Combined application of bacterial biofertilizers, seaweed, and Thymbra spicata extract enhanced productivity, nutritional value, and postharvest shelf life of cucumber (Cucumis sativus L.)

Abstract

Environmentally friendly biostimulants and biofertilizers have emerged as promising alternatives to synthetic agricultural inputs for improving crop productivity, fruit quality, and postharvest longevity. This study investigated the effects of Thymbra spicata L. extract at 0% (E0), 10% (TE1), and 20% (TE2), applied individually or in combination with seaweed biofertilizer (1%) (SB) and bacterial biofertilizer (10%) (BB), on the growth, yield, physiological responses, and postharvest characteristics of cucumber (Cucumis sativus L. var. eureka) under field conditions. The experiment was conducted as a factorial arrangement in a randomized complete block design with three replications. The results showed that the combined application of TE1 + SB + BB was the most effective treatment with respect to yield, vitamin C content, antioxidant activity, and postharvest longevity. Compared with the untreated control, this treatment increased fruit number by 29.32%, total yield by 43.23%, fruit firmness by 22.34%, vitamin C content by 33.25%, antioxidant activity by 72.93%, and shelf life by 121.67%, while reducing fruit weight loss by 45.67%. Although several other treatments improved specific physiological and biochemical traits, TE1 + SB + BB provided the greatest benefits for the major yield- and postharvest-related attributes evaluated in this study. These findings indicated that integrating 10% T. spicata extract with seaweed and bacterial biofertilizers enhanced cucumber yield, antioxidant capacity, and postharvest longevity, and could represent an effective and sustainable approach for cucumber production.

Introduction

Forecasts indicate that the global population will rise to 9.6 billion by 2050, requiring an 80–110% increase in food and agricultural production to meet rising demand while preserving soil health and addressing environmental challenges [1]. Strategies such as agricultural intensification, expanding cultivated land, adopting high-yield crop varieties, and applying chemical fertilizers are often proposed as solutions. Still, these approaches come with significant drawbacks, including biodiversity loss, soil degradation, water depletion, and groundwater pollution, as well as practical limitations [2, 3]. Moreover, most crops are highly vulnerable to postharvest spoilage caused by physical damage, microbiological contamination, and chemical reactions, which adversely affect their appearance, flavor, firmness, and aroma, rendering them unfit for consumption. Consequently, it is imperative to adopt strategies that not only increase crop yields but also reduce postharvest losses, extend shelf life, and maintain freshness throughout the supply chain, ensuring high-quality produce from farm to fork [4, 5].

Cucumber (Cucumis sativus L.), a member of the Cucurbitaceae family, is an economically significant vegetable and an important source of vitamins and minerals in the human diet [6]. Unlike many other crops, cucumbers reach harvest maturity rapidly, and their fruits are widely consumed fresh, in salads, pickles, and various culinary preparations [7]. With rapid economic development improving living standards, cucumber producers increasingly prioritize both yield and quality to meet market demands [8]. However, cucumbers are highly perishable due to their high water content, making them prone to water loss, mechanical damage, shriveling, and yellowing during postharvest handling and storage, which significantly limits their shelf life and marketability [9].

The excessive use of chemical fertilizers and pesticides in agriculture has led to significant adverse environmental and ecological impacts. As a result, there is an increasing shift toward the use of biostimulants, biofertilizers, and naturally occurring compounds, which are known to alleviate the negative effects of environmental stress, control pests and diseases, and notably enhance crop yield per unit area [10]. While chemical fertilizers have been widely adopted as a quick solution to address soil nutrient deficiencies, sustainable agricultural practices necessitate reducing their application and replacing them with biological fertilizers. This approach mitigates environmental issues, enhances yield and quality, and supports the production of organic crops [11].

Biofertilizers are derived from various soil microorganisms, including bacteria, fungi, and algae, with bacteria being the most widely utilized [12]. A specific group of beneficial rhizosphere bacteria, referred to as plant growth-promoting bacteria (PGPB), plays a crucial role in improving crop yields and extending post-harvest shelf life [13]. These bacteria function directly by fixing nitrogen, producing growth-regulating compounds, and enhancing nutrient uptake [14]. They also provide indirect benefits by producing cell wall-degrading enzymes to target pathogenic fungi and by inducing resistance to abiotic stresses [15].

Seaweeds have a long history of agricultural use as compost, mulch, and in the form of extracts to improve crop yields through seed priming, soil amendments, root immersion, foliar application, and fertigation [16]. Seaweed fertilizers are particularly effective in reducing physiological disorders caused by mineral deficiencies, improving crop yields, and increasing resistance to biotic and abiotic stresses. Rich in nitrogen, mineral elements, and plant hormones, seaweed extract promotes plant growth and yield [17]. It also strengthens plant cell walls, thereby reducing fruit weight loss during storage and extending post-harvest shelf life [18]. Furthermore, the antioxidants and amino acids in seaweed fertilizers reduce soluble solids while increasing organic acids during the post-harvest period. Their antifungal metabolites also contribute to extended crop shelf life [19, 20].

Growing environmental concerns and the adverse effects associated with the excessive use of synthetic agrochemicals have stimulated increasing interest in medicinal plants and their bioactive extracts as sustainable and eco-friendly alternatives for improving crop productivity, quality, and postharvest performance [21]. Medicinal plant extracts are widely recognized as bioactive agents capable of mitigating the negative effects of environmental stress on crops and controlling post-harvest diseases, due to their rich content of antimicrobial, antioxidant, and defense-eliciting phytochemicals that can reduce pathogen incidence and enhance crop quality during storage [22, 23].

Thymbra spicata L. is a Mediterranean aromatic and medicinal plant widely distributed in the eastern Mediterranean and Western Asia, where it typically grows in dry, rocky, and semi-arid habitats. Belonging to the Lamiaceae family, it is well known for its strong antibacterial, antifungal, antioxidant, and antiseptic activities, which are mainly attributed to its rich profile of bioactive secondary metabolites, including phenolic acids such as rosmarinic acid, monoterpenoids like thymol and carvacrol, flavonoids, as well as volatile compounds such as terpinen and p-cymene [24, 25]. In parallel, extracts derived from thyme species (Thymus spp.) have been shown to effectively promote plant growth and enhance yield, suppress microbial spoilage, preserve key quality attributes during storage, and extend the post-harvest shelf life of perishable horticultural crops, underscoring their potential as natural alternatives to chemical fertilizers and synthetic preservatives [26–28].

Given the growing importance of biostimulants in improving crop productivity, supporting sustainable and organic agriculture, mitigating pathogen pressure, and maintaining post-harvest quality, the present study aimed to evaluate the effects of T. spicata extract, applied alone and in combination with seaweed and bacterial biofertilizers, on cucumber (Cucumis sativus L. var. eureka). Particular emphasis was placed on fruit yield and postharvest shelf life in response to different treatment regimes, while endogenous salicylic acid was evaluated as a biochemical marker to explore its potential association with the observed physiological and postharvest responses.

Materials and methods

Geographical characteristics of the study site

The study was conducted at the research farm of the Department of Horticultural Science, Faculty of Agriculture, Ilam University, Iran. The site is located approximately 1,410 m above sea level, at 46°22′37″ E longitude and 33°39′26″ N latitude, with an average annual temperature of 15 °C.

Experimental treatments

The experimental treatments were organized as follows:

1. T. spicata L. ethanolic extract (TE): Dried aerial parts of the plant were purchased from Bahar Herbal Market, a local herbal supplier in Ilam, Iran, on 20 April 2022. The dried plant material was visually inspected for uniformity, color, and the absence of visible impurities, then ground into a fine powder. The samples were stored under dry, cool, and dark conditions until extraction to preserve the stability of bioactive compounds. To prepare the extract, 40 g of the powdered material was soaked in 70% ethanol for 72 h. The extract was filtered using Whatman No. 1 filter paper, and the ethanol was evaporated by exposing the filtrate to room temperature for two days. The resulting extract was used to prepare treatments containing no extract (E0), 10% extract (TE1), and 20% extract (TE2). The study complied with relevant institutional and national guidelines for research involving plant materials. Since the plant material was obtained from a commercial herbal supplier and no wild plant collection was conducted, no collection permit was required. Because dried commercial plant material was used, taxonomic verification by a specialist and the preparation of a voucher specimen were not feasible, and no voucher specimen was deposited.

2. Seaweed biofertilizer (SB) treatment: Purchased from Coda Spain, E-Dalgin containing pure Ascophyllum nodosum extract. To prepare a 1% V/V solution, 10 mL of the biofertilizer was adjusted to 1 L using dH₂O in a graduated cylinder.

3. Bacterial biofertilizer (BB): To prepare this biofertilizer, the following were procured from Green Biotech Incorporation, Ltd. (Tehran, Iran). First, 20 mL of each biofertilizer was combined in a beaker to achieve a total volume of 100 mL and then was diluted to a final volume of 1 L by adding dH₂O.

   1. Liquid AzotoBARVAR-1 biofertilizer: This fertilizer contains Pantoea agglomerans strain O4, with a 10⁷ to 10⁸ CFU/mL concentration and efficiently converts atmospheric nitrogen into ammonium ions, which are easily absorbed by plants.
   2. PhosphoBARVAR-2 biofertilizer: This fertilizer contains phosphate-dissolving bacteria, including Pseudomonas putida strain P13 and Pantoea agglomerans strain P5, with a concentration of 10⁷ to 10⁸ CFU/mL. These bacteria enhance phosphorus availability to plants by secreting organic acids and producing phosphatase enzymes, which decompose insoluble phosphorus compounds in the soil into forms that plants can readily absorb.
   3. PotaBARVAR-2 biofertilizer: This fertilizer contains potassium-dissolving bacteria, including Pseudomonas koreensis strain TE14 and Pseudomonas vancouverensis strain TE19, with a concentration of 10⁷ to 10⁸ CFU/mL. These bacteria secrete organic acids in the rhizosphere, which break down insoluble potassium compounds in the soil. This decomposition process releases potassium ions, enabling plants to absorb them more efficiently and supporting their growth and development.
   4. SulfoBARVAR-1 biofertilizer: This fertilizer contains Thiobacillus strains T40 and T95, with concentrations of 10⁷ to 10⁸ CFU/mL. These sulfur-oxidizing soil bacteria increase soil acidity by oxidizing elemental sulfur to sulfuric acid (H₂SO₄), thereby decreasing soil pH. This acidification process facilitates the dissolution of essential nutrients such as phosphorus (P), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), making them more bioavailable for plant uptake and promoting optimal growth and development.
   5. FerrozincBARVAR biofertilizer: This fertilizer contains siderophore-producing bacteria, specifically Pseudomonas japonica strains Fz.29 − 1 and Fz.21 − 1, with concentrations of 10⁷ to 10⁸ CFU/mL. These bacteria enhance nutrient uptake by producing siderophores—iron-chelating compounds that bind to and solubilize iron from the soil, making it more bioavailable for plants. This process not only improves iron absorption but also supports overall plant health, vigor, and resistance to abiotic stress.

The 10% and 20% of the plant extract solutions were prepared as weight/volume (w/v) concentrations by diluting the concentrated extract with distilled water, corresponding to 10 and 20 g of dry plant material equivalent per 100 mL of the final solution, respectively. Complete ethanol removal after rotary evaporation was qualitatively verified by the absence of ethanol odor and by reaching a constant extract weight, defined as less than 2% variation between two consecutive weighings performed at a 6-h interval. The pH of the extract solutions was adjusted to 6.5–7.0 using phosphate buffer before foliar application. In addition, a preliminary phytotoxicity test was conducted on a small number of cucumber plants before the main experiment, and no visible symptoms of chlorosis or necrosis were observed. Based on the initial concentrations provided by the manufacturer (10⁷–10⁸ CFU/mL for each commercial formulation), the final bacterial concentration in the diluted biofertilizer mixture was approximately 10⁶–10⁷ CFU/mL. Each plant received 50 mL of the suspension per application, corresponding to approximately 5 × 10⁷–5 × 10⁸ CFU plant⁻¹. Over the three application events, the cumulative bacterial dose was approximately 1.5 × 10⁸–1.5 × 10⁹ CFU plant⁻¹. According to the manufacturer, all bacterial formulations are compatible when mixed, and no antagonistic interactions have been reported among the constituent strains.

Experimental design

The experiment was conducted during the 2022 growing season (2 May–20 August 2022) as a factorial randomized complete block design (RCBD) with three replications to account for field heterogeneity. Two experimental factors were evaluated: (i) Thymbra spicata L. extract at three concentrations, 0% (E0), 10% (TE1), and 20% (TE2), and (ii) biofertilizer at four levels: control (C), seaweed biostimulant (SB, 1%), bacterial biofertilizer (BB, 10%), and their combination (SB + BB). The concentrations of T. spicata extract were selected based on previous studies demonstrating their effectiveness under horticultural conditions [29, 30]. The factorial arrangement resulted in 12 treatment combinations, which were randomly assigned to the experimental plots within each block.

Field layout and crop management

The experimental field was prepared by plowing with a reversible plow followed by disk harrowing to obtain a uniform seedbed. The experimental area was divided into three blocks, each consisting of 12 experimental plots, giving a total of 36 plots. Each experimental plot consisted of a single 3-m row. Adjacent blocks were separated by 2.0 m, while neighboring rows within each block were spaced 1.5 m apart. Cucumber seeds (Cucumis sativus L. var. eureka; Pakan Seed Co., Isfahan, Iran) were sown directly on 2 May 2022 at a depth of 3 cm. Rows were spaced 1.5 m apart, with 0.60 m between plants within each row. To minimize border effects, the first and last 0.30 m of each row were left unplanted, providing an effective row length of 2.4 m in which five plants were established per experimental plot. Consequently, the net harvested area of each experimental plot was 3.6 m² (2.4 m × 1.5 m). Each treatment consisted of five plants per replication (15 plants per treatment), resulting in a total of 180 plants across the experiment.

Before planting, well-decomposed farmyard manure was incorporated into the soil at a rate of 540 kg over the 215 m² net experimental area (equivalent to approximately 25 t ha⁻¹). No supplemental fertilizers were applied during the experiment, and all treatments received identical conventional crop management except for the assigned experimental treatments. Irrigation was supplied using a drip tape system every 2–3 days for approximately 3 h, with irrigation scheduling adjusted according to the soil field capacity. Weed control was performed manually throughout the growing season, and no chemical herbicides or pesticides were applied.

During the experimental period, climatic conditions gradually changed from relatively mild to hot and dry. Maximum air temperature increased from approximately 22 °C in May to 41 °C in August, while rainfall declined to nearly zero during July and August, resulting in typical summer growing conditions for the region.

Treatment application

The first bacterial inoculation and foliar applications were performed when plants reached the four- to six-leaf stage (31 May–1 June 2022). The second applications were carried out at the beginning of flowering (14–15 June 2022), and the third applications were performed at the onset of fruit set (25–26 June 2022). Harvesting commenced on 2 July and continued until 20 August 2022.

At the four-leaf stage, plant roots were inoculated with the bacterial biofertilizer (BB) by injection into the soil around the root zone, while foliar applications of the seaweed biostimulant (SB) and T. spicata extract (TE) were initiated simultaneously. For each BB application, three holes were carefully made around the root zone of each plant to avoid root injury. A total of 50 mL of a 10% BB suspension was equally divided among the three holes and injected using a syringe. To maintain close contact between the bacterial inoculum and the actively developing root system, the distance of the injection holes from the plant stem was gradually increased from 5 cm at the four- to six-leaf stage (first application) to 10 cm at flowering (second application) and 15 cm at the onset of fruit development (third application). After each BB application, the injection holes were gently covered with soil to minimize evaporation and microbial contamination.

Foliar applications of SB and TE were carried out between 17:00 and 19:00 h using a hand sprayer until runoff. The exact spray volume per plant was not recorded, as all treatments were applied uniformly under the same spraying conditions. No surfactant was added to the spray solutions. To minimize spray drift between adjacent plots, applications were performed under calm weather conditions with little or no wind, and the spray nozzle was carefully directed toward the target plants while maintaining adequate spacing between experimental plots.

Soil characteristics

To assess the soil’s physicochemical properties, three soil samples were randomly collected from the experimental site before planting. These samples were sent to the Soil Science Laboratory at Ilam University for analysis. The results of the analysis are presented in Table 1. This baseline characterization ensured that the experimental conditions were consistent and suitable for evaluating the impact of the treatments.

Table 1.

Physiochemical characteristics of the field soil

Moisture percentage (%)	Sand (%)	Clay (%)	Silt (%)	Soil pattern	Acidity	EC (dS m⁻¹)	Organic carbon (%)	Total nitrogen (%)	Phosphorus (ppm)	Potassium (ppm)
33	24	11	65	Loam-Silt	7.2	0.91	0.432	0.05	37.07	340.10

Sampling and trait measurement

Sampling was conducted in two stages to align with the research objectives: morphological traits were measured during the flowering stage, whereas physiological, biochemical, and post-harvest traits were evaluated during the fruiting period. To preserve tissue quality, plant samples were immediately transported from the field to the laboratory in ice packs. Fresh tissue samples were gently blotted with tissue paper to remove surface moisture and immediately frozen in liquid nitrogen before biochemical analyses.

Morphological traits

Morphological traits were assessed by selecting five cucumber plants from the center of each experimental plot. Plant height, measured from the soil surface to the tip of the main stem, was recorded using a tape measure with a precision of 1 mm. To determine leaf area, three leaves were sampled from each plant, and their dimensions were measured using grid paper for accurate estimations. The number of lateral branches emerging from the main stem was recorded at two distinct growth stages: the early development phase and as the plants approached senescence. This approach ensured a comprehensive evaluation of growth dynamics throughout the crop’s lifecycle.

Physiological and biochemical traits

Leaf relative water content (RWC) and electrolyte leakage (EL)

To determine RWC and EL, six leaf disks (1 cm in diameter) were collected from fully developed young leaves. The fresh weight (FW) of the leaf disks was recorded using a digital scale (SAS-3031, Tehran, Iran). The disks were then immersed in distilled water (dH₂O) and refrigerated for 4 h to allow them to reach full turgor. After this period, the disks were carefully removed from the water, blotted dry between two layers of filter paper to eliminate surface moisture, and their turgid weight (TW) was measured. Subsequently, the samples were oven-dried at 70 °C for 48 h to determine their dry weight (DW). RWC was calculated using the following equation provided by González and González-Vilar [31]:

Electrolyte leakage (EL) was quantified following the method described by Karray-Bouraoui et al. [32]. This dual approach enabled a detailed assessment of plant water status and membrane integrity under different treatments.

Total chlorophyll content

To determine total chlorophyll content, a 500 mg leaf tissue sample was homogenized in 5 mL of 80% acetone. The resulting homogenate was centrifuged at 4 °C for 15 min at 13,000 rpm using a PrismR centrifuge (Labnet, USA). The supernatant was collected, and its volume was adjusted to 10 mL with 80% acetone. Absorbance was measured at 646.6 and 663.6 nm using a spectrophotometer (Specord 50, Analytik Jena AG, Germany). Total chlorophyll content was calculated according to the equations of Lichtenthaler [33] and expressed as mg g⁻¹ fresh weight (FW).

Total phenol and flavonoid contents

The total phenolic content in cucumber leaves was determined using the Folin-Ciocalteu method. Fresh leaf tissue (0.1 g) was homogenized in 95% methanol, and 200 µL of the extract was mixed with 800 µL of 1 N Folin-Ciocalteu reagent. After incubating the mixture at room temperature for 5 min, 3 mL of 15% sodium carbonate was added, followed by a 30-minute incubation at room temperature. Absorbance was measured at 765 nm using a spectrophotometer. For the preparation of the standard curve, serial solutions of gallic acid were prepared in the concentration range of 0–100 µg/mL. Total phenolic content was then calculated from this curve and expressed as mg/g FW according to Ainsworth and Gillespie [34].

Total flavonoid content was determined following the method described by Ceylan et al. [35]. Serial quercetin solutions in the range of 0–50 µg/mL were used to generate the standard curve, and flavonoid concentrations were expressed as µg/g FW. These assays provided reliable estimates of total phenolic and flavonoid contents, which were used to evaluate the biochemical responses of cucumber leaves to the applied treatments.

Leaf enzyme activities

Phenylalanine ammonia-lyase (PAL) activity in cucumber leaves was assayed using frozen leaf tissue. Briefly, 5 g of frozen samples were homogenized for 2 min in 15 mL of 100 mM borate buffer (pH 8.8) supplemented with polyvinylpyrrolidone (PVPP, 6 g), 5 mM β-mercaptoethanol, and 2 mM EDTA to prevent phenolic interference and enzyme degradation. The homogenate was centrifuged at 14,000 rpm for 15 min at 4 °C, and the resulting supernatant was used as the crude enzyme extract for activity determination.

PAL activity was determined by incubating 0.5 mL of enzyme extract with 2.0 mL of 0.1 M borate buffer (pH 8.8) containing 3 mM L-phenylalanine. The reaction mixture (2.5 mL) was incubated at 30 °C for 60 min, and the increase in absorbance was measured at 290 nm using a spectrophotometer. Enzyme activity was calculated from a cinnamic acid standard curve and expressed as nmol cinnamic acid equivalents (CAE) min⁻¹ g⁻¹ FW according to Habibi et al. [36].

CAT activity was determined according to Aebi [37]. The reaction mixture consisted of 2.9 mL phosphate buffer containing H₂O₂ and 0.1 mL enzyme extract (final volume 3.0 mL). The decrease in absorbance due to H₂O₂ decomposition was monitored at 240 nm immediately after mixing at 25 °C.

Antioxidant activity

To evaluate the antioxidant potential of experimental samples, free radical inhibition was quantified by monitoring the change in optical density of DPPH radicals. Following the method described by Thaipong et al. [38], 0.2 mL of sample extract was diluted with methanol, and 2 mL of DPPH solution (0.05 mM) was added to the mixture. The reaction mixture was allowed to stand in the dark at room temperature for 30 min to ensure complete interaction between the DPPH radicals and the sample antioxidants. The absorbance was then measured at 517 nm using a spectrophotometer. The antioxidant activity, expressed as the percentage of DPPH radical inhibition, was calculated using the following equation:

Salicylic acid (SA) content

Cucumber leaf samples were flash-frozen in liquid nitrogen and ground into a fine powder to preserve biochemical integrity. Salicylic acid (SA) was extracted by homogenizing 50 mg of the powdered tissue in 3 mL of chloroform, followed by centrifugation at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and maintained on ice until analysis. For SA quantification, 100 µL of the chloroform extract was mixed with 2.9 mL of freshly prepared 0.1% ferric chloride solution. After the formation of the colored complex, absorbance was measured at 540 nm using a spectrophotometer. SA concentration was determined from a standard curve prepared with authentic salicylic acid standards. The colorimetric determination of SA was performed according to the ferric chloride assay described by Warrier et al. [39], and the results were expressed as µg mL⁻¹.

Leaf nutrients

To analyze leaf nutrient composition, 15–20 leaves at the fruiting stage (50 days after sowing) were collected from mature branches located in the middle section of treated cucumber plants. The samples were carefully washed to remove surface contaminants and dried at 70 °C for 48 h. The dried leaves were prepared for digestion using a fresh oxidation method involving salicylic acid, 96% sulfuric acid, selenium, and hydrogen peroxide (H₂O₂). A 0.3-g sample of dried plant material was weighed and transferred to a digestion tube. To this, 2.5 mL of the acid mixture was added, ensuring thorough wetting of all particles through gentle shaking. The samples were then incubated in the digestion tubes for 2 h before being heated at 100 °C for an additional 2 h. After cooling, 1 mL of H₂O₂ was added incrementally three times, with careful stirring after each addition to ensure the reaction with H₂O₂ was complete. The tubes were reheated, and the temperature was gradually increased to 330 °C. The digestion was considered complete when the solution turned colorless or pale yellow. Following digestion, the mixture was allowed to cool, and 48.3 mL of deionized water was added to the tubes. The contents were thoroughly stirred and filtered to remove any insoluble particles. The filtered solution was then analyzed for nutrient content. Nitrogen (N) was determined using the Kjeldahl method [40], phosphorus (P) was quantified colorimetrically using a spectrophotometer, and potassium (K) was measured via flame photometry. Ca and Fe concentrations were analyzed using atomic absorption spectroscopy. The concentrations of N, P, and K were expressed as percentages, while Ca and Fe contents were reported in parts per million (ppm), following the procedures described by Da Silva et al. [41]. This comprehensive approach ensured accurate quantification of essential macronutrients and micronutrients in the treated plant samples.

Post-harvest traits

Total yield, firmness, weight loss, and post-harvest shelf life

The number of marketable cucumber fruits (70–120 g) was recorded at each harvest throughout the harvesting period. For each experimental plot (five plants), the cumulative number and weight of marketable fruits from all harvests were determined. The mean number of fruits per plot was calculated from the three replicate plots for each treatment. Fruit number per plant was then obtained by dividing the mean fruit number per plot by five. Fruit yield per hectare (t ha⁻¹) was reported as a standardized yield estimate, calculated from the mean fruit yield per plant using a standard planting density of 10,000 plants ha⁻¹.

Firmness was assessed immediately after harvest using a hand-held penetrometer equipped with an 8-mm cylindrical probe. Measurements were taken at three equatorial points on each cucumber, avoiding damaged or previously tested areas. The probe was applied perpendicular to the fruit surface with a gradual force until it penetrated 2–3 mm into the flesh. Firmness values were recorded in kg/cm², and the average was calculated for each fruit. All measurements were conducted at room temperature (20–25 °C), with the probe cleaned between samples.

Weight loss was calculated by weighing fruits at harvest (W₁) and reweighing them at the end of shelf life (W₂) using a digital scale. The percentage of weight loss was determined using the formula:

Fruits were harvested at the commercial maturity stage and stored in plastic bags under controlled conditions, with a constant temperature of 25 ± 2 °C and relative humidity of 55 ± 5%. The commercial maturity stage of the fruit was determined based on criteria including uniform green color, firmness ≥ 6 kg cm⁻², and fruit length ranging from 15 to 18 cm. To assess quality changes during the postharvest period, parameters such as fruit firmness, total soluble solids (TSS), fruit weight at harvest (day 0), and weight loss were measured at 24-hour intervals until the appearance of spoilage symptoms, including wrinkling, indentation, or softening. This approach allowed precise monitoring of fruit quality and determination of the postharvest shelf life.

Total soluble solids (TSS), titratable acidity (TA), and TSS/TA ratio

The TSS content was measured at room temperature in °Brix using an N1 Atago manual refractometer [42]. To determine titratable acidity (TA), 2 mL of fruit juice was diluted with 10 mL of distilled water and titrated with 0.1 N sodium hydroxide until the pH reached 8.2 ± 0.1 [43]. Titratable acidity (%) was calculated according to the following equation:

where V_NaOH is the volume (mL) of 0.1 N NaOH used for titration, N_NaOH is the normality of NaOH, E_acid is the equivalent weight of citric acid (0.064 g meq − 1), and V_sample is the fruit juice volume (2 mL). The TSS/TA ratio (ripening index), an objective indicator of fruit taste quality reflecting the balance between sweetness and acidity, was subsequently calculated by dividing TSS (°Brix) by TA (%). All measurements were performed three days after storage.

For postharvest quality assessments, five healthy fruits of uniform size and appearance were randomly selected from each experimental plot at the end of the storage period. The measurements for each trait were averaged to obtain a single representative value for each experimental unit (plot). These mean values were then used for statistical analysis to evaluate the effects of treatments relative to the control.

Fruit enzyme activity and vitamin C content

Catalase (CAT) enzyme activity in fruit was assessed according to the method described in the previous section for leaf enzyme activities. For ascorbate peroxidase (APX) activity, the reaction mixture contained 250 µL of 100 mM phosphate buffer (pH 7.0), 250 µL of 1 mM ascorbate, 250 µL of 0.4 mM EDTA, 190 µL of distilled water, 10 µL of 10 mM H₂O₂, and 50 µL of enzyme extract. The total reaction volume was 1 mL, and the reaction was carried out at room temperature (approximately 25 °C). The decrease in absorbance at 290 nm was recorded immediately after initiation of the reaction and after 1 min. APX activity was calculated based on the oxidation of ascorbate and expressed as µM oxidized ascorbate min⁻¹ g⁻¹ FW according to Yoshimura et al. [44]. The enzyme extract concentration was approximately 0.11 g FW mL⁻¹, based on the tissue-to-extraction volume ratio. Vitamin C content was determined using the method outlined by Ranganna [45] and expressed in mg/100 g FW.

Data analysis

Data were analyzed using a two-way ANOVA, appropriate for the factorial RCBD design, in SAS (version 9.4). Before ANOVA, the normality of residuals was assessed using the Shapiro-Wilk test, and homogeneity of variances was verified using Levene’s test (or Bartlett’s test, as appropriate for the RCBD design). This analysis evaluated both the main and interaction effects of the treatments while considering blocks as a random factor to account for environmental variability. Statistical significance of the effects was assessed using the F-test, and when significant differences were detected, means were separated using the LSD test at a 5% significance level. The results were subsequently summarized and presented in tables and figures to clearly and effectively illustrate the findings. Principal component analysis (PCA) and correlation analysis (heatmap) were performed in RStudio (version 1.3.959) to evaluate relationships among traits and treatment dispersion.

Results

Morphological traits

The application of the biostimulants and biofertilizers significantly influenced the vegetative growth parameters of cucumber plants. Compared to the control (123.67 cm height, 8.67 lateral branches, 7.49 cm² leaf area), various treatments demonstrated marked improvements. Notably, the TE1 treatment alone resulted in the most substantial increase in plant height (52.82% over control), reaching 189.00 cm at the 10% TE concentration. While TE1 alone was optimal for height, the combination of TE2 + SB yielded the highest number of lateral branches (19.00), representing a 119% enhancement compared to the control. Furthermore, the most significant boost in leaf area was observed with the TE1 + BB treatment, achieving 18.27 cm² (a 143.93% increase from the control) (Table 2). These findings highlight the differential efficacy of specific biostimulant and biofertilizer combinations in promoting distinct aspects of plant growth, underscoring the potential for tailored applications to optimize crop development.

Table 2.

Effect of thyme extract (0, 10 and 20%), seaweed biofertilizer (1%) and bacterial biofertilizer (10%) treatments on the morphological characteristics of the cucumber plant (Cucumis sativus L. var. eureka)

Extracts (%)	Biofertilizer	Plant height (cm)	No. of lateral branches	Leaf area (cm2)
	Control	123.67 ± 8.6e	8.667 ± 0.5ef	7.49 ± 0.97h
No Extract (E0)	Seaweed (SB)	159.17 ± 13.8bcd	14.00 ± 1.8bc	8.27 ± 0.27gh
	Bacteria (BB)	143.33 ± 6.9de	10.66 ± 1def	10.89 ± 0.44cde
	Seaweed + Bacteria (SB + BB)	166.33 ± 12.8abcd	15.00 ± 1.8b	9.79 ± 1.24efg
	Control	189.00 ± 6.3a	15.00 ± 0.9b	10.55 ± 0.5def
Extract 10 (TE1)	Seaweed (SB)	155.75 ± 10.1bcd	9.66 ± 1.9def	14.86 ± 0.72b
	Bacteria (BB)	173.00 ± 5.8abc	11.33 ± 1.9cde	18.27 ± 1.54a
	Seaweed + Bacteria (SB + BB)	151.50 ± 4.9cd	12.33 ± 0.5bcd	12.13 ± 1.02c
	Control	147.33 ± 8.8de	11.33 ± 2.3cde	12.29 ± 0.36c
Extract 20 (TE2)	Seaweed (SB)	178.67 ± 15.4ab	19.00 ± 0.9a	11.55 ± 1.85cd
	Bacteria (BB)	143.67 ± 27.1de	8.00 ± 1.8f	9.00 ± 0.1fgh
	Seaweed + Bacteria (SB + BB)	168.17 ± 17.3abcd	11.33 ± 1.9cde	15.23 ± 0.9b

*Means with at least one common letter in each column indicate non-significance at the 5% probability of the LSD test

Physiological and biochemical traits

Leaf relative water content (RWC) and electrolyte leakage (EL)

Regarding physiological traits, results indicated significant variation among treatments. The highest RWC was observed in the TE2 + BB treatment, showing a 27.27% increase compared to the control (Fig. 1A). While TE2 + SB + BB and TE1 + SB + BB also exhibited higher RWC values than the control, they remained lower than the peak recorded in TE2 + BB. In terms of membrane integrity, the lowest EL was associated with the TE2 treatment, which resulted in a 80.44% decrease compared to the control (Fig. 1B). Significant reductions in EL relative to the control were also observed in TE2 + SB, TE2 + SB + BB, and TE1 + BB, whereas SB + BB and TE1 + SB + BB treatments demonstrated more moderate reductions in leakage.

Fig. 1.

The effect of thyme extract (0, 10 and 20%), seaweed biofertilizer (1%), and bacterial biofertilizer (10%) on the physiological and biochemical traits of cucumber (Cucumis sativus L. var. eureka): (A) relative water content (RWC), (B) electrolyte leakage (EL), (C) total chlorophyll, (D) total phenol, (E) flavonoid, (F) phenylalanine ammonia-lyase (PAL) activity, (G) catalase (CAT) activity, (H) antioxidant activity, and (I) salicylic acid content. E0: without thyme extract; TE1: thyme extract 10%; TE2: thyme extract 20%; C: control; SB: seaweed biofertilizer; BB: bacterial biofertilizer; SB+BB: seaweed+bacterial biofertilizer. Different lowercase letters indicate significant differences among treatments at the 5% significance level using the LSD test

Chlorophyll content

The highest total chlorophyll content was recorded in the SB + BB treatment without extract, showing a 151% increase relative to the control. The TE1 + SB + BB and TE2 + SB + BB treatments also exhibited elevated chlorophyll contents, although their values did not reach those observed in SB + BB. The BB treatment alone, without extract, showed lower chlorophyll content than SB + BB, while the lowest chlorophyll levels were observed in the control and in treatments containing TE2 without biofertilizer (Fig. 1C).

Total phenol and flavonoid contents

Data analysis revealed that the BB treatment increased the total phenol content by 99.8% compared to the untreated control. The SB + BB and TE1 + SB + BB treatments also displayed higher phenol levels than the control (Fig. 1D). Regarding total flavonoid content, the highest values were observed in the TE1 + SB + BB treatment, showing an 9.14% increase relative to the control. Additionally, the TE1 + BB and TE2 + SB + BB treatments exhibited increased flavonoid content, whereas the lowest levels were observed in the control and in treatments containing TE2 without bacterial inoculation (Fig. 1E).

Leaf enzyme activity

According to the comparison of means, the TE2 + SB + BB treatment enhanced PAL activity by 12.23% (Fig. 1F). The TE1 + SB + BB and TE2 + SB treatments also showed an increase in PAL activity compared to the control. In terms of CAT activity, the highest levels were observed in the TE2 treatment (without biofertilizer), with a 5.7% increase relative to the untreated control. Treatments TE2 + SB and TE2 + BB also exhibited higher CAT activity than the control, whereas SB + BB and TE1 + SB + BB treatments showed lower CAT activity compared to the control (Fig. 1G).

DPPH antioxidant activity

The results showed that DPPH antioxidant activity was highest in the TE1 + SB + BB treatment, with a 72.93% increase relative to the untreated control. The TE2 + SB + BB treatment also exhibited high DPPH activity, close to that of TE1 + SB + BB. The lowest DPPH activity was observed in the control and in treatments lacking extract and bacterial inoculation, such as SB alone (Fig. 1H).

Salicylic acid (SA)

The results revealed that the SA content was significantly affected by the treatments used. The BB treatment (without extract) recorded the highest SA content, showing a 366.66% increase compared to the control. This was followed by the SB treatment, which was associated with a 341.17% increase (Fig. 1I).

Leaf nutrients

Based on the comparison of mean values, plants treated with SB + BB had 24.2% higher N content and 78.26% higher P content than the control. Plants treated with TE2 + SB + BB showed a 244% increase in potassium content and a 31.11% increase in calcium content, while those treated with TE1 + BB had a 254.34% increase in iron content compared to the control plants (Table 3).

Table 3.

The effect of thyme extract (0, 10 and 20%), seaweed biofertilizer (1%), and bacterial biofertilizer (10%) on the element content of cucumber leaves (Cucumber sativus L. var. eureka)

Extracts (%)	Biofertilizer	Nitrogen (%)	Phosphorus (%)	Potassium (%)	Calcium (ppm)	Iron (ppm)
	Control	3.02 ± 0.126d	1.84 ± 0.085d	1.02 ± 0.054j	415.66 ± 5.391g	3.57 ± 0.076h
No Extract (E0)	Seaweed (SB)	3.11 ± 0.135d	2.17 ± 0.108c	2.09 ± 0.076d	416.66 ± 8.066g	5.49 ± 0.117de
	Bacteria (BB)	3.31 ± 0.148c	1.48 ± 0.094g	1.61 ± 0.072g	488.33 ± 11.673cd	5.20 ± 0.045ef
	Seaweed + Bacteria (SB + BB)	3.75 ± 0.148a	3.28 ± 0.112a	2.28 ± 0.063c	504.33 ± 2.683b	5.39 ± 0.108ef
	Control	3.52 ± 0.126b	1.88 ± 0.09d	1.52 ± 0.059h	423.66 ± 9.423fg	6.84 ± 0.076c
Extract 10 (TE1)	Seaweed (SB)	3.30 ± 0.135c	1.66 ± 0.09e	2.45 ± 0.063b	369.66 ± 9.423h	4.70 ± 0.094g
	Bacteria (BB)	3.35 ± 0.148c	1.11 ± 0.085h	2.24 ± 0.059c	461.33 ± 8.066e	13.95 ± 0.13a
	Seaweed + Bacteria (SB + BB)	3.69 ± 0.157a	2.55 ± 0.068b	1.99 ± 0.059e	491.33 ± 6.282bc	5.82 ± 0.058d
	Control	3.03 ± 0.135d	1.61 ± 0.121ef	1.86 ± 0.054f	475.33 ± 7.174de	5.14 ± 0.112f
Extract 20 (TE2)	Seaweed (SB)	3.71 ± 0.148a	1.64 ± 0.081e	2.26 ± 0.049c	432.66 ± 8.532f	12.62 ± 0.081b
	Bacteria (BB)	3.03 ± 0.148d	1.55 ± 0.076f	1.40 ± 0.068i	103.66 ± 5.391i	4.71 ± 0.162g
	Seaweed + Bacteria (SB + BB)	3.67 ± 0.143a	2.59 ± 0.103b	3.51 ± 0.072a	545.00 ± 13.456a	12.65 ± 0.094b

*Means with at least one common letter in each column indicate non-significance at the 5% probability of the LSD test

Post-harvest traits

Total yield, firmness, weight loss, and post-harvest shelf life

Data analysis indicated that the TE1 + SB + BB treatment increased fruit number by 29.32%, total yield by 43.23%, and post-harvest shelf life by 121.67%, while reducing fruit weight loss by 45.67% compared with the untreated control. For fruit firmness, no significant differences were observed among BB, TE1, and TE1 + SB + BB, which exhibited increases of 25.46%, 25.46%, and 22.34%, respectively, compared with the untreated control (Table 4).

Table 4.

The effect of thyme extract (0, 10 and 20%), seaweed biofertilizer (1%) and bacterial biofertilizer (10%) on the postharvest traits of the cucumber fruit (Cucumis sativus L. var. eureka)

Extracts (%)	Biofertilizer	Mean fruit number/plot (5 plants)	Total yield (t ha⁻¹)	Shelf life (days)	Firmness (kg cm⁻²)	Fruit weight loss (%)
	Control	237.00 ± 4.025bc	37.64 ± 1.80ef	4.66 ± 0.5f	6.40 ± 0.089cd	8.43 ± 1.2a
No Extract (E0)	Seaweed (SB)	263.00 ± 2.862abc	46.61 ± 2.40cd	7.00 ± 0.1d	5.86 ± 0.635cd	6.40 ± 0.8abcd
	Bacteria (BB)	235.33 ± 8.475bc	47.69 ± 0.05c	8.00 ± 0.2c	8.03 ± 0.186a	4.78 ± 0.9d
	Seaweed + Bacteria (SB + BB)	228.33 ± 0.179cd	42.96 ± 1.40de	6.66 ± 0.5d	6.50 ± 0.268cd	6.72 ± 1.5abc
	Control	184.67 ± 3.309de	34.09 ± 0.22fg	9.00 ± 0.2b	8.03 ± 0.423a	4.91 ± 0.8cd
Extract 10 (TE1)	Seaweed (SB)	278.00 ± 2.504ab	40.93 ± 2.23de	6.00 ± 0.4e	5.96 ± 0.361cd	6.93 ± 0.8abc
	Bacteria (BB)	177.00 ± 2.504ef	32.93 ± 0.62g	8.00 ± 0.1c	6.46 ± 1.446cd	5.20 ± 0.3cd
	Seaweed + Bacteria (SB + BB)	306.50 ± 10.89a	53.91 ± 1.92a	10.33 ± 0.5a	7.83 ± 0.314ab	4.58 ± 0.3d
	Control	133.00 ± 1.968f	27.84 ± 1.63bc	6.00 ± 0.2e	7.00 ± 0.78abc	6.87 ± 0.7abc
Extract 20 (TE2)	Seaweed (SB)	219.33 ± 1.789cde	39.76 ± 2.02ef	7.00 ± 0.1d	5.80 ± 0.447d	6.21 ± 1.3bcd
	Bacteria (BB)	286.00 ± 4.204a	47.76 ± 1.73c	5.00 ± 0.1f	6.56 ± 0.186cd	8.27 ± 0.8ab
	Seaweed + Bacteria (SB + BB)	183.67 ± 0.626e	37.80 ± 0.90ef	9.00 ± 0.5b	6.73 ± 0.609bcd	5.83 ± 0.6cd

*Means with at least one common letter in each column indicate non-significance at the 5% probability of the LSD test

TSS, titratable acidity (TA), and TSS/TA ratio

Based on the comparison of means, the BB treatment produced the highest TSS, increasing this trait by 102.83% compared with the untreated control. The highest TSS/TA ratio was observed in TE1 + SB + BB, representing an increase of 199.66%, whereas the lowest TA was recorded in TE1, corresponding to a 61.90% reduction relative to the untreated control (Table 5).

Table 5.

The effect of thyme extract (0, 10 and 20%), seaweed biofertilizer (1%) and bacterial biofertilizer (10%) on the activity of catalase (CAT), ascorbate-peroxidase (APX) and vitamin C enzymes in the cucumber fruit (Cucumis sativus L. var. eureka)

Extracts (%)		Biofertilizer		Total soluble solids (°Brix)	Titratable acidity (%)		TSS/TA ratio		CAT (µmol/min/g FW)	APX (µmol/min/g FW)	Vitamin C (mg/100 g FW)
		Control		4.93 ± 0.258cd	0.42 ± 0.032a		11.78 ± 1.30h		0.056 ± 0.0007d	41.95 ± 0.745i	8.00 ± 0.258c
No Extract (E0)		Seaweed (SB)		5.26 ± 0.894c	0.18 ± 0.010e		29.22 ± 4.95b		0.029 ± 0.0004h	75.71 ± 0.301c	10.35 ± 0.894a
		Bacteria (BB)		10.00 ± 1.033a	0.35 ± 0.018bc		28.57 ± 1.06bc		0.042 ± 0.0007f	70.70 ± 0.83d	8.66 ± 1.033bc
		Seaweed + Bacteria (SB + BB)		4.06 ± 0.77e	0.18 ± 0.052e		22.55 ± 0.12d		0.179 ± 0.0008a	78.61 ± 0.166b	10.00 ± 0.77ab
		Control		4.33 ± 0.894de	0.16 ± 0.027e		27.06 ± 0.24c		0.131 ± 0.0007b	57.24 ± 0.242h	8.66 ± 0.894bc
Extract 10 (TE1)		Seaweed (SB)		3.90 ± 1.366e	0.32 ± 0.090c		12.18 ± 0.71g		0.064 ± 0.0006c	61.20 ± 0.548g	10.00 ± 1.366ab
		Bacteria (BB)		4.06 ± 0.894e	0.18 ± 0.026e		22.55 ± 1.53d		0.034 ± 0.0006g	68.07 ± 0.341e	8.66 ± 0.894bc
		Seaweed + Bacteria (SB + BB)		7.06 ± 1.693b	0.20 ± 0.089e		35.30 ± 3.54a		0.024 ± 0.0009i	79.14 ± 0.346b	10.66 ± 1.693a
		Control		4.00 ± 0.516e	0.30 ± 0.029bc		12.12 ± 2.01g		0.029 ± 0.0006h	28.22 ± 0.377j	10.00 ± 0.516ab
Extract 20 (TE2)		Seaweed (SB)		3.53 ± 0.54e	0.34 ± 0.036bc		10.38 ± 0.59i		0.021 ± 0.0007i	62.53 ± 0.489f	9.00 ± 0.54bc
		Bacteria (BB)		5.20 ± 0.447c	0.37 ± 0.005b		14.05 ± 2.00f		0.050 ± 0.0003e	93.92 ± 0.525a	9.00 ± 0.447bc
		Seaweed + Bacteria (SB + BB)		4.06 ± 0.258e	0.25 ± 0.037d		16.24 ± 0.59e		0.032 ± 0.0003gh	62.79 ± 0.314f	10.00 ± 0.258ab

*Means with at least one common letter in each column indicate non-significance at the 5% probability of the LSD test

Fruit enzyme activity and vitamin C content

The comparison of means revealed that the SB + BB treatment increased fruit CAT activity by 219.64%, the TE2 + BB treatment increased fruit APX activity by 123.88%, and the TE1 + SB + BB treatment increased vitamin C content by 33.25% compared to the control (Table 5).

PCA and correlation heatmap analysis

The explained variance of the principal components (Fig. 2A) showed that the first two principal components (PC1 and PC2) accounted for 24.6% and 15.0% of the total variance, respectively, explaining a cumulative 39.6% of the overall variation. The inclusion of the third and fourth principal components increased the cumulative explained variance to 62.8%, indicating that the dataset was distributed across multiple dimensions. However, the remaining components contributed negligibly, and the first two principal components captured the dominant pattern of treatment differentiation. The correlation matrix and hierarchical clustering dendrogram (Fig. 2B and C) revealed the relationships among the measured traits. EL exhibited significant negative correlations with most desirable traits, whereas total yield was positively correlated with nutrient contents, particularly N and K, as well as chlorophyll content. CAT and APX activities also showed strong positive correlations with vitamin C content and DPPH antioxidant activity. The two-dimensional PCA biplot based on PC1 and PC2 (Fig. 2D) showed that the combined treatment TE1 + SB + BB was positioned closest to the vectors representing desirable traits, including fruit number, total yield, fruit firmness, vitamin C content, antioxidant activity, P and Fe contents, shelf life, and reduced fruit weight loss, indicating that it was the most effective treatment for improving yield, fruit quality, nutritional attributes, and postharvest performance. The two-dimensional PCA biplot based on PC1 and PC2 (Fig. 2D) showed that the combined treatment TE1 + SB + BB was positioned closest to the vectors representing desirable traits, including fruit number, total yield, fruit firmness, vitamin C content, antioxidant activity, P and Fe contents, shelf life, and reduced fruit weight loss, indicating that it was the most effective treatment for improving yield, fruit quality, nutritional attributes, and postharvest performance. The PCA variables plot (Fig. 2E) further illustrated the relationships among the measured traits and their contributions to PC1 and PC2. In contrast, the control treatment and the treatments receiving the higher concentration of TE2 were located in the opposite direction of the desirable trait vectors in the PCA biplot (Fig. 2D). Finally, the heatmap (Fig. 2F) further supported the synergistic effect of the TE1 + SB + BB treatment, demonstrating that this combined treatment simultaneously improved both the vegetative–nutritional and quality–antioxidant trait groups. In contrast, the other treatments primarily influenced only one of these two trait groups.

Fig. 2.

Scree plot (A), correlation heatmap with hierarchical clustering (B), correlation matrix (C), PCA biplot (D), PCA variables plot (E), and heatmap of traits and treatments (F) for the morphological, physiological, biochemical, and postharvest characteristics of cucumber (Cucumis sativus L. var. eureka) under different treatments of Thymbra spicata L. extract (E0: no extract, TE1: 10%, and TE2: 20%) and biofertilizers (C: control, SB: seaweed biofertilizer, BB: bacterial biofertilizer). Abbreviations: FN, fruit number; TY, total yield; RWC, relative water content; NLB, number of lateral branches; PH, plant height; FF, fruit firmness; TSS, total soluble solids; TA, titratable acidity; TSS/TA, total soluble solids to titratable acidity ratio; EL, electrolyte leakage; Ch.T, total chlorophyll; LPAL, leaf PAL activity; LCAT, leaf CAT activity; DPPH, antioxidant activity; TPC, total phenolic content; TFC, total flavonoid content; FVC, fruit vitamin C content; LA, leaf area; SL, shelf life; FWL, fruit weight loss; K, leaf potassium; N, leaf nitrogen; P, leaf phosphorus; Fe, leaf iron; Ca, leaf calcium; FAPX, fruit APX activity; FCAT, fruit CAT activity; and SA, leaf salicylic acid content

Discussion

The differential responses of cucumber vegetative traits indicated that individual biostimulants and biofertilizer combinations optimized specific aspects of plant growth rather than producing identical physiological effects. TE1 was most effective in increasing plant height, TE2 + SB produced the highest number of lateral branches, and TE1 + BB resulted in the greatest leaf area, suggesting that each treatment combination promoted plant development through complementary physiological processes. Similar responses have been reported for natural biostimulants, which improve plant growth by modulating metabolic activity and physiological processes involved in plant development [46]. Plant-derived extracts can stimulate antioxidant responses and secondary metabolism, whereas seaweed-based biostimulants and beneficial bacteria enhance nutrient acquisition, plant growth, and physiological performance, collectively contributing to sustainable crop production [47–50]. The larger leaf area observed in TE1 + BB may also have resulted from improved nutrient uptake, particularly K, which plays an important role in osmotic regulation, photosynthetic activity, and maintenance of plant growth [51]. Likewise, the minerals, amino acids, vitamins, polysaccharides, and other bioactive compounds present in SB have been reported to stimulate root development, nutrient acquisition, photosynthesis, and biomass production [52–54]. The superior vegetative growth observed in different treatment combinations therefore likely resulted from complementary rather than independent physiological effects.

Treatment-specific differences were also evident in plant water relations and membrane stability. TE2 + BB produced the highest RWC, whereas TE2 alone resulted in the lowest EL, indicating that different combinations preferentially improved either water retention or membrane integrity. Excessive ROS accumulation disrupts membrane stability through lipid peroxidation, whereas enhanced antioxidant capacity limits oxidative damage and preserves membrane function [55]. The antioxidant compounds thymol and carvacrol present in TE may have contributed to the enhanced antioxidant capacity of treated plants, thereby reducing oxidative damage and contributing to the lower EL observed in TE-treated plants [56]. In contrast, the superior RWC obtained with TE2 + BB was likely associated with the complementary effects of TE and PGPB, whereby TE may have enhanced antioxidant protection while BB improved root growth and water acquisition [57]. Exopolysaccharides produced by PGPB have also been associated with improved membrane stability and stress tolerance [58]. Although BB and SB markedly increased endogenous SA content, TE1 + SB + BB produced the longest shelf life, indicating that improved postharvest performance cannot be attributed to SA alone. Instead, enhanced membrane stability, improved water status, and greater antioxidant capacity may have collectively contributed to the observed responses [59].

The chlorophyll response further demonstrated that different combinations affected specific physiological processes. SB + BB produced the highest chlorophyll content, whereas treatments containing TE2 without biofertilizers showed comparatively lower values. Because chlorophyll biosynthesis depends on adequate mineral nutrition, particularly Fe and Mg, improved nutrient availability following SB and BB application may have supported chlorophyll accumulation and photosynthetic activity [60–62]. Improved P availability may have further enhanced chloroplast function and carbon assimilation [63]. Bioactive compounds present in SB have also been associated with improved root development, nutrient uptake, and photosynthetic efficiency [64]. The superior chlorophyll content observed in SB + BB, therefore likely reflected the complementary contribution of improved nutrient acquisition and enhanced physiological activity, ultimately supporting vegetative growth.

Distinct treatment combinations also maximized different components of the antioxidant system. BB alone produced the highest phenolic content, whereas TE1 + SB + BB resulted in the greatest flavonoid accumulation. Similarly, TE2 + SB + BB showed the highest PAL activity, while TE2 alone produced the greatest CAT activity. These contrasting responses suggest that individual antioxidant-related traits were regulated through different physiological pathways. Phenolic and flavonoid biosynthesis has frequently been associated with enhanced PAL activity and improved nutrient availability [65–67]. Consequently, the higher phenolic and flavonoid contents observed in BB- and TE-containing treatments may have resulted from improved secondary metabolism supported by better nutritional status and complementary physiological interactions among the applied biostimulants [68].

The increased PAL and CAT activities observed in the present study were also consistent with improved physiological performance. Adequate Fe availability is essential for the activity of numerous metabolic enzymes [69], whereas SB supplies soluble sugars, amino acids, betaine, proteins, and other bioactive compounds that may enhance enzymatic activity [70]. PGPB present in BB also produce metabolites that support plant metabolism and stress tolerance [71]. Likewise, TE contains antioxidant compounds such as terpenoids, flavonoids, isoprenoids, and carvacrol that may further strengthen antioxidant defense [72, 73], while sulfated polysaccharides and other constituents of SB have been associated with enhanced antioxidant capacity [53, 74]. Improved nutrient acquisition by BB may also have contributed to higher antioxidant enzyme activities through better maintenance of plant nutritional status and ionic balance [75, 76].

DPPH antioxidant activity was greatest in TE1 + SB + BB, whereas TE2 + SB + BB produced a comparable response, indicating that the simultaneous application of TE, SB, and BB enhanced antioxidant capacity more effectively than individual treatments. The complementary action of antioxidant compounds supplied by TE, together with the improved nutritional status and physiological activity promoted by SB and BB, may therefore explain the superior antioxidant performance of the combined treatments. This integrated response likely contributed to the overall improvement in plant growth and physiological performance observed throughout the study.

The endogenous SA content responded differently from most postharvest traits. BB alone produced the highest SA concentration, followed by SB, whereas the longest shelf life and highest yield were obtained with TE1 + SB + BB. These findings indicate that although SA may have contributed to improved defense responses, enhanced postharvest performance was unlikely to be mediated solely by SA. SA is widely recognized as a signaling molecule involved in plant defense, growth, and postharvest physiology [77, 78]. Components of seaweed extracts, including laminarin and alginate oligosaccharides, have been reported to influence endogenous SA signaling pathways [79–82], while Pseudomonad-based biofertilizers may stimulate systemic acquired resistance [83]. The superior performance of TE1 + SB + BB therefore was more likely associated with the complementary action of improved antioxidant capacity, membrane stability, nutrient acquisition, and physiological performance than with increased SA concentration alone.

Leaf nutrient composition further supported the complementary effects of the combined biostimulants. SB + BB produced the highest N and P concentrations, TE2 + SB + BB maximized K and Ca accumulation, and TE1 + BB resulted in the highest Fe content. These treatment-specific responses suggest that different combinations optimized the acquisition of individual nutrients rather than producing a uniform nutritional response. Beneficial microorganisms may improve N availability through biological nitrogen fixation [84–86], while phosphate- and potassium-solubilizing bacteria increase the availability of P and K, supporting photosynthesis, metabolism, and plant growth [87–91]. Growth-promoting bacteria may also improve Ca availability and enhance Fe acquisition through siderophore production [92–94]. In addition, SB provides essential macro- and micronutrients together with oligosaccharides that promote root growth, nutrient absorption, and photosynthetic activity [95–97]. The superior nutrient status observed in the combined treatments therefore likely resulted from complementary improvements in nutrient availability and uptake rather than from a single physiological mechanism.

The improvement in total yield and post-harvest performance was most evident in TE1 + SB + BB, which simultaneously increased fruit number, total yield, and shelf life while reducing fruit weight loss. In contrast, fruit firmness did not follow the same response pattern, as no significant differences were observed among BB, TE1, and TE1 + SB + BB, indicating that this trait responded differently from the other post-harvest characteristics. Seaweed extracts have been associated with enhanced photosynthesis, assimilate production, fruit development, and yield [98–101], whereas PGPB improve nutrient availability, water uptake, and the production of growth-promoting compounds that support plant productivity [102, 103]. The superior yield obtained with TE1 + SB + BB therefore was likely associated with the complementary effects of TE, SB, and BB, where improved nutrient acquisition, greater photosynthetic capacity, enhanced antioxidant protection, and improved water relations collectively promoted fruit production and postharvest performance. Secondary metabolites present in TE may also have contributed to improved fruit quality because phenolic compounds possess antioxidant activity and may reduce oxidative damage during fruit development and storage [104, 105]. Previous studies have suggested that TE extracts may influence ethylene biosynthesis and the activity of enzymes involved in cell wall metabolism [106, 107]. Such mechanisms may have contributed to the improved fruit firmness and extended shelf life observed in TE-containing treatments. Likewise, reduced ethylene production together with lower activities of polygalacturonase, lipoxygenase, cellulase, and pectin-methylesterase may have delayed fruit softening and respiration during storage [108–112]. Although these mechanisms were not directly examined in the present study, the improved water status, membrane stability, antioxidant capacity, and overall physiological condition observed in TE-containing treatments provide plausible biological explanations for the enhanced postharvest performance. The biofertilizers used in the present study also appeared to contribute to delayed fruit softening and improved storage performance. Natural hormones, amino acids, antioxidants, and other bioactive compounds present in SB may have helped maintain fruit firmness while slowing metabolic changes during storage [113, 114]. The combined action of SB and BB may therefore have complemented the physiological effects of TE, contributing to lower fruit weight loss and longer shelf life in the combined treatments rather than through a single biochemical pathway alone.

TSS, TA, and the TSS/TA ratio responded differently to the applied treatments, indicating that the same physiological processes did not necessarily regulate fruit taste attributes and overall fruit quality. Although the BB treatment produced the highest TSS and the TE1 treatment resulted in the lowest TA, the combined treatment TE1 + SB + BB achieved the highest TSS/TA ratio and provided the best overall postharvest performance. This finding suggests that the TSS/TA ratio was determined by the balance between soluble sugar accumulation and organic acid metabolism rather than by either parameter alone. TSS reflects the accumulation of soluble sugars resulting from photosynthesis and assimilate translocation, whereas TA depends largely on the concentration of organic acids that gradually decline during ripening and storage because of respiratory metabolism [115]. The higher TSS observed in the BB treatment may have been associated with improved photosynthetic performance and enhanced carbohydrate accumulation in developing fruits. Although BB alone maximized TSS, its combination with seaweed biostimulant and the 10% T. spicata extract produced a more favorable balance between soluble sugars and organic acids, leading to the highest TSS/TA ratio. Such complementary responses are consistent with the ability of biofertilizers to enhance nutrient availability, increase leaf area, and improve assimilate production and translocation [116, 117]. Likewise, the lower TA observed in the TE1 treatment may indicate changes in organic acid metabolism during fruit development and storage, potentially associated with altered respiratory activity and fruit maturation [118, 119].

Although the TSS/TA ratio is widely used as an indicator of the balance between sweetness and acidity, this parameter should not be considered a comprehensive measure of fruit quality. The present study demonstrated that maximizing a single fruit quality attribute did not necessarily result in the greatest overall fruit quality. While the BB treatment produced the highest TSS and the TE1 treatment exhibited the lowest TA, the combined TE1 + SB + BB treatment integrated favorable taste characteristics with superior agronomic and postharvest performance by increasing total yield, extending shelf life, reducing fruit weight loss, and enhancing vitamin C content and antioxidant activity. These findings emphasize that treatment performance should be evaluated using multiple agronomic, physiological, nutritional, and postharvest attributes rather than relying solely on the TSS/TA ratio.

Fruit antioxidant responses also differed among treatments. SB + BB produced the highest CAT activity, whereas TE2 + BB resulted in the greatest APX activity, indicating that antioxidant enzyme responses were optimized by different combinations of biostimulants and biofertilizers. Extracts from medicinal plants have been reported to influence fruit physiology and postharvest metabolism [120, 121]. Likewise, PGPB present in BB treatment have been associated with enhanced antioxidant enzyme activity, potentially through improved physiological status and stress tolerance [122, 123]. Improved nutrient availability following SB and BB application may also have supported antioxidant enzyme activity because many antioxidant enzymes require mineral cofactors for optimal function [75, 124, 125]. The diverse mineral composition and bioactive constituents of SB, together with the nutrient-solubilizing capacity of PGPB, therefore may have collectively enhanced antioxidant metabolism by improving plant nutritional status and ionic balance [75].

Vitamin C generally declines during storage because increased oxidative metabolism accelerates the accumulation of ROS and the degradation of ascorbic acid [126]. Antioxidant systems help limit this process, with APX utilizing vitamin C as a substrate during ROS detoxification [127]. In the present study, TE-containing treatments, particularly TE1 + SB + BB, maintained the highest vitamin C content, whereas SB + BB and TE2 + BB maximized CAT and APX activities, respectively. These findings suggest that vitamin C preservation was associated with the complementary effects of TE, SB, and BB rather than with a single antioxidant mechanism. Reduced oxidative damage together with improved nutrient availability, especially P and K, may have supported vitamin C biosynthesis and accumulation [128–130]. Furthermore, the bioactive compounds naturally present in seaweed extracts, including carotenoids, phenolic compounds, flavonoids, and vitamins, may also have contributed to preserving ascorbic acid during storage [131].

The multivariate analyses further strengthened the physiological interpretation of the experimental results. Although different treatments maximized individual traits, PCA clearly demonstrated that TE1 + SB + BB provided the best overall performance by simultaneously improving fruit number, total yield, fruit firmness, vitamin C content, antioxidant activity, nutrient accumulation, shelf life, and reducing postharvest weight loss. Correlation analysis further supported these responses by revealing strong positive associations among nutrient status, chlorophyll content, antioxidant activity, vitamin C, and yield, whereas EL showed negative correlations with most desirable traits. These relationships indicate that improvements in nutritional status, physiological performance, antioxidant capacity, and postharvest quality were closely interconnected rather than acting independently. Collectively, these results highlight the value of integrating plant-derived biostimulants with microbial and seaweed-based biofertilizers as a sustainable strategy for simultaneously enhancing plant productivity, fruit quality, and storage life.

Conclusions

This study demonstrated that the combined application of TE1 + SB + BB provided the best overall performance under the tested field and postharvest conditions. Although some individual or partially combined treatments were superior for specific physiological or vegetative traits, PCA confirmed that TE1 + SB + BB achieved the most balanced and integrated improvement by simultaneously enhancing fruit number, total yield, antioxidant capacity, vitamin C content, postharvest shelf life, and reducing fruit weight loss. These overall improvements were likely associated with the complementary physiological effects of thyme extract, seaweed biofertilizer, and bacterial biofertilizer on plant nutrition, antioxidant status, and postharvest performance. Therefore, TE1 + SB + BB can be considered a promising pre-harvest biostimulant strategy for improving cucumber productivity, fruit quality, and storage performance under the conditions evaluated in this study. However, further validation across different growing seasons, cultivars, environments, and commercial storage conditions is required before broader recommendations for sustainable cucumber production can be made.

Authors’ contributions

Bahareh Karami: Conceptualization, Validation, Project administration, Investigation, Data curation, Writing the original draft. Majid Bagnazari: Writing – review & editing, Methodology, Supervision. Meisam Mohammadi: Data analysis, Review & editing. Afsaneh Azizi: Writing – original draft, Review & editing, Visualization, Methodology. All authors reviewed the manuscript.

Funding

Partial financial support was received from the Vice-Chancellor for Research and Technology at Ilam University, Ilam, Iran.

Data availability

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

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.