Enhancing growth and physiological performance of Theobroma cacao plantlets with biostimulant extracts from Acanthophora spicifera and Ulva sp

The establishment of Theobroma cacao L. plantlets in the field is a critical stage in the cultivation process for ensuring successful crop development. Perennial crops such as cacao may benefit from biostimulants that enhance early establishment and support long-term growth and productivity. The objective of this study was to investigate the effects of algal-based biostimulant extracts derived from Acanthophora spicifera and Ulva sp. on the growth and physiological performance of T. cacao plantlets. A grafted CCN-51 plantlets from approximately 2 to 3 months old before acclimatation period for two weeks, were used for transplantation and later treatment initiation. The extracts of seaweeds were obtained using water, 25% of aqueous ethanol and 50% aqueous ethanol were applied at concentrations of 2.5%, 5% and 7.5% (V/V). The treatments were applied for eight consecutive weeks, where plants physiological traits were evaluated at 15-day intervals. Treatment effects depended on the algal species, extraction medium, and mainly application concentration. Selected Ulva sp. extracts, particularly the 50% ethanol extract applied at a low concentration (doses at 2.5%), increased the Chlorophyll Content Index compared with other treatments up to 43.1 ± 1.19 CCI. Leaf number also varied among treatments across assay, whereas plant height and leaf length were not significantly affected. These findings indicate that selected seaweed extracts can modulate the early physiological performance of cacao plantlets, although their effects are dose and time dependent.

Cacao (Theobroma cacao L.) is a crop of major global economic and cultural importance, and international demand for cocoa products continues to increase. In Ecuador, cacao is one of the country’s leading non-oil agricultural exports and plays an important economic, agricultural, and gastronomic role. Ecuadorian cocoa beans are internationally recognized for their distinctive flavor and aroma and generate substantial export income, primarily as fermented and dried beans.

In 2024, approximately 612,453 ha were planted with cacao in Ecuador, producing 403,699 metric tons of dry cocoa beans (Instituto Nacional de Estadística y Censos (INEC), 2026). Cacao production is largely based on smallholder farming systems; approximately 87% of production units cultivate less than 5 ha. Many of these producers operate with limited access to financial resources, technical assistance, and agricultural inputs, which restricts the implementation of essential agronomic practices and contributes to yields below the crop’s potential (Vázquez‐deCastro et al., 2024).

Cacao production in Ecuador faces several interconnected challenges, including losses caused by pests and diseases, cadmium contamination of cocoa beans, inadequate plantation management, limited availability of productive genetic material adapted to local conditions, and insufficient production traceability. In addition, the mixing of Nacional and CCN-51 cocoa beans can reduce quality consistency and limit the differentiation of sensory profiles and value-added products. Addressing these challenges requires innovative and sustainable strategies that improve productivity and quality while supporting small-scale producers (García-Briones et al., 2021).

Plant biostimulants represent a promising complementary strategy for addressing some of these production limitations. Yakhin et al. (2017), defined a plant biostimulant as a formulated product of biological origin that improves plant productivity through the novel or emergent properties of its complex constituents. Unlike conventional fertilizers, biostimulants do not act primarily as direct sources of nutrients. Instead, they may modulate processes such as cell division, root and shoot elongation, photosynthesis, nutrient uptake, and stress responses. These properties are particularly relevant to cacao cultivation because biostimulants could improve plant establishment, nutrient-use efficiency, and resilience to environmental stress. Nevertheless, biostimulants should be considered complementary inputs and not replacements for balanced plant nutrition and appropriate agronomic management.

Among the different categories of plant biostimulants, seaweed-derived products have shown considerable potential. Seaweeds have been used to improve soil fertility and crop productivity for several decades (Craigie, 2011; Dutta et al., 2023; Khan et al., 2009). Since the development of liquid seaweed extracts in the 1950s (Michalak and Chojnacka, 2015), numerous commercial formulations have become available for agricultural and horticultural applications. Seaweed extracts contain diverse bioactive constituents, including polysaccharides, phenolic compounds, amino acids, minerals, vitamins, pigments, and hormone-like molecules. These constituents can influence physiological processes such as respiration, photosynthesis, nucleic acid metabolism, and ion uptake (Blunden et al., 1996; Craigie, 2011; Crouch and Van Staden, 1993; Khan et al., 2009; Rayorath et al., 2008).

Reported effects of seaweed-based biostimulants include improved root development, photosynthetic activity, nutrient acquisition, biomass accumulation, yield, and resistance to some pathogens and environmental stresses (Sharma et al., 2014). Their application may also improve nutrient-use efficiency and, under some production conditions, reduce dependence on mineral fertilizers (Sunarpi et al., 2010). However, these responses depend on the seaweed species, extraction method, application concentration, plant species, and growing conditions.

Although brown seaweeds have traditionally dominated the commercial biostimulant market, green macroalgae of the genus Ulva (Chlorophyta) are emerging as promising alternatives because of their abundance, rapid growth, and biochemical composition. The biostimulatory activity of Ulva spp. has been associated partly with ulvan, a water-soluble sulfated polysaccharide composed mainly of rhamnose, glucuronic acid, iduronic acid, and xylose (Lahaye and Robic, 2007). In addition to its structural function in the algal cell wall, ulvan can act as a bioactive elicitor that activates physiological and biochemical responses in plants. Ulva biomass also contains macro- and micronutrients, amino acids, vitamins, and other metabolites that may contribute to plant nutrition and metabolic regulation (Kidgell et al., 2019).

Extracts obtained from Ulva spp. have produced growth-promoting responses in several plant species. For example, the application of 0.07 mg mL −1 crude ulvan extract increased root length by 174%, root dry weight by 83%, shoot dry weight by 54%, and total plant dry weight by 62% in Arabidopsis thaliana under specific experimental conditions (Shefer et al., 2022). Seaweed applications have also modified substrate properties and promoted growth in horticultural crops such as tomato (Espinosa-Antón et al., 2023). These responses have been associated with improved nutrient uptake, chlorophyll accumulation, photosynthetic performance, and biomass production.

In addition to promoting growth, Ulva -based extracts may improve plant tolerance to environmental stress. In common bean, for example, Ulva lactuca extract improved growth under salinity and increased chlorophyll content, antioxidant enzyme activity, and the accumulation of osmoprotective compounds (Nhhala et al., 2025). Such responses may reduce oxidative damage and improve plant performance under unfavorable growing conditions. Nevertheless, the effects of seaweed extracts are species- and concentration-dependent and must be evaluated directly in each target crop (Rabhi et al., 2025).

Red seaweeds also represent valuable sources of biostimulant compounds. Among them, Acanthophora spicifera is a tropical red seaweed with potential agricultural applications. Species of Acanthophora contain chemically diverse constituents, including sulfated polysaccharides, phenolic compounds, pigments, minerals, and plant growth-related metabolites (Guillén et al., 2022). Extracts of A. spicifera applied to tomato and sweet pepper have been shown to modulate genes involved in photosynthesis, nutrient metabolism, hormonal signaling, plant growth, and defense responses. These changes were accompanied by improved growth and reduced severity of bacterial and fungal diseases (Ali et al., 2022, 2023). The occurrence of A. spicifera along the Ecuadorian coast provides an opportunity to investigate this locally available biomass as a potential source of agricultural biostimulants.

The biological activity of seaweed extracts depends strongly on the extraction procedure. Solvent polarity, concentration, temperature, extraction time, and biomass pretreatment determine which bioactive constituents are recovered and may consequently influence plant responses (Kadam et al., 2013). For A. spicifera, lyophilization has been reported as an effective drying method for preserving phenolic and flavonoid compounds, while extraction with 50% ethanol produced high antioxidant activity compared with other solvent systems (Valdez et al., 2023). Water and aqueous ethanol may recover different groups of compounds; therefore, comparing these extraction systems can help determine which fractions provide the strongest biostimulant response.

Despite the reported effects of Acanthophora and Ulva extracts in other plant species, their influence on the early growth and physiological performance of cacao remains poorly understood. In this study, extracts from the red seaweed A. spicifera and the green seaweed Ulva sp., collected from coastal areas of Ecuador, were prepared using water and two aqueous ethanol concentrations (25% and 50%). The extracts were evaluated at different application concentrations to determine their effects on the growth and physiological performance of T. cacao plantlets. We hypothesized that plant responses would depend on the algal species, extraction solvent, and application concentration, and that selected extract treatments would improve cacao plantlet performance compared with the untreated control. This study evaluates the potential of locally sourced seaweed biomass as a sustainable input for improving early cacao establishment.

Experimental overview

A repeated-measures experiment was conducted to evaluate the effects of foliar applications of seaweed extracts on the growth and physiological performance of Theobroma cacao L. plantlets. Extracts were obtained from the red seaweed Acanthophora spicifera and the green seaweed Ulva sp. Samples were extracted using three extraction media: 25% aqueous ethanol, 50% aqueous ethanol, and Milli-Q water. Each extract was evaluated at application concentrations of 2.5%, 5%, and 7.5% (v/v). Plants sprayed with filtered water served as controls.

Seaweed collection and storage

The red seaweed A. spicifera was manually collected from the intertidal zone of San Pedro, Santa Elena, Ecuador (1°57′19.5″ S, 80°43′46.5″ W), the green seaweed Ulva sp. was collected from Puerto El Morro, Guayas, Ecuador (2°36’0” S80°16’60” W). The collected biomass was thoroughly washed with tap water to remove sand, debris, epiphytes, and other surface contaminants. Larger materials, including shells and stones, were removed manually. The cleaned biomass was placed in sealed polyethylene bags and stored at −20 °C until extract preparation.

Preparation of seaweed extracts

Seaweed extracts were prepared at a biomass-to-solvent ratio of 1:10 (w/v), following Sunarpi et al. (2010) and Garai et al. (2021), with modifications. For A. spicifera, 200 g of thawed and cleaned biomass was homogenized with 2 L of the corresponding extraction medium: Milli-Q water, 25% aqueous ethanol, or 50% aqueous ethanol. The mixture was homogenized for 2 min using a blender and subsequently macerated for 24 h in darkness at 4 °C. After maceration, the extract was filtered through two layers of muslin cloth and centrifuged at 3,500 rpm for 15 min at 5 °C. The recovered supernatant was designated as the undiluted stock extract. For Ulva sp., 100 g of thawed and cleaned biomass was homogenized with 1 L of Milli-Q water, 25% aqueous ethanol, or 50% aqueous ethanol. Each mixture was macerated for 24 h in darkness at 4 °C and subsequently centrifuged at 3,000 rpm for 15 min. The supernatant was collected and designated as the undiluted stock extract.

Plant material and growing conditions

Grafted T. cacao plantlets of the CCN-51 clone were obtained from a commercial nursery located in La Troncal canton, Cañar, Ecuador. At the beginning of the acclimation period, the plants were approximately 2–3 months old. The plantlets were acclimated for two weeks under shade-house conditions and subsequently transplanted into containers (45x45cm) filled with a plant growth substrate. During the experiment, the plants were maintained at an average temperature of ~29 °C. Plants were irrigated daily, and no fertilizer was applied during the experiment. Plants were maintained for 10 days after transplanting before the first treatment application.

Experimental design and treatment application

The experimental factors were algal species (A. spicifera and Ulva sp.), extraction medium (25% aqueous ethanol, 50% aqueous ethanol, and water), application concentration (2.5%, 5%, and 7.5% v/v), and time. A filtered-water treatment was included as the control. Plants were randomly assigned to treatments according to a completely randomized design. Each treatment consisted of 30 individual plants, and each plant was considered an experimental unit. The experiment included a total of 450 plants. Immediately before application, the stock extracts were diluted with filtered water to final concentrations of 2.5%, 5%, or 7.5% (v/v). Control plants were sprayed with an equivalent volume of filtered water. Treatments were applied weekly for eight consecutive weeks using a handheld sprayer. Approximately 3.79 L of treatment solution was applied to each group of 45 plants, corresponding to approximately 84 mL plant −1 or until uniform leaf coverage was achieved. Applications were conducted on the same day each week. Plants from different treatments were spatially separated during application to prevent spray drift and cross-contamination. Measurements were recorded immediately before the first treatment application (week 0) and at weekly intervals thereafter for eight measurement occasions. Measurements were taken before the corresponding weekly application.

Growth and chlorophyll measurements

The evaluated variables were plant height, number of leaves, leaf length, and relative chlorophyll content. Plant height was measured from the substrate surface to the apical meristem using a measuring tape and expressed in centimeters. The number of 3 leaves on each plant was recorded. Leaf length was measured from the point of petiole insertion to the leaf apex and expressed in centimeters. Three fully expanded and similarly positioned leaves were selected and marked on each plant before the experiment. Leaf length and chlorophyll measurements were recorded repeatedly on these leaves, and their mean was used as the plant-level observation. When a marked leaf was lost or damaged, it was not replaced. Relative chlorophyll content was measured non-destructively using a CCM-200 Chlorophyll Content Meter (Opti-Sciences, Hudson, NH, USA). Measurements were taken at greenhouse and expressed as Chlorophyll Content Index (CCI). All measurements were recorded in the same day to reduce variation associated with time of day.

Statistical analysis

Because all response variables were measured repeatedly on the same experimental units (individual plants), observations are inherently non-independent. Standard one-way or factorial ANOVA, which assumes independence of residuals, is therefore inappropriate for this design. Accordingly, we employed a mixed-effects modeling framework that partitions the total variance into fixed effects (treatment, dose, and time) and a plant-level random effect to properly account for within-subject correlation across measurement occasions. The plant-level random intercept absorbs pre-existing differences in baseline physiology among individual plants, isolating the effects of treatment and dose from uncontrolled plant-to-plant variation.

Continuous physiological traits, total chlorophyll content and plant height, were analyzed with a Linear Mixed-Effects Model (LMM) fitted via restricted maximum likelihood (REML) using the lme4 package (Bates et al., 2015) in R (R Core Team, 2024). The full model included the three-way interaction among treatment group (Trt), dose (D), and time (t) as fixed effects, with an individual plant-level random intercept to capture baseline heterogeneity and within-plant correlations over time as shown in Equation 1 for chlorophyll content (CCI) and plant height:

where y i j k is the observed value of the response variable for treatment i, dose j, time point k, and plant l; μ is the overall intercept; Trt, Dj, and tk denote the main fixed effects; (T r t × D × t) i j k is the three-way interaction term; u l ∼ N (0, σ u 2) is the random intercept for plant l; and ϵ i j k l ∼ N (0, σ 2) is the residual error. Missing values were handled via the na. exclude option, which excludes incomplete records from model fitting while retaining those plants which are qualified in the different observation weeks, thereby preserving the full longitudinal structure.

Leaf count data are non-negative integers and typically exhibit overdispersion relative to a Poisson distribution, rendering a Gaussian LMM invalid for this response. The number of leaves per plant was therefore modeled with a Generalized Linear Mixed-Effects Model (GLMM) using a negative binomial error distribution (NB2 parameterization) and a log link function, fitted with the glmmTMB package (Equations 2, 3 model leaf count data): (Brooks et al., 2017).

Preliminary inspection of the data confirmed that the variance in leaf counts substantially exceeded the mean in most treatment-dose groups, supporting the use of the negative binomial distribution over the simpler Poisson model.

where β 0 is the intercept on the log scale, all fixed and random effects are as defined above, and θ is the negative binomial dispersion parameter estimated from the data.

By Day 45, four treatment-dose combinations produced complete defoliation (100% leaf loss), reducing within-group variance to zero for those groups and causing convergence failures in the repeated-measures models. To handle this structural discontinuity, the longitudinal mixed-effects analysis was restricted to the growth phase (Days 0–30), and Day 45 data from the surviving treatment groups were analyzed separately using cross-sectional one-way ANOVA (chlorophyll and height) and a negative binomial GLM (leaf counts); the defoliated groups were excluded from continuous trait analyses but represented as zero-leaf groups in the count model.

For the LMMs, fixed-effect significance was assessed via Type III sums of squares with Kenward–Roger degrees-of-freedom approximation using the lmerTest package (Kuznetsova et al., 2017), which provides reliable F -tests and p -values even in unbalanced designs. For the GLMM, Type III Wald χ 2 tests were obtained with the car package (Fox and Weisberg, 2019).

Significant higher-order interaction terms were decomposed through pairwise estimated marginal means (EMMs) computed with the emmeans package (Lenth, 2023). Comparisons were performed separately within each time point to characterize the temporal evolution of treatment effects. P -values were adjusted for multiple comparisons using Tukey’s honest significant difference (HSD) method, and treatment groups were labeled with compact letter displays (CLD) such that groups sharing a common letter do not differ significantly at α = 0.05. To explore the overall multivariate phenotypic response to the algae-based treatments across all measured traits, a Principal Component Analysis (PCA) was performed on z-score-standardized trait means per plant. Standardization was necessary to place all variables on a common scale prior to covariance decomposition. Biplots of the first two principal components were generated to visualize treatment-level clustering and the direction of trait contributions. All multivariate analyses and visualizations were conducted using the factoextra package (Kassambara and Mundt, 2020). This analysis was intended as exploratory and complementary to the hypothesis-driven mixed-effects models; it does not test statistical hypotheses but instead synthesizes the multivariate physiological response in a single visual display.

All figures were created with ggplot2 (Wickham, 2016) following Nature Publishing Group aesthetic guidelines. Statistical analyses and figures were produced in R (R Core Team, 2024). A two-tailed significance threshold of α = 0.05 was applied throughout.

Effect of algae-based biostimulants on chlorophyll content

The linear mixed-effects model detected a significant interaction between treatment-dose combination and time for Chlorophyll Content Index (CCI; F 26,804 = 2.094, p = 0.001; Supplementary Table 1). This interaction indicated that differences among treatment-dose combinations changed across measurement times. Significant effects of time (F 2,804 = 1110.581, p < 0.001) and treatment-dose combination (F 13,405 = 2.261, p = 0.007) were also detected. Because of the significant interaction, treatment comparisons were conducted separately within each measurement time. In biological terms, this means that the response of chlorophyll accumulation to each biostimulant formulation was not constant over time: some treatments promoted greater chlorophyll gains at specific evaluation points, while others showed negligible or delayed effects.

No detectable differences among treatment-dose combinations were detected at Day 0 (Tukey-adjusted p>0.05). whereas treatment-specific responses emerged during the subsequent evaluations (Figure 1A). By Day 45, the largest estimated CCI value was observed for 2.5% UEt50 (43.1 ± 1.19 CCI), while several low-to-moderate concentrations of Ulva sp. extracts produced relatively high CCI values during the later evaluations.

Effect of algae-based biostimulants on leaf proliferation

The negative binomial generalized linear mixed-effects model detected a significant interaction between treatment-dose combination and time (Wald χ² 26 = 46.31, p = 0.008). A significant effect of time was also detected (χ² 2 = 10.72, p = 0.005), whereas the overall effect of treatment-dose combination was not significant (χ² 13 = 12.28, p = 0.505). Because of the significant interaction, treatment comparisons were conducted separately within each measurement time. This implies that the capacity of each biostimulant formulation to stimulate leaf production was contingent upon the time at which it was measured during the experiment. Some treatments exhibited early gains that were not sustained, while others demonstrated effects that manifested only later.

Leaf number was similar among treatments at baseline, but leaf number differed significantly among treatments during subsequent evaluations (Tukey-adjusted p<0.05; Figure 1B). By Day 45, a leaf abscission was observed in four treatment combinations (Ulva sp. 25% ethanol at 2.5% dose (UEt25 2.5%), Ulva sp. 50% ethanol at 2.5% dose (UEt50 2.5%), Ulva sp. 50% ethanol at 7.5% dose (UEt50 7.5%), and A. spicifera 25% ethanol at 7.5% dose (AEt25 7.5%)),indicating a treatment specific phytotoxic response after prolonged exposure.

Effect of algae-based biostimulants on plant height and leaf length

Plant height increased over time in all treatment groups (F 2,779 = 15.341, p < 0.001). However, no significant overall effect of treatment-dose combination was detected (F 13,406 = 0.884, p = 0.570). The interaction between treatment-dose combination and time was also not significant (F 25,779 = 0.924, p = 0.572), providing no evidence that the temporal change in plant height differed among treatment-dose combinations. Leaf length did not differ significantly among treatment-dose combinations or across their interactions with time, leaf-length results are presented in (Supplementary Data Sheet 1; Figure 2B).

Multivariate phenotypic profiling

Temporal responses of cocoa plantlets to seaweed extract treatments over four weeks are presented (Figure 2) including plant height, leaf length, number of leaves, and chlorophyll content measured weekly over a four-week period under six treatments. To synthesize the overall physiological response across all measured traits, a Principal Component Analysis was performed on standardized mean trait values per plant (Figure 3). The biplot revealed a first principal component (Dim1, 46.4% of variance) driven mainly by chlorophyll content, along which Ulva sp. ethanol-based treatments, particularly UEt50 at low doses, clustered towards positive scores associated with superior photosynthetic and foliar performance. A. spicifera treatments at high ethanol concentrations and doses tended to project toward the opposing pole, consistent with their comparatively lower chlorophyll accumulation and the phytotoxic defoliation observed at Day 45. Plant height contributed minimally to the first two principal components, corroborating its statistical non-significance in the mixed-model analyses. Collectively, the multivariate profile confirms that Ulva sp. 50% ethanol extract at low-to-moderate doses (2.5–5.0%) provides the most beneficial and stable biostimulant effect on the overall plant physiological condition throughout the experimental period.

Algae-based biostimulants, including extracts from microalgae and seaweed, are increasingly recognized for their ability to enhance plant growth, stress tolerance, and physiological traits such as chlorophyll content. Numerous studies across diverse crops—including beans, tomatoes, wheat, broccoli, basil, potatoes, and others—demonstrate that algae-derived biostimulants can significantly increase leaf chlorophyll levels under both optimal and stress conditions (Aboulthana et al., 2026; Lam et al., 2025; Melo et al., 2024; Rachidi et al., 2020; Sosnowski et al., 2019; Munaro et al., 2024; Mystkowska, 2022; Shayen et al., 2023). Despite the extensive research conducted on various crops, scientific research on cacao is yet to be performed.

The findings of this study demonstrate that marine algae extracts can exert a distinct and time-dependent influence on the early development of plants, particularly in traits related to foliar growth and chlorophyll accumulation. Although plant height and leaf size remained unaffected, the marked changes in leaf number and chlorophyll content suggest that these extracts act first at a physiological level rather than through structural growth. This pattern is consistent with prior reports showing that seaweed-derived biostimulants tend to trigger metabolic and photosynthetic adjustments before morphological changes become visible (Craigie, 2011). Several seaweed extracts (Ascophyllum nodosum, Codium fragile, Kappaphycus alvarezii, Ulva lactuca, Galaxaura rugosa) have been applied to various crops, resulting in observed changes in chlorophyll levels and improvements in growth, including tomato, grapes, sugar cane, apple, mango and several cereals (Blunden et al., 1996; Li and Mattson, 2015; Morales-Sierra et al., 2023; Rossini et al., 2024; Sabir et al., 2014; Yang et al., 2023; Abdel-Sattar et al., 2024; Ali et al., 2019, 2021; Al-Musawi et al., 2025). Numerous seaweed species have been tested in previous studies, however, there are no reports, to this date, of the application of Acanthophora spicifera and Ulva sp. extracts used as biostimulants for cacao seedlings. Nonetheless, biostimulants derived from seaweed combined with other biomaterials have been applied on cacao plants for plant nursery support, disease control and enhance cocoa yield, there is no strong evidence regarding the effect of these biostimulants on chlorophyll content (Guamán Villa et al., 2022; Ramdani et al., 2025; Rodriguez Arrobo et al., 2023).

Chlorophyll content showed the clearest and most consistent physiological response, particularly under Ulva ethanol extracts. The fact that differences emerged only after the third week reinforces the idea that algae-derived compounds may progressively influence chlorophyll synthesis, nitrogen metabolism, and photosynthetic efficiency (Battacharyya et al., 2015). The enhanced performance observed with the low-dose Ulva 50% ethanol extracts is consistent with reported findings that Ulva species are rich in bioactive sulfated polysaccharides, betaines, and mineral cofactors known to enhance chlorophyll formation at moderate concentrations (Arioli et al., 2015). The possible presence of cytokinins and betaines that have been previously identified in Ulva species, and may be related to the increase in chlorophyll content on the leaves of the cacao plants (Ali et al., 2019; El Boukhari et al., 2021; Sridhar and Rengasamy, 2012; Whapham et al., 1993). Cytokinins represent a class of phytohormones that play a pivotal role in various physiological processes, including the promotion of cell division and the modulation of shoot and root growth, cell differentiation, and senescence. Cytokinin has been demonstrated to function as a modulator of chlorophyll synthesis and chloroplast function, regulating key steps in chlorophyll biosynthesis (Cortleven and Schmülling, 2015). In a recent study done by Choez-Guaranda et al. (2025), they measured the concentration of kinetin (cytokinin), indole acetic acid and gibberellic acid, from three different extraction methods of Ulva intestinalis (Supplementary Table 2). Although the Ulva species used in this study was not identified, it was collected near from the location of the study conducted by Choez and colleagues. Consequently, a correlation between both specimens can be established. The high concentration of cytokinin in the 50% ethanolic extracts from U. intestinalis supports the enhanced performance of the low-dose Ulva 50% ethanol extracts observed in our study. Conversely, the reduced effect of higher doses of A. spicifera ethanol extract reflects the dose-dependent nature of biostimulants, where excessive concentrations may inhibit photosynthetic pigment accumulation due to osmotic or metabolic overload (Calvo et al., 2014). This variable effect has also been observed in basil when sprayed with an extract from Kappaphycus alvarezii. The authors of the study suggested that the efficacy of biostimulants can vary depending on the crop species, resulting in divergent morphological and biochemical outcomes (Nunes et al., 2026). Auquilla’s (2022) study (Auquilla Yangua, 2022), conducted in Loja, Ecuador, examined the effect of an algae-based treatment on the physiological characteristics of different cacao cultivars (including CCN51), including chlorophyll content. The study reported no significant increment in chlorophyll content among the cultivars evaluated. This discrepancy may be attributed to the type and origin of the biostimulants applied. Auquilla applied a commercial product, ALGA-Q, which is a natural, organic biostimulant formulated from marine algae such as Ascophyllum nodosum and Sargassum (according to the manufacturer). Li and Mattson's (2015) study yielded similar results when they applied extracts from Ascophyllum nodosum on ‘Bravo White’ petunia and ‘Celebrity’ tomato plants. Following a weekly foliar application over a period of six weeks, a significant increase in chlorophyll was observed in petunia, while no such change was detected in the other species. Conversely, Ali et al. (2019) applied A. nodosum extract to a different tomato variety, Hybrid-61 tomato seedling, at 10-day intervals for 30 days. This treatment resulted in a significant increase in chlorophyll content, reaching nearly 15% more than the control groups. The outcomes showed by these studies imply that the effects of biostimulants are dependent on factors such as the origin of the biostimulants, their concentration, and the specific crop system employed. Additionally, the observed increase in chlorophyll in our study could also be attributed to the composition and concentration of algae, which differed significantly between experiments. ALGA-Q claims to be composed of 10% algae extract and other organic components, while Ulva sp. extract is only composed of 2.5%, 5% and 7.5% on alcoholic extractions. The present study is the first report of increased chlorophyll content in T. cacao L. seedling by the application of the Ulva sp. extracts.

Taken together, the results highlight that algae extracts exert their strongest influence on leaf proliferation and chlorophyll enhancement rather than on structural growth, at least during early weeks of development. The contrasting behaviors between species and solvents underscore the importance of refining extraction methods and dose ranges to achieve stable, long-term benefits. This study contributes to the growing body of evidence that seaweed-based biostimulants can enhance plant performance when applied under appropriate conditions, supporting their use as sustainable tools for improving crop growth and physiological vigor (du Jardin, 2015; Yakhin et al., 2017).

The different concentrations and seaweed extracts from A. spicifera and Ulva sp. induced in time physiological responses in CCN-51 cacao plantlets, primarily affecting chlorophyll content (estimated) and leaf growth, while plant height and leaf length show that they were not significantly affected. Low concentrations of Ulva sp. extracts obtained with 50% ethanol produced the strongest chlorophyll responses; however, complete leaf abscission observed in some treatment with dose combinations after prolonged exposure highlights, this result shows the need to optimize extract concentration and application frequency before their potential agronomic use.