Biostimulant Extracts Obtained from the Brown Seaweed Cystoseira barbata Enhance the Growth, Yield, Quality, and Nutraceutical Value of Soil-Grown Tomato

Abstract

The use of seaweed-derived biostimulants has gained attention as a sustainable strategy to enhance crop production. Brown seaweeds, in particular, are rich in bioactive compounds that can improve plant growth, yield, and quality parameters. This study investigated the biostimulant potential of extracts derived from Cystoseira barbata for promoting tomato growth and improving fruit quality. Three different extracts (water, alkali, and acid), applied as soil drenches, were tested on a determinate tomato cultivar under greenhouse conditions. In young plants, alkali and acid extracts increased stem length by 40% and 60%, respectively, while water and acid extracts accelerated early flowering. Alkali and acid extracts also improved fruit yield by approximately 65%. Additionally, all extracts enhanced fruit quality by increasing fruit EC and Brix values, soluble carbohydrate levels, total phenolic content, total antioxidant capacity, lycopene and β-carotene concentrations, and vitamin C content, albeit to varying degrees. Along with increases in fruit K concentration in response to water and alkali extracts, all seaweed extract-treated groups showed elevated fruit S concentrations, accompanied by increases in reduced glutathione levels. These results indicate that C. barbata extracts can enhance plant performance while improving the nutritional and nutraceutical properties of tomato fruits. The observed effects were strongly influenced by the extraction method, which alters the extract composition. Extracts from sustainably sourced C. barbata may contribute to improved productivity and quality in horticulture; however, further research is needed to enable the standardized production of C. barbata, optimize biostimulant formulations, and validate their effectiveness under field conditions.

1. Introduction

The human population is projected to reach approximately 11.2 billion by 2100 [1], and this rapid growth is expected to drive a substantial increase in the demand for food production and supply [2]. At the same time, the climate crisis has emerged as a major threat, disrupting agricultural production, food security, and related industries [3]. Addressing these challenges requires immediate and innovative solutions, including the adoption of sustainable agricultural practices [4]. It is becoming increasingly evident that alternative approaches are needed to enhance resource-use efficiency, reduce losses due to abiotic and biotic stresses, and maximize productivity [5]. Modern agricultural techniques must also strike a balance between economic profitability for producers and minimal environmental impact [6]. As consumer preferences continue to shift toward more nutritious food options, the importance of sustainable agricultural production continues to rise [7].

According to the EU Regulation 2019/1009, a plant biostimulant is defined as a fertilizing product which can, independently of its nutrient content, improve nutrient use efficiency, availability of nutrients in the soil or rhizosphere, tolerance to abiotic stress, and quality traits [8]. Under the growing threat of global climate change, the importance of biostimulants in sustainable agriculture is growing rapidly as they can enhance the climate resilience of crops by boosting their tolerance to abiotic stressors [9]. Even though marine macroalgae, also called seaweeds, have been used as a source of organic matter and fertilizer in agriculture since ancient times, their biostimulant effects have been documented relatively recently, which has led to the increasing commercialization of a diverse set of seaweed-based products [10,11]. Today, seaweed extracts (SWEs) represent a major category of organic non-microbial biostimulants. Brown macroalgae (Phaeophyta) are the most commonly used seaweeds as sources of biostimulants; however, red (Rhodophyta) and green (Chlorophyta) seaweeds have also been extensively studied in this context and have made their way into commercial formulations. Seaweed extracts have already been adopted by farmers all over the world, and they are being increasingly used to concurrently enhance plant health, vigor, yield, and quality in a sustainable manner [12].

The complexity of SWE-based biostimulants makes it challenging to directly link their composition to observed benefits [13]. They typically contain, depending on the species and extraction method, varying amounts of essential or beneficial mineral nutrients, pigments, vitamins, amino acids, betaines, and other compatible solutes such as mannitol, phenolic compounds including phlorotannins with antioxidant and metal-chelating activities, phytohormones, and phytohormone-like compounds, as well as soluble bioactive polysaccharides, the most important of which include fucoidans, laminarins, and alginic acid [14,15,16]. Some of the bioactive compounds found in SWEs are unique to specific seaweed taxa, highlighting the importance of the species from which they are obtained [10,17].

Various extraction processes are applied on fresh or dried seaweed biomass to disrupt the cells and release the organic and inorganic beneficial constituents into the aqueous phase. While alkali extraction is the most widely used method for the industrial production of SWEs, acid and neutral extraction methods are also commonly employed and are known to yield effective biostimulants [14,18]. Neutral methods typically involve extraction with water, either at elevated temperatures below the boiling point or in an autoclave [19,20]. Recently, there has also been growing interest in other types of extracts, including polysaccharide-enriched extracts [20,21] and minimally processed homogenates [16]. The extraction processes have a significant impact on the composition of the extracts, which, in turn, exhibit altered biological activity on plants [22].

Tomato is the most widely produced non-starchy vegetable crop, with a total production of 192 million tons on 5.4 million hectares of land [23]. With a total production of 13.3 million tons in 2023, Türkiye ranked third among the world’s major tomato-producing countries, following only China and India. Tomato is not only a good dietary source of several micronutrients, including but not limited to vitamin C and potassium, but also rich in health-promoting nutraceuticals with antioxidant properties, such as phenolic compounds, lycopene, and β-carotene, making it a functional food with potential in the prevention of chronic diseases, including various cancers and cardiovascular conditions [24,25].

An increasing number of studies have explored the biostimulant effects of SWEs on tomato under field [26,27,28,29] or greenhouse conditions [30,31,32,33]. The vast majority of all publications dealing with SWEs as biostimulants in tomato cultivation focused on brown seaweeds from several genera, including but not limited to Ascophyllum, Durvillaea, Ecklonia, Padina, Sargassum, Fucus, and Bifurcaria [20,30,32,34].

Both foliar and soil (or substrate) applications of SWEs have been widely tested in studies evaluating their effects on tomato performance. In one of the earliest studies on this topic, Kumari et al. [26] found that soil drench applications were generally at least as effective as foliar applications in enhancing the growth, yield, and quality traits of field-grown tomatoes, though their relative effectiveness depended on SWE concentration. Similarly, Ali et al. [35] documented the comparable effectiveness of foliar and soil applications in a field experiment. However, several studies have reported that soil applications can surpass foliar treatments in promoting tomato growth under greenhouse conditions [19,36]. These findings may be attributed to (i) the role of algal polysaccharides in improving soil aggregate stability, aeration, and water-holding capacity, (ii) the stimulation of beneficial microbial populations essential for nutrient cycling, plant growth promotion, and disease suppression, (iii) prolonged mineral nutrient availability in soil compared to the short-term bursts provided by foliar sprays, and (iv) direct root exposure to SWEs, enhancing root activity and resilience [32,36,37].

Cystoseira barbata (C. barbata), also known as Gongolaria barbata, is a long-lived brown seaweed species that belongs to the genus Cystoseira sensu lato (the Cystoseira complex) within the family Sargassaceae [38,39]. This littoral species, considered relatively tolerant to eutrophication, thrives in rocky, shallow, sheltered, and well-lit marine environments, typically at depths of less than 1 m. During winter, it develops floating secondary branches with aerocysts, forming a three-dimensional habitat that supports marine biodiversity. Even though different Cystoseira species, including C. foeniculacea, C. humilis, C. ericoides, and C. tamariscifolia, have recently been used to produce SWEs, which have shown promising results as biostimulants for tomatoes under various abiotic stress conditions [40,41,42], C. barbata has not yet been investigated in this context. With the exception of a study by Demir et al. [43], which tested the effects of C. barbata suspensions on the germination of several vegetable crops without discussing them as biostimulants, our previous publication is the only article addressing the effects of C. barbata extracts on crop production [44].

Despite the widespread use of brown SWEs as plant biostimulants, the genus Cystoseira remains largely understudied in this context. To our knowledge, this is the first study to examine the biostimulant effects of C. barbata extracts on tomato plants. Another key contribution of this research to the literature on biostimulants is the comparative evaluation of three distinct extracts from this species. This study assesses the effects of the soil applications of these extracts on tomato growth, yield, and selected sensory and nutritional quality parameters of the fruits under greenhouse conditions in the absence of any deliberate stress application.

2. Materials and Methods

2.1. Collection and Storage of C. barbata

Cystoseira barbata was harvested in late winter from shallow waters (depth < 100 cm) along the coast of the Marmara Sea at the following coordinates: 40°49′30″ N, 29°16′44″ E. After rinsing with deionized water, half of the fresh biomass was stored at −80 °C, whereas the rest was dried at 70 °C and then finely ground.

2.2. Preparation and Composition of C. barbata Extracts

The preparation of three distinct extracts followed the protocols described below as previously detailed by Mutlu-Durak et al. [44]. The mineral and bioactive compound concentrations, antioxidant capacities, and hormone-like activities of the experimental SWEs are also reported therein.

First, 100 g of dried, ground samples were combined with deionized water to achieve a final volume of 1 L and incubated at 70 °C for three hours with continuous stirring. The mixture was filtered through cheesecloth and then centrifuged at 8000× g for 15 min to obtain the supernatant used as water extract.

Then, 500 g of the fresh sample was homogenized in 1 L of deionized water, adjusted to pH 3.0 ± 0.1 using H₂SO₄, and incubated at 45 °C for 30 min. After centrifugation at 8000× g for 15 min and subsequent filtration, the pH of the supernatant was adjusted to 7.0 ± 0.1 using KOH and used as an acid extract.

The alkali extraction method followed the same protocols as acid extraction. After the last incubation at 45 °C, KOH was added to adjust the pH to 12.0 ± 0.1. The mixture underwent incubation at 80 °C for 3 h, followed by centrifugation at 8000× g for 15 min, filtration, and neutralization to a pH of 7.0 ± 0.1 with H₂SO₄ for use as an alkali extract.

2.3. Plant Material and Growing Conditions

The effects of the SWEs were tested on tomato (Solanum lycopersicum L. cv. Cuma F1) plants grown in soil-filled pots under greenhouse conditions between April and August 2021. Cuma F1 is a hybrid, determinate, and early maturing cultivar, producing dark red, slightly flattened, high-quality fruits. Inside the climate-controlled Venlo-type glasshouse, the temperature was kept at 25 ± 3 °C during the day and 20 ± 3 °C at night.

Seedlings were grown in a calcareous (20% CaCO₃) and alkaline (pH 8.0) clay loam soil containing only 1% organic matter. Each pot was filled with 3.5 kg of air-dried soil. Prior to the transplantation of commercial seedlings (one per pot), 100 mg N per kg soil in the form of commercial calcium nitrate and 50 mg P per kg soil as monopotassium phosphate (corresponding to ~63 mg K per kg soil) were homogenously incorporated into the soil. The same fertilizers at the same rates were applied again in the form of concentrated solutions with the irrigation water 35 days after transplantation, effectively increasing the total rates of N, P, and K to 200, 100, and 125 mg per kg soil, respectively. Additionally, 1% (w:v) monopotassium phosphate was sprayed twice on the foliage of plants at a rate of 30 mL per plant to avoid any risk of P or K deficiency.

Plants were irrigated daily with deionized water. The pots were kept on their saucers throughout the experimental period to prevent random leaching losses of nutrients. The experiment was terminated 100 days after transplantation.

2.4. Experimental Design

The experiment was designed as a single-factor experiment with 4 treatment groups. In addition to the negative control group, which was not treated with any SWE and is hereafter referred to as the control group, there was one group of pots for each of the 3 experimental C. barbata extracts. Extracts were applied directly to the soil without further dilution. Each extract was applied to the soil at a single rate: 100 mL per pot for the water extract, 30 mL per pot for the alkali and acid extracts. The SWEs were applied to the pots one week after transplantation for the first time. The first application was made one week after transplantation, and subsequent applications were performed at 15- to 21-day intervals for a total of six applications. Each treatment was represented by 5 pot replicates.

2.5. Plant Growth Measurements, Fruit Harvest, and Sample Storage

When the plants were at the flowering stage (BBCH scale 60–69), 35 days after transplantation, the following parameters were recorded: stem length, number of inflorescences, and number of flowers. Stem length was defined as the sum of the length of the main axis and the lengths of the branches. Flower clusters were referred to as inflorescences. Only fully open flowers were counted to determine the total number of flowers at anthesis.

Fully ripened tomatoes were harvested periodically until the experiment was terminated. Total soluble solids (Brix), electrical conductivity (EC), and pH measurements were conducted on freshly harvested fruit samples. All fruits harvested throughout the experimental period were immediately frozen at −80 °C. The pooled tomatoes harvested from each pot were chopped into small cubes while still frozen. For each plant, a subsample of chopped tomatoes was dried at 70 °C and then ground into a fine powder for mineral analysis as described below. The remaining cubes were homogenized using a blender. Multiple subsamples from each homogenized sample were again stored at −80 °C for subsequent spectrophotometric quality analyses.

2.6. Determination of Total Soluble Solids (TSS, Brix), EC, and pH of Tomato Fruits

Total soluble solids of homogenized fruit samples were determined with a digital refractometer (MASTER-53M, Atago Co., Tokyo, Japan) in degrees Brix. After the centrifugation of the homogenates, the supernatants were used to determine their pH (Sensor InLab^® Routine Pro-ISM, Mettler-Toledo International Inc., Greifensee, Zürich, Switzerland) and EC (Seven2Go™ Pro, Mettler-Toledo International Inc., Greifensee, Zürich, Switzerland).

2.7. Mineral Analysis

Dried and ground fruit samples were digested in a closed-vessel microwave system (Mars 6, CEM Corporation, Matthews, NC, USA) using nitric acid and hydrogen peroxide, as described by Ceylan et al. [45]. The concentrations of essential macro- (Ca, K, Mg, P, and S) and micronutrients (B, Cu, Fe, Mn, Zn) were determined using inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent 5800 VDV, Agilent Technologies, Santa Clara, CA, USA).

2.8. Determination of Total Phenolic Content Using the Folin–Ciocalteu Assay

The total phenolic contents (TPCs) of the fruit samples were determined using the Folin–Ciocalteu assay as described by Singleton et al. [46] and Mannino et al. [47]. For this purpose, 5 g of the homogenized fruit sample was extracted with 5 mL of 70% methanol. Gallic acid was used as a standard. Absorbance was recorded at 760 nm using a UV/Vis spectrophotometer (Peak Instruments C-7100S, Houston, TX, USA). Results were expressed as the mg of gallic acid equivalent (GAE) per 100 g of fresh weight (FW).

2.9. Determination of Total Antioxidant Capacity

The methanol extracts of the homogenized fruit samples, prepared for the TPC assay, were also used for the determination of total antioxidant capacity with 3 different methods, as follows:

1-

The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay was conducted according to the method described by Re et al. [48].

2-

The second assay conducted for this purpose was the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [49,50].

3-

Finally, the ferric-reducing antioxidant power (FRAP) assay was carried out according to the method described by Benzie and Strain [51].

Different concentrations of Trolox [(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid], a vitamin E analog, were used to plot standard curves. The results were presented as µmol Trolox equivalent (TE) per 100 g FW.

2.10. Determination of Lycopene and β-Carotene Concentrations

Lycopene and β-carotene concentrations in tomato homogenates were quantified by the method originally described by Nagata and Yamashita [52] and then modified by Fernández-Garcí et al. [53]. Next, 1 g of homogenized fruit juice was extracted with 10 mL of acetone/hexane mixture (2:3). Absorbance was read at 663, 645, 505, and 453 nm. The concentrations were calculated with the formulas given below:

-

Lycopene (mg per 100 g FW) = –0.0458 A₆₆₃ + 0.204 A₆₄₅ + 0.372 A₅₀₅ − 0.0806 A₄₅₃

-

β-Carotene (mg per 100 g FW) = 0.2160 A₆₆₃ − 1.220 A₆₄₅ − 0.304 A₅₀₅ + 0.452 A₄₅₃

2.11. Vitamin C Assay

Total and reduced ascorbic acid (AsA) concentrations in fruit samples were determined spectrophotometrically [54,55]. For this purpose, homogenized fruit juice was extracted with a 5% metaphosphoric acid solution in a ratio of 1:5 (w/v). The absorbance of samples and standard solutions was measured at 525 nm. Results were reported as mg total/reduced AsA per 100 g FW.

2.12. Determination of Reduced Glutathione (GSH) Concentration

The extraction was conducted according to Kampfenkel et al. [54]. Then, the GSH concentrations in the samples were determined using the method described by Zhou et al. [56]. The standard curve was plotted using GSH solutions with known concentrations, and results were expressed in mg GSH per 100 g FW.

2.13. Determination of Total Soluble Carbohydrate Concentration

The method used to measure soluble carbohydrates through UV spectroscopy was based on the method described by Yemm and Wills [57] and Mengutay et al. [58], with some minor modifications. Dried and ground fruit samples were extracted in a ratio of 1:100 (w/v) with 80% EtOH. The standard curve was constructed using different concentrations of D-glucose. Soluble carbohydrate concentrations were expressed as mg g⁻¹ dry weight (DW).

2.14. Statistical Analysis

The JMP software (version 18) was used for statistical analysis. One-way analysis of variance (ANOVA) was used to evaluate the overall significance of the SWE applications on the reported traits. Significant differences between means, indicated by different letters in tables and figures, were then determined using Fisher’s protected least significant difference (LSD) test at 5% significance.

2.15. Generative AI

During the preparation of this manuscript, ChatGPT (developed by OpenAI, model GPT-4o) was used solely for generating visual icons included in the graphical abstract, based on author-provided prompts. These AI-generated components were then assembled, annotated, and finalized by the authors using Microsoft PowerPoint. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

3. Results

3.1. Plant Growth and Yield Parameters

The stem length, number of inflorescences, and number of flowers were determined 5 weeks after transplantation when the experimental plants reached the flowering stage (

Table 1. Effect of seaweed (C. barbata) extracts on stem length, number of inflorescences, and number of flowers of tomato (Solanum lycopersicum L. cv. Cuma F1) plants 35 days after transplantation.

Seaweed Extracts	Stem Length (cm)	Number of Inflorescences (plant−1)	Number of Flowers (plant−1)
Control	60 ± 10 b	3.8 ± 0.4 b	9 ± 2 b
Water	65 ± 12 b	4.8 ± 1.5 b	14 ± 3 a
Alkali	84 ± 7 a	5.5 ± 1.3 ab	10 ± 2 b
Acid	97 ± 12 a	6.8 ± 1.7 a	14 ± 2 a

Values are means and standard deviations of five replicates. Different letters indicate significant differences between means according to Fisher’s protected LSD test (p < 0.05).

). Application of alkali and acid extracts significantly increased the stem length by 40% and 62%, respectively, whereas the water extract did not affect this parameter. At this stage, neither the number of inflorescences nor the number of flowers was significantly enhanced by the alkali extract. However, the water and acid extracts resulted in a 56% increase in the number of flowers, with the acid extract also inducing a 79% increase in the number of inflorescences.

At the tested rates, soil treatments with the experimental C. barbata extracts led to significant increases in total fruit yield per plant over the course of 100 days after transplantation (Figure 1). Treatments with alkali and acid extracts were associated with a relatively higher yield increase by 64%, while the positive effect of the water extract application remained at 20%.

The stem, leaf, and total shoot (vegetative) DWs were overall positively affected by SWE applications (Figure 2). Although no significant differences were observed for these vegetative growth traits among the different groups treated with the three experimental extracts, the highest values were found for plants grown in soil treated with the water extract of C. barbata. Plants produced 45–60% more vegetative aboveground biomass under any extract treatment compared to the control 100 days after transplantation.

3.2. Brix, EC, and pH

All SWE applications reduced the fruit pH compared to the control group (

Table 2. Effect of seaweed (C. barbata) extracts on pH, EC, and Brix of tomato (Solanum lycopersicum L. cv. Cuma F1) fruits.

Seaweed Extracts	pH	EC (μS cm−1)	Brix (°Bx)
Control	4.8 ± 0.1 a	3409 ± 110 d	5.4 ± 0.5 c
Water	4.2 ± 0.1 b	5739 ± 140 a	7.5 ± 0.5 a
Alkali	4.3 ± 0.0 b	4913 ± 219 b	6.9 ± 0.7 ab
Acid	4.3 ± 0.1 b	4381 ± 155 c	6.2 ± 0.3 b

Values are means and standard deviations of five replicates. Different letters indicate significant differences between means according to Fisher’s protected LSD test (p < 0.05).

). The EC of homogenized tomato juice markedly increased when tomato plants were grown in soil treated with the experimental acid extract. Further significant increments were observed with the alkali and then with the water extract treatments. A similar trend was observed in Brix values in response to the SWE treatments. Compared to control, the Brix values of tomatoes harvested from acid, alkali, and water extract-treated plants were significantly improved by 15, 28, and 39%, respectively.

3.3. Mineral Concentration

The C. barbata extract applications did not cause any significant changes in the P and Mg concentrations of tomato fruits (

Table 3. Macronutrient (P, K, Ca, Mg, and S) concentrations of tomato (Solanum lycopersicum L. cv. Cuma F1) fruits in response to seaweed (C. barbata) extract applications.

Seaweed Extracts	P (mg g−1)	K (mg g−1)	Ca (mg g−1)	Mg (mg g−1)	S (mg g−1)
Control	3.0 ± 0.3 a	25 ± 1 b	3.1 ± 0.2 a	1.2 ± 0.1 a	0.8 ± 0.1 c
Water	2.6 ± 0.1 a	27 ± 0 a	2.1 ± 0.5 b	1.2 ± 0.1 a	1.2 ± 0.1 b
Alkali	3.0 ± 0.2 a	27 ± 1 a	2.3 ± 0.1 b	1.3 ± 0.1 a	1.4 ± 0.1 a
Acid	2.9 ± 0.3 a	25 ± 1 b	2.4 ± 0.1 b	1.2 ± 0.1 a	1.2 ± 0.1 b

Values are means and standard deviations of five replicates. Different letters indicate significant differences between means according to Fisher’s protected LSD test (p < 0.05).

). Water and alkali extracts significantly increased the fruit K concentrations, albeit by less than 8% over the control. Regardless of the type of extract, plants grown in treated soils produced fruits with 23–32% less Ca and 50–75% more S than the control plants.

The micronutrient concentrations of tomato fruits in response to SWE applications are reported in

Table 4. Micronutrient (Fe, Zn, Mn, Cu, and B) concentrations of tomato (Solanum lycopersicum L. cv. Cuma F1) fruits in response to seaweed (C. barbata) extract applications.

Seaweed Extracts	Fe (mg kg−1)	Zn (mg kg−1)	Mn (mg kg−1)	Cu (mg kg−1)	B (mg kg−1)
Control	32 ± 2 a	5.6 ± 0.5 a	8.6 ± 0.7 b	3.2 ± 0.2 a	8.8 ± 0.6 b
Water	31 ± 4 a	5.5 ± 0.6 a	8.8 ± 0.9 b	3.1 ± 0.4 a	11.1 ± 1.9 a
Alkali	35 ± 3 a	5.7 ± 0.7 a	11.0 ± 0.5 a	3.5 ± 0.6 a	9.0 ± 0.9 b
Acid	33 ± 3 a	5.6 ± 0.5 a	10.4 ± 0.6 a	3.2 ± 0.3 a	9.5 ± 0.4 b

Values are means and standard deviations of five replicates. Different letters indicate significant differences between means according to Fisher’s protected LSD test (p < 0.05).

. Among the analyzed micronutrients, Fe, Zn, and Cu were unaffected by the treatments. The alkali and acid extract applications elevated Mn concentration in fruits by 25%, whereas the water extract application led to 26% higher fruit B concentration.

3.4. Phenolic and Antioxidant Activity

The concentrations of phenolics in the tested fruit samples and their antioxidant activities, as determined using ABTS, DPPH, and FRAP assays, are presented in Figure 3. All SWEs significantly elevated the TPC of tomato fruits to a similar extent (Figure 3A). The total antioxidant capacity assays showed results consistent with the TPC assay (Figure 3B–D). According to the ABTS and FRAP assays, all tested SWEs significantly enhanced the antioxidant capacity of tomato fruits to nearly the same level (Figure 3B–D). However, according to the DPPH assay, the water extract was even more effective than the other extracts in this respect (Figure 3C).

3.5. Carotenoids

Lycopene and β-carotene concentrations in the fruits are presented in Figure 4. Compared to the control group, a significant increase in lycopene concentration was observed only in plants treated with the water extract of C. barbata (Figure 4A). The β-carotene levels in fruits from water- and alkali extract-treated plants nearly doubled, whereas those in fruits from acid extract-treated plants remained unaffected (Figure 4B).

3.6. Vitamin C

Both the total and reduced vitamin C concentrations were enhanced by the tested C. barbata extract applications (Figure 5). Significant increases of up to 50% in total AsA concentration of tomato fruits were observed upon water and acid extract treatments. The increases observed for reduced AsA were even more pronounced and ranged between 80% and 130%, with the highest concentration measured in fruits harvested from water extract-treated plants.

3.7. Reduced Glutathione

Seaweed extract treatments had significant positive effects on the GSH concentration in tomato fruits, as shown in Figure 6. Regardless of the seaweed extraction method, the GSH concentrations in fruits increased by 24% compared to the control group.

3.8. Soluble Carbohydrate

The total soluble carbohydrate concentrations of tomato fruits are shown in Figure 7. When tomato plants were grown in the soil treated with water or alkali extract of C. barbata, fruits had 39% more soluble carbohydrates than control fruits. However, in the acid extract-treated group, the total soluble carbohydrate concentration remained unchanged.

4. Discussion

Recent agricultural research increasingly focuses on sustainable methodologies to enhance crop productivity and ensure food security. Recently, different extracts of the understudied brown seaweed C. barbata were shown to enhance wheat seedling growth when applied as seed or substrate treatments [44]. Building on these promising findings, the same water, alkali, and acid extracts of C. barbata were applied in the present study to the soil of pot-grown greenhouse tomatoes to assess their effects on yield and quality parameters.

The compositions and concentrations of both the experimental and commercial SWEs tested as soil drench or fertigation treatments for tomatoes vary widely in the literature. These variations stem from differences in seaweed species, extraction and processing methods, and additional components in the formulations [16,31,59]. As a result, directly comparing dilution ratios, application rates, and application frequencies across studies may not always be meaningful. Nevertheless, higher application rates and frequencies increase the costs associated with SWE use, making economic feasibility a key consideration. The practical applicability of SWEs in commercial tomato farming ultimately depends on their net economic returns [30,60]. Commonly tested soil application frequencies for SWEs vary widely, ranging from twice daily via drip irrigation [32] to once a week [19,36] or once every two weeks as soil/substrate drench treatments [13,26,34]. In many such studies, soil drench applications were repeated five to seven times. The methodology of the present study, in which C. barbata extracts were applied at 15- to 20-day intervals for a total of six applications over the 100-day cultivation period after transplantation, is, therefore, consistent with previous research. The application rates for the experimental water, alkali, and acid extracts of C. barbata in this study were determined based on the findings by Mutlu-Durak et al. [44], who reported that the best rate for the water extract prepared from dried seaweed biomass was two-to-four-times higher than the best rates for the alkali and acid extracts prepared from fresh biomass.

In the presented research, the C. barbata extracts generally improved both vegetative and generative growth parameters, as well as the fruit yield of tomato (Table 1, Figure 1 and Figure 2). At the tested rates, the alkali and acid extracts, which maximized stem length during early growth (Table 1), also resulted in the highest fruit yields by the end of the study (Figure 1). Since the tomato cultivar used in this study was determinate, lateral branches were retained to preserve the cultivar’s natural bushy growth habit and avoid introducing artifacts from cultural practices, unlike in the case of the single-stem cultivation typically applied to indeterminate cultivars. Consequently, a higher total stem length, which includes the lengths of the lateral branches, reflects enhanced seedling vigor during early growth and is not the result of excessive internode elongation, which would not be a desirable trait. The rate of crop development, and the onset and rate of flowering in particular, are key parameters that determine yields of crops, such as determinate cultivars of tomatoes [61]. In this context, the increased flower count on plants treated with the water and acid extracts 35 days after transplantation is an important finding that may have contributed to the yield benefits (Table 1 and Figure 1). Supporting our finding, the expression levels of six major flowering genes were upregulated in tomato foliarly treated with brown SWEs, which was discussed as the molecular basis for enhanced flowering induced using SWEs [62]. Extracts from various brown seaweed species have been reported to induce early and/or enhanced flowering in tomatoes, leading to an increased number of fruits and higher fruit yield [32,33]. Seaweed-based plant biostimulants can also accelerate tomato ripening, enabling earlier harvests and higher economic returns [47,60]. This is most likely associated with the phytohormone-like effects of SWEs.

The acidity and TSS (measured in degrees Brix) of tomato juice are among the most important quality traits for both table (fresh market) and processing tomatoes [63,64]. These parameters are not only critical determinants of sensory properties of tomatoes but also key elements of industrial quality in processing applications. Tomato ripening is typically associated with a decrease in acidity, i.e., a rise in pH due to degradation of organic acids such as citric acid, and an increase in TSS [65]. However, the pH of ripe, high-quality tomatoes typically remains below 4.6, which is important not only for achieving a balanced flavor but also for maintaining sufficient acidity to ensure food safety in processing applications without the need for drastic thermal treatments [66,67]. In the present study, fruits collected from SWE-treated groups exhibited lower pH and markedly higher EC and Brix values compared to the control fruits (Table 2). Consistently, several studies have reported significant increases in Brix values in tomatoes following SWE treatments, indicating an enhancement in fruit quality [28,59].

The main soluble solids in tomato juice affecting the Brix value include soluble carbohydrates, primarily glucose and fructose, as well as organic and amino acids such as citric acid, malic acid, glutamic acid, and ascorbic acid [64,67,68]. Therefore, an increase in Brix value during ripening, despite the expected decline in citric acid, results from the accumulation of soluble carbohydrates. Notably, the Brix value of tomatoes correlates well with total soluble sugar content [63,69]. In the present study, both the Brix value and the total soluble carbohydrate concentration exhibited similar responses to the different SWE applications, with the most and least pronounced increases observed in response to the water and acid extracts, respectively (Table 2 and Figure 7). A similar trend was also observed for the EC of tomato juice.

Mineral analysis revealed changes in the macro- and micronutrient profiles of tomato fruits following SWE treatments (Table 3 and Table 4). Given that K is the most abundant mineral in the tested SWEs [44], the most commonly discussed essential macronutrient for tomato quality [70,71], and an important dietary component whose deficiency is highly prevalent in modern diets [72], any effect of the SWEs on K concentration in tomato fruits deserves particular attention. The significant increases in fruit K concentrations observed following the application of water and alkali extracts of C. barbata (Table 3) are modest compared to the increases in tomato K concentrations reported in response to some other SWE treatments in the literature [27,31]. On the other hand, the significantly lower Ca concentrations in fruits from SWE-treated plants may result from an antagonistic interaction between K and Ca ions at the level of uptake as well as translocation [73].

However, it is important to note that the mineral concentrations presented in this study are expressed on a dry matter basis. Although changes in the dry matter ratio of tomato fruits in response to SWE treatments were not directly measured, previous studies have shown that, in tomatoes, the Brix value is highly positively correlated with the dry matter ratio, which equals the sum of the total soluble and insoluble solids [74]. If tomato fruits with significantly different Brix values and dry matter ratios have the same mineral concentration on a dry weight basis, those with higher Brix values will inherently have higher mineral concentrations on a fresh weight basis. Based on this relationship, and considering the significantly higher Brix values measured in tomatoes from all SWE-treated plants, including those treated with the acid extract, it can be confidently concluded that these fruits had markedly higher K concentrations on a fresh weight basis compared to the control fruits (Table 2 and Table 3).

The most pronounced change in the concentration of any essential mineral in the tomato fruit in response to SWE treatments was observed for S (Table 3). This can be attributed to the high S concentrations in tested extracts, second only to their K concentrations [44]. While the water extract was rich in K and S due to the intrinsic abundance of K ions and S-containing soluble compounds in C. barbata, such as fucoidans, the even higher K and S concentrations in the alkali and acid extracts are attributable to the addition of potassium hydroxide and sulfuric acid during their preparation. In addition to the direct S fertilizer effects of the tested SWEs, the metabolic shifts caused by their bioactive constituents, including S-containing sugars like fucoidans, may involve enhanced N and S assimilation as reported and discussed by several researchers before [75,76,77]. Sulfur is, on one hand, potentially important for the flavor of tomato fruit, which contains multiple aroma-active volatile S compounds [78]. On the other hand, S is required for the synthesis of cysteine, which, along with glutamic acid and glycine, constitutes the tripeptide glutathione [79]. Consistent with this, the higher S concentrations in fruits from SWE-treated plants were accompanied by significantly elevated glutathione concentrations (Table 3 and Figure 6). The ascorbate–glutathione cycle, which is central to the redox metabolism of plants with all its enzymatic and non-enzymatic components, plays a critical role in tomato fruit ripening by regulating the availability of ROS and reducing power [80]. Furthermore, given the pivotal role of glutathione in maintaining redox balance in human physiology and growing evidence of abnormally low glutathione levels in patients with various chronic diseases, glutathione is being investigated both as a biomarker and as a potential therapeutic target in these conditions [81]. While the bioavailability of dietary glutathione is debated, plant-based foods rich in glutathione or its precursors are considered important to support the glutathione status of the body.

Several studies in the literature have reported the positive effects of SWE applications on the concentrations of metallic micronutrients, such as Cu, Fe, Mn, and Zn, in tomato fruits [27,47]. However, in the present study, the only significant changes observed in fruit micronutrient concentrations were a modest increase in Mn in response to the applications of the alkali and acid extracts and a modest increase in B in response to the application of the water extract of C. barbata (Table 4). The optimization of B nutrition is critical for tomato growth and quality attributes, including flavor and nutritional profile [82].

The total phenolic concentration in tomato fruit increased significantly in response to SWE applications (Figure 3A), in agreement with previous studies in which soil and/or foliar applications of extracts from different brown seaweeds, including Ascophyllum nodosum, and Sargassum johnstonii led to tomatoes with higher phenolic contents [26,83]. An increase in phenolic content is considered a positive response because (i) phenolic compounds are secondary metabolites that help protect plants against both abiotic and biotic stressors [84,85], and (ii) increasing evidence suggests that dietary (poly)phenols are beneficial for human health due to their protective effects against various chronic diseases [86]. Phenolic compounds are the most potent contributors to the antioxidant activity of fruits and vegetables, and, therefore, there is typically a strong positive correlation between the TPC and the total antioxidant activity of these foods [87]. Indeed, all tested SWEs significantly boosted the total antioxidant activity of tomato (Figure 3B–D). However, Mannino et al. [47] also reported significant biostimulant-induced increases in the total antioxidant capacity of tomato, as measured using the ABTS and DPPH assays, despite the lack of a significant increase in TPC. The observed increases in the TPC and, thus, the total antioxidant activity of tomatoes harvested from SWE-treated plants can possibly be attributed to three different effects. Firstly, polysaccharides found in SWEs, including alginates, fucoidan, and laminarin, can act as elicitors and enhance the activity of phenylalanine ammonia-lyase (PAL), a crucial enzyme in phenolic production [18,88,89]. Secondly, SWEs contain substantial concentrations of various solutes, including but not limited to osmolytes like mannitol, which may elicit mild and temporary osmotic stress in treated plants, effectively activating secondary metabolism as a defense response [13,44]. Finally, the applied C. barbata extracts are inherently rich in phenolic compounds [44], which can be taken up by the plants and/or influence endogenous signaling pathways, further contributing to the total phenolic content measured in the treated plants.

Lycopene, the red pigment that is responsible for the color of ripe tomatoes, is the immediate precursor of all cyclic carotenoids, including β-carotene, which is both an accessory photosynthetic pigment with potent antioxidant activity and typically the most important provitamin A in the diet [90]. Both pigments are considered key parameters for both sensory and nutritional quality of tomato. Applications of various SWEs have been associated with significantly higher lycopene concentrations in tomatoes [26,28,59]. In the presented research, only the water extract of C. barbata significantly enhanced the lycopene concentration (Figure 4A), whereas both the water and alkali extracts led to higher β-carotene concentration in tomato fruits (Figure 4B). Concurrent increases in lycopene and β-carotene contents in tomatoes in response to seaweed-based biostimulant application were also reported by Sidhu et al. [91].

Tomato is generally considered a good source of vitamin C, although the exact amount per serving depends on the type, cultivar, and growing conditions, as well as on processing and storage conditions. Since vitamin C concentration is an important nutritional quality attribute of tomato, several studies on the effects of seaweed-based biostimulants on tomato quality have reported vitamin C levels, albeit with inconsistent findings. Significant increases in the total ascorbic acid concentration of tomato were observed in response to SWE applications in many studies [26,28,30,35,92] but not in others [31,47]. The present study is in agreement with the former set of references, as all tested SWEs led to increased total vitamin C levels (Figure 5). Moreover, the concentration of reduced ascorbate increased to an even greater extent in response to the biostimulant applications, with the benefit provided by the water extract being particularly pronounced.

The yield-quality dilemma is a well-recognized phenomenon, addressing the fact that conditions favoring yield may not always align with those that enhance quality, and that there may be a trade-off between yield and quality [71]. Although SWEs used as biostimulants can often enhance the growth, yield, and quality of tomatoes concurrently [32,59], this is not always the case. For instance, Di Stasio et al. [31] reported that the nutritional quality of tomatoes could be enhanced by two Ascophyllum nodosum extracts, one of which did not affect the growth or yield, whereas the other reduced fruit fresh weight. In the current work, all tested C. barbata extracts resulted in higher yield while enhancing most quality parameters compared to the control. However, the water extract maximized various quality traits, including the Brix value, total antioxidant capacity as determined using the DPPH assay, lycopene concentration, and reduced vitamin C concentration in tomatoes (Table 2 and Figure 3, Figure 4 and Figure 5). Nevertheless, it lagged behind the alkali and acid extracts in terms of yield under our experimental conditions (Figure 1).

5. Conclusions

Cystoseira barbata, a brown seaweed with substantial biomass in shallow waters along the coasts of the Marmara Sea and the Black Sea, holds great potential as a local bioeconomy resource for the production of extracts with biostimulant properties. Different types of extracts, each with distinct compositions, can be applied to soil to simultaneously enhance tomato yield and quality. The evidence presented here indicates that the observed beneficial effects are not solely attributable to improvements in the mineral nutritional status of plants, although the tested SWEs can directly or indirectly contribute to the mineral nutrition of tomato. Further research is needed to elucidate the individual contributions of the bioactive constituents in the SWEs to the observed benefits; however, increasing evidence in the literature suggests that the complex interactions and possible synergies between these constituents are likely behind the biostimulant properties of SWEs rather than isolated components. Since the tested C. barbata extracts contained substantial amounts of minerals such as K and S, very much like other brown SWEs studied in the literature, it is important to test their effects on well-fed plants to focus on just the non-nutrition effects. While our results are promising, commercial formulations should be developed and tested under field conditions to validate the effects at larger scales and to determine optimal application rates for cost-effectiveness. However, the commercial farming of C. barbata needs to be established to enable sustainable harvesting, prevent collection from the wild, and thereby protect eulittoral habitats along with the biodiversity they support. Seaweed-based biostimulants offer a sustainable approach in horticulture, with the added benefit of improving the nutritional and nutraceutical profiles of healthy fruits and vegetables.