Enhanced Salt Stress Tolerance in Maize Using Biostimulant and Biosurfactant Applications

Abstract

This research was conducted to investigate effects of biostimulants containing plant growth-promoting rhizobacteria and enriched biosurfactants, which were tested individually and in combination (biostimulant + enriched biosurfactant) on plant growth parameters, physiological and biochemical properties of maize seedlings under different salinity conditions (0, 100, 200 mM NaCl). In the experiment, biostimulant (B: 0.3 g/L), enriched biosurfactant (E-S: 3 mL/L), and their combination (B + E-S) were applied by foliar spray at each NaCl level. Salt stress negatively affected the growth and physiological traits of maize seedlings, while biostimulant and enriched biosurfactant improved these parameters. Under severe salinity stress (200 mM NaCl), the biostimulant, enriched biosurfactant, and their combined application markedly mitigated oxidative and osmotic damage. Compared with the untreated 200 mM NaCl group, these treatments (B, ES, B + ES) reduced proline accumulation by 65%, 52%, and 70%; hydrogen peroxide (H₂O₂) level by 53%, 39%, and 58%; and malondialdehyde (MDA) content by 72%, 50%, and 73%, respectively. These reductions indicate a substantial decrease in oxidative stress and membrane lipid peroxidation. In conclusion, biostimulant and enriched biosurfactant applications may be a promising approach to reduce the negative effects of salinity stress on maize.

1. Introduction

Salinity is considered to be among the abiotic stresses that affect all croplands, especially arid and semiarid areas, and has the most negative impact on plant growth and development [1]. Human-induced factors such as improper irrigation systems and over fertilization are among the main reasons that increase salinity. In particular, climate change has led to increases in temperature and decreases in precipitation, thus reducing water quality and increasing the salt content of groundwater [2]. Approximately 20% and 33% of total cultivated and irrigated agricultural lands are saline soils, respectively [3]. Soil salinization is a global threat affecting 1100 Mha of soil, representing approximately 7% of the world’s land surface [4]. Salt toxicity has effects such as tissue, root and shoot death; leaf shedding; decreased stomatal conductance; disruption of cellular metabolism; and decreased photosynthetic activity in plants [5]. Under salt stress, stomatal conductance decreases mainly due to osmotic-driven reductions in guard cell turgor, ionic toxicity (particularly Na⁺ and Cl⁻ accumulation), and ABA-mediated signalling, all of which promote stomatal closure and restrict gas exchange [6]. In addition to reducing agricultural production of most crops, it also disrupts the physicochemical properties of the soil and the ecological balance of the region [7].

Plant response to salinity stress is one of the most important issues in agricultural and environmental sciences. This response varies between sensitive, moderately sensitive, moderately tolerant and tolerant depending on the plant species, stress level and environmental conditions [8,9]. The tolerance level of plants to salinity stress affects all physiological and metabolic changes, starting from germination, including the photosynthesis process and other biosynthetic processes [10,11].

Different approaches have been studied and developed to increase salinity tolerance. The use of salt-tolerant genotypes, application of different irrigation techniques [3,12], and use of organic biostimulants such as bio-organic fertilizers [13,14], proline [15], biochar [16], compost [17], humic substances [18] and ascorbic acid [19] are the leading practices that increase crop performance in saline soils.

Biostimulants facilitate the absorption of nutrients, promote growth, and increase stress resistance [20,21]. It has been proven that the use of biostimulants can increase resistance to abiotic stresses (drought, high temperatures, salinity, heavy metals, etc.), increase yield, or improve quality traits to maintain good yield and harvest quality under these conditions [22]. In recent years, there has been an increase in the use of biostimulants to increase the tolerance of plants to salt stress [15]. Although several studies report the beneficial effects of biostimulants on maize under salt stress [1,23], the combined use of biostimulants and biosurfactants remains largely unexplored. Different originated biostimulants have been reported to mitigate effects of abiotic and biotic stresses [24,25].

Recently, surfactants have been studied regarding their possible use in agriculture. They are amphiphilic molecules with wide application in the food, pharmaceutical, agricultural and detergent industries [26]. They can be divided into two basic groups: synthetic surfactants and biosurfactants. Biosurfactants have special advantages over synthetic surfactants, such as higher biodegradability, lower toxicity, higher foaming capacity, and higher activity at extreme temperatures, pH and salinity [27]. Studies have shown that biosurfactant-producing bacteria can be used to accelerate the remediation of organic and metal-contaminated sites [28]. These properties have led to the study of biosurfactants in recent years as a promising option to replace a number of synthetic surfactants [29]. However, very limited research has examined whether enriched biosurfactants can directly influence plant physiological responses under salt stress, and no studies have evaluated their interaction with biostimulants in maize. This represents a significant gap in the current understanding of plant–surfactant–biostimulant interactions.

Maize, a native plant of the American continent, is currently considered the third most important cereal with the highest nutritional value in the world [30]. There is a cultivation area of 196,982 thousand hectares in the world, mostly in the temperate zone [31]. Maize yield is expected to double by 2050 with rapid population growth, changes in human nutrition, and strong demand for animal products [32]. However, abiotic stresses such as drought and salinity caused by global climate change and water scarcity threaten future maize supply [33]. This is because approximately 70% of maize cultivation is carried out in arid and semiarid regions where soil salinization occurs [34]. Maize is known to be moderately sensitive to salt stress [35]. Salt levels reaching 100 mmol NaCl reduce maize growth by approximately 50% at the seedling stage [36]. Salinity has a negative influence on seed germination, germination rate, seedling emergence, and the vigour index of maize [37]. Similarly, earlier studies proved that salinity stress negatively affected ion homeostasis, water uptake, photosynthetic activity, yield and biomass of maize, increased reactive oxygen species (ROS) level, proline, H₂O₂ and MDA [38,39,40]. Maize plants employ several adaptive mechanisms to survive in saline soil conditions. The major strategies include (i) maintaining ion homeostasis and compartmentalization to exclude toxic ions; (ii) synthesizing osmo-protectants and compatible solutes to support antioxidant defense; (iii) activating key enzymes and producing antioxidant compounds; (iv) regulating hormonal pathways to adjust hormonal balance; (v) inducing apoplastic acidification; (vi) synthesizing polyamines; and (vii) generating nitric oxide (NO) [41].

Studies have shown that the use of biostimulants reduces the negative impacts caused by salt stress in maize. D’Amato and Del Buono [1] pointed out that the plant markedly alleviated the adverse effects of salt stress on shoot length, fresh weight, and chlorophyll and carotenoid levels while also reducing Na⁺ uptake by the plants. In terms of oxidative status, biostimulant-treated seedlings had lower concentrations of H₂O₂ and MDA. These findings can be explained by the biostimulant’s ability to limit Na⁺ accumulation, which in turn led to reduced levels of H₂O₂ and MDA and overall antioxidant activity.

New technologies are being developed worldwide to enhance plant resilience in regions influenced by salinity. Surfactants, which act as wetting agents, are being used now to reduce the surface tension of water and enable it to spread more effectively through the soil. Earlier studies showed that surfactants ameliorated the negative effects of salinity stress on plant growth and yield of some plants [42,43]. Ali et al. [44] showed that a PGPR (plant growth-promoting rhizobacteria) with genes that produce biosurfactants increased the resistance of maize plants under elevated salt concentrations.

However, little research has been performed on the combined use of biostimulants and biosurfactants under salinity stress. While biostimulants are mainly studied for improving plant growth and stress tolerance, biosurfactants are mostly examined for soil cleanup, leaving their possible interaction in plant stress physiology largely unexplored. While biostimulants are mainly studied for improving plant growth and stress tolerance, biosurfactants are mostly examined for soil clean-up, leaving their possible interaction in plant stress physiology largely unexplored. Therefore, understanding whether these two inputs can work synergistically to mitigate salt stress represents an important and underexplored research question. The current study was designed based on the hypothesis that biostimulants and enriched biosurfactant applications, individually or in combination, may alleviate the detrimental effects of salinity by improving physiological and biochemical responses in maize. Accordingly, the objectives were (i) to evaluate the separate and combined effects of biostimulant and enriched biosurfactant treatments under 100 and 200 mM NaCl conditions; (ii) to determine their influence on growth, physiological and biochemical parameters; and (iii) to assess whether combined applications offer superior mitigation compared to individual treatments.

2. Materials and Methods

The study was carried out as a pot study in greenhouse conditions in Atatürk University in Turkey between September 2024 and December 2024, and maize (Zea mays L. cv. Dekalp 6442) was used as plant material. Dekalp 6442 variety, which was obtained from Bayer Crop Science Türkiye, is an early silage maize seed suitable for Erzurum and similar ecologies (short vegetation period), reaching harvest maturity in 90 days. Relative humidity was 60–70% throughout the study, and temperature was 25 ± 2 °C during the day and 18 ± 2 °C at night. Seeds were planted in 2 L pots filled with a mixture of garden soil, peat, and sand, and 7 seeds were planted in each pot at a depth of 2–3 cm. After emergence, seedlings were thinned to four uniform plants per pot. The research was conducted in a completely randomized design with three replications, and six pots (Karasu Plastik LLI. Istanbul, Türkiye) were used in each replication. Applications were started on plants that reached the seedling stage (V1). The biostimulant and surfactants (Kiana Agriculture B.V. (Singel 542, 1017 AZ Amsterdam, The Netherlands)) used in the study are given in

Table 1. Composition of the biostimulant (B) and enriched surfactant (E-S) formulations used in the study.

Product	Component Category	Components	Quantitative Composition
Biostimulant (B)	Microorganisms	Paenibacillus polymyxa, Pseudomonas fluorescens, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Azotobacter chroococcum, Azospirillum brasilense	1 × 109 cfu/mL (total viable cell concentration)
	Enzymes	Protease,	Protease 300 U/g,
		Xylanase,	Xylanase 1700 U/g,
		α-amylase,	α-amylase 1750 U/g,
		Cellulase + Hemicellulase,	Cellulase + Hemicellulase 200 U/g,
		Phytase	Phytase 500 U/g
	Organic Acids	Fulvic acid	100 ppm
	Hormones (microbial origin)	Auxins (IAA), Cytokinins, Gibberellic acid	Produced in situ by microbial metabolism
Enriched-Surfactant (E-S)	Surfactant base	Trisiloxane alkoxylate (trioksisilan)	0.2% (v/v)
	Enzymes	Protease,	Protease 300 U/g,
		Lipase,	Lipase 150 U/g,
		Cellulase + Hemicellulase	Cellulase + Hemicellulase 200 U/g
	Organic Acids	Fulvic acid	100 ppm
	Microorganisms	Same bacterial consortium as B	1 × 109 cfu/mL

. The experimental design consisted of four treatments applied at three salinity levels (0, 100, and 200 mM NaCl). The treatments included (i) a control treatment without any biostimulant or biosurfactant, (ii) biostimulant applied alone, (iii) biosurfactant applied alone, and (iv) the combined application of biostimulant and biosurfactant. All treatments were evaluated at each salinity level to determine the individual and combined effects of these applications on maize seedlings (Figure S1). Solutions of biostimulant (B) and enriched surfactants (E-S) were prepared at concentrations of (B) 0.3 g/L, (E-S) 3 mL/L. These doses were selected based on preliminary trials and previous studies indicating that they are effective concentrations for enhancing plant physiological responses without causing phytotoxicity. The applications were started 7 days after emergence, and three foliar applications were made with seven-day intervals. To maintain normal growth, the seedlings were irrigated with 1/2 Hoagland solution [45] every three days. A reduced-strength nutrient solution was used to avoid nutrient-driven interference and to maintain a low, stable osmotic background, thereby enabling a more precise evaluation of salinity-induced ionic disruptions (Na⁺/K⁺ imbalance and Cl⁻ accumulation).

To establish appropriate irrigation, a soil moisture meter (WET Sensor, Delta-T Devices, Cambridge, UK) was used to ensure pots were adequately watered. Salinity treatments consisted of 0 mM NaCl (control) and 100 mM NaCl. To minimize the impact of abrupt salinity stress, the NaCl concentration was gradually increased over 2–4 irrigation cycles until reaching the desired level. The experiment ended 35 days after plant emergence. Each measurement was repeated three times, and samples were taken from the middle leaves.

To understand how salinity and treatments affected maize seedlings, we measured several key parameters such as plant height (PH); fresh and dry weights of both the plant (PFW, PDW) and root (RFW, RDW); leaf area (LA); chlorophyll, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), proline, hormone (ABA, IAA, GA) and plant nutrient element content; and antioxidant enzyme activities (CAT, POD, SOD).

All measurements were conducted using established scientific methods.

2.1. Plant Height, Fresh Weight, and Dry Weight Measurements

Plant height was measured from the soil surface to the tip of the youngest fully expanded leaf using a digital ruler. For biomass measurements, plants were carefully harvested and separated into shoots and roots. Fresh weight (PFW, RFW) was recorded immediately after harvesting using a precision analytical balance. For dry weight determination (PDW, RDW), plant tissues were placed in paper envelopes and dried in a forced-air oven at 70 °C for 72 h until a constant weight was achieved. Dry weights were then measured using the same analytical balance. All measurements were performed on three biological replicates per treatment.

2.2. Leaf Area

The leaf area was determined by an area meter (LI-3100, LICOR, Lincoln, NE 68504 USA).

The biostimulant (B) and enriched surfactant (E-S) used in this study are not commercial products; they were formulated under laboratory conditions exclusively for research purposes. All enzymes included in the formulations were naturally produced and purified by microorganisms and supplied by Kiana Agriculture B.V. (Singel 542, 1017 AZ Amsterdam, The Netherlands). Their activities are quantitatively provided in Table 1 (protease 300 U/g, xylanase 1700 U/g, α-amylase 1750 U/g, cellulase + hemicellulase 200 U/g, phytase 500 U/g). Microorganisms were obtained in lyophilized form, activated prior to experiments, and standardized to a viable cell density of 1 × 10⁹ cfu/mL at the time of application. Plant growth hormones were not externally added; they originated from the natural metabolic activities of Pseudomonas, Bacillus, Azospirillum, and Azotobacter species. The fulvic acid concentration in both formulations was 100 ppm. The surfactant used in the E-S formulation was a trisiloxane alkoxylate with a final concentration of 0.2% (v/v). As the aim of this study was to evaluate the combined effects of the formulations, no individual controls were designed for enzymes, microorganisms, or hormone components.

2.3. Chlorophyll Content

Chlorophyll content in leaf samples was determined spectrophotometrically using a Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, Ratastie, Finland) at wavelengths of 645 nm and 663 nm. Chlorophyll concentration was calculated in milligrams per gram of fresh weight based on the method described by [46]:

Total Chl: A652 × 27:8 × 20/mg leaf weight;

Chl a: (11.75 × A662–2.35 × A645) × 20/mg leaf weight;

Chl b: (18.61 × A645–3.96 × A662) × 20/mg leaf weight.

2.4. Antioxidant Enzymes

For antioxidant enzyme activities, fresh leaf samples were homogenized in the extraction solution following the protocols outlined by Angelini et al. [47] and Angelini and Federico [48]. The resulting supernatant was used to assess enzyme activities. Superoxide dismutase (SOD) activity was measured at 560 nm, catalase (CAT) at 240 nm, and peroxidase (POD) at 436 nm using spectrophotometric methods detailed by Angelini et al. [48,49].

2.5. Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) content was determined using a colorimetric assay. Leaf tissues (200 mg) were homogenized in cold 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged, and the supernatant was reacted with potassium phosphate buffer and potassium iodide. The absorbance of the resulting solution was measured at 390 nm. H₂O₂ concentration was quantified by comparing the absorbance to a standard curve generated using known H₂O₂ concentrations [50].

2.6. Malondialdehyde

Lipid peroxidation, a form of oxidative damage to cell membranes, was assessed by measuring malondialdehyde (MDA) levels. MDA is a by-product of lipid breakdown. The concentration of MDA was determined spectrophotometrically using an extinction coefficient of 155 mmol L⁻¹ cm⁻¹ [51].

2.7. Proline

Proline content was determined using a ninhydrin assay. Frozen leaf samples (50 mg) were ground to a fine powder in liquid nitrogen and extracted with 3% 5-sulfosalicylic acid. The homogenate was filtered, and 2 mL of the filtrate was reacted with ninhydrin and glacial acetic acid at 100 °C for 1 h. After cooling, the reaction was stopped, and the proline concentration was measured spectrophotometrically at 520 nm [52].

2.8. Hormones

Hormones—including abscisic acid (ABA), indole acetic acid (IAA), and gibberellic acid (GA)—were extracted and purified according to the method described in [53]. Hormone quantification was performed using high-performance liquid chromatography (HPLC) with a Zorbax Eclipse-AAA C-18 column (Agilent Technologies, Santa Clara, CA, USA). Hormone peaks were detected by measuring absorbance at 265 nm in a UV detector, following the procedures outlined in [54].

2.9. Plant Nutrient Elements

The quantification of essential macro (K, Ca) and microelements (Na, Cl) was performed in accordance with [55].

2.10. Statistical Analysis

To analyse the data, a two-way analysis of variance (ANOVA) was performed using IBM SPSS Statistics version 24. The least significant difference (LSD) test at a significance level of 0.05 was used to identify significant differences between group means. Graphical visualizations, including the polar heat map, were generated using OriginLab 2025b (OriginLab Corporation, Northampton, MA, USA).

3. Results

Treatments significantly influenced all measured physiological and biochemical responses, including plant growth, pigment concentrations, oxidative stress indicators, osmolyte accumulation, antioxidant enzyme activities, and hormone levels (

Table 2. Analysis of variance (ANOVA) results for measured variables under different treatments.

Variable	Treatment (df = 11)	Error (df = 24)
PFW (g/plant)	425.299 **	0.283
PDW (g/plant)	4.400 **	0.000
RFW (g/plant)	168.625 **	0.063
RDW (g/plant)	1.751 **	0.001
LA (cm2/plant)	152.102 **	1.648
Chl a (mg/g)	4.708 **	0.002
Chl b (mg/g)	1.492 **	0.004
Total Chl (mg/g)	10.261 **	0.010
H2O2 (mmol/kg)	63,344.490 **	84.548
MDA (mmol/kg)	42,765.357 **	19.940
SOD (eu/g leaf)	5,920,878.870 **	17,103.526
CAT (eu/g leaf)	669,171.923 **	1157.594
POD (eu/g leaf)	199,872,189.966 **	106,248.279
IAA (ng/mg tissue)	1223.810 **	2.175
GA (ng/gDW)	81,917,286.817 **	70,215.063
ABA (ng/gDW)	4,134,422.278 **	4949.818
Proline (%)	0.769 **	0.001
Sucrose (%)	4.910 **	0.043
L-K (%)	2.266 **	0.003
L-Ca (%)	1.485 **	0.004
L-Na (%)	74,688.227 **	57.944 **
L-Cl (mg/g)	87,044.816 **	315.093
R-K (%)	0.684 **	0.001
R-Ca (%)	0.275 **	0.000
R-Na (mg/g)	153,174.538 **	174.727
R-Cl (mg/g)	6464.997 **	113.090

**: p < 0.01; PFW: Plant fresh weight; PDW: Plant dry weight; RFW: Root fresh weight; RDW: Root dry weight; LA: Leaf area; Chl a: Chlorophyll a; Chl b: Chlorophyll b; Total Chl: Total chlorophyll; H₂O₂: Hydrogen peroxide; MDA: Malondialdehyde; SOD: Superoxide dismutase; CAT: Catalase; POD: Peroxidase; IAA: Indole-3-acetic acid; GA: Gibberellic acid; ABA: Abscisic Acid, L: Leaf, R: Root, K: Potassium, Ca: Calcium, Na: Sodium and Cl: Chloride.

).

Briefly, 100 and 200 mM NaCl caused pronounced salinity stress, leading to decreases in PFW, PDW, RFW, RDW, and LA (Figure 1 and Figure 2). In the 100 mM NaCl treatment, the recorded values were 12.76 g for PFW, 0.22 g for PDW, 7.09 g for RFW, 0.68 g for RDW, and 14.02 cm² for LA. Increasing NaCl concentration to 200 mM resulted in a significant reduction in plant growth, evidenced by a PFW of 9.19 g, PDW of 0.13 g, RFW of 5.90 g, RDW of 0.38 g, and LA of 9.10 cm² (Figure 3). Biostimulants (B and ES) notably enhanced plant growth in both normal and saline environments. B and ES, when applied individually, raised PFW to 36.81 g and 36.71 g, respectively, in contrast to 33.28 g observed in the control group. Additionally, the combination of biostimulants with NaCl mitigated the negative impacts of salinity stress (Figure 1). The treatment with B + 100 mM NaCl resulted in a PFW of 13.84 g, while the combination of B + ES + 100 mM NaCl further increased PFW to 15.62 g, in contrast to 12.76 g observed with 100 mM NaCl treatment alone. Similar trends were noted at 200 mM NaCl, where the combination of B + ES + 200 mM NaCl yielded a PFW of 11.45 g, significantly more significant than the 9.19 g recorded with 200 mM NaCl alone. The combined application of B and ES (B + ES) yielded the highest performance, with a PFW of 37.64 g and a LA of 33.43 cm² in non-saline conditions (Figure 3).

Treatments included no B/E-S (control), B (biostimulant alone), E-S (biosurfactant alone), and B/E-S (combined biostimulant + biosurfactant). Each salinity level received all four treatments, and plants were grown under controlled greenhouse conditions.

Salt stress and other treatments significantly influenced chlorophyll content. The Chl a content in 100 mM NaCl was measured at 1.93 mg/g, and Chl b was 1.96 mg/g, yielding a total chlorophyll level of 3.36 mg/g. At a salt concentration of 200 mM NaCl, there was a significant reduction in both Chl a (1.34 mg/g) and Chl b (1.19 mg/g), resulting in a total chlorophyll content of 2.59 mg/g. The control group exhibited Chl a and Chl b levels of 2.30 mg/g and 1.20 mg/g, resulting in a total chlorophyll concentration of 3.50 mg/g (Figure 3). Using biostimulants (B) resulted in a notable enhancement of chlorophyll levels, with Chl a and Chl b measuring 5.46 and 3.75 mg/g, respectively, leading to a cumulative chlorophyll content of 9.29 mg/g. Applying biostimulants in conjunction with NaCl (e.g., B + 100 mM NaCl) alleviated the negative impacts of salt stress, resulting in intermediate chlorophyll concentrations relative to treatments with salt alone (Figure 3).

Hydrogen peroxide (H₂O₂) and MDA serve as established indicators of oxidative stress in plants. In a 100 mM NaCl solution, H₂O₂ concentrations were measured at 267.83 mmol/kg, whereas MDA concentrations were recorded at 236.86 mmol/kg. At a concentration of 200 mM NaCl, the levels of H₂O₂ and MDA increased significantly, reaching 551.87 and 431.69 mmol/kg, respectively. The results indicate enhanced oxidative damage in response to severe salt stress. In contrast, the biostimulant treatment (B + ES) significantly lowered oxidative stress markers, resulting in H₂O₂ and MDA levels of 41.53 and 14.69 mmol/kg, respectively. The combination of biostimulants with salt stress (e.g., B + ES + 100 mM NaCl) resulted in some alleviation of oxidative stress, as evidenced by H₂O₂ levels of 140.48 and MDA levels of 56.14 mmol/kg, suggesting a limited protective effect (Figure 4).

Enzymatic activity for SOD, CAT, and POD differed among treatments, indicating different antioxidant responses to salt stress and biochemical applications (Figure 5). SOD (1232.22 U/mg), CAT (818.64 U/mg), and POD (929.28 U/mg) increased somewhat with 100 mM NaCl. At 200 mM NaCl, SOD (3354.33 U/mg) and POD (1374.67 U/mg) activity increased, indicating enhanced oxidative stress (Figure 5). In treatments with bacterial inoculation (B), SOD activity was 786.33 U/mg, and CAT activity was 964.80 U/mg, compared to the control values of 916.01 and 355.87, respectively. SOD (2220.87 U/mg) and POD (2262.75 U/mg) activity increased considerably when bacteria were treated with 100 mM NaCl (B + 100 mM NaCl). Bacterial inoculation with 200 mM NaCl (B + 200 mM NaCl) boosted SOD, CAT, and POD activities to 4538.18, 1603.750, and 5474.50 U/mg, respectively, showing that it reduces oxidative stress under high salinity. Exogenous substances (ES) increased SOD (1055.03 U/mg) and POD (678.33 U/mg) little compared to the control group. SOD (4631.98 U/mg) and POD (22,809.37 U/mg) were most significant in the ES + 200 mM NaCl treatment, which combined ES with salinity stress. SOD (2522.72 U/mg) and POD (1278.72 U/mg) increased significantly with ES + 100 mM NaCl. B + ES treatments had modest antioxidant enzyme activity, with SOD (745.79 U/mg) and CAT (658.10 U/mg) being lower than salinity stress treatments. B + ES + 100 mM NaCl and B + ES + 200 mM NaCl increased antioxidant enzyme activity significantly when salinity stress was added. B + ES + 200 mM NaCl increased POD activity (21,791.30 U/mg), demonstrating the synergistic effects of bacterial and exogenous salinity stress treatments (Figure 5).

The link between salt stress and phytohormone concentrations was clear in the treatments. Levels of IAA and GA decreased with increasing concentrations of NaCl. Under 200 mM NaCl, IAA and GA levels were measured at 2.15 ng/mg and 18,060.06 ng/g, respectively, in contrast to 11.29 ng/mg and 2300.71 ng/g observed in the control group. Conversely, ABA, a hormone linked to stress responses, exhibited a significant increase in concentration under salt stress, reaching a maximum of 140.02 ng/g at 200 mM NaCl. Biostimulant treatments stabilized these hormone levels, whether administered alone (B + ES) or in conjunction with salt stress (B + ES + 100 mM NaCl). In the B + ES treatment, IAA and GA were measured at 62.28 ng/mg and 540.35 ng/g, respectively. Meanwhile, ABA levels decreased to 2963.98 ng/g, suggesting a possible function in alleviating salt-induced stress responses (Figure 6).

The 200 mM NaCl treatment had the highest proline accumulation at 1.88%, followed by the ES + 200 mM NaCl200 treatment at 0.88%. The proline content of control plants was found to be comparatively low at 0.24%. Significant divergence was seen in the sucrose content; the B + 100 mM NaCl treatment had the greatest value (5.50%), while the ES + 200 mM NaCl treatment had the lowest (1.34%) (Figure 7).

Figure 7. Effects of various treatments on (a) proline and (b) sucrose contents. The bars represent means ± SE (n = 3). The bars represent means ± SE (n = 3). The differences between means labeled with different letters are statistically significant at the 5% level, according to Fisher’s LSD test. NaCl: Sodium chloride; B: Biostimulant; ES: Enriched surfactant; B + ES: Combination of biostimulant and biosurfactantThe B treatment had the highest leaf potassium (L-K) content at 3.51%, while the lowest L-K value, 1.09%, was found at 200 mM NaCl. Calcium (L-Ca) values ranged from 2.74% in B + ES treatments to 0.26% in 200 mM NaCl. Under salt stress, the amounts of sodium (L-Na) and chloride (L-Cl) rose dramatically, peaking in the 200 mM NaCl treatment (L-Na: 474.81 mg/g, L-Cl: 584.59 mg/g) (Figure 8).

Figure 7. Effects of various treatments on (a) proline and (b) sucrose contents. The bars represent means ± SE (n = 3). The bars represent means ± SE (n = 3). The differences between means labeled with different letters are statistically significant at the 5% level, according to Fisher’s LSD test. NaCl: Sodium chloride; B: Biostimulant; ES: Enriched surfactant; B + ES: Combination of biostimulant and biosurfactantThe B treatment had the highest leaf potassium (L-K) content at 3.51%, while the lowest L-K value, 1.09%, was found at 200 mM NaCl. Calcium (L-Ca) values ranged from 2.74% in B + ES treatments to 0.26% in 200 mM NaCl. Under salt stress, the amounts of sodium (L-Na) and chloride (L-Cl) rose dramatically, peaking in the 200 mM NaCl treatment (L-Na: 474.81 mg/g, L-Cl: 584.59 mg/g) (Figure 8).

The B + ES treatment had much higher levels of root potassium (R-K) (1.42%) than the 200 mM NaCl treatment, which had the lowest concentration (0.10%). Root calcium (R-Ca) levels peaked in ES + 200 mM NaCl at 0.87% and reduced to 0.06% in 200 mM NaCl. Salt stress substantially impacted the quantities of sodium (R-Na) and chloride (R-Cl) in roots; the 200 mM NaCl treatment had the highest R-Na concentration at 801.94 mg/g (Figure 9).

Figure 10 presents a heatmap of the Pearson correlation based on the relationships among plants’ various physiological and biochemical parameters. Significant positive correlations have been observed between plant fresh weight (PFW) and plant dry weight (PDW) (r = 0.98), proving a direct and proportional relationship among the components of overall plant biomass. Root dry weight (RDW) exhibited a significant positive correlation with both plant fresh weight (PFW) (r = 0.99) and plant dry weight (PDW) (r = 0.99), highlighting the related growth of these variables. A strong positive correlation (r = 0.99) between plant fresh weight (PFW) and root dry weight (RDW) suggests a direct relationship between root development and overall plant biomass. The significant correlation between leaf area (LA) and root fresh weight (RFW) (r = 0.96) points out the synchronized growth of leaves and roots. The observed moderate positive correlation between total chlorophyll (Total Chl) and leaf area (r = 0.47) indicates a relationship between the accumulation of photosynthetic pigments and leaf morphology. Photosynthetic pigments exhibited significant correlations and in particular a strong positive relationship between chlorophyll a (Chl a) and total chlorophyll (Total Chl) (r = 0.97). This suggests a coordinated build-up of photosynthetic pigments. Leaf area (LA) exhibited a moderate correlation with total chlorophyll (r = 0.47) and a strong correlation with root fresh weight (RFW) (r = 0.96), indicating a relationship between photosynthetic capacity and structural growth.

Stress-related indicators, such as H₂O₂ and MDA, demonstrated a robust positive correlation (r = 0.97), emphasizing their significance as primary markers of oxidative stress. Negative correlations were observed between H₂O₂ and antioxidant enzymes, specifically SOD (r = −0.67) and CAT (r = −0.44), suggesting the role of these enzymes in reducing oxidative damage.

Indole-3-acetic acid (IAA) exhibited a strong positive correlation with ABA (r = 0.85), indicating their coordinated roles in stress response mechanisms. Gibberellic acid (GA) exhibited a significant negative correlation with PFW (r = −0.86) and a positive correlation with H₂O₂ (r = 0.97), suggesting its role in growth regulation and stress signalling. Moreover, a significant and negative correlation was found between growth characteristics and Na and Cl content, while a positive correlation was found between K and Ca content. Once more, a positive correlation was determined between these and the proline amount.

The Principal Component Analysis (PCA) recommended detailed insights into the relationships and contributions of different physiological and biochemical variables. The first principal component (PC1) was identified as the most significant, representing 66.73% of the total variance. This component was mainly linked to growth-related traits, exhibiting the highest positive loadings from root fresh weight (RFW, 0.23), leaf potassium (L-K, 0.23), and root potassium (R-K, 0.23). In contrast, the most significant negative loadings in PC1 were recorded for hydrogen peroxide (H₂O₂, −0.23), leaf sodium (L-Na, −0.23), and gibberellic acid (GA, −0.23). The results suggest that PC1 predominantly reflects plant biomass accumulation and nutrient uptake efficiency, exhibiting a negative correlation with oxidative stress and hormonal signals (

Table 3. Principal Component Analysis (PCA) loadings, eigenvalues, and variance contributions for variables.

Variable	PC1	PC2	PC3	PC4
PFW (g/plant)	0.22	0.20	0.03	0.04
PDW (g/plant)	0.21	0.21	0.11	0.01
RFW (g/plant)	0.23	0.16	0.07	−0.05
RDW (g/plant)	0.22	0.18	0.06	−0.01
LA (cm2/plant)	0.22	0.11	0.03	−0.19
Chl a (mg/g)	0.17	−0.22	0.28	0.28
Chl b (mg/g)	0.12	−0.31	0.20	0.15
Total Chl (mg/g)	0.17	−0.22	0.32	0.29
H2O2 (mmol/kg)	−0.23	0.17	0.11	0.06
MDA (mmol/kg)	−0.21	0.23	0.09	0.14
SOD (eu/g leaf)	−0.18	−0.08	0.35	−0.14
CAT (eu/g leaf)	−0.08	−0.53	0.12	−0.07
POD (eu/g leaf)	−0.09	−0.21	0.23	−0.53
IAA (ng/mg tissue)	0.20	−0.03	0.28	0.19
GA (ng/gDW)	−0.23	0.08	0.09	0.07
ABA (ng/gDW)	0.22	0.13	0.20	0.15
Proline (%)	−0.20	0.22	0.22	0.13
Sucrose (%)	0.01	−0.20	−0.28	0.44
L-K (%)	0.23	0.06	0.03	−0.02
L-Ca (%)	0.22	−0.13	0.14	−0.07
L-Na (%)	−0.23	0.02	0.13	0.13
L-Cl (mg/g)	−0.22	0.14	0.17	0.14
R-K (%)	0.23	0.17	0.00	0.12
R-Ca (%)	0.16	0.18	0.29	−0.26
R-Na (mg/g)	−0.21	0.15	0.15	0.20
R-Cl (mg/g)	−0.20	0.05	0.35	−0.03
Eigenvalue	17.35	2.74	1.87	1.64
Percentage of Variance	66.73%	10.52%	7.19%	6.29%
Cumulative	66.73%	77.25%	84.44%	90.73%

PFW: Plant fresh weight; PDW: Plant dry weight; RFW: Root fresh weight; RDW: Root dry weight; LA: Leaf area; Chl a: Chlorophyll a; Chl b: Chlorophyll b; Total Chl: Total chlorophyll; H₂O₂: Hydrogen peroxide; MDA: Malondialdehyde; SOD: Superoxide dismutase; CAT: Catalase; POD: Peroxidase; IAA: Indole-3-acetic acid; GA: Gibberellic acid; ABA: Abscisic acid, L: Leaf, R: Root, K: Potassium, Ca: Calcium, Na: Sodium and Cl: Chloride.

).

PC2 accounted for an additional 10.52% of the variance and was defined by variables associated with photosynthetic pigments and enzymatic activities. PC2 exhibited the highest positive loadings for malondialdehyde (MDA, 0.23) and proline (0.22), indicating oxidative and stress-related responses. The biggest negative loadings were linked to catalase (CAT, −0.53), chlorophyll b (Chl b, −0.31), and total chlorophyll (combined Chl a and b, −0.22) (Table 3). The findings indicate a trade-off between stress responses and photosynthetic activity in PC2.

PC3 accounted for 7.19% of the variance and predominantly represented the variation in antioxidant enzyme activities. Superoxide dismutase (SOD) exhibited the highest positive loading at 0.35, followed by root chloride (R-Cl) at 0.35 and total chlorophyll at 0.32. These findings underscore the significance of oxidative stress regulation and photosynthetic adjustments. The most significant negative loading observed was for sucrose (−0.28) (Table 3), suggesting an inverse correlation between metabolic reserves and stress enzyme activity.

The treatment with 200 mM NaCl alone shows a strong association with stress-related parameters such as H₂O₂, SOD, proline, and MDA, as well as Na and Cl contents, indicating an enhanced oxidative and osmotic stress response (Figure 11). This clustering highlights the detrimental effects of high salinity stress on plant physiology. The treatment with B alone shows a close association with parameters like photosynthetic pigments (Chl a, Chl b, and Total Chl) and IAA, while ES and control treatments indicate a close association with growth parameters (Figure 11).

The treatment for 200 NaCl + B is linked to variables including plant biomass indicators (PFW and RFW) and chlorophyll concentration (Chl a, Chl b, and Total Chl) (Figure 11). According to this, adding biostimulant (B) under high salinity stress substantially reduces the negative effects of salt by preserving photosynthetic efficiency and encouraging growth, most likely through increased metabolic activity and food availability.

On the other hand, 200 NaCl + ES shows a substantial correlation with osmolytes (proline and sucrose) and antioxidant enzymes (SOD and POD) (Figure 11). This suggests that applying ES to plants under salinity stress strengthens their defences by promoting osmotic adjustment and the antioxidant system, which lessens oxidative damage from elevated salt concentrations.

4. Discussion

Soil salinization, a multifactorial stress type, is a problem that occurs through natural geochemical processes and secondary anthropogenic activities, causing serious reductions in crop production [56]. Maize (Zea mays L.) is an important C4 plant from the Poaceae family and is moderately sensitive to salt stress [57]. In our study, it was found that salinity stress negatively affected the growth of maize seedlings (Figure 1 and Figure 2). The suppressive effects of salinity on maize seedlings have been reported in many existing studies [1,58]. It is known that shoot and root growth in maize under salt stress is strongly inhibited [59,60]. Indeed, the results obtained from our study showed that PFW, RFW, PFW, RDW, PH and LA decreased by 61.27–72.38%, 66.50–71.28%, 92.08–95.32%, 67.31–80.97%, 30.97–37.53%, 50.80–66.04%, respectively, in 100 mM and 200 mM NaCl conditions. When plants are exposed to salty environments, they encounter a series of negative impacts. Their cells dehydrate, their outer membranes become damaged, and essential digestive enzymes escape. This cellular disruption hinders growth and leads to loss of plant firmness [61]. The reduced growth rate under salinity stress can be attributed to several factors, including decreased water availability, nutrient imbalances, and toxic levels of sodium.

Photosynthetic activity is considered one of the major factors controlling plant growth [62]. Salinity indirectly slows down photosynthesis in plants, and photosynthesis is directly related to stomatal conductance, chlorophyll content, transpiration, and water potential [63]. Leaf photosynthesis may decrease due to reduced stomatal conductance as a result of water imbalance under salt stress [64]. The findings of the study showed that salinity stress caused significant decreases in chlorophyll a, chlorophyll b, and total chlorophyll, which is in agreement with the literature (Figure 4) [65]. The depreciation of chlorophyll content under salinity stress can be attributed to the inhibition of chlorophyll synthesis and its degradation due to oxidative stress [66]. Moreover, a decrease in chlorophyll content and photosynthetic efficiency has been observed under salinity stress in various plant species [67,68,69]. The combined B + ES treatment at 200 mM NaCl partially fixed chlorophyll a, chlorophyll b, and total chlorophyll levels. This improvement is closely linked to enhanced antioxidant enzyme activities (SOD, CAT, POD), which reduce ROS accumulation and protect chloroplast structures from oxidative damage. Similar findings have been reported in maize and other crops treated with PGPR-based biostimulants under salinity stress [44,70,71].

Salt stress leads to higher ROS accumulation, which disrupts cellular redox homeostasis and leads to oxidative damage [58]. Indeed, our findings showed that salinity stress conditions in the control group caused an increase in MDA and H₂O₂ levels, as expected, and caused oxidative stress in maize seedlings (Figure 4). When plants experience oxidative stress, free radicals are generated. These highly reactive molecules can attack lipid membranes, triggering a process known as lipid peroxidation. Malondialdehyde (MDA) is a common end product of this process. Studies have shown that increased MDA levels are indicative of stress, particularly salt stress, as demonstrated in tomatoes [72]. The final products of lipid peroxidation include aldehydes like MDA and various hydrocarbons [73,74].

Salinity tolerance can be attributed to increasing antioxidant enzyme activity and thereby reducing oxidative damage [75]. The results obtained from the study showed that salinity stress increased SOD and CAT activity (Figure 6). Previous studies have reported that salinity stress increased antioxidant enzyme (SOD, CAT) activities in maize (Maize L.) [58,65,76]. Among the ROS detoxification activities, CAT and SOD are called frontline defences [77]. These factors play a significant role in enhancing plant tolerance to salt stress [20]. One of the key mechanisms for detoxifying ROS generated during salt stress is the induction of ROS-scavenging enzymes, such as SOD and CAT [78]. Biostimulants help plants manage oxidative stress in several ways. Fulvic and humic substances enhance the activity of antioxidant genes (SOD, CAT, POD), strengthening the system that removes reactive oxygen species [79]. The PGPR strains in the biostimulant, such as Paenibacillus polymyxa, Bacillus amyloliquefaciens, and Pseudomonas fluorescens, produce ACC deaminase, phytohormones (IAA, GA, CK), and exopolysaccharides. These compounds lower ethylene levels, help plants retain water, and reduce the build-up of ROS during salt stress [80]. Biosurfactants also stabilize plant cell membranes, enhance nutrient uptake, and increase the availability of metal cofactors, thereby supporting antioxidant enzymes [81]. Together, these effects explain why H₂O₂ and MDA levels were lower in this study under the B and ES treatments.

A relationship between antioxidant capacity and NaCl tolerance has been demonstrated in numerous plant species [82]. Additionally, plant hormones such as ABA and IAA play a crucial role in plant responses to abiotic stress [83,84]. Our findings show that salt stress increases ABA levels in maize seedlings, suggesting its importance in salt tolerance (Figure 6). Elevated ABA levels can activate sucrose nonfermenting 1-related protein kinases [85]. Additionally, salt stress can inhibit IAA synthesis, leading to growth inhibition [86]. These results align with previous research demonstrating that salt stress induces increased ABA and decreased IAA levels [87]. This hormonal imbalance, characterized by high ABA and low cytokinin and IAA, likely reflects the plant’s adaptive strategy to prioritize salt resistance over growth.

In this study, an increase in proline and sucrose content was observed, aligning with its established roles in osmoregulation and ROS scavenging (Figure 8). Proline’s ability to preserve cellular hydration is crucial for maintaining macromolecular structure and function, particularly enzymes. Furthermore, intracellular proline accumulation enhances salinity stress tolerance and serves as a valuable nitrogen source during stress recovery [88]. The proline content of corn seedlings under salt stress is higher than that of unstressed corn seedlings. Osmotic regulation is one of the most important mechanisms by which plants tolerate salinity stress by maintaining cell water content and turgor [89]. It is known that the increase in the concentration of proline in the cell is considered an indicator against stress and constitutes the first step of metabolic reactions that initiate the plant’s defence mechanism [90]. Studies reveal that proline always increases more significantly than other amino acids in tobacco plants under salinity and drought stresses [91]. Binzel et al. [92] reported that proline represents approximately 80% of the total amino acid pool in tobacco cells under NaCl stress, and it has been found that proline is one of the most abundant amino acids in plants exposed to salinity [93,94]. Proline helps maintain the structure of important cellular components, such as enzymes, by preserving water content in the cytoplasm. It plays a crucial role in salt tolerance and serves as a nitrogen source during stress recovery [88].

Salinity stress disrupts plant mineral nutrition by impacting nutrient availability and transport [95]. In our study with maize seedlings, exposure to 100 mM NaCl led to decreased levels of essential nutrients such as potassium and calcium in leaves and roots compared to control conditions. Conversely, sodium and chloride levels significantly increased. This nutrient imbalance may be attributed to competition between salinity stress and essential nutrients such as potassium, calcium, and nitrate for uptake and translocation within the plant [96].

The combined biostimulant and biosurfactant treatment (B + ES) enhanced the uptake of key nutrients, including K, Ca, Mg, and P, while reducing Na and Cl accumulation. K⁺ is central to osmotic balance, enzyme activation, and stomatal regulation, so its retention is essential for salinity tolerance [97]. Ca²⁺ stabilizes cellular membranes and improves ion selectivity under NaCl stress [98] Mg²⁺ is crucial for chlorophyll biosynthesis and ATP formation, supporting photosynthesis under salt stress [99]. Increased P supports energy metabolism and ATP-dependent stress responses [100]. These mineral enhancements together contribute to better ionic homeostasis and greater resilience in maize seedlings exposed to salinity.

The findings of our research corroborate the previously documented mechanisms through which the employed biostimulants mitigate plant stress and promote growth [20,101,102]. Our research demonstrated that salinity stress had detrimental effects on the growth, physiological processes, and biochemical composition of tomato seedlings. However, the application of B and ES significantly improved the plant’s resistance to salinity stress by activating a range of physiological and biochemical defence mechanisms. Specifically, external application of B + ES promoted the growth of maize seedlings under saline conditions (Figure 1 and Figure 2). All enzymes (protease, xylanase, α-amylase, cellulase+hemicellulase, and phytase) included in the formulations in the study were naturally produced and purified by microorganisms. The synergistic effects of the B + ES treatment result from complementary actions of microbial biostimulants and biosurfactants [103]. PGPR strains support hormone synthesis, root growth, and the reduction of stress ethylene, while biosurfactants improve nutrient solubilization, root–soil contact, and microbial colonization efficiency [104]. PGPR also activate antioxidant gene networks through ABA-dependent and MAPK signaling pathways [105]. Together, these mechanisms enhance mineral balance, antioxidant capacity, and physiological stability under salt stress.

Biostimulants have been shown to play a significant role in enhancing plant tolerance to abiotic stresses through a process known as induced systemic tolerance (IST) [106]. Biostimulants, derived from diverse natural sources, offer a promising approach in sustainable agriculture. By enhancing plant vigour and resilience, they minimize the reliance on synthetic fertilizers while bolstering tolerance to environmental stresses such as drought and salinity [107]. These beneficial compounds improve plant water management by increasing root water uptake and optimizing water use efficiency, thereby mitigating stress-related losses [108]. Biosurfactants, produced by microorganisms, represent an eco-friendly alternative to chemically synthesized surfactants. They exhibit numerous advantages, including enhanced soil health through the degradation of pollutants. In agricultural applications, biosurfactants stimulate plant growth by increasing the availability of nutrients and promoting beneficial microbial activity [109].

The application of B and ES helped to alleviate the negative impacts of salinity stress on the chlorophyll content of maize seedlings (Figure 3). Numerous studies have demonstrated the potential of biostimulants to mitigate the adverse effects of salinity on chlorophyll content through a variety of mechanisms. These include the activation of plant’s antioxidant defence system and improving the photosynthetic efficiency, chlorophyll content, production of osmolytes, and water use efficiency [110].

Biostimulant applications reversed the salt stress-induced increases in H₂O₂ and MDA content of maize seedlings (Figure 4). These findings suggest that some bioactive substances such as fulvic acid, hormones, enzymes etc. play a crucial role in the antioxidant protection observed in plants treated with the B and B + ES under salt stress. This likely occurs through the scavenging of reactive oxygen species (ROS) and the subsequent inhibition of oxidative stress.

Biostimulants are a valuable tool in mitigating the adverse impacts of salinity stress because they increase antioxidant enzyme activity [93]. Indeed, the results of the study showed that B and SE treatments resulted in an extra increase in SOD, POD and CAT activity (Figure 5). Biostimulants have been demonstrated to upregulate the expression of genes involved in antioxidant defence systems. This includes genes encoding enzymes such as superoxide dismutase, catalase, and peroxidase. This enhanced gene expression enables plants to more effectively scavenge ROS that accumulate under stress conditions, thereby mitigating oxidative damage to cellular components [111].

Furthermore, the external application of B and ES was observed to enhance the levels of IAA and GA, while simultaneously reducing the ABA content in maize seedlings, both under conditions of salinity stress and in the absence of stress, as shown in Figure 6. Various phytohormones function as bioactive compounds, exerting significant influence on the stress response pathways in crops. Phytohormones are organic molecules that play an important role in tolerance to abiotic stress of plants [112]. Moreover, the application of microbial biostimulants, seaweed extracts, and humic substances increased the levels of auxin, GA, and cytokinin (CK) in sorghum plants under salt stress conditions [113]. Previous studies have demonstrated that biostimulants can upregulate the production of phytohormones in horticultural crops, enabling them to adapt to abiotic stress. Notably, various seaweed extracts and botanicals have been shown to exhibit activities similar to CK and auxins [114]. Furthermore, the application of humic substances has been reported to stimulate the endogenous production of auxins and GA [115].

The incorporation of B and ES into the maize seedling regimen resulted in a significant increase in proline and sugar content levels under both stress and non-stress conditions (Figure 7). Proline plays a multifaceted role within plant cells, acting as an osmoprotectant for osmotic regulation, scavenging ROS, stabilizing macromolecules to mitigate stress-induced damage [116], and contributing to the osmotic balance necessary for leaf growth and expansion [117].

In this study, the external application of biostimulants significantly increased the mineral content in maize seedlings under both salinity stress and non-stress conditions, with the notable exception of Na and Cl (Figure 8 and Figure 9). Furthermore, biostimulant treatments effectively reduced Na and Cl concentrations in seedlings subjected to salinity stress. Similarly, studies reported that biostimulant treatments increased plant nutrient uptake of plants except for Na and Cl [118,119]. Biostimulants have been shown to influence the levels of key plant hormones, including auxin, cytokinin, and abscisic acid [120]. This hormonal influence contributes to enhanced nutrient uptake, particularly of nitrogen (N), phosphorus (P), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), by stimulating root growth [121]. One key mechanism of action is their ability to increase the bioavailability of nutrients and facilitate the interaction between hydrophobic substrates and microbial cells. This enhances nutrient uptake and microbial activity, leading to improved plant growth. Furthermore, biosurfactants contribute to plant health by stimulating the production of plant hormones, enhancing microbial interactions, and increasing resistance to both biotic and abiotic stresses [122]. Emerging evidence suggests that biostimulants can induce epigenetic changes in plants. These changes, which alter gene expression without modifying the underlying DNA sequence, can lead to improved stress tolerance and adaptive responses. Biostimulants have been reported to modulate phytohormone signalling, enhancement of nutrient uptake, and utilization of plants [110].

5. Conclusions

This groundbreaking study examines the effects of biostimulants and enriched surfactants on maize plants subjected to salinity stress. This study concludes that applications of biostimulant (B), enriched biosurfactant (ES), or their combination, are highly effective in protecting maize seedlings from severe salinity stress. Salinity drastically reduced biomass and chlorophyll content. The treatments, especially B + B-S, restored these parameters, sustaining biomass accumulation and protection photosynthetic apparatus. Notably, under severe salinity (200 mM NaCl), the combined treatment increased plant fresh weight from 9.19 g (200 mM NaCl) to 11.45 g (B + ES + 200 mM NaCl). Under the same severe salinity conditions, oxidative and osmotic damage were significantly reduced; compared with the untreated 200 mM NaCl group, B, ES, and B + ES lowered proline accumulation by 65%, 52%, and 70%, respectively. They decreased H₂O₂ by 53%, 39%, and 58% and lowered MDA by 72%, 50%, and 73%, indicating a notable reduction in oxidative stress and membrane lipid peroxidation. Salinity disrupted nutrient balance by increasing toxic Na and Cl while reducing essential elements’ uptake. The B + ES application improved essential elements for plant uptake and limited Na and Cl uptake, which are critical for salinity tolerance. In conclusion, B and B + ES applications offer a sustainable and effective strategy through which to improve crop performance in saline environments by conferring comprehensive protection across physiological, biochemical, and mineral–nutrient levels. To enhance the practical relevance of these findings, future research should include large-scale field trials to evaluate treatment effectiveness in real agricultural settings, analyse performance across various crop species with different salt sensitivities, and optimize application dosages and schedules for the best results. Additionally, molecular and omics-based methods could help to identify the specific signalling pathways responsible for the observed physiological gains.