The use of biostimulants in agriculture is currently emerging as a resource to increase crop productivity and quality. Additionally, the concurrent application of other bioactive materials or bioregulators alongside biostimulants can prove valuable in mitigating oxidative stress induced by various abiotic stresses. However, in some cases, these compounds applied at inadequate doses produce toxic effects under non-stressful conditions. Therefore, the objective of this study was to analyze the effects of applying various doses of a seaweed-derived biostimulant containing additional bioactive compounds (Cytolan® Stress) under optimal growth conditions. Lettuce plants were grown under control conditions without biostimulants (control plants) and with two biostimulants Cytolan® (seaweed-derived biostimulant with proline) and Cytolan® Stress applied foliarly at three doses (150, 300, and 500 mL/hL). Growth and quality parameters and physiological processes within primary and secondary metabolism were analyzed. The results identified the 300 mL/hL dose of Cytolan® Stress as the best treatment. Thus, it improved biomass production, induced photosynthetic activity, increased phytohormone, amino acids (AAs), and mineral nutrient profiles, and stimulated N assimilation. Besides, it substantially enhanced the antioxidant capacity and the concentration of various antioxidant compounds, leading to increased lettuce quality and potential stress tolerance. However, the 500 mL/hL Cytolan® Stress caused phytotoxic effects, underscoring the importance of applying the biostimulant at the proper dose.

1 INTRODUCTION

The regulation of plant growth and the mitigation of the negative effects of environmental stresses during ontogenesis are important factors determining the productivity of agricultural crops. Abiotic stress can be prevented by optimizing plant growth conditions by providing water, nutrients, and growth regulators. In addition to these conventional approaches, biostimulants are increasingly being incorporated into production systems to enhance productivity and bolster tolerance to the growing prevalence of stresses in the current climate change scenario (Jardin, 2012; Brown and Saa, 2015; Zulfiqar et al. 2020; Ma et al. 2022; Rakkammal et al. 2023).

Biostimulants represent a novel category of agricultural products distinct from fertilizers because their function and role in plant growth are independent of their nutrient content. They have recently been included in the new EU regulation on fertilizer products (Soppelsa et al. 2020). The EU officially recognizes plant biostimulants as a separate class of agricultural inputs (Carmody et al. 2020) and defines them as any product that stimulates plant growth and plant nutritional processes regardless of nutrient content to improve one or more of the following characteristics of the plant or plant rhizosphere: nutrient use efficiency, abiotic stress tolerance, quality characteristics, and nutrient availability (Caradonia et al. 2020; Poberezny et al. 2020). According to different studies, the global market is projected to exceed four billion dollars in turnover by 2025, and significant growth is anticipated on a global scale in the coming years (Carmody et al. 2020).

At present, the characteristics of biostimulants' bioactive components, the elucidation of their action mechanisms, and the plant responses at different levels, including morphological, biochemical, and metabolic levels, are the focal points of attention and research among scientists worldwide (Kocira et al. 2020). Biostimulants exhibit diverse action mechanisms, encompassing the activation of nitrogen (N) metabolism and the release of phosphorus (P) in the soil, stimulation of microbial activity, promotion of root growth, and enhancement of physiological processes such as germination, photosynthesis, nutrient absorption, growth hormone synthesis, and reduction of senescence. Furthermore, biostimulants have the capacity to mitigate the negative effects of abiotic stress factors in plants by inducing defense responses, including the activation of the antioxidant system or the synthesis of osmoprotective compounds in plants (Francesca et al. 2020; Zulfiqar et al. 2020; Franzoni et al. 2022; Ma et al. 2022; Ziaei and Pazoki, 2022).

Among the different types of existing biostimulants, those based on algae extracts are currently among the most used. Algae are generally considered an attractive biological material that provides a large number of direct and indirect benefits to the development of most crops (Ma et al. 2022). Algae extracts have gained interest as biostimulants because of their biochemical profile as they usually contain a wide variety of bioactive compounds and signaling molecules such as cytokinins, auxins, gibberellins (GAs), abscisic acid (ABA), vitamins, amino acids (AAs), polysaccharides, and mineral nutrients (Mosa et al. 2022; El Khattabi et al. 2023).

In addition, the concurrent application of other bioactive materials or bioregulators alongside biostimulants can prove valuable in mitigating the oxidative stress induced by various abiotic stresses. Consequently, the use of phytohormones, nitrogenous compounds, sugars, and polyols serves to diminish the generation of reactive oxygen species (ROS), regenerate oxidized molecules, detoxify ROS, and function as osmoprotectors. Moreover, these compounds positively regulate the expression of genes associated with stress resistance responses (Zulfiqar and Ashraf, 2021; Rodrigues de Queiroz et al. 2023). The advantageous effects of these bioactive materials have been observed in plants exposed to stress conditions. However, under optimal growth conditions, these effects may not be as conspicuous and, in some cases, negative responses that hinder plant growth have been observed. For example, in several species, the application of compounds such as ascorbate (AsA), proline, melatonin, and tocopherol during normal growth conditions has been shown to inhibit plant growth (Rodrigues de Queiroz et al. 2023).

The primary objective of this study was to analyze the effects of applying the biostimulant Cytolan® Stress, developed by Promisol S.A., under optimal growth conditions. This biostimulant not only includes an extract of Ascophyllum nodosum algae but also comprises various bioactive compounds. This study assesses the impact of different doses of two biostimulants (Cytolan®, seaweed-derived biostimulant with proline, and Cytolan® Stress) by examining growth and quality parameters and investigating fundamental physiological processes within primary and secondary metabolism. These processes encompass assessing photosynthetic efficiency, hormone, total phenols, flavonoids and anthocyanin concentrations, and nutritional status.

2 MATERIALS AND METHODS

2.1 Plant material and growing conditions

Lettuce plants (Lactuca sativa cv. Maravilla de Verano) were used. The seeds of these plants germinated and grew for 45 days in a tray with cells (cell size, 3 cm x 3 cm x 10 cm) at Saliplant SL (Carchuna, Granada, Spain). Subsequently, the seedlings were transferred to a growth chamber of the Department of Plant Physiology of the University of Granada under controlled conditions with temperatures of 25°C/15°C (day/night), relative humidity 60–80%, and a 16 h/8 h photoperiod with a PPFD (photosynthetic photon-flux density) of 350 μmol⁻² s⁻¹ (measured with an SB quantum 190 sensor, LI – COR Inc.).

Under these conditions, the plants grew in individual pots (13 cm upper diameter, 10 cm lower diameter, 12.5 cm high, and a volume of 2 L) filled with a perlite:vermiculite mixture. Fertilization consisted of a complete Hoagland-type nutrient solution, with small modifications for lettuce cultivation, composed of 4 mM KNO₃, 2 mM Ca(NO₃)₂, 2 mM MgSO₄, 1 mM KH₂PO₄, 1 mM NaH₂PO₄, 125 μM Fe-EDDHA, 50 μM H₃BO₃, 2 μM MnCl₂, 1 μM ZnSO₄, 0.25 μM CuSO₄, and 0.1 μM Na₂MoO₄ with a pH of 5.8. The amount of nutrient solution applied each day will be approximately 50 mL per pot, never exceeding the drainage volume by 10%.

2.2 Treatments description and experimental design

The effect of foliar application of Cytolan® and Cytolan® stress products from Promisol S.A. was analyzed. The doses applied were 150, 300, and 500 mL/hL. The biostimulant Cytolan® is composed of an alkaline extract of the seaweed Ascophyllum nodosum and the amino acid proline, whereas Cytolan® Stress is composed of an alkaline extract of the seaweed Ascophyllum nodosum and proline, to which AsA, α-tocopherol, salicylic acid (SA), and mannitol have been added. These treatments were foliarly applied using a sprayer. A control treatment without the application of the biostimulant was arranged.

The application of the different treatments started 7 days after transplanting the plants in the growth chamber, and the products were applied twice with a periodicity of 15 days between each application. The experimental design consisted of a complete randomized block with 8 plants per treatment arranged in individual pots with the treatments distributed randomly in the growth chamber.

2.3 Plant sampling and biomass measurement

Sampling of the aerial part was performed 7 days after the last application of the treatments. All plants from each treatment were processed instantly for subsequent analysis. Sampling was performed 29 days after transplantation.

The plant material was rinsed and subsequently dried on filter paper to obtain the fresh weight (FW). Half of the fresh or frozen samples at −40°C were used for the analysis of the following parameters: leaf area, chlorophyll (Chl) a fluorescence (Fv/Fm, RC/ABS, PI(Abs), pigment concentration, photosynthetic efficiency (Infra-Red Gas Analyzer, IRGA-LiCOR 6400), hormonal and AA profiles, proteins and soluble sugar concentrations, FRAP and TEAC antioxidant tests, and concentration of antioxidant compounds, ascorbate, and nitrates. The remaining half of the plant materia was freeze-dried and used to determine the dry weight (DW) and the concentration of mineral nutrients. To obtain plant biomass, fresh and dry plant leaves were weighed on a laboratory scale, and their weight was expressed as g.

2.4 Plant analysis

2.4.1 Leaf area

The leaves of all plants were separated and leaf area was measured using a LI-COR optical reader, model LI-3000A. Leaf area was expressed as cm².

2.4.2 Analysis of Chl a fluorescence

Plants were dark-acclimated for 30 min before conducting measurements. During this process, a specialized leaf clip was affixed to each leaf. Chl a fluorescence kinetics were assessed using a Handy PEA Chlorophyll Fluorimeter (Hansatech Ltd.). The OJIP phases were initiated by exposure to red light at 650 nm, with a light intensity of 3000 μmol photons m⁻² s⁻¹. Subsequently, the OJIP fluorescence phases were evaluated using the JIP test, as detailed by Strasser et al. (2000). These measurements were performed on fully mature leaves located in the central part of the plant. To examine energy flow and photosynthetic activity, we relied on the parameters obtained from the JIP test: initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv = Fm-Fo), maximum quantum product of primary photochemistry (ΦPo = Fv/Fm), performance index (PIabs), and proportion of active reaction centers (RC) (RC/ABS) (Strasser et al. 2000).

2.4.3 Concentration of photosynthetic pigments

The concentration of photosynthetic pigments was analyzed by the method of Wellburn (1994) with certain modifications. Plant material (0.1 g) was macerated and mixed with 1 mL of methanol, then centrifuged for 5 min at 5000 g. The absorbance of the supernatant was measured at 3 different wavelengths: 666 nm, 653 nm, and 470 nm, and the following calculations were made based on the following equations:

Chl a = 15.65 x A666nm – 7.34 x A653nm
Chl b = 27.05 x A653nm – 11.21 x A666nm
Carotenoids = (1000 x A470nm – 2.86 x Chl a – 129.2 X Chl b) / 221

Chl a and Chl b concentrations were expressed as mg g⁻¹ FW and carotenoids concentration was expressed as μg g⁻¹ FW.

2.4.4 Analysis of gas exchange parameters

We used a LICOR 6800 Portable Photosynthesis System Infrared Gas Analyzer to collect measurements. The middle leaves were positioned within the measurement chamber to ensure that they were exposed to the best growth conditions. To ensure accuracy, the instrument was prepped for 30 min and underwent calibration before use. Measurements were performed using standard optimal cuvette conditions at 400 μmol mol⁻¹ CO₂ concentration, 500 μmol m² s⁻¹ photosynthetically active radiation (PAR), 60% relative humidity, and leaf temperature at 30°C. The net photosynthetic rate (A), intercellular CO₂ (C_i) transpiration rate (E), and stomatal resistance (r) were recorded simultaneously. Data were stored on the LICOR device and analyzed using “Photosyn Assistant” software. The units for gas exchange parameters were as follows: A (μmol m⁻² s⁻¹), Ci (μmol mol⁻¹), r (s cm⁻¹), E (mmol m⁻² s⁻¹), and WUE (μmol mmol⁻¹).

2.4.5 Non-structural carbohydrates concentration

To determine the concentration of non-structural carbohydrates (glucose + fructose + sucrose), 0.5 g of plant material was homogenized in 5 mL of 96% ethanol. The insoluble fraction of the extract was washed with 5 mL of 70% ethanol. The extract was centrifuged at 3500 g for 10 min and the supernatant was stored at 4°C for the determination of sugars according to the method described by Irigoyen et al. (1992).

2.4.6 Hormonal profile analysis

Indoleacetic acid (IAA), gibberellins (GA, GA1 + GA3 + GA4), cytokinins [CKs: trans-zeatin (tZ) + isopentenyl adenine (iP)], ABA, aminocyclopropane carboxylic acid (ACC, as precursor of ethylene), SA, and jasmonic acid (JA) were analyzed as described by Navarro-Morillo et al. (2023) using a U-HPLC-MS system. The concentration of phytohomones was expressed as ng g⁻¹ DW.

2.4.7 NO₃⁻ determination

To determine the concentration of soluble NO₃⁻, an aqueous extraction was performed following the method of Cataldo et al. (1975). The determination of NO₃⁻ was based on the colorimetric reaction formed by the union of NO₃⁻ with salicylic acid in a basic medium (Cataldo et al. 1975). NO₃⁻ was expressed as mg g⁻¹ FW.

2.4.8 Nutrient concentration, N content, and nutrient utilization efficiency (NUtE)

The determination of the concentration of mineral nutrients was performed using ICP-OES. The leaf samples were subjected to a mineralization process following the Wolf (1982) method. Dry leaves (0.2 g) were taken and subjected to digestion with 30% HNO₃ and H₂O₂ at 300°C and the mineralized product obtained was used for the analysis of ionic elements. For the determination of N concentration, 0.2 g of dry leaves and roots were ground and mineralized with 98% H₂SO₄ and 30% H₂O₂ at a temperature of 300°C, and the mineralized product was used for N analysis. The total N concentration was determined by colorimetry based on the Berthelot reaction, according to the method described by Krom (1980). The N content was calculated by multiplying the N concentration by the dry weight. To calculate nitrogen utilization efficiency (NUtE), the equation described by Xu et al. (2012) was used: NUtE = Dry biomass (g) / mg N. NUtE was expressed as g DW mg⁻¹ N.

2.4.9 Determination of aminogram and soluble proteins

AAs were extracted and analyzed using a UHPLC system equipped with a fluorescence detector following the method described by Navarro-Morillo et al. (2023). AAs concentrations were expressed as μg g⁻¹ DW.

To determine soluble proteins, approximately 0.5 g of plant material was homogenized with 5 mL of 50 mM phosphate buffer, pH 7.0. The homogenate was filtered with 4 layers of gauze and subsequently centrifuged at 12360 g for 15 min. The supernatant was used for the quantification of the soluble proteins. To a 0.1 mL aliquot of the supernatant, 0.9 mL of phosphate buffer was added to 50 mM pH 7.0 and 5 mL of Coomassie blue. After 20 min, the samples were measured at a wavelength of 595 nm against an albumin standard curve (Navarro-León et al. 2016). Protein concentration was expressed as mg g⁻¹ FW.

2.4.10 Phenols, flavonoids, anthocyanins, and AsA concentration

The phenols present in the plant samples were obtained based on Rivero et al. (2001). A total of 0.1 g of the plant material was macerated, to which 0.5 mL of methanol, 0.5 mL of chloroform, and 0.25 mL of 1% NaCl were added. Subsequently, it was centrifuged at 2000 g for 10 min, obtaining the methanolic phase to which 180 μL of H₂O, 240 μL of Na₂CO₃ and 90 μL of the 50% Folin–Ciocalteu reagent were added. After the addition of all the compounds, the mixture was shaken and incubated for 1 h at room temperature, after which the absorbance of its content was measured at 725 nm.

The total flavonoid concentration was determined according to the colorimetric method described by Kim et al. (2003) with some modifications. The same extraction process used in the determination of phenols was carried out for the plant samples. 85 μL of the methanolic phase was extracted, to which 340 μL of H₂O and 26 μL of NaNO₂ were added, stirred, and allowed to rest for 5 min in the dark. Afterwards, 26 μL of AlCl₃ was added, turning the samples yellowish, and 170 μL of NaOH, turning them pink, stirred again, and kept in the dark for 15 min. The absorbance of the samples was measured at 415 nm.

The concentration of anthocyanins was determined by applying the differential pH method according to Giusti and Wrolstad (2001). Macerated plant material (0.1 g) was mixed with 1 mL of 1% acidified methanol was added. Subsequently, the material plus methanol was centrifuged for 15 min at 2000 g, 250 μL of the supernatant and 1 mL of potassium chloride were added, measured in the spectrophotometer at 460 nm and, secondly, 250 μL of the supernatant to which 1 mL of sodium acetate was added was measured at an absorbance of 710 nm. The following formula developed to measure the concentration of anthocyanins in cauliflower was applied:

\left[\left(\left(\mathsf{A}\mathsf{460}\hbox{--} \mathsf{A}\mathsf{710}\right)\ \mathsf{x}\ \mathsf{449.2}\ \mathsf{x}\ \mathsf{0.2}\ \mathsf{x}\ \mathsf{1,000}\right)/\mathsf{26,900}\right]

The method of Law et al. (1983) was used to quantify the AsA concentration.

Phenols, flavonoids, anthocyanins, and AsA concentrations were expressed as mg g⁻¹ FW.

2.4.11 Antioxidant capacity: FRAP and TEAC tests

The ferric-reducing antioxidant power (FRAP) assay was performed using the FRAP reagent. A 100 μL extract obtained by homogenizing leaves in 10 mL of methanol was added to 2 mL of FRAP reagent. Finally, absorbance was measured at 593 nm (Benzie and Strain, 1996). FRAP was expressed as mg g⁻¹ FW.

The trolox equivalent antioxidant activity (TEAC) test was performed using the Cai et al. (2004) method. The previous extracted sample for the FRAP test was mixed with 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) reagent (Sigma-Aldrich), and then the absorbance of the mixture was recorded at 734 nm. TEAC was expressed as mM g⁻¹ FW.

2.5 Statistical analysis

We conducted the analyses three times, and the results were subjected to statistical analysis of variance (ANOVA) with a 95% confidence interval. To compare the means of different treatments, we employed Fisher's least significant differences (LSD) test at a 95% probability level. The significance levels were denoted as follows: * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and “NS” indicating no significance. Statgraphics Centurion XVI software was used to perform statistical analysis.

3 RESULTS AND DISCUSSION

3.1 Plant growth

One of the primary criteria that must define a biostimulant is the absence of phytotoxic effects and, conversely, the promotion of plant growth and productivity (Jardin, 2012). In this study, to verify the biostimulant effect of Cytolan® and Cytolan Stress®, we analyzed parameters such as the production of fresh and dry biomass of the shoot and leaf area, which are indicative of plant growth (Brown and Saa, 2015; Rakkammal et al. 2023).

Foliar application of all doses of Cytolan® produced positive effects, increasing both fresh and dry biomass compared with control plants. Regarding the application of Cytolan® Stress, we observed an improvement in the growth of lettuce plants with the application of 150 and 300 mL/hL doses. More specifically, the optimal dose for plant growth was 300 mL/hL, and Cytolan® was better than Cytolan® Stress for biomass enhancement, with increases of 35% for fresh biomass and 62% for dry biomass with respect to the data obtained in control plants (Table 1; Figure S1). Other studies also found an increase in biomass after the application of seaweed-derived biostimulants in soybean, apple, tomato, and other species (Tandon and Dubey 2015; Ma et al. 2022; Mosa et al. 2022; El Khattabi et al. 2023).

TABLE 1. Effect of Cytolan® and Cytolan® Stress biostimulants on fresh weight, dry weight, and leaf area of leaves of lettuce plants.

Treatment	Fresh weight (g)	Dry weight (g)	Leaf area (cm²)
Control	36.63 ± 1.13e	1.77 ± 0.04e	405.25 ± 12.58de
Cytolan® 150 mL/hL	38.67 ± 1.63d	2.08 ± 0.06d	518.66 ± 40.76c
Cytolan® 300 mL/hL	49.26 ± 1.02a	2.86 ± 0.06a	559.81 ± 31.49a
Cytolan® 500 mL/hL	41.76 ± 1.89c	2.25 ± 0.09c	391.04 ± 5.29e
Cytolan® Stress 150 mL/hL	41.43 ± 2.02c	2.14 ± 0.04c	433.31 ± 35.78d
Cytolan® Stress 300 mL/hL	46.85 ± 0.73b	2.52 ± 0.16b	512.08 ± 19.44b
Cytolan® Stress 500 mL/hL	32.48 ± 0.93f	1.36 ± 0.15f	335.37 ± 14.13f
p-value	***	***	***

All data values represent means ± standard error (n = 8 data). The level of significance was represented as.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

On the other hand, the highest dose of Cytolan® Stress (500 mL/hL) resulted in a significant decrease in the shoot growth of lettuce plants (Table 1; Figure S1). Cytolan® Stress contains bioactive compounds such as AsA, α-tocopherol, proline, SA, and mannitol. However, most of the beneficial effects resulting from the application of these bioactive compounds have been observed in plants subjected to stress conditions. In plants grown under optimal conditions, these effects have not been very evident, and even negative responses have been observed, reducing plant growth. For instance, in Arabidopsis thaliana, Pistacia vera, Malus domestica, Prunus avium, and Beta vulgaris plants, the application of these compounds under normal growth conditions decreased plant growth (Rodrigues de Queiroz et al. 2023). Our results confirm these observations, suggesting that under adequate growth conditions without the presence of any type of stress, the application of Cytolan® Stress at high doses would eliminate the biostimulant effect of this product; therefore, its use at this dose would not be recommended.

Regarding the leaf area values, the 150 and 300 mL/hL doses of Cytolan® and the 300 mL/hL dose of Cytolan® Stress increased the leaf area compared with the control plants. Therefore, the application of Cytolan® and Cytolan® Stress, both at doses of 300 mL/hL, could significantly increase the photosynthetic capacity of plants by increasing the light capture area. Again, the phytotoxic effect of applying Cytolan® Stress at 500 mL/hL was observed in this parameter as a significant reduction in leaf area was observed (Table 1).

3.2 Photosynthesis-related parameters

Other studies have reported positive effects of biostimulant application on the photosynthetic process and pigments (Francesca et al. 2020; Ali et al. 2021; Franzoni et al. 2022). To establish a connection between enhanced plant growth and the induction/activation of various fundamental physiological processes in plants, we examined carbon metabolism by analyzing photochemical activity and photosynthesis efficiency. Thus, it has been demonstrated that Chl a fluorescence mirrors plant photochemical conditions and photosynthetic alterations resulting from stress factors. When metabolic disruptions occur, the plant generates fluorescence to dissipate excess energy and safeguard against stress-induced damage (Strasser et al. 2000).

Table 2 shows the Chl a fluorescence results. The Fv/Fm index in plants supplied with Cytolan® at all doses and Cytolan® Stress at low and intermediate doses presented values around 0.87, which indicates that the application of these treatments did not give rise to phytotoxicity (Abdeshahian et al. 2010). In general, the application of the biostimulant did not affect the Chl a fluorescence parameters. However, the application of Cytolan® Stress applied at the highest dose (500 mL/hL) produced a significant reduction in the Fv/Fm, RC/ABS, and PI(Abs) parameters (Table 2). The reduction in these parameters indicates that the use of this treatment produces an imbalance in the photochemical activity, increasing the emission of fluorescence by lettuce plants and reducing the energy available for the photosynthetic process (Strasser et al. 2000). These results, in turn, could explain the reduction in growth that occurs in plants receiving this treatment (Table 1).

TABLE 2. Effect of Cytolan® and Cytolan® Stress biostimulants on Chl a fluorescence parameters of lettuce plants.

Treatment	Fv/Fm	RC/ABS	PI(Abs)
Control	0.865 ± 0.001a	0.32 ± 0.002ab	1.92 ± 0.10a
Cytolan® 150 mL/hL	0.868 ± 0.005a	0.30 ± 0.01b	1.91 ± 0.28a
Cytolan® 300 mL/hL	0.875 ± 0.002a	0.31 ± 0.01ab	1.96 ± 0.16a
Cytolan® 500 mL/hL	0.871 ± 0.004a	0.32 ± 0.01a	1.96 ± 0.28a
Cytolan® Stress 150 mL/hL	0.868 ± 0.005a	0.32 ± 0.02ab	2.16 ± 0.18a
Cytolan® Stress 300 mL/hL	0.865 ± 0.002a	0.30 ± 0.005b	1.97 ± 0.17a
Cytolan® Stress 500 mL/hL	0.831 ± 0.005b	0.21 ± 0.004c	1.45 ± 0.29b
p-value	*	**	*

Variable fluorescence/maximum fluorescence ratio (Fv/Fm), proportion of active reaction centers (RC/ABS), performance index (PIabs). Values are means ± standard deviation (n = 9 data). The levels of significance were represented as.
* (p < 0.05) and.
** (p < 0.01). Values with different letters indicate significant differences according to Fisher's least significant differences test.

The concentration of Chl a, b, and carotenoids, as well as the parameters of Chl a fluorescence, are indicative of photosynthetic activity and photochemical efficiency (Li et al. 2010). In the present study, the application of Cytolan® and Cytolan® Stress at the highest doses resulted in a decrease in the foliar concentration of these pigments, which was confirmed especially in the case of Cytolan® Stress at a dose of 500 mL/hL (Table 1). Concerning the rest of the treatments, the application of Cytolan® and Cytolan® Stress at low and intermediate doses (150 and 300 mL/hL) produced a positive effect by increasing the foliar concentration of Chls, although it did not affect the carotenoids concentration with respect to the values obtained in control plants (Table 3). The increase in Chls concentration suggests a beneficial action of these biostimulants applied at these doses, improving light capture and thus the transfer of light energy to chemical energy (Brotosudarmo et al. 2018).

TABLE 3. Effect of Cytolan® and Cytolan® Stress biostimulants on the concentration of Chl a, Chl b, and carotenoids of lettuce plants.

Treatment	Chl a	Chl b	Carotenoids (μg g⁻¹ FW)
Treatment	(mg g⁻¹ FW)	(mg g⁻¹ FW)	Carotenoids (μg g⁻¹ FW)
Control	0.20 ± 0.00d	0.11 ± 0.002b	24.56 ± 5.19ab
Cytolan® 150 mL/hL	0.24 ± 0.01a	0.14 ± 0.004a	23.79 ± 1.01b
Cytolan® 300 mL/hL	0.25 ± 0.01a	0.13 ± 0.004a	23.90 ± 1.13b
Cytolan® 500 mL/hL	0.18 ± 0.01c	0.11 ± 0.002b	18.86 ± 1.12c
Cytolan® Stress 150 mL/hL	0.23 ± 0.01b	0.13 ± 0.004a	28.04 ± 4.61a
Cytolan® Stress 300 mL/hL	0.22 ± 0.00b	0.12 ± 0.001b	23.89 ± 0.93b
Cytolan® Stress 500 mL/hL	0.16 ± 0.00e	0.10 ± 0.002c	13.49 ± 0.55d
p-value	***	***	***

Values are means ± standard deviation (n = 9 data). The levels of significance were represented as.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

The analysis of gas exchange parameters related to photosynthesis showed that all the treatments applied, except Cytolan® Stress at a dose of 500 mL/hL, increased A and C_i levels, presenting the maximum values in plants supplied with Cytolan® and Cytolan® Stress at 300 mL/hL, respectively. However, the highest dose of Cytolan® Stress reduced the values of these parameters (Table 4), which could contribute to the lowest biomass production of plants that received this treatment (Table 1). Regarding the parameters indicative of plant transpiration, Cytolan® applied at all doses increased r and, thereby, reduced E, whereas the effect of Cytolan® Stress depended on the dose. Thus, the 300 mL/hL dose reduced r and increased E, whereas the 500 mL/hL dose produced the opposite effect with plants having the lowest E (Table 4). These results lead to a higher WUE in plants supplied with the biostimulants compared with control plants, which could be useful under water deficit conditions by allowing greater CO₂ assimilation with less water loss through the stomata (Hatfield and Dold 2019). Nevertheless, the excessive closure of stomata by Cytolan® Stress 500 mL/hL could be a rapid mechanism of adaptation to stress that reduces intracellular CO₂ influx, leading to a reduction in photosynthesis and, thus, to a lack of the endogenous electron acceptor NADP⁻, which ultimately results in ROS formation (Nxele et al. 2017).

TABLE 4. Effect of Cytolan® and Cytolan® Stress biostimulants on gas exchange parameters of lettuce plants.

Treatment	A (μmol m⁻² s⁻¹)	C_i (μmol mol⁻¹)	r (s cm⁻¹)	E (mmol m⁻² s⁻¹)	WUE
Control	5.68 ± 0.01f	240.00 ± 1.67e	7.63 ± 0.02d	2.72 ± 0.05b	2.12 ± 0.09f
Cytolan® 150 mL/hL	6.45 ± 0.03d	293.71 ± 0.17b	8.23 ± 0.04c	2.65 ± 0.01c	2.43 ± 0.01d
Cytolan® 300 mL/hL	7.21 ± 0.01a	289.81 ± 0.16c	8.19 ± 0.02c	2.61 ± 0.02c	2.76 ± 0.02c
Cytolan® 500 mL/hL	6.32 ± 0.02e	273.15 ± 0.28d	11.00 ± 0.18b	2.13 ± 0.00d	2.96 ± 0.01b
Cytolan® Stress 150 mL/hL	6.52 ± 0.02c	295.05 ± 0.19b	7.61 ± 0.003d	2.72 ± 0.01b	2.40 ± 0.01d
Cytolan® Stress 300 mL/hL	6.94 ± 0.04b	299.05 ± 0.46a	7.10 ± 0.01e	3.03 ± 0.00a	2.29 ± 0.01e
Cytolan® Stress 500 mL/hL	4.77 ± 0.01 g	232.03 ± 1956f	19.83 ± 0.18a	1.41 ± 0.01e	3.37 ± 0.02a
p-value	***	***	***	***	***

Net photosynthetic rate (A), intercellular CO₂ (C_i) transpiration rate (E), stomatal resistance (r), water use efficiency (WUE). Values are means ± standard deviation (n = 9 data). The level of significance was represented as.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

The induction of photosynthetic activity in plants usually induces carbon metabolism and the synthesis of sugars for plant growth (Cho et al. 2015). In general, the treatments of this study produced a significant increase in the concentration of soluble sugars in lettuce plants, with the highest values found in plants sprayed with 300 mL/hL dose (Figure 1). This result is directly proportional to the highest A (Table 4), and the highest biomass production observed in these plants (Table 1). In contrast, the minimum values of soluble sugars were presented in plants supplied with Cytolan® Stress (500 mL/hL dose) (Figure 1), which also supports the phytotoxic effect of this treatment by reducing photosynthesis and carbon metabolism.

Details are in the caption following the image — **FIGURE 1**
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Effect of Cytolan® and Cytolan® Stress biostimulants on soluble sugar concentration in leaves of lettuce plants. Columns represent the means and error bars standard error (n = 9 data). Values with different letters indicate significant differences according to Fisher's least significant differences test.

3.3 Phytohormone profile

One of the beneficial effects of the application of algae extracts as a biostimulant is the exogenous supply of hormones that usually act by stimulating plant growth (Mosa et al. 2022; El Khattabi et al. 2023). The analysis of the hormonal profile is useful to explain the biostimulant effect of any compound or prototype applied to plants (Blázquez et al. 2020). In the present study, this analysis showed that both Cytolan® and Cytolan® Stress increased the concentration of the different growth hormones studied (IAA, tZ, iP, GA1, GA3, and GA4) compared with control plants, but no differences between the different applied doses were observed (Table 5).

TABLE 5. Effect of Cytolan® and Cytolan® Stress biostimulants on the hormonal profile (ng g⁻¹ DW) in lettuce plants.

Treatment	Control	Cytolan 150 mL/hL	Cytolan 300 mL/hL	Cytolan 500 mL/hL	Cytolan Stress 150 mL/hL	Cytolan Stress 300 mL/hL	Cytolan Stress 500 mL/hL	p-value
IAA	1.50 ± 0.10b	1.99 ± 0.23a	1.96 ± 0.12a	1.87 ± 0.22a	1.82 ± 0.22a	1.89 ± 0.20a	1.86 ± 0.26a	*
tZ	686.19 ± 13.60b	824.42 ± 19.86a	817.40 ± 18.88a	806.71 ± 16.32a	809.02 ± 77.30a	809.11 ± 33.58a	815.01 ± 62.34a	*
IP	0.53 ± 0.07b	0.62 ± 0.03a	0.60 ± 0.04a	0.62 ± 0.05a	0.60 ± 0.06a	0.63 ± 0.07a	0.61 ± 0.07a	*
GA1	0.30 ± 0.02b	0.49 ± 0.07a	0.51 ± 0.06a	0.47 ± 0.03a	0.48 ± 0.07a	0.43 ± 0.03a	0.44 ± 0.03a	*
GA3	0.13 ± 0.02b	0.18 ± 0.03a	0.21 ± 0.02a	0.18 ± 0.02a	0.18 ± 0.03a	0.19 ± 0.03a	0.20 ± 0.03a	*
GA4	0.27 ± 0.03b	0.46 ± 0.06a	0.44 ± 0.04a	0.40 ± 0.04a	0.42 ± 0.02a	0.41 ± 0.04a	0.42 ± 0.04a	*
ABA	58.84 ± 1.59b	59.57 ± 1.73b	59.84 ± 1.74b	61.79 ± 1.79b	62.02 ± 2.65b	60.71 ± 0.81b	81.83 ± 5.27a	*
ACC	15.13 ± 0.48b	14.71 ± 0.12b	14.89 ± 0.56b	15.17 ± 0.22b	15.25 ± 0.84b	15.41 ± 0.65b	22.39 ± 2.06a	*
JA	407.85 ± 10.68	404.71 ± 7.07	396.85 ± 8.81	400.10 ± 4.40	402.53 ± 11.68	408.98 ± 7.89	409.44 ± 6.47	N.S.
SA	3023 ± 105b	3118 ± 81b	3114 ± 65b	3133 ± 213b	3014 ± 141b	3078 ± 158b	4082 ± 231a	*

Indoleacetic acid (IAA), gibberellin (GA), trans-zeatin (tZ), isopentenyl adenine (iP), abscisic acid (ABA), aminocyclopropane carboxylic acid (ACC), jasmonic acid (JA), salicylic acid (SA). Values are means ± standard deviation (n = 9 data). The levels of significance were represented as NS (p > 0.05) and * (p < 0.05). Values with different letters indicate significant differences according to Fisher's least significant differences test.

Regarding the stress-related hormones (ABA, ACC as a precursor of ethylene, and SA), there were no significant differences between treatments except for an increase in plants supplied with the highest dose of Cytolan® Stress. In contrast, we did not observe changes in JA concentration between the different treatments (Table 5). Cytolan® Stress contains SA as one of its bioactive compounds, which in this experiment only increased in plants supplied with the 500 mL/hL dose (Table 5). SA accumulation regulates the level of other hormones by inducing the accumulation of ABA and ethylene (Blázquez et al. 2020). The higher accumulation of ABA in the Cytolan® Stress treatment at 500 mL/hL could contribute to the lower E (Table 4), and the increase in ACC as ethylene could favor ROS accumulation (Sharma et al. 2019), which supports the phytotoxic effect of this treatment (Table 1).

3.4 Nitrogen parameters, AAs, and protein concentrations

Some studies have shown that applying biostimulants can improve the growth and productivity of plants by influencing N metabolism (Di Mola et al. 2020; Navarro-León et al. 2022). In the present study, the application of both Cytolan® and Cytolan® Stress at all doses reduced foliar NO₃⁻ concentrations but there were no differences between the different applied doses of both products (Table 6). NO₃⁻ is the predominant source of N for plants, and its decrease is usually indicative of an increase in N assimilation processes (Maathuis, 2009; Vidal et al. 2020). Alternatively, given that the biostimulants contain proline, the degradation of this AA may produce glutamate and aspartate that could act by reducing the assimilation processes and NO₃⁻ absorption due to an accumulation of organic N compounds (Iqbal et al. 2020; The et al. 2021). Indeed, this result agrees with the higher concentration of these two AAs in our experiment (Table 7). Furthermore, high NO₃⁻ levels in lettuce leaves increase the risk of certain diseases after human consumption (Mensinga et al. 2003). Therefore, the use of Cytolan® and Cytolan® Stress would improve the nutritional quality of lettuce plants intended for human consumption.

TABLE 6. Effect of Cytolan® and Cytolan® Stress biostimulants on Total N Concentration and N Utilization Efficiency (NUtE) of lettuce plants.

Treatment	NO₃⁻	N content	NUtE	Soluble proteins
Treatment	(mg g⁻¹ FW)	(mg N)	(g DW mg⁻¹ N)	(mg g⁻¹ FW)
Control	10.05 ± 0.90a	66.86 ± 1.20e	0.047 ± 0.001e	0.48 ± 0.06c
Cytolan® 150 mL/hL	6.07 ± 0.40b	86.62 ± 1.29c	0.050 ± 0.001d	0.89 ± 0.03b
Cytolan® 300 mL/hL	6.89 ± 0.30b	114.45 ± 3.37a	0.072 ± 0.001a	0.97 ± 0.01a
Cytolan® 500 mL/hL	6.84 ± 0.17b	91.59 ± 3.77c	0.056 ± 0.001c	0.92 ± 0.04ab
Cytolan® Stress 150 mL/hL	6.83 ± 0.44b	81.49 ± 1.56d	0.056 ± 0.002c	0.95 ± 0.05a
Cytolan® Stress 300 mL/hL	7.05 ± 0.17b	98.67 ± 5.20b	0.065 ± 0.002b	0.98 ± 0.12a
Cytolan® Stress 500 mL/hL	6.00 ± 0.39b	52.46 ± 4.14f	0.035 ± 0.002f	0.48 ± 0.04c
p-value	***	***	***	***

Nitrogen utilization efficiency (NUtE). Values are means ± standard deviation (n = 9 data). The level of significance was represented as.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

TABLE 7. Effect of Cytolan® and Cytolan® Stress biostimulants on the aminogram (μg g⁻¹ DW) in lettuce plants.

Treatment	Control	Cytolan 150 mL/hL	Cytolan 300 mL/hL	Cytolan 500 mL/hL	Cytolan Stress 150 mL/hL	Cytolan Stress 300 mL/hL	Cytolan Stress 500 mL/hL	p-value
Alanine	8.90 ± 0.40c	11.53 ± 1.12b	15.20 ± 0.43a	15.09 ± 0.84a	11.91 ± 0.60b	14.51 ± 0.65a	14.74 ± 1.36a	***
Arginine	2.53 ± 0.16c	3.20 ± 0.21b	4.34 ± 0.12a	4.72 ± 0.33a	3.38 ± 0.36b	4.20 ± 0.24a	4.68 ± 0.57a	***
Aspartate	34.37 ± 2.23d	38.10 ± 1.50c	42.03 ± 1.70b	41.61 ± 2.08b	41.12 ± 0.76b	45.00 ± 0.14a	45.78 ± 1.82a	***
Glutamate	40.31 ± 0.85d	45.22 ± 1.80c	50.97 ± 0.68b	51.79 ± 1.58b	51.05 ± 0.68b	56.99 ± 1.00a	56.36 ± 1.78a	***
Histidine	3.16 ± 0.14	3.43 ± 0.41	3.39 ± 0.33	3.27 ± 0.30	3.22 ± 0.19	3.49 ± 0.43	3.36 ± 0.15	N.S.
Isoleucine	618.43 ± 5.22	621.50 ± 8.55	627.23 ± 4.36	626.06 ± 8.76	625.95 ± 5.90	623.75 ± 9.70	617.28 ± 5.11	N.S.
Leucine	418.05 ± 5.06	426.89 ± 4.57	416.56 ± 5.44	427.01 ± 4.21	415.05 ± 4.09	425.46 ± 3.90	424.67 ± 8.48	N.S.
Lysine	3.59 ± 0.13	3.63 ± 0.06	3.59 ± 0.41	3.63 ± 0.28	3.41 ± 0.13	3.22 ± 0.26	3.38 ± 0.33	N.S.
Phenylalanine	322.30 ± 4.13c	360.18 ± 3.00b	398.01 ± 7.82a	395.45 ± 4.60a	356.06 ± 7.88b	404.47 ± 5.59a	400.09 ± 11.16a	***
Proline	56.58 ± 2.22d	65.97 ± 3.09c	82.97 ± 3.39b	83.53 ± 2.96b	78.81 ± 1.15b	96.98 ± 2.17a	96.24 ± 3.46a	***
Serine	15.62 ± 1.22c	19.86 ± 0.53b	25.90 ± 0.82a	26.84 ± 0.30a	20.67 ± 1.39b	26.31 ± 0.46a	24.67 ± 0.58a	***
Threonine	10.33 ± 0.86	10.57 ± 0.40	10.88 ± 0.60	10.40 ± 0.35	10.83 ± 0.30	10.44 ± 0.95	10.91 ± 1.58	N.S.
Tryptophan	81.60 ± 2.11c	91.06 ± 0.67b	103.10 ± 3.13a	103.55 ± 3.00a	93.68 ± 1.75b	106.95 ± 1.70a	106.76 ± 2.17a	***
Tyrosine	94.25 ± 3.62	92.01 ± 1.08	96.94 ± 2.33	95.65 ± 1.92	94.42 ± 0.99	93.75 ± 0.70	94.65 ± 2.77	N.S.
Cysteine	6.21 ± 0.24	6.53 ± 0.37	5.90 ± 0.24	5.86 ± 0.64	5.78 ± 0.53	6.62 ± 0.18	5.98 ± 0.07	N.S.
TOTAL	1716 ± 7d	1799 ± 18c	1887 ± 10b	1894 ± 19b	1815 ± 13b	1922 ± 15a	1909 ± 25a	***

Values are means ± standard deviation (n = 9 data). The levels of significance were represented as NS (p > 0.05) and.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

The application of biostimulants also has the objective of increasing NUtE, improving production with lower N fertilization (Laurent et al. 2020; Navarro-León et al. 2022). In our experiment, the application of Cytolan® and Cytolan® Stress increased the total N concentration and content and NUtE compared with the control plants, except for the 500 mL/hL dose of Cytolan® Stress, which reduced these parameters with respect to the control (Table 6 and S1). The maximum values for N content and NUtE occurred with the application of 300 mL/hL of Cytolan® (Table 6). These results, together with the induction of photosynthetic efficiency (Table 2) and the increase in the concentration of growth-promoting hormones (Table 5), could explain the positive effect of Cytolan® and Cytolan® Stress applied at 300 mL/hL in increasing plant growth (Table 1). Furthermore, the use of these biostimulants could reduce the application of N fertilizers without impairing crop yields, which would reduce costs and environmental impact.

As we delve further into the examination of various nitrogenous compounds, the analysis of AAs profile becomes crucial, not only because of their significance in plant growth but also because of their regulatory influence on N absorption and assimilation processes (Iqbal et al. 2020; The et al. 2021). The application of Cytolan® product and Cytolan® Stress at all dosage levels increased the total concentration of soluble AAs, with the highest values occurring in plants supplied with the Cytolan® Stress (300 and 500 mL/hL doses) (Table 7). The greater presence of glutamate and aspartate in plants supplied with the biostimulants could have a positive effect on plant growth since these AAs are nitrogenous compounds that transport to growing areas through the phloem and also act as regulators of N absorption by the roots (Iqbal et al. 2020; The et al. 2021; Trovato et al. 2021).

In addition to soluble AAs, the foliar concentration of soluble proteins in our experiment showed an increase in all Cytolan® and Cytolan® Stress treatments, except for Cytolan® Stress at a high dose (500 mL/hL) (Table 6). The increase in soluble proteins, especially with the application of the dose of 300 mL/hL for Cytolan® and Cytolan® Stress, would explain the increase in plant growth caused by these treatments (Table 1) since soluble proteins represent the levels of cellular enzymes involved in essential processes such as N assimilation, photosynthesis, carbon metabolism, etc. (Iqbal et al. 2020; The et al. 2021). The concentration of soluble proteins also indicates that the application of Cytolan® Stress at a high dose is not suitable for inducing plant growth because a similar soluble protein concentration was observed in this treatment compared with control plants (Table 6).

3.5 Mineral element concentrations

Another positive effect of biostimulant application is the improvement of the nutritional state of plants through the increased accumulation of essential mineral elements (Jardin 2012; Papa et al. 2022). The concentration of mineral nutrients in the leaves is presented in Figure 2 and Table S1. In general, the values obtained can be considered normal, and nutrients were not in a range of excess or deficit that could limit the normal growth of plants (Epstein and Bloom, 2005). There were no differences in the concentration of any nutrient between the Cytolan® and Cytolan® Stress treatments, but K, Ca, and micronutrients concentrations were higher compared to control plants with the application of both biostimulants (Figure 2 and Table S1). The increment in K and Ca could be significant given their crucial physiological functions and the fact that they contribute to tolerance to stresses such as salinity stress (Capula-Rodríguez et al. 2016; Navarro-León et al. 2020). Furthermore, the increase in micronutrient concentration can contribute to the enhanced functionality of antioxidant enzymes, photosynthesis, and N assimilation, as these nutrients play pivotal roles in these processes (Epstein and Bloom, 2005; Hänsch and Mendel, 2009).

3.6 Antioxidant compounds

Several studies have also demonstrated that the application of biostimulants can increase the accumulation of compounds with antioxidant activity in plants (Francesca et al. 2020; Franzoni et al. 2022; Ma et al. 2022). The results showed that the application of Cytolan® Stress, which represents an extra contribution of a phenolic compound such as SA and of AsA, determined a higher foliar concentration of total phenols and AsA. In addition, the maximum values were presented in the Cytolan® Stress treatment at a dose of 500 mL/hL. Likewise, the application of Cytolan® also increased the foliar concentration of these compounds compared with control plants, but to a lesser extent than the application of Cytolan® Stress (Table 8).

TABLE 8. Effect of Cytolan® and Cytolan® Stress biostimulants on the concentration of total phenols, flavonoids, anthocyanins and AsA in lettuce plants.

Treatment	Total phenols	Flavonoids	Anthocyanins	AsA
Treatment	(mg g⁻¹ FW)	(mg g⁻¹ FW)	(mg g⁻¹ FW)	(mg g⁻¹ FW)
Control	1.27 ± 0.05e	0.98 ± 0.05f	0.44 ± 0.01e	0.21 ± 0.002e
Cytolan® 150 mL/hL	1.29 ± 0.06e	1.12 ± 0.08e	0.54 ± 0.01c	0.23 ± 0.04e
Cytolan® 300 mL/hL	1.42 ± 0.13d	1.26 ± 0.07d	0.54 ± 0.01c	0.29 ± 0.04d
Cytolan® 500 mL/hL	1.61 ± 0.05c	1.34 ± 0.14d	0.49 ± 0.02d	0.31 ± 0.03d
Cytolan® Stress 150 mL/hL	2.45 ± 0.25c	1.69 ± 0.08c	0.68 ± 0.05b	0.41 ± 0.02c
Cytolan® Stress 300 mL/hL	3.21 ± 0.22b	2.07 ± 0.15b	0.73 ± 0.02b	0.48 ± 0.003b
Cytolan® Stress 500 mL/hL	4.32 ± 0.31a	3.31 ± 0.14a	0.96 ± 0.08a	0.60 ± 0.02a
p-value	***	***	***	***

Ascorbate (AsA). Values are means ± standard deviation (n = 9 data). The level of significance was represented as.
*** (p < 0.001). Values with different letters indicate significant differences according to Fisher's least significant differences test.

The results clearly suggest that the application of Cytolan® Stress was more effective in inducing the antioxidant capacity of lettuce plants because the application of all doses showed a very significant increase in both the values of the FRAP and TEAC tests. The maximum values were found in plants supplied with Cytolan® Stress applied at 500 mL/hL. Likewise, the application of Cytolan® at all doses increased the values of FRAP and TEAC to a lesser extent than Cytolan® Stress (Figure 3). Therefore, another additional positive effect of the assayed biostimulants could be the attainment of plants that are more adept at facing oxidative stress (Rodrigues de Queiroz et al. 2023). Furthermore, Cytolan® application could deliver plants with enhanced nutritional quality for human consumption; AsA and phenolics being of significant interest in preventing specific diseases (Baslam et al. 2013).

In conclusion, this study identified the application of Cytolan® Stress at a dose of 300 mL/hL as the most advantageous treatment for lettuce cultivation. Thus, this biostimulant substantially improved lettuce growth, probably by inducing photosynthetic activity and enhancing phytohormone, AAs, and mineral nutrient accumulation. Furthermore, the biostimulant improved NutE; thereby, this product could allow the reduction of N fertilizers without impairing the yield of the crops, reducing costs and environmental impact. Additionally, it substantially augmented the antioxidant capacity and concentration of various antioxidant compounds, leading to increased lettuce quality and potential stress tolerance. However, it is worth noting that applying Cytolan® Stress at doses exceeding 300 mL/hL, although still elevating antioxidant capacity, eliminated the beneficial effect on plant biomass production, underscoring the importance of adhering to recommended dosages (Figure 4).

AUTHOR CONTRIBUTIONS

JMR and BB conceived and designed the research. DVC and SAC conducted experiments. ENL, SAC, and DVC analyzed the data. JMR and ENL wrote the manuscript. JMR and BB did a critical revision of the article. All authors read and approved the manuscript.

FUNDING INFORMATION

This work was supported by the PAI program (Plan Andaluz de Investigación, Grupo de Investigación AGR282).

Open Research

DATA AVAILABILITY STATEMENT

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Supporting Information

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Citing Literature

Volume176, Issue1

January/February 2024

e14193

This article also appears in:

Biostimulants in Agriculture

Filename	Description
ppl14193-sup-0001-FigureS1.tifTIFF image, 625.2 KB	Figure S1. Photographs showing the appearance of the lettuce plants subjected to the different treatments at the end of the experiment.
ppl14193-sup-0002-TableS1.docxWord 2007 document , 18.6 KB	Table S1. Effect of Cytolan® and Cytolan® Stress biostimulants on AA profile (μg g⁻¹ DW) of leaves of lettuce plants.

Is the application of bioactive anti-stress substances with a seaweed-derived biostimulant effective under adequate growth conditions?

Abstract