Synergistic Biofertilization by Marine Streptomyces sp. and Leonardite Enhances Yield and Heatwave Resilience in Tomato Plants

Abstract

Humic substances and beneficial microorganisms are key biostimulants for sustainable agriculture and global food security in the face of climate change. Marine bacteria are emerging as a promising source of plant-beneficial microbes, tapping into a microbial diversity as immense as the oceans themselves. However, their potential, limitations, and mechanisms of action––especially in combination with other biostimulants––remain largely unexplored. In this study, we isolated the Streptomyces sp. LAP3 strain from the giant limpet Scutellastra mexicana. We evaluated the efficacy of the marine bacterium, applied alone or in combination with the humic product Leonardite hydrolate (L), in enhancing tomato performance under field conditions. Treatments included: (1) marine Streptomyces (MS), (2) Leonardite hydrolate (L), (3) both biostimulants (MS + L), and (4) a control (CTRL). We assessed growth, photosynthetic performance, antioxidant responses, and fruit yield and quality. Both biostimulants individually improved plant performance, but their combination had a significant synergistic effect, markedly boosting tomato productivity, thermotolerance, and resilience during a heatwave. Enhanced photosynthetic efficiency and antioxidant enzyme activity were associated with improved agronomic traits. These results highlight the potential of combining Streptomyces sp. LAP3 and Leonardite hydrolate as an eco-friendly strategy to increase crop productivity, strengthen stress resilience, promote sustainable agriculture, and reduce reliance on agrochemicals.

1. Introduction

Promoting sustainable agriculture and reducing agrochemical dependency are fundamental for the resilience of farming systems to climate change, as well as for meeting increasing consumer demands for environmentally sustainable, high-quality food products [1]. Plant-microorganism interactions offer promising solutions to achieve these goals [2]. Plant growth-promoting bacteria (PGPBs) are a diverse group of microorganisms that establish beneficial relationships with plants, enhancing their survival and productivity [3]. When applied as microbial biofertilizers to seeds, roots, or foliage, PGPBs improve nutrient use efficiency, growth, and yield [4,5]. PGPBs facilitate nutrient acquisition by fixing nitrogen (N₂) and solubilizing insoluble minerals (e.g., phosphorus (P), potassium (K), iron (Fe), and zinc (Zn)) through the production of organic acids, siderophores, transport proteins, and enzymes. Moreover, some of them can produce growth regulators or stress-responsive phytohormones to mitigate abiotic stressors (e.g., drought, high soil salinity, and extreme temperatures) through multiple mechanisms [5]. PGPBs also activate antioxidant enzymes and detoxification mechanisms, improving plant health under stressful conditions [6].

Similar to plant–microbe interactions in terrestrial environments, microalgae growth-promoting bacteria (MAGPBs) form symbiotic relationships with microalgae, which rely on them for growth and survival [7,8]. Notably, crops (e.g., tomato, tobacco) treated with marine bacteria have enhanced growth and stress resilience [9,10]. This heterologous symbiosis elicits stress tolerance mechanisms analogous to natural plant-microbe interactions [11]. Marine ecosystems thus represent an untapped reservoir of microbial symbionts with the potential to confer stress resilience and enhance crop yields [12,13,14]. The phylum Actinomycetota stands out for its potential in this context. Marine Streptomyces strains isolated from salt marshes have been shown to confer salt tolerance to tomato plants [15,16], while the deep-sea actinomycete Dermacoccus abyssi has also been shown to mitigate salt stress in the same species [17].

The present study evaluates a marine Streptomyces strain isolated from turf algae growing on the shells and surrounding habitat of the giant Mexican limpet, Scutellastra mexicana. This limpet species cultivates algal gardens in wave-exposed intertidal zones [18,19]. The stable algal epibiota on its shell and their algal gardens suggest complex ecological interactions, potentially mediated by microbial symbionts, making it a promising source for novel plant-beneficial bacteria.

Recent studies have shown that combining beneficial microorganisms with humic substances amplifies the positive effects of both, resulting in increased plant growth, productivity, and yield [20,21,22]. Humic substances, derived from decomposed organic matter, directly improve plant physiology and support rhizosphere microorganisms [21,23]. One such humic substance is Leonardite, a highly oxidized form of lignite that is particularly rich in humic and fulvic acids, known to enhance nutrient uptake, stimulate root development, and improve soil structure and microbial activity [24]. As a result, Leonardite-derived formulations are widely used in agriculture [25,26].

This research aims to investigate the plant growth-promoting properties of a bacterial strain isolated from the epibiont algae of S. mexicana, both alone and in combination with a humic substance derived from the hydrolysis of Leonardite. We hypothesized that combining microbial-based and organic residue-based biostimulants would have a synergistic effect on enhancing tomato plant performance under field conditions. To test this hypothesis, plant growth, photosynthetic performance, enzymatic and non-enzymatic antioxidant systems, as well as fruit yield and quality were compared across four treatments: (1) Marine Streptomyces alone (MS), (2) Leonardite hydrolate alone (L), (3) both biostimulants (MS + L), and (4) control (CTRL).

2. Materials and Methods

2.1. Isolation and Identification of Shell-Associated Bacteria in Scutellastra mexicana

The marine actinomycete strain was isolated from turf samples collected from the shell and associated turf gardens of Scutellastra mexicana. Samples were obtained from three individuals in the rocky intertidal zone of Coastecomates Beach, Bahía Navidad, Jalisco, Mexico (19°14′17” N, 104°47′32” W) in January 2023. Turf was removed using a chisel and hammer, placed in sterile bags, and stored at 4 °C during transport. No specimens were removed from the wild.

Actinomycete strains were isolated using the dry stamping technique [27]. The turf samples were washed with 70% ethanol to remove epiphytic microorganisms, rinsed three times with sterile seawater, and dried in open Petri dishes inside a laminar flow hood for 72 h. Dried samples were then ground in a mortar and the resulting powder was used to inoculate a solid isolation medium (agar (18.0 g/L, soluble starch (1.0 g/L), casamino acids (0.1 g/L), bactopeptone (0.2 g/L), yeast extract (0.4 g/L), cycloheximide (100 μg/mL), gentamicin (5 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA), and artificial seawater via cotton-rube impregnation. Inoculated plates were then incubated at 28 °C for four weeks. The bacterial colonies that grew separately were purified on A1 medium (antibiotic-free 10× isolation medium), which consisted of soluble starch (10.0 g/L), bactopeptone (2.0 g/L), yeast extract (4.0 g/L), agar (16.0 g/L), and artificial seawater [28]. Gram-positive strains with morphological characteristics typical of the phylum Actinomycetota were isolated from the samples and cryopreserved in a 20% glycerol solution at −70 °C for further studies.

Initial identification of the bacterial strains was based on colony morphology and their specific requirement for seawater to support growth, followed by confirmation through 16S rRNA gene sequencing [29,30]. The most recurrent and abundant colony type observed on the shells and turf algae associated with Scutellastra mexicana was selected for further analysis and designated as LAP3.

2.2. Molecular Identification of Strain LAP3

DNA was extracted from a LAP3 colony cultured in liquid A1 medium using the DNeasy^® Blood and Tissue Kit (Qiagen, Germantown, MD, USA). The 16S rRNA gene was subsequently amplified using primers FC27 and RC1492 with DreamTaq Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: initial denaturation at 95 °C for 15 min; 32 cycles of 95 °C for 1 min, 61 °C for 1 min, and 72 °C for 1 min; followed by a final extension at 72 °C for 7 min [30]. PCR products were purified using the Wizard^® SV Gel and PCR Clean-Up Kit (Promega, Madison, WI, USA), sequenced at UNAM Biotechnology Institute (Mexico City), and analyzed using BLAST (Basic Local Alignment Search Tool) available on the NCBI website (http://www.ncbi.nlm.nih.gov/). A neighbor-joining phylogenetic tree was constructed in MEGA with 1000 bootstrap replicates. The sequence was deposited in the GenBank database under accession number PQ555187 (http://www.ncbi.nlm.nih.gov/genbank, accessed on 10 August 2025).

2.3. In Vitro Evaluation of Plant Growth-Promoting Activity of LAP3 Strain

To assess the production of Auxin and Auxin-like molecules, the LAP3 bacterial strain was cultured in tryptic soy broth supplemented with 1 g/L tryptophan at 30 °C with shaking at 210 rpm for 4 days. Subsequently, 5 mL of the culture was centrifuged at 600 rpm for 20 min. A 1 mL aliquot of the supernatant was mixed with 1 mL of Salkowski reagent, incubated in the dark for 30 min, and the absorbance was measured at 530 nm [31]. The assay was performed in triplicate, and absorbance values were interpolated against a standard curve of Indoleacetic acid [32].

For evaluation of ACC deaminase activity, the LAP3 strain was inoculated on agar plates of Dworkin and Foster (DF) salt medium, where ammonium sulfate (NH₄)₂SO₄ was replaced with 1-aminocyclopropane-1-carboxylic acid as the sole nitrogen source and incubated at 30 °C for 4 days. Bacterial growth in this medium indicates a positive result [33]. Phosphate solubilization was assessed in modified Pikovskaya (PVK) agar medium using two different phosphate sources: aluminum phosphate (AlPO₄) and calcium phosphate (Ca)₃(PO₄)₂ [34]. Siderophore production was determined using chromeazurol S (CAS) agar medium, following the method described by Schwyn and Neilands [35], with modifications suggested by Louden et al. [36]. In both the phosphate solubilization and siderophore tests, the strain was spot-inoculated onto agar plates, and the appearance of a transparent halo around the colony indicated a positive result. Nitrogen fixation capacity was qualitatively evaluated by the ability of LAP3 to grow on a nitrogen-free solid medium (NFB) [37].

To assess the enzymatic capacity of LAP3 to degrade cellulose-derived polymers, the strain was also spot-inoculated onto agar plates containing specific cellulose-derived substrates as the main carbon source. Amylase activity was tested using starch as the carbon source [38]. Exoglucanase activity was evaluated using microcrystalline cellulose [39] and endoglucanase activity was assessed using carboxymethyl cellulose as the carbon source [40]. Plates were incubated for 3 days for the endo- and exoglucanase assays and 8 days for amylase activity. Positive enzymatic activity was inferred from observable changes in the substrate surrounding the bacterial colony, representing the substrate hydrolysis. After incubation, all plates were stained with iodine to confirm substrate hydrolysis.

2.4. Plant Material

Solanum lycopersicum seeds (cv. Rio Fuego; Kristen Seed, San Diego, CA, USA) were thoroughly washed with 10% (v/v) soap solution and rinsed with water. After washing, seeds were surface-sterilized with 3% sodium hypochlorite (NaOCl) for 2 min and rinsed three times with distilled water. Sterilized seeds were sown in germination trays filled with a 1:1 (v/v) mixture of peat moss and vermiculite, and maintained in a bioclimatic chamber for germination and growth during 20 days under a 16 h photoperiod at 30 °C followed by an 8 h dark period at 22 °C, to produce healthy tomato seedlings. The seedlings were then transplanted into bags containing 4 kg of a 2:1 (v/v) mixture of agricultural soil and tezontle (a porous volcanic rock) and placed outdoors for acclimatization and growth.

2.5. Biostimulant Preparation

Leonardite hydrolate was obtained through the chemical hydrolysis of Leonardite powder and potassium hydroxide, as described by García et al. [41]. The resulting hydrolate contained 7% humic substances (6% humic acid, 0.7% fulvic acid) and 7% organic matter (EC = 19 µS cm⁻¹; pH = 9.5). The marine Streptomyces sp. LAP3 inoculum was prepared from a culture of the isolated Streptomyces strain grown in 30 mL of A1 liquid medium at 28 °C under constant shaking (210 rpm) for 3 days, (OD_600nm) of 0.8–1.0.

2.6. Experimental Design

A controlled field experiment was conducted from March to June 2024 to evaluate the effect of biostimulant treatments on tomato (Solanum lycopersicum L.) performance under natural environmental conditions. Healthy tomato plants were grown in substrate-filled bags arranged in a completely randomized design (CRD) on open ground fully exposed to light, temperature, and humidity. During April and May, temperatures peaked between 38 and 40 °C, resulting in temporary drought and heat stress during the critical periods of flowering and fruit development (Figure S1).

The experimental design included four treatments: (1) control group (no biostimulant), (2) Leonardite hydrolate (L), applied diluted 1:100 in water, (3) marine Streptomyces strain LAP3 (MS), applied as a 1:10 dilution of culture; and (4) a combined treatment (MS + L) at the same respective dilutions. Treatments were applied via irrigation with 250 mL per plant, to six plants per treatment (24 experimental units in total) on days 23, 42, and 73 after sown. All plants received Steiner nutrients every week and water daily (Figure 1).

2.7. Plant Growth and Photosynthetic Performance Assessment in Response to Biostimulant Inoculatio

The effects of biostimulants on plant growth were evaluated 72 days after sowing at the end of the vegetative phase by measuring manually, using a measuring tape, the shoot length, leaf number, leaf width, and leaf height. Briefly, shoot length was measured from its base at the soil surface to the apical meristem. The total number of leaves per plant was counted. Leaf height was measured on the fourth fully expanded compound leaf from the apex, from the petiole base to the terminal leaflet tip. Leaf width was measured at the widest point of the same leaf, perpendicular to the midrib. Biomass responses to the treatments were assessed by determining the dry weights of shoots and roots.

Chlorophyll fluorescence was measured using a Junior-PAM fluorometer on leaves dark-adapted for 20 min. Rapid light curves were performed using actinic light intensities ranging from 5 to 1500 µmol photons m⁻²s⁻¹, following the manufacturer’s recommendations. PSII quantum efficiency (F_V/F_M) [32,33], maximum electron transport rate (ETR_MAX), and non-photochemical quenching (NPQ) were quantified to assess the impact of biostimulants on overall photosynthetic performance [42,43].

2.8. Evaluation of Antioxidant Enzyme Activity and Total Phenolic Content in Response to Biostimulant Inoculation

Total protein and enzyme extracts were prepared immediately before assaying antioxidant enzymes and total phenolics. Briefly, 1 g of leaf tissue was ground in ice-cold phosphate buffer (50 mM, pH 7.0) containing 1 mM EDTA, 0.1% (v/v) Triton X-100, and 1% (w/v) polyvinylpyrrolidone (PVP). Homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected for analysis. The total protein concentration was determined by infrared spectroscopy (Direct Detect), using bovine serum albumin (BSA) as the standard. For Ascorbate peroxidase (APX) assays, the extraction buffer was supplemented with 2 mM ascorbate.

Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured according to Beyer and Fridovich [44], based on the inhibition of nitroblue tetrazolium (NBT) photoreduction. The 2 mL reaction mixture comprised 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1% (w/v) Triton X-100, 13 mM methionine, 75 µM NBT, 2 µM riboflavin, and 50 µL of enzyme extract. Tubes were illuminated under a 20 W fluorescent lamp at 25 °C for 10 min; controls included a complete reaction without enzyme (maximum color development) and a non-illuminated reaction mixture (blank). One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT reduction by 50% at 560 nm.

Ascorbate peroxidase (APX; EC 1.11.1.11) activity was measured following the method of Nakano and Asada [45] using a 1 mL reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 15 µL of enzyme extract, and 2 mM H₂O₂ added to initiate the reaction. Enzyme activity was determined by monitoring the decrease in absorbance at 290 nm (ε = 2.8 mM⁻¹ cm⁻¹) corresponding to the oxidation of ascorbate.

Guaiacol peroxidase (GPX; EC 1.11.1.7) activity was assayed according to the method of [46] using a 3 mL reaction mixture consisting of 100 mM sodium phosphate buffer (pH 5.8), 7.2 mM guaiacol, 11.8 mM H₂O₂ (initiating the reaction), and 100 µL of enzyme extract. The reaction was monitored by measuring the increase in absorbance at 470 nm (ε = 26.6 mM⁻¹cm⁻¹) resulting from tetraguaiacol formation.

Total phenolics were quantified using the Folin–Ciocalteu method [47], with gallic acid as the standard. Leaf powder was extracted with 80% methanol (1:9, w/v) by sonication for 10 min, followed by centrifugation at 3500 rpm for 10 min. The supernatant (1 mL) was mixed with 250 µL of 2 N Folin–Ciocalteu reagent and 750 µL of 20% (w/v) Na₂CO₃ solution. After 1 h incubation at room temperature, absorbance was measured at 760 nm. Results were expressed as micrograms of gallic acid equivalents (GAE) per gram of dry weight.

2.9. Effect of Biostimulants on Tomato Yield and Quality During the Reproductive Stage

Mature tomatoes (fully ripened red fruits) were harvested on days 90, 96, and 105 after sowing. Yield, fruit size, weight, and the incidence of blossom-end rot lesions (BER; characterized by a dark basal lesion) were recorded for each of the six plants per treatment group.

Synergistic effects were assessed using Colby’s method, as described by Soller and Wedemeier [48], using Equations (1) and (2), where E_Colby represents the expected effect of two combined treatments, P denotes the percentage effect of each biostimulant relative to the control, and SF is the synergistic factor. A synergistic interaction is confirmed when SF > 1. If SF = 1, the effect is considered additive, while when SF < 1, the interaction is considered antagonistic. Synergy is considered to occur when the combined treatment performs significantly better than the sum of the individual effects.

$$E_{Colby} = P_1 + P_2 - {\displaystyle \frac{P_1{P}_2}{100}}$$

(1)

and

$$SF = {\displaystyle \frac{E_{measured}}{E_{Colby}}}$$

(2)

2.10. LAP3 Strain Root Colonization Visualization

Microbial colonization was visualized in the roots of tomato seedlings grown from LAP3 inoculated seeds on agar plates. Seeds were surface-sterilized using established protocols. Sterilized seeds were immersed for 30 min in 5 mL of LAP3 bacterial suspension (prepared as described in Section 2.1) or A1 growth medium (control) within 15 mL falcon tubes. Seeds were then transferred to plates containing Gamborg’s B-5 basal medium (Sigma-Aldrich, St. Louis, MO, USA) solidified with 0.6% (w/v) phytagel. Plates were incubated at 28 °C under a 16:8 h light/dark photoperiod (200 µmol photons m⁻²s⁻¹) for 8 days to promote root growth suitable for visualization. Roots were fixed with paraformaldehyde, bleached with NaOH/H₂O₂ solution, and stained with methyl blue dye [49], mounted on glass slides in water and covered with coverslips. Visualization was performed using a Primo Star compound light microscope (Carl Zeiss, Göttingen, Germany).

2.11. Statistical Analysis

For statistical analysis, data were tested for normality (Shapiro–Wilks test) and for homogeneity of variances (Levene’s test). When normality and homogeneity assumptions were met, data were analyzed using a one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test to compare means at a significance level of p < 0.05. If the assumptions were not satisfied, a one-way permutational ANOVA (PERMANOVA) based on Euclidean distance metrics, was performed, followed by Pairwise comparisons using 9999 permutations to assess differences among treatments. PERMANOVA was chosen due to its robustness against non-normality and heteroscedasticity. Analyses were conducted in Sigma Plot v. 12 (Systat Software, Inc., Chicago, IL, USA) and PRIMER + PERMANOVA software (Version 7.0.24; PRIMER-e, Plymouth, UK).

3. Results

3.1. Isolation, Identification, and Characterization of the Streptomyces sp. LAP3 Strain from S. mexicana

The LAP3 strain was isolated from shell and turf algae associated with S. mexicana. It was selected for further analysis for exhibiting the highest colonial growth on the isolation media. The strain exhibited a distinctive colonial morphology characterized by the formation of cream-colored aerial hyphae, which darkened to a deep brown color after six weeks of incubation. Additionally, the underside of the culture exhibited brown pigmentation. Gram staining confirmed the strain as Gram-positive, and microscopical examination of the hyphal structures was consistent with those observed in actinomycetes (Figure 2A,B).

Phylogenetic analysis based on 16S rRNA gene sequencing demonstrated that LAP3 belongs to the genus Streptomyces, within the family Streptomycetaceae and Phylum Actinomycetota. The strain exhibited 100% sequence identity with the reference strain Streptomyces cellulosae (GenBank Accession No. DQ442495) as well as with other Streptomyces strains isolated from diverse terrestrial and marine environments, including rhizosphere soil (GenBank Accession No. KY511722), lake sediments (GenBank Accession No. OQ271419), riverbank soil (GenBank Accession No. LC514431), marine sediments (Genbank Accession No. KX352811), and marine sponges (GenBank Accesion No. MN339888) (Figure 3). LAP3 exhibited noteworthy PGPB biochemical characteristics when evaluated in vitro. LAP3 tested positive for ACC-deaminase activity, phosphate solubilization, siderophore production, and N₂ fixation. Moreover, LAP3 showed the capacity to degrade and utilize cellulose and its derivatives as carbon sources (

Table 1. Biochemical plant growth-promoting (PGPB) traits of Streptomyces sp. LAP3.

Streptomyces PGP Properties	Activity
Auxin like molecules	5.7 ± 1.02 µg/mL
ACC deaminase	+
Phosphate solubilization	+
Siderophores	+
Nitrogen-fixing	+
Cellulolytic capacity	Hydrolysis index
Amylase	HI = 2.88 ± 0.05
Exoglucanase	HI = 4.46 ± 0.39
Endoglucanase	HI = 4.16 ± 0.32

The hydrolysis index (HI) was calculated using the formula HI = D/d, where D represents the diameter of the clear (hydrolyzed) zone and d the diameter of the bacterial growth zone. (+): activity detected by qualitative assay.

; Figure S2). None of the other isolated strains from S. mexicana samples exhibited as many biochemical characteristics of plant growth promotion as LAP3.

3.2. The Marine Bacterium Streptomyces LAP3 Colonizes Roots

To determine whether the selected strain could establish a symbiotic relationship with tomato plants, we first tested it in vitro in a sterile environment using Gamborg medium as a substrate. As shown in Figure 2C, clear colonization of the roots by Streptomyces was observed from the central part to the tip, confirming a close ecological interaction that could enable LAP3 to function as a plant growth-promoting bacterium.

3.3. LAP3 Enhances Biomass Accumulation in Tomato Plants

Significant variations in plant growth were observed in response to the biostimulant treatments, as shown in Figure 4. Compared to the control, plants treated with biostimulants L, MS, or MS + L exhibited a higher leaf number. Notably, the treatment (MS + L) resulted in a statistically significant 26.5% increase in leaf number relative to the control group. Leaf width increased by approximately 15% in all biostimulant-treated plants. Leaf height varied among treatments, with significantly larger leaves observed in MS and MS + L plants (Figure 4). Despite the differences found in leaf morphology, plant height (shoot size) remained similar.

In terms of biomass, the MS and MS + L treatments showed significant increases in shoot and root dry weight. The MS + L treatment showed the most significant effect, with shoot and root dry weights 60% and 78% higher, respectively, than the control. Overall, biomass accumulation was enhanced in plants treated with LAP3, whether applied alone or in combination with Leonardite hydrolate, primarily due to increased leaf mass and root system rather than shoot elongation.

3.4. Marine Streptomyces Inoculation Enhances Photoprotective Capacity and Induces an Increase in Chlorophyll Content

Photosynthetic performance was assessed to investigate the mechanism by which biostimulants promote growth. Maximum quantum efficiency (F_V/F_M) values in control plants (≈0.79) were not statistically different from those in L and MS treated plants (F_V/F_M ≈ 0.80 for both treatments; ANOVA, p > 0.5). However, the MS + L treatment showed a significantly higher quantum yield (F_V/F_M = 0.82 ± 0.003) than the other treatments, suggesting a photoprotective effect of the combined biostimulants on PSII (Figure 5A).

The effects of biostimulants on ETR_MAX differed notably from those observed for F_V/F_M. Average ETR_MAX values in plants treated with L (≈117.4), MS (≈124.7), and MS + L (≈118.6) were significantly higher than in control plants (ETR_MAX = 104.37 ± 4.74). The percentage increase in ETR_MAX compared to the control was 12.4% for L, 19.5% for MS, and 13.6% for MS + L, respectively.

The effect of biostimulants on NPQ followed a similar pattern to that observed for ETR_MAX. NPQ values in plants treated with L, MS, and MS + L increased by approximately 18%, 25.5%, and 24.8%, respectively, compared to control plants, suggesting that all biostimulant treatments enhanced photoprotective capacity via NPQ. Overall, ETR_MAX and NPQ were the most sensitive photosynthetic parameters to biostimulant application (Figure 5B,C). Notably, the combination of biostimulants has a significant effect on PSII photoprotection, as indicated by the higher F_V/F_M values of the MS + L treatment.

The total concentration of photosynthetic pigments chlorophyll a, chlorophyll b, and β-carotene was not significantly affected by the application of Leonardite alone. However, both chlorophyll a and b increased by over 26% and 21%, respectively, in response to LAP3 inoculation and the combined treatment with Leonardite. β-carotene concentration also rose significantly, showing increases of 24% and 26% in MS and MS + L treatments, respectively. These results indicate that pigment accumulation is a key physiological response to marine bacterial inoculation, whether applied alone or in combination with Leonardite (Figure 6).

3.5. Marine Streptomyces Inoculation Promotes Antioxidant Activity

Antioxidant enzymatic and non-enzymatic systems are key biochemical mechanisms that help prevent oxidative damage under stress conditions. To assess the impact of biostimulants on these protective systems, we evaluated the activity of antioxidant enzymes and the total phenolic content in tomato plants (Figure 7). The application of Leonardite alone did not significantly affect the activity of any antioxidant enzyme in tomato leaves. However, treatment with marine Streptomyces, either alone or in combination with Leonardite, significantly altered the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), as well as total phenol content, compared to control plants.

SOD activity increased by 95.63% and 61.06% in MS and MS + L treatments, respectively. Interestingly, APX activity decreased by approximately 50% in Leonardite-treated plants, while it increased by 33% and 39% in the MS and MS + L, respectively. A similar trend was observed for GPX activity: Leonardite alone had no effect, whereas MS and MS + L increased by 99.48% and 44.10%, respectively, relative to the control. Regarding total phenol content, only the MS + L treatment produced a significant increase, reaching 17% above control levels. No statistically significant changes were observed in plants treated with Leonardite or Streptomyces alone (Figure 7).

Among the evaluated treatments, MS and MS + L had the most notable effects on the antioxidant system of tomato plants, significantly promoting the activities of SOD, APX, GPX, as well as total phenolic content. Notably, treatment with LAP3 alone (MS) led to the most pronounced increases in enzyme activity, with SOD and GPX levels rising by over 95%, and APX increasing by 33%. The combined treatment MS + L also enhanced antioxidant responses, though to a slightly lesser extent. In contrast, Leonardite alone had no significant effect on SOD or GPX and even reduced APX activity by about 50%. These results highlight a strong impact of the microbial treatment, especially when applied alone, on enhancing both enzymatic and non-enzymatic antioxidant defenses (Figure 7), in contrast to other parameters measured in which the application of both biostimulants was synergistic.

3.6. Effect on Tomato Fruit Yield and Quality During Reproductive Stage

Following the analysis of growth, photosynthesis, antioxidant enzymes, and pigments at the end of the vegetative phase, the impact of biostimulant treatments on fruit yield and quality was evaluated. The results revealed significant differences in fruit number, size, and quality across treatments (Figure 8). Independent application of Leonardite and marine Streptomyces led to notable increases in average fruit yield, by up to 26% and 50%, respectively, compared to the non-inoculated control. Remarkably, the combined application of marine Streptomyces and Leonardite resulted in the highest fruit yield, with an increase of up to 77% over the control. According to the Colby test, this combination produced a synergistic effect on fruit production (SF = 1.2 > 1).

Interestingly, blossom-end rot (BER) was observed in some plants from both the Leonardite and control groups, with an average of 2.00 ± 0.63 and 0.83 ± 0.40 affected fruits per plant, respectively. In contrast, no BER symptoms were observed in plants treated with marine Streptomyces, either alone or in combination with Leonardite. Additionally, fruits from these treatments showed improved quality, characterized by a uniformly healthy and vibrant appearance. Overall, the combined treatment of marine Streptomyces and Leonardite proved most effective in enhancing plant growth, increasing fruit yield, and reducing the incidence of BER.

4. Discussion

This study reports the isolation, identification, and characterization of the marine Streptomyces sp. LAP3 strain from the shells and turf algae of the giant Mexican limpet (Scutellastra mexicana), with a focus on its plant growth-promoting (PGP) potential, both alone and in combination with a Leonardite-based hydrolate. The biofertilizer effects were evaluated through parameters such as plant growth, photosynthetic efficiency, antioxidant capacity, and fruit yield responses, showcasing a promising role of marine-derived microbes in enhancing plant productivity. Moreover, by combining marine-derived microbes with humic substances, more robust and sustainable agricultural systems can be developed that support long-term soil health.

Streptomyces sp. LAP3 was consistently the most abundant actinomycete isolated from both Scutellastra shell and garden turfs, which shared identical algal compositions, an indication of the limpet’s ecological strategy. Phylogenetic analysis placed LAP3 alongside Streptomyces cellulosae, a halotolerant strain adapted to saline environments. In vitro assays confirmed its ability to produce ACC deaminase, auxins, siderophores, and phosphate-solubilizing compounds, supporting its function as a metabolically versatile PGPB. Streptomyces genus is among the most prevalent in soils, where it plays a central role in organic matter degradation and nutrient cycling [50]. Many species within this genus function as endophytic symbionts with plant growth-promoting (PGP) attributes [51]. Given its metabolic versatility and ecological adaptability, LAP3 strain emerges as a promising plant growth-promoting rhizobacterium (PGPR) and microbial biofertilizer, with the potential to persist in soil. Notably, when evaluated in combination with a Leonardite-based hydrolate, its effectiveness was not only maintained but enhanced––particularly in terms of fruit yield––suggesting a synergistic interaction between the two components. While humic substances like Leonardite hydrolate are widely trusted among growers, awareness of the benefits of microbial biofertilizers remains limited. Combining Leonardite with beneficial microbes in experimental trials could therefore help bridge this knowledge gap and promote broader adoption of microbial technologies in agriculture.

Some of the findings of this study were unexpected. No significant differences were observed in physiological or biochemical indicators between the control (CTRL) and Leonardite (L) treatments, contrary to previous reports describing Leonardite as a plant growth-promoting substance that enhances physiological and biochemical functions [52]. Leonardite and other humic substances are generally known to improve soil organic matter, stimulate microbial activity, and enhance soil structure, thereby facilitating the gradual release of key nutrients such as nitrogen (N), phosphorus (P), potassium (K), iron (Fe), calcium (Ca), and zinc (Zn) [53]. However, nutrient release from humic substances is highly variable, depending on their physicochemical characteristics, abiotic conditions (e.g., pH, temperature, moisture, clay content), and the native soil microbiota responsible for their decomposition into bioactive compounds. In our study, the limited response to Leonardite may have been due to insufficient microbial activity in the experimental soil, which likely impeded the breakdown of Leonardite hydrolate into plant-available forms at effective concentrations.

In contrast, plants treated with MS and MS + L exhibited significant differences in most physiological and biochemical markers compared to CTRL and L-treated plants, explaining their higher growth and fruit yield. However, no significant differences were detected between MS and MS + L groups for most parameters, with the exception of total phenolics and fixed biomass (shoot and root dry weight). MS + L plants showed elevated values in these metrics, along with a numerically higher (though statistically non-significant) quantum efficiency. The increased fixed biomass and fruit yield in MS + L may be attributed to marginally reduced PSII damage, likely due to enhanced phenolic antioxidant capacity. Since PSII repair under stress is energy-intensive, even a slight improvement in PSII efficiency (indicated by a modest increase in F_V/F_M) could significantly influence biomass accumulation and yield.

The presence of Streptomyces strain LAP3—a potential decomposer of humic substances—may explain the synergistic enhancement of plant growth and tomato yield in the MS + L treatment. Members of the Streptomyces genus are known to decompose organic matter into bioactive molecules and nutrients [54]. For instance, Streptomyces rochei can break down humic substances into small aromatic and aliphatic compounds, amplifying the plant growth-promoting (PGP) effect [55]. Additionally, humic derivatives stimulate the growth of soil and symbiotic bacteria [56], increasing organic acid secretion in the rhizosphere and improving mineral nutrient uptake (e.g., Ca and Fe), as has been shown in tomato roots [57].

Calcium deficiency is a key factor in blossom-end rot (BER) development in tomatoes. While Leonardite hydrolate alone did not reduce BER incidence, both MS and MS + L treatments provided protection. This suggests that Streptomyces may enhance Ca absorption from both soil and Leonardite hydrolate. Furthermore, Streptomyces spp. are prolific producers of antimicrobial metabolites [58], which may have suppressed fungal colonization of necrotic tissue in BER lesions. Previous studies report Streptomyces cellulosae as an effective biocontrol agent against fungal pathogens [59,60], supporting the hypothesis that Streptomyces-derived metabolites could contribute to BER mitigation.

The experimental results presented here underscore the potential of marine Streptomyces, particularly when combined with Leonardite hydrolate, as an effective microbial biofertilizer. While individual applications of Leonardite hydrolate or marine Streptomyces did not significantly alter plant height, the combined treatment led to an increase in leaf number and fruit yield relative to control plants. It is likely that the synergistic enhancement of vegetative growth is linked to a greater photosynthetic surface area as well as increased root system.

Plants rely on a resilient photosynthetic apparatus to support growth and productivity, particularly under stressful conditions. Heatwaves and dry spells—like those observed during this study (Figure S1) can significantly reduce crop productivity. To evaluate the impact of biofertilizer treatments under these conditions, key indicators of photosynthetic performance were analyzed. In healthy, non-stressed plants, the maximum quantum efficiency of PSII (F_V/F_M) typically hovers around 0.83 [61], whereas lower values suggest photoinhibition or damage to PSII [62]. Such reductions are linked to diminished productivity, as evidenced by decreased maximum electron transport rates (ETR_MAX) [63].

In this work, only plants treated with both marine Streptomyces and Leonardite showed an increase in F_V/F_M, indicating that their combination effectively protects PSII from oxidative stress and prevents F_V/F_M decline. It is possible that in this scenario NPQ activation by biofertilizer treatments, enhances the capacity to dissipate excess light energy as heat, reducing oxidative pressure in PSII and maintaining the high quantum efficiency (F_V/F_M) observed.

To cope with oxidative stress often intensified by adverse environmental conditions plants primarily rely on the activation of antioxidant enzymes [64]. Microbial interaction is thought to maintain high F_V/F_M, by stimulating antioxidant enzyme activity [65]. In this study, the application of marine Streptomyces alone or combined with Leonardite consistently enhanced the activities of SOD, APX, and GPX, indicating a robust stimulation of the enzymatic antioxidant system for relieving oxidative pressure. Microbial activation of enzymatic antioxidant systems may also explain the higher concentration of photosynthetic pigments, and total carotene. As reducing the oxidative pressure prevents chlorophyll bleaching, this likely explains a higher concentration of these pigments in microbial-treated plants. As plants treated solely with Leonardite hydrolate had no significant effect on SOD or GPX activities and even exhibited a reduction in APX activity, suggesting that bacterial inoculation was the primary driver of the observed antioxidant boost.

Previous research has shown that biostimulants can upregulate antioxidant defenses [66], particularly through the role of SOD in converting superoxide radicals (O₂•⁻) into less harmful molecules such as H₂O₂ [67]. In a work on Vitis vinifera cv. Antão Vaz, plants inoculated with a marine bacterial consortium (including Aeromonas aquariorum SDT13, Bacillus methylotrophicus SMT38, and B. aryabhattai SMT48) significantly enhanced SOD levels [68] under thermal stress. Similar SOD activation has been reported in response to terrestrial bacteria like Micrococcus luteus [69] and Bacillus subtilis [70], as well as non-bacterial biostimulants used under phosphorus-deficient and water-limited conditions [71].

The promotion of APX and GPX activities by biostimulants was less consistent than that observed for SOD. Both APX and GPX scavenge H₂O₂, with APX using ascorbate as an electron donor [72], and GPX relying on guaiacol [73]. Treatments with marine Streptomyces inoculation increased APX and GPX activities in tomato plants, whereas Leonardite alone did not enhance GPX activity and even suppressed APX activity. Previous works assessing the effect of biostimulants on APX and GPX activity have found contrasting results [68,71,74]. Carreiras et al. [68] observed higher GPX activity in control plants of Vitis vinifera compared to those treated with rhizobacterial consortia. Kaya et al. [71] showed that Leonardite and sulfur soil addition in Zea mays L. (cv. DKC-5789) reduced peroxidase activity under phosphorus deficiency and water stress, while Yıldıztekin et al. [74] found that the concentration of humic acids from Leonardite influences the activity of the peroxidase enzyme in plants under salt stress. Interestingly, APX activity was lower in plants treated with Leonardite alone than in control plants. The potential interactions between non-microbial biostimulants and H₂O₂ signaling pathways remain largely unclear. However, given the role of H₂O₂ in boosting plant stress tolerance [75,76], further investigation is needed to clarify its link to reduced APX as well as to determine the optimal application rates for non-microbial biostimulants.

Marine microorganisms have been shown to decrease free radical content in plants subjected to abiotic stress. Inoculation of tomato plants with Dermacoccus, reduced H₂O₂ levels under salt stress [77]. Similarly, Streptomyces sp. C1-2 [78] and Planococcus maritimus [79] have revealed the ability to reduce DPPH radicals. These findings highlight a promising role for microbial biofertilizers in enhancing plant tolerance to abiotic stress and support their use to mitigate the adverse effects of climate change on crop production [80].

Using biofertilizers can promote ecological sustainability and improve food quality while mitigating abiotic stress caused by climate change [23,81]. The positive effects of marine Streptomyces offer a promising alternative to reduce reliance on synthetic fertilizers and pesticides, enhancing crop yield and resilience [82]. Combining bacterial inoculation with Leonardite application demonstrated synergistic benefits. It increased fruit yield, reduced blossom end rot, and boosted photosynthetic efficiency and antioxidant activity. Since direct effects of the Leonardite hydrolate alone in tomato plants were scarce compared to positive effects of the marine bacteria or when both biostimulants were combined, it is possible that the molecular size or the complexity of the humic substances in the Leonardite hindered their absorption and thus their plant benefits that have been largely demonstrated in previous works [83,84,85,86]. It has been shown that reducing size and complexity of humic substances by microbial biodegradation enhances their positive effects on plants [55]. Specifically in this work, the combination of Leonardite hydrolate and the marine bacteria that resulted in the higher tomato yield could be related to the biodegradation of Leonardite hydrolate by the marine Streptomyces, releasing smaller and more bioactive humic molecules. Future research efforts will clarify the signaling and metabolic pathways affected by both types of biostimulants, as well as their interactions with soil microbiota. Finally, assessing the scalability and economic feasibility of producing these biofertilizers for broader agricultural use is still necessary.

5. Conclusions

An urgent shift towards sustainable practices is now more critical than ever, as the overuse of fertilizers and agrochemicals has impacted farm soils and surrounding ecosystems, with broader knock-on effects. Here, we have demonstrated that microbial and humic-based biostimulants, particularly when used in combination, offer a promising approach to increasing crop productivity and quality while enhancing plant resilience to environmental stress. In this work, Leonardite hydrolate alone did not significantly influence physiological or biochemical parameters of tomato plants, possibly due to insufficient microbial decomposition. However, its combination with the marine Streptomyces LAP3 (MS + L) enhanced phenolic content, increased shoot and root biomass, and enhanced fruit yield, suggesting a synergistic interaction. Nutrient mobilization aided by Streptomyces may be one of the causes. In addition, MS and MS + L treatments effectively reduced blossom-end rot (BER), indicating improved calcium uptake and potential antifungal effects from Streptomyces LAP3. These findings highlight the context-dependent efficacy of Leonardite, emphasizing the importance of microbial activity in unlocking its growth-promoting potential. Further research should optimize the application strategies to maximize agricultural benefits. Moreover, the LAP3 Streptomyces strain isolated in this study holds a significant potential for biofertilizer development in combination with Leonardite hydrolate. Importantly, this study underscores the value of exploring marine ecological niches to uncover new beneficial microorganisms for agriculture.