Evaluating a Natural-Based Solution for Its Stimulation in Cucumis sativus Plants and Fruits

Abstract

The current study researched the biostimulant impacts of a natural-based solution (NBS) that contained eucalyptus and rosemary essential oils on cucumber crops. The effects of NBS (one time-NBS1; two times-NBS2) application on plant development and physiological attributes (chlorophylls, stomatal conductance), total phenolics, non-enzymatic and enzymatic antioxidant activities, leaf minerals content, cucumber quality attributes at harvest and after one-week storage were assessed through experiments. NBS1 spraying was less effective than NBS2 application because it resulted in a decrease in mineral accumulation (like reduced nitrogen) and other physiological characteristics (like chlorophylls). The plants’ enhanced oxidative stress and activation of several enzymatic antioxidant systems were reflected in the use of a commercial solution (CS) based on amino acids and biostimulants, which also boosted stomatal conductance, reduced nitrogen, calcium, and magnesium accumulation, and antioxidant capacity. No differences were found in plant height, number of leaves, plant biomass, chlorophyll fluorescence, total phenols, and various fruit quality attributes, including firmness, fresh weight, respiration rates, total soluble solids, ascorbic acid, decay, and marketability among the treatments. In fact, the effects of both CS and NBS treatment on cucumber plants and fruits were less pronounced, suggesting that more than two applications should be explored in the future.

1. Introduction

The worldwide shortage of food is a major concern for human beings, given that by 2050, there will be 9.5 billion people on Earth. One of the most significant issues confronting the agricultural industry is securing and optimizing yields of crops in the context of climate change [1]. The supply of biostimulants to boost agricultural productivity is one area of research aimed at assuring the food supply [2,3]. Biostimulants are a wide variety of chemicals and/or microorganisms employed in agriculture to improve soil fertility, plant stress resistance, plant development, and yield of high-quality crops [2,3,4]. Moreover, biostimulants affect the shelf life of fresh produce by modulating the plant growth and stress responses of plants, resulting in higher fresh produce quality [5,6,7].

In horticulture, biostimulants derived from marine algae and amino acids have been used extensively [8] due to their abundance in polyphenol-based compounds and phytohormones [2,9]. In recent years, materials including silicon, protein hydrolysate, humic and fulvic acids, and others have been studied with encouraging results to either preserve yield under adverse conditions or boost it under favorable environments [1,5,10]. Furthermore, food production may be supported with biostimulants, by the use of plant growth-promoting rhizobacteria, Trichoderma spp., and arbuscular mycorrhizal fungus, particularly in low-input agriculture (e.g., decreased nitrogen-N and phosphorus-P fertilizer utilization) [3,11].

The beneficial impacts of using biostimulants in fresh produce have been shown in a number of research studies. For example, lettuce has been tested with marine algae (Ascophyllum nodosum)-based substances, which revealed higher yields [12] and mitigated the adverse impacts of potassium deficit throughout plant growth and processed product storage [13]. The impacts of biostimulants on plants under drought stress were also investigated [9,14]. The significance of employing biostimulants in dry areas to boost plant productivity and nutritional content was brought to light by Pereira et al.’s findings [14]. Applying plant-derived protein hydrolysates to lettuce under salt condition in soil improved fresh and dry biomass [1]. The utilization of biostimulants stimulated the levels of phenolic antioxidants in spinach and broccoli heads [8,15]. However, when it came to their capacity to increase the polyphenols levels in two spinach varieties under drought conditions, the four tested biostimulant products did not show any encouraging findings [14]. Other trials examining the stimulatory role of bioactive molecules coming from smoke and seaweed Ecklonia maxima in spinach indicated enhanced plant development and nutritive value product [16]. According to Rady and Mohamed [17], the nutrient accumulation (N, P, potassium-K, and calcium-Ca) in bean leaves was increased with immersing bean seeds or applying foliar spraying containing salicylic acid or leaf extracts from Moringa oleifera. Furthermore, treated bean plants had greater quantities of free proline, ascorbic acid, total soluble sugars, total chlorophylls, and total carotenoids than the water-treated plants. Overall, the effects of biostimulants have been evaluated in terms of improving drought tolerance [18], increasing yield and nutritional value [19,20], and reducing oxidative stress and decreases in yield [21], and prolonging the shelf life of fresh produce [5,6,7]. Application of biostimulants affected lycopene content in mature fruits, which is related to an increase in the fruits’ red color intensity [22]. The protective role that biostimulants can have against stressors provides savings on the fruit antioxidant reservoir that are directly connected with fruit color and antioxidants [23,24]. Therefore, fruit quality-related attributes, including weight, firmness, color, soluble solids, polyphenols and antioxidants are important for evaluation after biostimulant applications [5,7,22,23].

The possible utilization of essential oils (EOs) as biostimulants has received less research, even though there are already many published articles on biostimulant use and effects on various crops [25,26]. Previous studies revealed that rosemary (Rosmarinus officinalis L.) EO use on tomato seedlings improved plant growth and nutrient accumulation [27]. Essential oils are composed of many constituents that might have the potential to function as biostimulants, and this needs further exploitation [28]. Furthermore, many studies are focusing on biostimulant effects on plant growth and yield, whereas little is understood about how biostimulants affect various secondary metabolites and enzyme activities.

Essential oils derived from rosemary (R. officinalis L.) and eucalyptus (Eucalyptus globulus L.) are abundant in 1,8-cineole (also referred to as eucalyptol), with a variety of biocidal properties against insects [29], bacteria [30,31], and fungi [32], as well as having herbicidal [33], antiallergic, antitumoral and gastroprotective actions, as stated by Caldas et al. [34]. The objectives of this study were to assess the biostimulant impacts on (i) cucumber growth and yield by using a natural-based product that contained eucalyptus and rosemary EOs, (ii) fruit quality, and (iii) storability.

2. Materials and Methods

2.1. Plants Used and Experimental Design

This research took place at Cyprus University of Technology, Limassol, Cyprus, during spring–summer of 2020 in a greenhouse. Cucumber (Cucumis sativus cv. Fenomeno) seedlings were purchased from a commercial nursery. Seedlings were transplanted in pots filled with soil (the soil had a clay–loam texture, with 1.42% organic matter; total CaCO₃ 24.27%; pH 7.72; electrical conductivity (EC: 0.69 mS cm⁻¹); reduced nitrogen (N 0.41 g kg⁻¹).

A natural-based solution (NBS; named “Agriculture Green-tech E”, Meydan Solution Ltd., Larnaca, Cyprus) consisting of R. officinalis L. and Eucalyptus crabra L. EOs were employed, according to previous reports [35]. In brief, the main components of R. officinalis (rosemary) oil were isoborneol (30.29%), α pinene (25.71%), α terpineol (14.89%), and 1,8-cineole (10.81%), while the main constituents for E. crabra (eucalyptus) oil were 1,8-cineole (26.51%), α pinene (24.12%), and δ-3 carene (20.10%) [35]. The NBS product was a mixture of the eucalyptus:rosemary EO (2:1 v/v), and it also contained vinegar < 5% (w/w), and emulsifier-treated water (<80%). A commercial solution (CS) (Razormin Atlantica agricola SA, Alicante, Spain) was used at 0.25% (v/v) as a positive control. Razormin contains free amino acids, polysaccharides, iron, manganese, zinc, copper, boron, and molybdenum, which are all water-soluble [35].

2.2. Preliminary Test

A preliminary test was implemented to identify the levels of the NBS after application to cucumber plants and the possible phytotoxic effects had occurred. Cucumber seedlings were grown in soil in pots (5 L) for 25 days and then sprayed with seven NBS concentrations (0.0–0.5-1.0-1.5-2.0-2.5-3.0%, based on v/v), while the 0% NBS was water-sprayed plants and considered a control treatment. Three replications were used for each concentration. Plants were recorded for phytotoxic marks (spots) every other day for 10 days. According to the outcomes of phytotoxic spots (Figure S1; symptoms of blotch were superficial and irregular in shape and size), the NBS levels of 2.0% (v/v) were selected to be tested on the cucumber crop.

2.3. Main Experiment

Cucumber seedlings (84 in total) were transplanted in single 9-L pots filled with soil. The pots were arranged in twin rows, with plants spaced 0.45 m from each other within the row, at 0.7 m within twin rows, and 1.2 m between rows [36]. Cucumber plants were trained on a string according to the single pruning scheme (the main stem grew vertically, lateral shoots and flowers removed to 1.0 m height, and then lateral shoots allowed to grow and pruned after the 2nd fruit setting) and were grown for 13 weeks. Plants were drip-irrigated (daily 5 min/time at an early stage of plant development; every second day after the 4th week with 10 min/time, or according to crop needs) using a timer and recording water drainage from the pot’s holes. Fertigation (EC: 2.5 mS cm⁻¹; 200 mL per plant) took place once 10 days after transplanting (DAT) with commercial (i.e., 20-20-20) fertilizers. Climatic conditions (day and night temperature and relative humidity) were recorded and are presented in Figure S2.

Plants were subjected into different treatments: (i) plants sprayed with water as a control; (ii) foliar spray with the Razormin commercial solution (CS) at 2.5 mL L⁻¹ at 31 DAT and 51 DAT, for a total of two applications; (iii) foliar spray with the NBS at 2.0% once (NBS1) at 31 DAT; and (iv) foliar spray with the NBS at 2.0% applied twice (NBS2) at 31 DAT and 51 DAT. The first application was at the first fruit setting. Each treatment was replicated in three plots and each plot had seven plants in a completely randomized design set-up.

2.3.1. Plant Growth and Physiological Parameters

Cucumber plant height, leaf number, and plant fresh and dry biomass were recorded in six replicates/treatment. Plant yield as the total harvested fruits from each plant (21 plants/treatment) was recorded throughout the harvesting period, and yield was computed as kg of fruits/plant.

Leaf stomatal conductance was measured on the 4th–5th leaf from the top of the plant (2 leaf measures/plant), by using a ΔT-Porometer AP4 (Delta-T Devices-Cambridge, UK) according to the manufacturer’s instructions [37]. Leaf chlorophyll fluorescence (Fv/Fm) was measured with an OptiSci OS-30p Chlorophyll Fluorometer (Opti-Sciences). Chlorophylls (chlorophyll a-Chl a, chlorophyll b-Chl b, and total chlorophylls-total Chls) content was measured in dimethyl sulfoxide extracts, as described by Chrysargyris et al. [37].

2.3.2. Total Phenols and Antioxidant Activity

Total phenols content in leaves (four replicates/treatment; each replicate was a pooled leaf sample) was determined with the Folin–Ciocalteu method at 755 nm, according to Marinou et al. [38], and results were expressed as equivalents of gallic acid per gram of fresh weight (mg of GAE g⁻¹ fresh weight). The antioxidant activity was determined with ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods [39], and results were expressed as trolox equivalents (mg of trolox g⁻¹ fresh weight).

2.3.3. Hydrogen Peroxide, Lipid Peroxidation, and Antioxidative Enzyme Activities

Hydrogen peroxide (H₂O₂) content in leaves was determined as described previously [39], the absorbance was measured at 390 nm, and the results were expressed as μmol H₂O₂ g⁻¹ fresh weight. Lipid peroxidation was assessed (in terms of the malondialdehyde content-MDA) [39], the absorbance was measured at 532 nm, and the results were expressed as nmol of MDA g⁻¹ fresh weight.

Enzyme antioxidant activities in leaves was determined [39]. Catalase activity (CAT), superoxide dismutase activity (SOD) and peroxidase activity (POD) were assayed following the methods described previously [39]. In brief, CAT was assayed in a reaction mixture at 240 nm, SOD was assayed at 560 nm, POD was assayed at 430 nm, and the results were expressed in units mg⁻¹ of protein.

2.3.4. Leaf Nutrient Content

The nutrient uptake in leaves (four replicates/treatment) was determined. Leaves were dried (at 65 °C for four days). Extracts following acid digestion of the ashed samples were used for nutrient determinations [40]. Potassium and sodium (Na) were determined photometrically (Flame photometer, Lasany Model 1832, Lasany International, Panchkula, India), phosphorus was determined spectrophotometrically (Multiskan GO, Thermo Fischer Scientific, Waltham, MA, USA), and magnesium (Mg) and calcium by an atomic absorption spectrophotometer (PG Instruments AA500FG, Leicestershire, UK), following Chrysargyris et al. [40]. Reduced nitrogen content was determined by the Kjeldahl method (BUCHI, Digest automat K-439 and Distillation Kjelflex K-360, Flawil, Switzerland) [40]. Data were expressed in g kg⁻¹ of dry weight.

2.3.5. Fruit Quality

Cucumbers harvested from the 15th–25th internodes were considered for quality assessment. At least four to six biological replicates were used in each treatment. Fresh weight for each fruit was recorded. Fruit firmness was assessed at two points on each cucumber’s shoulder using a texture-meter FT 011 (TR Scientific Instruments, Forli, Italy) with an 8 mm plunger. The amount of force (in Newtons) needed to break through the cucumber’s pericarp (i.e., surface) in six replicates was measured at room temperature [41].

Color was measured at two points of each cucumber (and the average value was used as one replicate), for six individual fruits, by using the Hunter Lab System and a Minolta colorimeter model CR400 (Konica Minolta, Osaka, Japan). Individual L* (lightness), a* (redness), and b* (yellowness) value, Chroma value (C), hue (h), and whiteness index (WI) were computed by the following equations C = (a*² + b*²)^1/2, h = b*/a* and WI = 100 − [(100 − L*)² + a*² + b*²]^1/2 [41].

The ethylene and carbon dioxide (CO₂) production were assessed in each fruit, previously placed in a 1 L plastic container for 1 h, at ambient temperature. A dual gas analyzer (GCS 250 Analyzer, International Control Analyser Ltd., Kent, UK) was used for CO₂ levels and the results were expressed as mL CO₂ kg⁻¹ h⁻¹ [35]. At the same container, an ethylene analyzer (ICA 56 Analyzer, International Control Analyser Ltd., Kent, UK) was employed for ethylene release by the fruit, and the results were expressed as μL ethylene kg⁻¹ h⁻¹ [35].

Total soluble solids (TSS) were measured in cucumber juice by digital refractometer (model Atago PR-101, Atago Co. Ltd., Tokyo, Japan) and expressed as a percentage. Titratable acidity (TA) was measured via potentiometric titration (Mettler Toledo DL22, Columbus, OH, USA) of fruit juice and the results were expressed in malic acid percentage. The ratio TSS/TA was computed as a sweetness/ripening indicator. Ascorbic acid (AA) was determined by the 2,6-Dichloroindophenol titrimetric method, as described previously [41], and the results were expressed as mg of AA per gram of fresh weight. Fruit total phenolic content and antioxidant activity were assayed, as described in Section 2.3.2. Similarly, fruit nutrient content of N, K, P, Na, Ca, and Mg were assayed, as described in Section 2.3.4.

Cucumber marketability, aroma, and appearance were recorded by using a 1–10 scale (1: not marketable quality (i.e., malformation, wounds, infection); 3: low marketable quality with malformation; 5: marketable with few defects i.e., small size, decolorization (medium quality); 8: marketable (good) quality; 10: marketable with no defects (extra quality)) [41]. Fruit decay was visually evaluated on the day of harvest. A commodity was considered decayed when the symptoms of mycelia or bacteria development were present. A scale from 1 to 10 showing the surface infection percentage as 1: 0–10% infection; 2: 11–20% infection; 3: 21–30% infection; 4: 31–40% infection; 5: 41–50% infection; 6: 51–60% infection; 7: 61–70% infection; 8: 71–80% infection; 9: 81–90% infection; and 10: 91–100% infection was assessed to estimate the degree of produce infection.

2.3.6. Fresh Produce Storability

Cucumber storability after the applications of CS or NBS products on plants was assessed by placing a batch of fruits in 5 L containers (two fruits/container; three replicate containers/treatment) and containers were then placed in a refrigerated chamber at 11 °C and 90% relatively humidity (RH), in the dark, for seven days [42]. Fruits were aerated (open container lids every two days) to avoid air composition abnormalities (i.e., decreased O₂ and increased CO₂ levels due to fruit respiration process). After seven days of storage, fruits were evaluated for weight loss, firmness, respiration rate, ethylene emission, color indices (L*, a*, b*, Hue, Color Index, and Chroma), ascorbic acid content, total phenolics, antioxidant activity (as assayed by DPPH and FRAP), TSS, TA, marketability, appearance, aroma, and decay, as described in previous sessions. Fruit weight was recorded on the harvesting day (day 0) and every other day, up to the last day of storage (day 7).

2.4. Statistical Methods

Data were statistically analysed with IBM SPSS version 29 comparing means (±standard error-SE), with one-way ANOVA and Duncan’s multiple range tests calculated at p < 0.05. Measurements were performed in four to six biological replicates/treatment (each replicate was a pooled sample).

3. Results

The effect of CS and NBS on fruit yield and plant growth is illustrated on

Table 1. Plant growth attributes and yield of cucumber plants with regard to the foliar application.

Plant Development	Control	CS	NBS1	NBS2
Height (m)	1.66 ± 0.01 a	1.73 ± 0.04 a	1.71 ± 0.03 a	1.72 ± 0.04 a
Leaf number	28.83 ± 0.31 a	26.66 ± 0.49 a	29.66 ± 0.76 a	29.50 ± 0.71 a
Plant biomass (g)	556.60 ± 38.52 a	585.08 ± 17.19 a	622.85 ± 42.02 a	623.18 ± 20.00 a
Plant dry weight (g)	67.88 ± 3.16 a	67.36 ± 2.38 a	69.38 ± 4.27 a	71.63 ± 1.65 a
Yield (kg plant−1)	2.74 ± 0.15 ab	2.87 ± 1.14 a	2.37 ± 0.09 b	2.51 ± 0.12 ab
Fruit number	11.76 ± 0.65 ab	12.33 ± 0.71 a	10.15 ± 0.52 b	10.90 ± 0.69 ab

Control: denotes water-based foliar application; CS: denotes the commercial solution; NBS1: denotes the natural-based solution applied once; NBS2: denotes the natural-based solution applied twice. According to Duncan’s MRT, means ± SE in the same row that are separated by distinct Latin letters deviate significantly (p = 0.05).

. Leaf number, plant height, and biomass (both fresh and dry) remained unaffected after the foliar applications. CS application increased yield (up to 16.5%) and fruit number (up to 17.7%) compared to the NBS1, while they remained at the same levels when the product was applied twice (NBS2), compared to the control or the CS application.

The impacts of the foliar spraying on the plants’ photosynthetic parameters are shown in

Table 2. Leaf stomatal conductance, chlorophyll fluorescence and content of chlorophyll a, chlorophyll b, and total chlorophylls of cucumber plants with regard to the foliar application.

Plant Physiology	Control	CS	NBS1	NBS2
Stomatal conductance (mmol m−2s−1)	315.75 ± 16.75 b	428.33 ± 38.09 a	340.66 ± 11.75 ab	340.91 ± 37.21 ab
Chlorophyll fluorescence (Fv/Fm)	0.79 ± 0.00 a	0.79 ± 0.00 a	0.78 ± 0.00 a	0.78 ± 0.00 a
Chlorophyll a (mg g−1 Fw)	0.81 ± 0.03 a	0.69 ± 0.05 ab	0.66 ± 0.04 b	0.72 ± 0.02 ab
Chlorophyll b (mg g−1 Fw)	0.22 ± 0.01 a	0.17 ± 0.02 ab	0.15 ± 0.01 b	0.18 ± 0.00 ab
Total Chlorophylls (mg g−1 Fw)	1.03 ± 0.04 a	0.86 ± 0.07 ab	0.81 ± 0.05 b	0.91 ± 0.03 ab

Control: denotes water-based foliar application; CS: denotes the commercial solution; NBS1: denotes the natural-based solution applied once; NBS2: denotes the natural-based solution applied twice. According to Duncan’s MRT, means ± SE in the same row that are separated by distinct Latin letters deviate significantly (p = 0.05).

. Stomatal conductance was increased by 26.3% after the application of the CS, in comparison to the control. The chlorophylls levels (a, b, and total) were decreased after the NBS1 application compared to the control, but remained similar to the control levels when the NBS was applied twice (NBS2). Chlorophyll fluorescence did not change among the treatments.

The antioxidant capacity (DPPH, FRAP) of the cucumber leaves was higher (up to 24.6%) in CS plants than in the control plants and the ones that were sprayed with NBS1 and NBS2 (Figure 1B,C). Total phenolics in cucumber leaves were unchanged after all treatments and were averaged at 1.69 mg of GAE per g of fresh weight (Figure 1A).

The application of the CS significantly increased (+15%) the levels of lipid peroxidation (in terms of MDA content), as regards to the control plants or the plants sprayed with the natural-based solution (NBS1 and NBS2), while the NBS application had no impact on this stress indicator, in comparison to the control plants, as the MDA levels did not differ (Figure 2A). Hydrogen peroxide was increased when plants were sprayed with NBS1 (up to 24.2%) and NBS2 (up to 27.2%), compared to the water-sprayed plants (control) or to the CS-sprayed plants (Figure 2B). The NBS1 application increased SOD activity compared to the CS-sprayed plants (Figure 2C) but there was no difference with the control plants. CAT activity was increased (Figure 2D) at the application of NBS1 compared to all other applications. POD was also increased when the CS was applied to the cucumber plants, followed by the NBS1 application, while the NBS2 kept the enzymatic antioxidant activity at the control levels (Figure 2E).

The foliar applications affected the levels of nutrients in cucumber leaves as they are shown in Figure 3. The application of CS increased (up to 10.4%) N levels, while NBS1 decreased (up to 18.8%) N levels, compared to the control (Figure 3A). Potassium was found to decrease by 20.2% when the NBS2 was applied (Figure 3B), compared to the control plants. Sodium levels remained unaffected after all foliar applications (Figure 3D), while the leaf P content decreased (up to 23.7%) after the application of all the tested products (Figure 3C). Calcium was increased after the CS application (Figure 3E) while Mg was found to be increased after the CS and NBS2 application (Figure 3F), compared to the control.

The impacts of the CS and NBS on cucumber fruit quality attributes are presented in

Table 3. Fruit quality-related attributes harvested from cucumber plants with regard to the foliar application.

Quality Attributes	Control	CS	NBS1	NBS2
Fruit fresh weight (Fw; g)	233.31 ± 4.98 a	235.09 ± 3.67 a	236.81 ± 5.96 a	236.85 ± 7.36 a
Firmness (in Newtons)	33.53 ± 2.25 a	33.63 ± 1.76 a	31.30 ± 2.13 a	31.76 ± 1.62 a
Respiration rates (mL CO2 kg−1 h−1)	22.68 ± 1.29 a	22.15 ± 2.27 a	21.47 ± 1.05 a	25.29 ± 2.41 a
Ethylene production (μL kg−1 h−1)	3.81 ± 0.37 a	3.99 ± 0.64 a	4.22 ± 0.42 a	4.73 ± 0.27 a
Lightness (L* value)	34.04 ± 0.61 b	34.35 ± 0.76 ab	36.73 ± 0.73 a	34.06 ± 1.08 b
Redness (a* value)	−9.38 ± 0.91 ab	−9.44 ± 0.56 ab	−11.70 ± 0.85 b	−9.05 ± 0.87 a
Yellowness (b* value)	11.95 ± 1.30 b	11.86 ± 0.84 b	15.82 ± 1.41 a	11.45 ± 1.31 b
Hue value	128.29 ± 0.37 a	128.63 ± 0.31 a	126.67 ± 0.45 b	128.60 ± 0.55 a
Chroma value	15.19 ± 1.58 ab	15.16 ± 1.01 ab	19.68 ± 1.64 a	14.60 ± 1.57 b
Color Index	−23.26 ± 0.72 b	−23.35 ± 0.74 b	−20.35 ± 0.72 a	−23.64 ± 1.88 b
TSS (%)	3.40 ± 0.10 a	3.07 ± 0.08 a	3.07 ± 0.28 a	3.15 ± 0.09 a
TA (malic acid g L−1)	1.51 ± 0.13 a	2.07 ± 0.32 a	1.75 ± 0.03 a	1.64 ± 0.02 a
AA (mg 100 g−1 Fw)	6.92 ± 0.70 a	6.65 ± 0.66 a	6.70 ± 0.48 a	7.88 ± 0.94 a
Phenols (mg GAE g−1 Fw)	0.21 ± 0.02 a	0.19 ± 0.01 a	0.20 ± 0.01 a	0.22 ± 0.01 a
DPPH (mg trolox g−1 Fw)	1.03 ± 0.07 a	1.08 ± 0.12 a	0.82 ± 0.07 a	0.96 ± 0.06 a
FRAP (mg trolox g−1 Fw)	0.32 ± 0.02 a	0.32 ± 0.00 a	0.30 ± 0.02 a	0.32 ± 0.03 a
Marketability (1–10)	8.72 ± 0.30 a	8.55 ± 0.20 a	8.61 ± 0.16 a	9.11 ± 0.14 a
Aroma (1–10)	9.66 ± 0.17 a	9.38 ± 0.26 a	9.44 ± 0.11 a	9.83 ± 0.07 a
Appearance (1–10)	8.78 ± 0.07 ab	8.89 ± 0.69 a	8.61 ± 0.05 bc	8.50 ± 0.07 c
Decay (1–10)	1.00 ± 0.00 a	1.00 ± 0.00 a	1.00 ± 0.00 a	1.00 ± 0.00 a

Control: denotes water-based foliar application; CS: denotes the commercial solution; NBS1: denotes the natural-based solution applied once; NBS2: denotes the natural-based solution applied twice. According to Duncan’s MRT, means ± SE in the same row that are separated by distinct Latin letters deviate significantly (p = 0.05).

. The parameter that was affected by the foliar applications tested was fruit appearance, where NBS2 treated fruits had the lowest values compared to the fruits from control plants. NBS1 application increased fruit lightness (L* value) and yellowness (b* value), and consequently increased Chroma and color index but decreased Hue value, in comparison to the control treatment. Both CS and NBS2 had similar effects to the control treatment on fruit color. All the other parameters tested remained unaffected by the different foliar applications of the tested products.

Fruits derived from plants that had been subjected to CS or NBS foliar applications and stored for 7 days at 11 °C and 90% RH revealed changes to their quality attributes, as presented in

Table 4. Foliar application on cucumber plants and impacts on fruit quality attributes following seven days of storage at 11 °C and 90% RH.

Quality Attributes	Control	CS	NBS1	NBS2
Fruit weight loss (%)	1.61 ± 0.06 a	1.75 ± 0.06 a	1.64 ± 0.07 a	1.59 ± 0.08 a
Firmness (in Newtons)	35.13 ± 1.97 a	35.82 ± 1.20 a	35.64 ± 1.75 a	34.69 ± 1.53 a
Respiration rates (mL CO2 kg−1 h−1)	13.69 ± 2.38 a	18.73 ± 0.62 a	12.95 ± 1.58 a	18.69 ± 2.49 a
Ethylene production (μL kg−1 h−1)	4.51 ± 0.66 ab	2.97 ± 0.47 b	5.93 ± 0.41 a	4.80 ± 0.51 a
Lightness (L* value)	35.27 ± 1.04 a	33.78 ± 0.77 a	35.25 ± 0.68 a	34.59 ± 1.25 a
Redness (a* value)	−11.91 ± 0.19 ab	−10.61 ± 0.49 a	−11.54 ± 0.76 ab	−12.78 ± 0.54 b
Yellowness (b* value)	15.93 ± 0.31 ab	14.12 ± 0.83 b	15.32 ± 1.21 ab	17.33 ± 0.96 a
Hue value	126.79 ± 0.13 a	127.01 ± 0.34 a	127.13 ± 0.40 a	126.52 ± 0.42 a
Chroma value	19.90 ± 0.36 ab	17.67 ± 0.96 b	19.19 ± 1.43 ab	21.54 ± 1.10 a
Color Index	−21.30 ± 0.64 a	−22.40 ± 0.70 a	−21.54 ± 0.70 a	−21.53 ± 0.81 a
TSS (%)	3.62 ± 0.13 a	3.07 ± 0.75 b	3.10 ± 0.10 b	3.10 ± 0.12 b
TA (malic acid g L−1)	1.76 ± 0.05 a	1.90 ± 0.05 a	1.74 ± 0.06 a	1.72 ± 0.04 a
AA (mg 100 g−1 Fw)	7.19 ± 0.48 a	6.91 ± 0.55 a	6.75 ± 0.75 a	7.27 ± 0.28 a
Phenols (mg GAE g−1 Fw)	0.23 ± 0.01 a	0.24 ± 0.00 a	0.22 ± 0.00 a	0.22 ± 0.00 a
DPPH (mg trolox g−1 Fw)	1.07 ± 0.06 a	1.21 ± 0.04 a	1.13 ± 0.06 a	1.25 ± 0.08 a
FRAP (mg trolox g−1 Fw)	0.32 ± 0.01 a	0.28 ± 0.01 b	0.26 ± 0.01 b	0.26 ± 0.01 b
Marketability (1–10)	7.94 ± 0.33 ab	8.1600.23 a	7.22 ± 0.33 b	7.77 ± 0.18 ab
Aroma (1–10)	9.05 ± 0.20 a	8.44 ± 0.22 b	7.66 ± 0.22 c	8.61 ± 0.10 ab
Appearance (1–10)	7.78 ± 0.07 b	7.78 ± 0.07 b	7.67 ± 0.00 b	8.16 ± 0.07 a
Decay (1–10)	1.00 ± 0.00 a	1.00 ± 0.00 a	1.00 ± 0.00 a	1.00 ± 0.00 a

Control: denotes water-based foliar application; CS: denotes the commercial solution; NBS1: denotes the natural-based solution applied once; NBS2: denotes the natural-based solution applied twice. According to Duncan’s MRT, means ± SE in the same row that are separated by distinct Latin letters deviate significantly (p = 0.05).

. Application of NBS (both once and twice) stimulated the ethylene production compared to the CS application, but did not differ with the control treatment. CS and NBS decreased TSS, antioxidant activity (as assayed by FRAP), and fruit aroma in comparison to the control treatment. NBS2-treated fruit revealed the highest score for appearance. NBS2-treated fruit increased greenish (lower negative a* value), yellowness (b* value), and chroma values compared with the CS application but did not change when compared with NBS1 and control.

4. Discussion

Biostimulant application in plants alters the metabolic process and final product quality [43]. Przygocka-Cyna and Grzebisz [44] linked the utilization of biostimulants to increased plant nutrient accumulation and hence higher nutrient content in the final products. In the current research, neither the NBS nor the CS affected plant height, leaf number, and plant fresh weight, whereas the yield decreased with the NBS1 application compared to the CS but not when compared to the control. This decrease is associated with the decreased number of fruits produced (Table 1) in plants treated with NBS1 rather than the mean fresh weight of the fruit itself (Table 3). Previous studies reported a positive effect of the applied NBS on tomato crops, as both the height and yield of tomatoes were increased [36]. Yet in earlier research, high rosemary EO treatment (10%) on tomato seedlings reduced plant height, which might be attributed to stress signaling molecules (e.g., ethylene) [27]. Indeed the lower examined levels in that study of 5% rosemary EO did not impact the tomatoes’ height [27], being in agreement with the current findings for cucumbers when 2% of the NBS (EOs mixture) was applied. The employed NBS seems not to observe any biostimulant impacts on cucumber plants, as growth-related parameters were unaffected compared to the control plants. However, prior reports had demonstrated that various organic substances like humic acids, amino acids, salicylic acid, and vitamins had stimulatory effects on different plant species [45,46,47]. When cucumber plants were sprayed with humic acid (3 g L⁻¹) and a biostimulant product (Ecormon 0.45 g L⁻¹), they revealed positive and significant responses on plant growth and yield [48]. This variation in findings among different studies highlights the complexity of natural-based compounds and their use/effectiveness in the agricultural sector.

Furthermore, the use of NBS1 reduced the chlorophyll content compared to the control application. Notably, CS treatment resulted in a rise in stomatal conductance. Photosynthesis and plant water relations were linked to stomatal conductance [49]. In this instance, applying CS to cucumber plants may enhance their absorption of water; however, this factor was not measured in the current study, and additional research is necessary to that direction. The present findings contradict the results observed for lettuce, as the use of foliar fertilizer combining macronutrients [12] and fish-derived protein hydrolysates [50] boosted the amount of chlorophyll and carotenoids in lettuce. The above-mentioned effectiveness of biostimulants tested might be related to leafy vegetables (i.e., lettuce) compared to fruity vegetables (i.e., cucumber), with different growing durations, application doses and products examined. The benefits of biostimulants based on amino acids have been previous reported for lettuce [51], which might justify CS effectiveness in the present outcomes. Other compounds that had biostimulant effects on the crops under study included chitosan, which increased photosynthesis in lettuce [52]. Indeed, the NBS2 application maintained chlorophylls levels and stomatal conductance to similar levels to the control, indicating the number of applications is important, and possibly more applications could benefit plant growth and physiology.

The application of NBS seems not to affect the total phenols content and antioxidant properties of cucumber plants, being in accordance with previous reports on tomato plants after spraying with NBS products [36]. However, the application of CS increased the antioxidant activity of cucumber plants, as determined by DPPH and FRAP. The use of amino acid-based biostimulants and amino acids in conjunction with A. nodosum considerably raised the amount of total phenols in broccoli [8], which is opposed to our findings. Furthermore, adding brown seaweed (A. nodosum) to spinach increased its phenolic content [15].

Cucumber leaf damage was increased when CS was applied (Figure 2A), as indicated by the increased MDA content. This increase affected the enzymatic antioxidant activity of the plants, as POD was increased. Notable is that the primary activation of antioxidant enzyme metabolism (first a rise in SOD, then in POD, which led to the detoxification of reactive oxygen species-ROS and therefore a drop-in the enzyme activity) may have been the cause of the lowered SOD levels at the CS treatments. Moreover, ROS detoxification can take place with the activation of the non-enzymatic mechanisms in plants, as assayed with increased DPPH and FRAP. Wozniak et al. [53] studied > 30 crops, and found that one of the primary effects of more than 50 biostimulants—which ranged from seaweed extracts, humic components, hydrolyzed proteins, live microbial inoculums, and synthetic compounds—were connected to the regulation of ROS overproduction and the preservation of cellular membranes. The plants’ elevated enzymes antioxidant activities and secondary metabolites demonstrated protecting strategies. However, further research is required to fully address this issue.

Cucumber leaf nutrients were impacted by NBS treatment. Consequently, when NBS1 was sprayed, the amount of N and P in the leaves dropped, whereas the NBS2 application decreased K and P but increased Mg levels, suggesting an antagonistic effect for the K and Mg. This increase in Mg might support the plant photosynthetic rates, whereas similar Mg increase was noted in a previous study when tomato seedlings were subjected to rosemary EO applications [27] and in cucumber treated with humic acid and biostimulants [48]. Furthermore, the nutrient content of bean plants was enhanced by the use of salicylic acid or M. oleifera leaf extract [17]. In previous studies, higher rosemary oil (0.1%) use stimulated the accumulation of minerals (N, K, Mg, iron-Fe, and zinc-Zn) in tomato seedlings [27]. This suggests that the beneficial effects of EOs depend on the specific levels of rosemary oil used as well as the plant developmental stage (mature plants versus seedlings) and tested plant species (cucumber versus tomatoes). Although biostimulants do not deliver minerals to plants (so they cannot be classified as fertilizers), they may indirectly aid in nutrient uptake by promoting plant and soil metabolic processes [54]. Previous reports indicated that rosemary EO application in tomato seedlings improved root fresh weight and root function but no reports in the literature exist regarding changes in mineral uptakes with the use of EOs [27]; however, measurements on roots were not implemented in the present study.

The commercial success of fresh produce (including fruit, vegetables, and herbs) depends on the commodity’s quality characteristics. According to this study, fruit quality was less affected by the CS or NBS applications at harvest, and in general maintained similar quality attributes as the control-treated fruits. For instance, fruit firmness, ethylene, and respiration rates were unaffected, indicating that the applied CS or NBS did not stimulate fruit metabolism at harvest. Only some differences in fruit color were observed with the NBS application, which finally negatively affected fruit appearance. After one week of storage in chilled conditions, the impacts of NBS were more evidenced as the ethylene production in fruits was increased, which reflected enhanced ripening metabolism and fruit senescence. This negatively affected the antioxidant capacity of the fruit and aroma. It is notable that cucumber appearance was improved after storage, following organoleptic tests. Therefore, as a result, cucumbers grown under NBS treatments may not last as long and they may need more postharvest preservation means (such as ozone or 1-Methylcyclopropene) to be applied and/or combined [55,56]. Similar to our observations, tomato fruit total phenolics and AA did not change once tomatoes were exposed to foliar treatments of protein hydrolysate, plant, and seaweed extracts [19]. However, the successful utilization of biostimulants in crops, especially when plants are subjected to various stresses, should not be ignored [8,9,57,58].

5. Conclusions

In this study, cucumber plants were used to assess the possible use of an NBS comprising EOs of rosemary and eucalyptus, both containing 1,8-cineole as the main EO component, as a putative biostimulant. Following application, measures related to plant production, quality, mineral accumulation, physiology, and enzyme activity were assessed. The application of NBS1 was less efficient than NBS2, as various physiological attributes (i.e., chlorophylls) and mineral accumulation (N) decreased. The application of CS stimulated stomatal conductance, N, Ca, and Mg accumulation and antioxidant capacity of the plants which mirrored the increased oxidative stress occurred, as well as the activation of several enzymatic antioxidant mechanisms. Indeed, both CS and NBS application had less effect on cucumber plants and fruits than in previous reports, indicating that a greater number (more than two) of applications should be tested in future. However, a possible phytotoxic effect should be also considered with the multiple applications and/or accumulation of stress effects. More study is needed to evaluate EOs mixtures for different crops and determine which components give EOs biostimulant attributes. A combination of different EOs can be tested, as the synergistic effects of the oil components is well characterised. Various plants species, including laurel and lavender, have considerable 1,8-cineole content compared to the tested EOs in the present work.