomass, which declined by approximately 20% under boron stress, although the reduction was not statistically significant. Consequently, the shoot-to-root ratio nearly doubled in boron-treated plants relative to the controls. No significant differences were observed in shoot biomass between treatments (

Table 1. Vegetative growth parameters in tomato plants irrigated with high boron concentration and treated with different formulations of algal extracts from L. digitata and P. tricornutum.

Algae	Treatment	Shoot Biomass (g ps)	Root Biomass (g ps)	Total Biomass (g ps)	PA:R
L. digitata	Control -B	23.8 cd	3.13 a	26.9 bc	8.05 b
	Control + B	20.2 d	1.32 c	21.5 c	15.8 a
	Alga1 + B	28.0 bc	3.02 ab	31.1 ab	10.9 b
	(Alga1 + mtd) + B	22.3 d	2.06 c	24.4 c	11.4 b
	(Alga1 + nut) + B	30.7 ab	1.86 c	32.6 a	16.6 a
	(Alga1 + mtd + nut) + B	33.9 a	2.16 bc	36.0 a	16.4 a
	ANOVA	***	***	***	***
P. tricornutum	Control -B	23.8 abc	3.13 a	26.9 a	8.05 b
	Control + B	20.2 bc	1.32 bc	21.5 bc	15.8 a
	Alga2 + B	24.5 abc	1.44 bc	25.9 ab	17.3 a
	(Alga2 + mtd) + B	19.3 c	1.11 c	20.4 c	18.0 a
	(Alga2 + nut) + B	25.0 ab	1.40 bc	26.4 ab	18.1 a
	(Alga2 + mtd + nut) + B	28.9 a	1.78 b	30.7 a	16.7 a
	ANOVA	**	***	**	**

ANOVA results: ** and *** indicate significant differences at p < 0.01 and p < 0.001, respectively. For each algal extract, different lowercase letters indicate significant differences among treatments at p < 0.05 according to Duncan’s multiple range test (n = 6).

).

For Laminaria digitata, the application of the algal extract alone (+B) promoted growth, increasing total biomass by around 40% and more than doubling root biomass compared with the boron control. When the extract was enriched with micronutrients (+nut + B) or with the combination of metalloids and micronutrients (+mtd + nut + B), total biomass further increased, reaching the highest values observed in the experiment, although differences were not statistically significant compared with the extract-only treatment. In contrast, Phaeodactylum tricornutum extract did not produce significant differences in any of the evaluated growth parameters compared with the boron control. Similarly, enriched formulations did not elicit significant positive effects, although a general trend towards increased total biomass was observed.

3.2. Gas Exchange Results

Tomato plants irrigated with excess boron exhibited a pronounced decline in photosynthetic activity. Net CO₂ assimilation (PN) decreased by more than 80% compared with plants grown without boron. In parallel, stomatal conductance (gs) and transpiration rate (E) were markedly reduced, by over 50% and 40%, respectively (

Table 2. Gas-exchange parameters in tomato plants irrigated with high boron concentration and treated with different formulations of algal extracts from L. digitata and P. tricornutum: PN (µmol CO₂ m⁻² s⁻¹), gs (mmol H₂O m⁻² s⁻¹), E (mmol H₂O m⁻² s⁻¹), Ci (µmol mol⁻¹), and WUE (µmol CO₂ mmol⁻¹ H₂O).

Algae	Treatment	PN	gs	E	Ci	WUE
L. digitata	Control -B	7.63 a	139.5 a	2.45 a	269.5 d	3.12 a
	Control + B	1.38 c	62.0 b	1.48 b	338.8 a	1.00 d
	Alga1 + B	5.00 b	108.8 a	2.45 a	292.3 c	2.05 bc
	(Alga1 + mtd) + B	1.48 c	38.0 b	0.91 c	318.3 b	1.63 c
	(Alga1 + nut) + B	5.45 b	127.0 a	2.39 a	298.5 c	2.28 b
	(Alga1 + mtd + nut) + B	5.00 b	132.8 a	2.46 a	307.5 bc	2.04 bc
	ANOVA	***	***	***	***	***
P. tricornutum	Control -B	7.63 a	139.5 a	2.45 a	269.5 c	3.12 a
	Control + B	1.38 c	62.0 c	1.48 b	338.8 a	1.00 d
	Alga2 + B	5.85 b	112.3 ab	1.99 a	282.5 c	2.93 ab
	(Alga2 + mtd) + B	2.88 c	85.0 bc	2.07 a	317.5 ab	1.41 d
	(Alga2 + nut) + B	5.28 b	106.0 b	2.39 a	283.0 c	2.27 bc
	(Alga2 + mtd + nut) + B	2.48 c	62.3 c	1.40 b	311.0 b	1.78 cd
	ANOVA	***	***	***	***	***

ANOVA results: *** indicates significant differences at p < 0.001. For each algal extract, different lowercase letters indicate significant differences among treatments at p < 0.05 according to Duncan’s multiple range test (n = 6).

). This stomatal closure was accompanied by an increase in intercellular CO₂ concentration (Ci) and a reduction in water-use efficiency (WUE).

For Laminaria digitata, application of the algal extract alone (+B) promoted recovery across all measured parameters (PN, gs, E, Ci, and WUE), although values comparable to the –B control were only reached for gs and Ci. Enriched formulations with metalloids and/or micronutrients did not outperform the effect of the extract-only treatment. In the case of Phaeodactylum tricornutum, application of the algal extract alone led to substantial recovery of photosynthetic parameters: PN increased more than four-fold compared with the +B control, while gs and E also rose markedly. WUE approached values observed in the –B control, and Ci declined toward optimal levels. Among enriched treatments, none produced stronger effects than the algal extract alone.

3.3. Chlorophyll Content in Middle and Upper Leaves

In plants irrigated with excess boron, a marked reduction in chlorophyll content was observed in middle leaves, with decreases of approximately 40–45% compared with plants grown without boron. A similar trend was detected in upper, fully expanded leaves, where chlorophyll content declined by about 30% under boron stress (Figure 1). No beneficial effects were detected for any of the treatments formulated with algal extracts or enriched with additional active compounds.

3.4. Mineral Analysis Results

Boron irrigation induced changes in the mineral composition of tomato leaves (

Table 3. Mineral composition of tomato leaves from plants irrigated with high boron concentration and treated with L. digitata and P. tricornutum.

		Mg	K		Ca		P
Algae	Treatment	g 100g−1 ps
L. digitata	Control -B	1.11 a	2.83 b		3.20 ab		0.48 c
	Control + B	0.81 bc	3.66 b		2.44 bcd		0.83 b
	Alga1 + B	0.50 d	3.01 b		1.74 d		0.81 b
	(Alga1 + mtd) + B	1.01 ab	4.94 a		3.68 a		1.25 a
	(Alga1 + nut) + B	0.73 cd	2.84 b		2.76 abc		0.63 bc
	(Alga1 + mtd + nut) + B	0.66 cd	2.97 b		2.26 cd		0.72 bc
	ANOVA	***	*		**		**
P. tricornutum	Control -B	1.11 a	2.83 bc		3.20 a		0.48 c
	Control + B	0.81 b	3.66 a		2.44 ab		0.83 a
	Alga2 + B	0.63 bc	2.93 ab		2.37 ab		0.79 ab
	(Alga2 + mtd) + B	0.44 c	2.29 bc		1.65 b		0.64 bc
	(Alga2 + nut) + B	0.58 c	2.10 c		2.09 b		0.53 c
	(Alga2 + mtd + nut) + B	0.64 bc	2.67 bc		2.52 ab		0.76 ab
	ANOVA	***	**		*		**
		Mn		Fe		Zn
Algae	Treatment	mg kg−1 ps
L. digitata	Control -B	143.4 c		171.3 b		26.9 c
	Control + B	120.2 c		135.1 b		34.9 c
	Alga1 + B	117.6 c		166.5 b		41.5 c
	(Alga1 + mtd) + B	211.3 b		261.8 a		51.8 c
	(Alga1 + nut) + B	342.0 a		234.2 a		329.3 b
	(Alga1 + mtd + nut) + B	346.4 a		241.5 a		456.1 a
	ANOVA	***		***		***
P. tricornutum	Control -B	143.4 bc		171.3 b		26.9 c
	Control +B	120.2 cd		135.1 b		34.9 c
	Alga2 +B	117.7 cd		158.5 b		46.1 c
	(Alga2 + mtd) +B	85.1 d		141.1 b		33.0 c
	(Alga2 + nut) +B	173.7 b		160.0 b		180.5 b
	(Alga2 + mtd + nut) +B	318.8 a		273.7 a		427.9 a
	ANOVA	***		***		***

ANOVA results: *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively. For each algal extract, different lowercase letters indicate significant differences among treatments at p < 0.05 according to Duncan’s multiple range test (n = 3).

). Magnesium content decreased by approximately 25% compared with –B plants, whereas phosphorus showed the opposite trend, increasing by nearly 70% under boron stress. No significant differences were observed in K, Ca, Mn, Fe, or Zn between the –B and +B controls.

For Laminaria digitata, application of the algal extract alone (+B) resulted in a marked reduction in Mg (0.50 vs. 0.81 g 100 g⁻¹ DW) compared with the +B control, while no significant differences were observed for the other nutrients. When the extract was supplemented with metalloids (mtd), concentrations of Mg, K, Ca, P, Mn, and Zn increased relative to the extract-only treatment. For Phaeodactylum tricornutum, no significant differences were detected between the algal extract (+B) and the +B control in any macro- or micronutrient. Among enriched treatments, P. tricornutum +mtd + B and +nut + B induced significant decreases in Mg and K. Regarding micronutrients, significant differences emerged with the +nut treatment, which increased foliar concentrations of Mn and Zn, and with the +mtd + nut treatment, which yielded the highest levels of Mn, Fe, and Zn, clearly differing from the algal extract alone (+B).

3.5. Boron Concentration

In leaves, boron-irrigated plants accumulated approximately 2200–3800 mg kg⁻¹ B, whereas –B plants reached only about 100 mg kg⁻¹. This represented more than a ten-fold increase in foliar B concentration. In roots, boron-treated plants registered values close to 300 mg kg⁻¹ compared with only 30–100 mg kg⁻¹ in –B plants, corresponding to up to a three-fold increase (Figure 2).

Application of products enriched with micronutrients (+nut) or with both metalloids and micronutrients (+mtd + nut) increased foliar B concentration for both algal species. However, in Laminaria digitata, the +mtd + nut treatment did not significantly differ from the other enriched formulations. In roots, within the L. digitata group, B concentration was similar across all algal treatments, and consistently higher than in the +B control (Figure 2). In contrast, application of Phaeodactylum tricornutum extract showed no significant differences in root B concentration among treatments.

3.6. Oxidative Stress Results

Leaf MDA concentrations were significantly higher in boron-irrigated plants compared with –B controls, with increases of approximately 30–40% (Figure 3). A similar trend was observed in roots, where boron-treated plants reached values of 38–42 nmol g⁻¹ DW compared with ~22 nmol g⁻¹ DW in –B plants, corresponding to about 40% greater MDA accumulation. Among boron-treated plants, no significant differences were detected between treatments in either the Laminaria digitata or Phaeodactylum tricornutum groups.

3.7. Metabolomic Analysis

The profiles of Ala, Gln, Pro, Cit, Glc, and Tri are presented in

Table 4. Metabolomic profile in tomato leaves from plants irrigated with high boron concentration and treated with L. digitata and P. tricornutum.

		Ala	Gln	Pro	Cit	Glc	Tri
Algae	Treatment	mg g−1 ps
L. digitata	Control -B	0.69 b	2.02 d	4.09 c	44.5 a	11.9 c	0.92 bc
	Control + B	1.08 a	5.08 a	2.86 d	44.9 a	24.0 b	0.81 c
	Alga1 + B	1.10 a	4.07 b	5.77 b	32.1 a	29.3 b	1.21 b
	(Alga1 + mtd) + B	0.89 ab	3.21 c	4.52 c	36.2 a	26.3 b	1.03 bc
	(Alga1 + nut) + B	0.08 c	0.33 e	0.59 e	1.31 b	4.65 d	0.14 d
	(Alga1 + mtd + nut) + B	1.06 a	3.76 bc	8.51 a	13.4 b	40.3 a	1.55 a
	ANOVA	***	***	***	***	***	***
P. tricornutum	Control -B	0.69	2.02 d	4.08 bc	44.5 a	11.9 c	0.92 c
	Control + B	1.08	5.08 a	2.86 c	44.9 a	24.0 b	0.81 c
	Alga + B	1.02	3.67 bc	5.79 ab	29.1 b	32.1 a	1.39 a
	(Alga2 + mtd) + B	0.79	3.92 b	2.99 c	27.9 b	29.5 ab	1.01 bc
	(Alga2 + nut) + B	0.73	2.69 cd	8.03 a	8.79 c	31.7 a	1.31 ab
	(Alga2 + mtd + nut) + B	0.89	3.25 bc	6.80 a	9.65 c	31.9 a	1.61 a
	ANOVA	ns	***	***	***	***	***

ANOVA results: ns indicates non-significant differences, while *** indicates significant differences at p < 0.001. For each algae, different lowercase letters indicate significant differences among treatments at p < 0.05 according to Duncan’s multiple range test (n = 4).

. In boron-irrigated plants, significant accumulation of Ala, Gln, and Glc was observed, together with a reduction in Pro compared with –B plants (Table 4).

For Laminaria digitata, application of the algal extract alone (+B) induced marked changes in the metabolic profile relative to the +B control: Pro, a key stress-response metabolite, increased significantly (5.77 vs. 2.86 mg g⁻¹ DW), as did Tri (1.21 vs. 0.81 mg g⁻¹ DW). In contrast, Gln (4.07 vs. 5.08 mg g⁻¹ DW) and Cit (32.1 vs. 44.9 mg g⁻¹ DW) declined, while Glc remained elevated without significant differences. Among enriched treatments, L. digitata +mtd + B significantly reduced Pro and Tri accumulation compared with the extract alone, thereby losing its protective effect. The L. digitata + nut + B treatment showed the weakest performance, with sharp decreases across all metabolites. Conversely, the triple formulation (+mtd + nut + B) further enhanced Pro (8.51 mg g⁻¹ DW, the highest in the study), Glc (40.3 mg g⁻¹ DW), and Tri (1.55 mg g⁻¹ DW), representing the most effective treatment for boosting osmolyte accumulation compared with the extract alone.

For Phaeodactylum tricornutum, alanine content did not differ significantly between treatments. Glutamine peaked in the +B control (5.08 mg g⁻¹ DW), whereas lower values were recorded in algal extract treatments (2.69–3.92 mg g⁻¹ DW). Proline levels increased in nutrient-supplemented treatments, reaching 8.03 and 6.80 mg g⁻¹ DW, compared with 2.86–4.08 mg g⁻¹ DW in the controls. Citrate was highest in the controls (44.5–44.9 mg g⁻¹ DW) and declined under all algal treatments (8.79–29.1 mg g⁻¹ DW). Glucose content ranged from 11.9 to 24.0 mg g⁻¹ DW in controls, but increased under algal treatments (29.1–32.1 mg g⁻¹ DW). Finally, Tri also rose with algal treatments, ranging from 1.01 to 1.61 mg g⁻¹ DW, compared with 0.81–0.92 mg g⁻¹ DW in controls.

3.8. Principal Component Analysis

To reduce the complexity of the dataset, a principal component analysis (PCA; Figure 4) was performed using physiological, mineral, metabolic, and oxidative stress variables, excluding growth-related variables (biomass, shoot, and root). The PCA generated two independent mathematical functions, PC1 and PC2, which together explained most of the variability. PC1 primarily captured variation associated with mineral nutrients (Mn, Fe, Zn, Ca), whereas PC2 predominantly integrated physiological variables related to gas exchange (PN, gs, E, WUE) and metabolites linked to stress tolerance (proline, tryptophan). Thus, PC1 can be interpreted as a “nutritional” axis, while PC2 represents a “physiological–metabolic” axis directly associated with the adaptive response to boron excess.

Correlation analysis between relative tolerance and PC1/PC2 scores revealed that PC1 was not significantly associated with relative tolerance (r = −0.12; p > 0.05), indicating that nutritional changes represented by this axis did not translate into biomass improvement. In contrast, PC2 showed a strong positive correlation with relative tolerance (r = 0.90; p < 0.05), demonstrating that treatments with higher photosynthesis, stomatal conductance, water-use efficiency, and accumulation of osmoprotective metabolites scored high on PC2 and were also those that best maintained growth under boron excess. These findings confirm that boron tolerance in tomato plants is primarily associated with primary metabolism and gas-exchange parameters.

4. Discussion

In this study, a high concentration of B (15 mg L⁻¹) in the nutrient solution exerted negative effects on tomato plants compared with -B controls, highlighting the susceptibility of tomato to B toxicity. Excess B significantly reduced growth, particularly inhibiting root development, which was reflected in a sharp increase in the shoot-to-root ratio. This effect was associated with high B accumulation in leaves (~2000 mg kg⁻¹ DW), which triggered toxic impacts on physiological and biochemical processes. At the physiological level, net photosynthesis (PN) dropped drastically, from 7.6 µmol CO₂ m⁻² s⁻¹ in -B controls to only 1.4 µmol CO₂ m⁻² s⁻¹ in +B plants, representing nearly an 80% reduction. This decline was accompanied by decreases in stomatal conductance and water-use efficiency, whereas Ci increased, suggesting that the high B concentration caused metabolic damage to the photosynthetic apparatus rather than stomatal limitations. At the metabolic level, B stress reduced proline levels (from 4.1 to 2.9 mg g⁻¹ DW), while glucose nearly doubled (from 11.9 to 24.0 mg g⁻¹ DW), evidencing osmotic imbalance and altered carbon metabolism. Nutritionally, although most elements remained within normal ranges, significant reductions in essential nutrients such as Mg (−27%), Ca (−24%), and Fe (−21%) were detected, exacerbating functional limitations. Overall, these findings confirm that excessive B in tomato induces a state of severe toxicity, impairing both growth and physiological–metabolic performance.

These results are consistent with previous reports. Pereira et al. [25] observed that tomato plants exposed to 6.9 mg L⁻¹ B in greenhouse conditions exhibited leaf necrosis, starch and sugar accumulation, and sharp declines in yield and fruit quality, effects mediated by enhanced ethylene biosynthesis and signaling. Similarly, Çelikkol and Acar [26] reported that exposure of tomato seedlings to 81 mg L⁻¹ B significantly reduced shoot and root length, water content, and biomass, while inducing ion leakage, increased malondialdehyde, and strong activation of antioxidant and ethylene-related genes, indicative of severe oxidative and metabolic damage. Cervilla et al. [27], working with nutrient solutions supplemented with 5.4–21.6 mg L⁻¹ B, found that B excess reduced water-use efficiency and nitrogen assimilation enzyme activity, while promoting accumulation of sugars, phenolics, and glutamine, thereby evidencing oxidative stress accompanied by foliar biomass loss. From an agronomic perspective, Francois [28] reported that tomato grown in sand culture exhibited a 3.4% decline in relative yield for each 1 mg L⁻¹ increase above 5.7 mg L⁻¹ B in irrigation water, in addition to negative impacts on fruit size and quality. Collectively, these findings underscore the urgent need to implement agronomic strategies that mitigate B toxicity while sustaining crop productivity. Among these, algal-based biostimulants have emerged as promising tools to enhance plant tolerance against abiotic stress.

Application of Laminaria digitata or Phaeodactylum tricornutum extracts, Alga1 + B and Alga2 + B, respectively, tended to improve the relative tolerance of tomato plants to excess B. For L. digitata, extract application significantly increased total biomass relative to the +B control, evidencing greater stress tolerance. This positive effect was associated with increases in PN, gs, transpiration, and WUE, together with reduced Ci. Although chlorophyll content (SPAD) did not show a complete recovery under boron toxicity, the improvement in photosynthetic rate (PN) after algal extract application can be attributed to enhanced stomatal conductance, CO₂ assimilation efficiency, and activation of photosynthetic enzymes rather than to increases in pigment concentration. This suggests that the extracts improved the functional performance of the existing photosynthetic apparatus, allowing plants to maintain higher photosynthetic activity despite reduced chlorophyll levels. Foliar and root B concentrations, as well as mineral nutrient levels, remained similar to the +B control, indicating that the improvement was not due to altered nutrient uptake but rather to metabolic adjustment, as reflected by increased proline and glutamine. For P. tricornutum, no significant increases in total biomass were detected compared with the +B control, although plants exhibited improved vegetative development, accompanied by higher PN, gs, transpiration, WUE, and Ci. Thus, the extract exerted a positive effect on plant physiology, supporting performance under B toxicity. In this case as well, B concentrations in leaves and roots were not altered, suggesting that tolerance resulted from reduced physiological toxicity rather than decreased B accumulation. Metabolically, the positive effect was supported by increased proline, glucose, and tryptophan, compounds acting as osmolytes and defense molecules that contribute to stress tolerance. Therefore, the increase in Mn, Fe, and Zn concentrations coincided with a reduction in citrate content, indicating a disturbance in the TCA cycle and a possible imbalance in energy metabolism under boron toxicity.

Direct evidence of algal extract application under B toxicity remains limited, but related studies in other crops and stress conditions provide useful insights. In citrus, Alebidi and Abdel-Sattar [29] tested the combined effect of boric acid and seaweed extract on Citrus sinensis (Valencia orange). Plants irrigated with 54 mg L⁻¹ B and treated foliarly with a water-based algal extract (10 g L⁻¹) showed improved growth and yield despite no reduction in tissue B, pointing to internal tolerance rather than exclusion mechanisms. Although chemical composition was not characterized, seaweed extracts typically provide polysaccharides, polyphenols, and hormone-like compounds. Similarly, Santaniello et al. [30] demonstrated in Arabidopsis thaliana that extracts of the brown alga Ascophyllum nodosum—rich in sulfated polysaccharides, mannitol, polyphenols, and natural phytohormones—enhanced drought tolerance by maintaining photosynthesis and activating antioxidant defenses. Lenart et al. [31] also reported that seaweed-derived biostimulants improved drought tolerance in blueberry (Vaccinium corymbosum) by promoting osmolyte accumulation and reducing oxidative damage. Collectively, these findings suggest that algal extracts confer tolerance mainly through mitigation of physiological toxicity (photosynthesis, water-use efficiency), metabolic adjustment (osmolytes, antioxidants), and maintenance of primary metabolism, rather than by reducing B accumulation in leaves or roots.

By contrast, enriched formulations did not outperform the simple algal extracts. For L. digitata, enrichment with metalloids or micronutrients failed to consistently improve growth compared with the extract alone. Although the full combination (mtd + nut) slightly increased biomass, this was not accompanied by significant improvements in gas exchange, chlorophyll content, B concentrations, or MDA levels, indicating limited additional benefits. This negative response could be explained by the fact that the simultaneous foliar application of metalloids (Se, Si) and micronutrients (Mn, Fe, Zn) under boron toxicity may have disrupted plant metabolic homeostasis. While these elements are known to enhance tolerance under moderate stress, their combined effect in the presence of high boron concentrations might have intensified oxidative and osmotic imbalances, interfering with carbon–nitrogen metabolism and reducing the accumulation of key osmoprotectants such as proline and glucose. Consequently, the enriched formulations induced metabolic disturbances that limited the adaptive responses observed with the simple algal extracts. Notably, micronutrient addition raised Mn, Fe, and Zn concentrations, but simultaneously reduced key metabolites such as proline, citrate, and glucose, suggesting a metabolic imbalance that could compromise tolerance. The integration of data from Table 3 and Table 4 and Figure 4 (PCA) clearly indicates that, for Laminaria digitata, the trace element-enriched formulation (+nut) drastically reduced key metabolites such as proline, citrate, glucose, and tryptophan, leading to the weakest overall effectiveness among treatments. This can be explained by the fact that excessive foliar uptake of transition metals such as Mn, Fe, and Zn under boron toxicity conditions may alter cellular redox homeostasis and interfere with key enzymatic processes involved in carbon and nitrogen metabolism. Although these micronutrients are essential cofactors, their accumulation beyond optimal levels can promote the generation of reactive oxygen species (ROS) and disturb ionic balance, forcing the plant to redirect metabolic resources toward detoxification and antioxidant defense. Consequently, the synthesis of primary osmoprotective metabolites such as proline, glucose, and citrate is reduced, leading to the metabolic imbalance observed in the enriched formulations. Similarly, for P. tricornutum, enriched formulations did not enhance B tolerance. Only the mtd+nut treatment slightly increased biomass compared with the extract alone, but without significant differences and without positive responses in gas exchange. In fact, PN decreased under both mtd and mtd + nut treatments. No effects were detected on chlorophyll content or B concentrations in leaves and roots. At the mineral level, the mtd + nut treatment strongly increased Mn, Fe, and Zn, but this coincided with reduced citrate, indicating an imbalance in energy metabolism.

In relation to oxidative stress, MDA levels did not significantly differ from the +B control in any of the algal treatments. However, the improvement in growth and physiological performance observed suggests that tolerance was mainly achieved through osmotic and metabolic adjustments rather than by direct reduction in lipid peroxidation. The unchanged MDA values likely reflect a balance between ROS generation and enhanced antioxidant activity, allowing plants to sustain growth despite persistent oxidative conditions.

In comparative terms, L. digitata extract proved more effective than P. tricornutum, as it significantly improved total biomass and promoted more pronounced physiological responses under excess B. These results highlight the potential of algal extracts, particularly L. digitata, as a promising agronomic strategy to mitigate B toxicity in tomato. The higher efficacy of L. digitata can be attributed to its characteristic chemical composition. This brown alga is especially rich in sulfated polysaccharides (laminarins, fucoidans, alginates), polyphenols, and mannitol, compounds associated with osmoprotective, antioxidant, and hormone-like signaling functions in plants [32,33]. Such metabolites likely contribute to the maintenance of photosynthesis and osmotic adjustment under B toxicity, as reflected in the enhanced biomass and physiological performance observed. In contrast, P. tricornutum, a diatom microalga, exhibits a distinct biochemical profile characterized by high levels of polyunsaturated fatty acids (EPAs), carotenoids such as fucoxanthin, proteins, and nitrogenous compounds [34]. Although these metabolites possess strong antioxidant properties, they appear to be less directly linked to osmotic adjustment and primary metabolism under B stress. This compositional difference may explain why L. digitata exerted a more pronounced effect on physiological and metabolic tolerance to B compared with P. tricornutum.

5. Conclusions

Overall, the results of this study confirm that tomato is highly susceptible to boron toxicity, exhibiting severe reductions in growth, photosynthesis, and metabolic balance. However, application of algal extracts from Laminaria digitata and Phaeodactylum tricornutum improved the relative tolerance of plants, primarily through the activation of physiological mechanisms (enhanced photosynthesis, stomatal conductance, and water-use efficiency) and metabolic adjustments (accumulation of proline, tryptophan, and glutamine), but without reducing boron accumulation in plant tissues. This effect is therefore associated with the mitigation of internal toxicity rather than exclusion mechanisms. In contrast, enriched mixtures with metalloids and micronutrients did not provide consistent additional benefits compared with the simple extracts, and in some cases induced metabolic imbalances (reductions in proline, citrate, and glucose) that may limit adaptive responses. Multivariate analysis (PCA) confirmed that relative tolerance was significantly associated with PC2, which integrates key physiological and metabolic variables, whereas PC1, dominated by nutritional variation, was not related to tolerance. These findings emphasize that improved resistance to excess boron in tomato relies primarily on the plant’s ability to sustain primary metabolism and gas exchange, rather than on modifications in mineral uptake, and highlight the potential of algal extracts as an agronomic strategy to mitigate this type of toxicity. Moreover, Laminaria digitata extract proved more effective than P. tricornutum, as it promoted greater growth and more pronounced physiological performance under excess B, underscoring its promise as a practical tool to alleviate boron toxicity in tomato cultivation. Future studies should include a detailed biochemical characterization of the algal extracts to better link their specific composition with the observed physiological and metabolic responses under boron toxicity.