Foliar Application of Iron and Zinc Affected Aromatic Plants Grown Under Conventional and Organic Agriculture Differently

Abstract

The utilization of organic fertilizers for the cultivation of wild edible and medicinal plants offers agronomic and ecological benefits, given their suitability to low-input and sustainable production systems. Under such conditions, these species may also benefit from targeted foliar applications of micronutrients to enhance their nutritional quality. This study examined the effects of a vinasse-based organic fertilizer and conventional fertilization regime, in combination with foliar applications of iron (Fe) and zinc (Zn), on the biomass, leaf photochemistry, and plant stress-related responses of Sideritis cypria and Origanum dubium. In S. cypria, organic fertilization resulted in a similar yield compared to conventional fertilization, while O. dubium showed a significant decrease in yield when using organic fertilizers. The impact of spraying with Zn on S. cypria dry matter content was related to the availability of nutrients, particularly nitrogen, while in O. dubium Zn spraying induced a decrease in dry matter. The total phenols content and antioxidant activity of S. cypria were elevated by conventional fertilization and foliar application of Fe, while the combination of organic fertilization and foliar application of Fe and Zn reduced lipid peroxidation. In O. dubium, foliar application of Fe and Zn led to a reduction in total phenols content, antioxidant capacity, and hydrogen peroxide content under adequate nutrition. In general, foliar spraying with Zn tended to improve water use efficiency under specific fertilization practices on both species, while the positive effect of conventional fertilization on nutrient use efficiency still requires further validation. Ultimately, the efficiency of organic fertilization was related to the examined species, inducing variations in leaf chlorophyll content. In addition, foliar application of Fe and Zn affected the antioxidant capacity and mineral content of the examined species. Thus, appropriate fertilization management is vital to fully realize the specific benefits of foliar micronutrient addition.

1. Introduction

Today’s agriculture faces enormous challenges in ensuring safe and healthy food production in regard to a constantly expanding population [1,2]. In parallel, the agriculture sector is one of the main key players in environmental constraints, natural resource deterioration, and decreased biodiversity of living organisms [3]. In order to preserve the availability of food, this concern necessitates not only a boost in worldwide crop production but also the implementation of strategies that guarantee food safety and nutritional value [4,5]. Therefore, crop yields may increase under intensive cultivation systems [6], which primarily depend on the use of overfertilization practices, mostly with inorganic fertilizers [7]. The literature has extensively documented the impact of cultivation methods on crops, since choosing the right ones enhances crop yield and quality [8,9,10]. However, the use of an inappropriate practice can lower crop yields, raise soil mineral content to harmful levels, lose minerals through leaching or mobilization, pollute water supplies, and deteriorate soil quality [11,12]. Farmers frequently choose intensive agricultural schemes, such as traditional crop production with high fertilizer, phytochemical, and water needs that result in large yields but not necessarily high-quality products. Nevertheless, this strategy has a number of negative effects on human health and the environment, such as soil deterioration, nutrient loss through leaching, and a rise in greenhouse gas emissions, as well as ultimately air, water, and soil contamination [4,11].

As part of sustainability, the preservation of biodiversity and natural resources and organic methods restrict the usage of inorganic fertilizers and instead employ environmentally friendly products like organic manure, natural-based products, and soil restoration practices [4,13,14]. A potentially environmentally friendly method of producing crops, organic agriculture strategies can yield high-quality products of nutritional value and qualities equivalent to those produced conventionally [15,16]. Organic farming is an integrated system under ecological and restoration principles. Since wild plant species have innate native adaptations to adverse soil and climate conditions, they have substantial ecological significance in this context, especially when it comes to soil deterioration [17]. Furthermore, organic farming has the ability to retain a smaller environmental impact while producing plant products that are healthier and of greater quality [7,18].

This is especially crucial for medicinal and aromatic plants (MAPs) since their quality and advantageous molecules are what really make them valuable [4,19]. Since these species are defined by minimal input needs (e.g., fertilizers, agrochemicals, watering), the incorporation of MAPs in organic agricultural production falls within its purview [20]. Although MAPs have comparatively poor yields, consumers’ interest in organically grown products and herbs has been steadily rising at the same time [16,21]. However, there is not much research that compares the conventional and organic methods of growing MAPs [9,22]. Prior reports indicated that although there were no appreciable changes in overall water usage, organic MAPs cultivation had better energy use and carbon footprints than conventional ones [16].

Awareness in using organic farming methods has grown as a result of the environmental impact of fertilizer and agrochemical inputs used in the production of MAPs, as well as a shortage of approved pesticides and herbicides for different MAPs [23,24]. Since MAPs have minimal nutrient requirements and are resilient to biotic and abiotic stresses, low-input cultivation techniques are appropriate for MAPs production. Aside from the environmental concerns, growing MAPs organically is linked to higher-quality final products than conventional methods, and using fewer chemical pesticides and fertilizers guarantees the safety of the final products because there are fewer heavy metals and chemical residues [22,25]. A further consideration to take into account is that commercial cultivation of MAPs improved access to the market year-round and allowed for the standardization of the final product while adhering to certain safety protocols. Moreover, MAPs cultivation is also lowering the possibility of genetic degradation brought on by irrational wild plant harvesting [24,26].

In agriculture, foliar mineral applications—especially with micronutrients such as zinc (Zn), manganese (Mn), and copper (Cu)—are one method of regulating mineral intake and needs through leaves in parallel with root absorption [27]. Foliar application may help to reduce oxidative stress or nutrient deficits caused by several abiotic and biotic causes [28,29,30] and act as a biofortification strategy to improve micronutrient uptake in plants [31]. Among the micronutrients, both iron (Fe) and Zn are essential components for plant growth and participate in several metabolic processes either as metal components of various enzymes or as structural, functional, or regulatory cofactors [32,33]. Iron and Zn appear to have a beneficial effect on plant development, as they stimulate chlorophyll content and photosynthetic rates, induce indole-3-acetic acid (IAA) biosynthesis (by Zn), contribute to protein biosynthesis and membrane stability, and enhance oxidation and reduction processes [34]. Previous research examined the impact of foliar administration of micronutrients on MAPs development and their essential oil production, including chamomile (Matricaria chamomilla L.) [35], sweet basil (Ocimum basilicum L.) [28], coriander (Coriandrum sativum L.) [36], and rosemary (Rosmarinus officinalis L.) [34].

Research into lesser-known local MAPs and their biological attributes is becoming increasingly popular [37,38]. With over 150 species, the Sideritis L. genus (Lamiaceae) is mostly distributed in the Mediterranean region [39]. One of them is the native Cypriot Sideritis cypria Post. [39], which has a phytochemical profile rich in secondary metabolites with potent cytotoxic and antioxidant properties linked to apigenin and apigenin derivatives [40] and antimicrobial properties towards Candida albicans and Gram-positive bacteria [39,41]. The principal components of the essential oil profile of S. cypria were β-phellandrene (25.11%), β-caryophyllene (22.52%), and α-pinene (11.92%) in leaves, which were linked with its biocidal activities [39].

Another important MAP species is known as Cypriot oregano, Origanum dubium Boiss., which is a perennial plant of the Origanum genus from the Lamiaceae family that thrives across the Mediterranean region and is well appreciated in Cyprus and in the eastern Mediterranean area. The plant favors great antioxidant and antiseptic/antimicrobial properties [42,43], is used against migraines, toothaches, and respiratory problems [44], and has been extensively utilized since ancient times. The principal components of the essential oil profile of O. dubium were carvacrol (44.59–68.67%), trans-sabinene hydrate (12.71–23.10%), p-cymene (2.97–9.01%), and γ-terpinene (3.53–8.22%) [45] with well-documented biocidal properties of the main components [43].

In the framework of putting sustainable agricultural techniques into reality, it is crucial to guarantee appropriate yields and improved quality variables, given the possibility for S. cypria and O. dubium to be commercial crop options, with the former being almost unexploited on a commercial scale. Recently their precise mineral needs in hydroponics were documented for the first time, with nitrogen-N, phosphorus-P and potassium-K of 150, 50, and 350 mg L⁻¹, respectively, for S. cypria [46] and with N, P, and K of 150, 75, and 350 mg L⁻¹, respectively, for O. dubium [45]. Moreover, the phytochemical profile of the selected species was varied, with S. cypria posing increased cytotoxic activities against breast cancer cells [47]. However, little knowledge exists on the cultivation practices of the above-mentioned species. Due to the growing demand for certification of the organic products, these approaches will assist in lessening the negative environmental effects of the present agriculture industry [48]. Consequently, the present research investigated the effects of a commonly accessible organic fertilizer on the development and quality characteristics of S. cypria and O. dubium plants cultivated in field conditions, compared to traditional inorganic fertilization. Furthermore, the impacts of micronutrient foliar application were also examined to further support the plants biomass production, mineral content, and antioxidant capacity.

2. Materials and Methods

2.1. Plant Species, Soil Characteristics, and Experimental Setup

S. cypria and O. dubium seedlings were purchased from the Cypriot National Centre of Medicinal/Aromatic Plants, Nicosia, in plastic trays, at the growth stage of 3–4 leaves and 5–6 cm height. Plants were transplanted in soil in a field of 1000 m² during spring of 2024 in a commercial organic farm, Anogyra, Limassol, Cyprus (34°44′16.53″ N; 32°44’40.86″ E, 428 m above sea level). The climate of the region during the experimental study had average minimum and maximum temperatures of 13.6 and 33.4 °C, respectively, air humidity ranged from 64 to 73%, and precipitation ranged from 0.5 to 34 mm.

Soil samples, collected from four points, were air-dried in an oven. Particle size distribution was determined by dry sieving with mesh sizes of 4.75, 2.00, 0.850, 0.425, 0.250, 0.150, and 0.075 mm. Additionally, a soil subsample was hand-sieved to pass a 2 mm mesh, and several soil physicochemical properties were determined as described previously [49]. Soil type was determined by the hydrometer method with the Bouyoucos scale. Calcium carbonate (CaCO₃) was determined using the Bernard calcimetry method, and results were expressed as a percentage. Organic matter was determined using the Walkley-Black method, and results were expressed as a percentage. Electrical conductivity (EC) and pH were determined using the 1:5 dilution method. Following extraction, P was determined using the Olsen sodium bicarbonate method spectrophotometrically, and K and sodium (Na) content were quantified using flame photometry. Nitrogen was determined by the Kjeldahl method (BUCHI, Digest automat K-439 and Distillation Kjeldahl K-360, Flawil, Switzerland). Magnesium (Mg) and calcium (Ca) were determined by an atomic absorption spectrophotometer (PG Instruments AA500FG, Leicestershire, UK) [9]. Mineral content in soil was expressed as grams per kilogram of dry weight (g kg⁻¹ DW).

Plants were arranged in single rows (inter-row of 1.5 m and intra-row of 0.33 m), resulting in a plant density of 20,368 plants ha⁻¹. Plants were grown for about six months in soil. The experimental farm was divided into three main plots with three planting rows each: (i) No fertilizers (NoFert), (ii) Organic fertilization (OrFert), and (iii) Conventional fertilization (CoFert). Each main plot was divided into three subplots, where the foliar applications took place as follows: (A) foliar with water (H₂O), (B) foliar with iron (Fe), and (C) foliar with zinc (Zn). Therefore, the experimental setup had nine treatments, and each treatment had 12 plants in each row (36 plants per treatment) for each of the examined species (324 plants for S. cypria and 324 plants for O. dubium).

The plants were irrigated every day for 10 min or according to the plant’s needs. The application of synthetic fertilizers [20-20-20 and 0-0-22+22S+11Mg (potassium magnesium sulfate-K₂SO₄·MgSO₄)] and the organic fertilizer (Phenix, Hello NATURE) took place three times (24 April 2024, 15 May 2024, and 28 May 2024). The fertilizers were applied on soil and directly incorporated in the soil to diminish any losses. Organic fertilization was carried out using Phenix (Hello Nature, Italy), a vinasse-based fertilizer with 6% N, 8% phosphorus pentoxide (P₂O₅), 15% potassium oxide (K₂O), and 3% magnesium oxide (MgO), and 50% organic matter content. Considering the chemical composition of the tested fertilizers, the amount of organic fertilizers was almost 1.5 times higher compared to the synthetic fertilizers to maintain a balanced mineral application. The cumulative amounts of N, P, K, and Mg were 75.21, 37.61, 138.95, and 22.49 kg ha⁻¹, respectively. The foliar application took place twice with 1.79 mM Fe and 1.74 mM Zn by using iron (II) sulfate (FeSO₄·H₂O) and zinc sulfate (ZnSO₄·H₂O) chemical fertilizers, respectively. No pesticides were used in the present study.

2.2. Plant Biomass and Photochemistry

Various measurements were implemented at the end of the experiment. Twelve replicated plants were harvested at 1 cm above soil, the upper fresh weight was weighed (g), dried at 42 °C to constant weight, and dry weight was recorded [45]. Dry matter content (%) was calculated.

Leaf photochemistry was also assessed by measuring the relative chlorophyll content by employing an optical chlorophyll meter (SPAD-502, Minolta, Osaka, Japan) [49]. Additionally, fresh plant tissue (six replications/treatment; each replication was a pool of two plant tissues; 0.1 g) was used for chlorophyll extraction [50]. The absorbance was then measured at 470, 653, and 666 nm (Multiskan GO, Thermo Fischer Scientific Oy, Vantaa, Finland). Photosynthetic leaf pigments, chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (total Chl), and carotenoid (Car) content were then calculated (mg g⁻¹ fresh weight). The ratio of chlorophyll a to chlorophyll b (Chl a/Chl b) and of carotenoids to total chlorophylls (Car/Total Chls) were also calculated.

2.3. Total Phenols, Total Flavonoids, and Antioxidant Capacity

Total phenols content was measured on the methanolic extracts from the different samples by using the Folin-Ciocalteu method; the samples were optometrically measured at 755 nm, and results were expressed as gallic acid equivalents (mg GAE g⁻¹ of fresh weight) as described previously [49]. Total flavonoid content was determined from the same extracts according to the aluminium chloride colorimetric method [51]; the absorbance of the solution was measured at 510 nm (Multiskan GO, Thermo Fischer Scientific Oy, Vantaa, Finland) and expressed as rutin equivalents (mg Rutin g⁻¹ of fresh weight).

The antioxidant activity of the methanolic plant extracts was determined by using three assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), as previously described [49], as well as the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, as described previously [52]. To give a more thorough and accurate evaluation of the plant’s antioxidant capacity, especially when various fertilization techniques are used, several radical scavenging tests were employed. It aids in taking into consideration the complexity of plant chemistry and the impact of various farming techniques; in fact, using at least two diverse approaches is advised [53]. The three spectrophotometric techniques that were used evaluated the capacity to decrease ferric ions (FRAP) and the free radical scavenging activity (DPPH, ABTS) [54]. Results were expressed as trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) equivalent (mg Trolox g⁻¹ of fresh weight).

2.4. Lipid Peroxidation and Hydrogen Peroxide Content

The content of hydrogen peroxide (H₂O₂) was assessed according to Loreto and Velikova [55], and lipid peroxidation was assessed according to De Azevedo Neto et al. [56]. The results were expressed as micromoles of H₂O₂ per gram of fresh weight, while lipid peroxidation was measured in terms of malondialdehyde (MDA) content and expressed as nanomoles of MDA per gram of fresh weight.

2.5. Nutrient Content in Plants

At the end of the experiment, samples from the upper parts of the plant (leaves and stems) were used to determine the mineral content in six replications per treatment (three pooled plants per replication). Plant tissue was dried to constant weight (at 42 °C), then was ash-burned at 450 °C for 6 h and acid-digested (2 M HCl) [45]. The nutrient content was determined, with K and Na measured photometrically (Flame photometer, Lasany Model 1832, Lasany International, Panchkula, India), and P measured spectrophotometrically (Multiskan GO, Thermo Fischer Scientific, Waltham, MA, USA). Nitrogen was determined by the Kjeldahl method (BUCHI, Digest automat K-439 and Distillation Kjeldahl K-360, Flawil, Switzerland). Data were expressed in g kg⁻¹ of dry weight [45].

2.6. Statistical Analysis

Statistical analysis was performed using IBM SPSS version 22. Data means were compared with one-way and two-way analysis of variance (ANOVA) and Duncan’s multiple range tests for comparisons of treatment means at p < 0.05. Measurements were done in four to six biological replications/treatments (each replication consisted of a pool of three individual measures/samples).

3. Results

3.1. Soil Analysis

Following analysis, the soil was characterized by a clay-loam texture, with almost 80% of the particles being lower than 4.75 mm. Additionally, the soil had 4.61% organic matter, available CaCO₃ of 60.67%, a pH of 7.64, and an EC of 1.08 mS cm⁻¹. Finally, the soil contained N 2.431 g kg⁻¹, P 0.054 g kg⁻¹, K 0.358 g kg⁻¹, Ca 6.298 g kg⁻¹, Mg 0.598 g kg⁻¹, and Na 0.126 g kg⁻¹.

3.2. Two-Way Anova Analysis

Table 1. Effect of different fertilization applications, foliar applications, and their interaction on Sideritis cypria and Origanum dubium plant biomass, physiology, nutrient content, antioxidant capacity, and stress response.

	Sideritis cypria			Origanum dubium
	Fertilization	Foliar	Fertilization × Foliar	Fertilization	Foliar	Fertilization × Foliar
Plant FW	***	ns	*	***	ns	*
Plant DW	ns	ns	ns	ns	ns	ns
Plant DM	***	*	ns	***	*	ns
SPAD	ns	ns	ns	ns	ns	ns
Chlorophyll a	ns	ns	ns	**	**	***
Chlorophyll b	ns	ns	ns	ns	*	***
Total chlorophylls	ns	ns	ns	ns	**	***
Carotenoids	ns	ns	ns	***	ns	***
Chl a/Chl b	*	ns	ns	**	ns	***
Car/Total Chls	ns	ns	ns	**	ns	***
N	***	***	***	***	***	***
P	***	***	***	***	***	***
K	***	***	***	***	***	***
Na	***	***	***	***	***	***
Phenols	***	***	***	*	***	***
DPPH	***	***	***	*	***	***
FRAP	***	**	***	**	***	**
ABTS	***	*	***	***	***	***
Flavonoids	***	*	***	***	***	***
MDA	***	***	***	***	***	***
H2O2	***	***	***	***	***	***

*** Significant at p < 0.001; ** significant at p < 0.01; * significant at p < 0.05; ns: not significant, according to two-way ANOVA. Fresh weight (FW); dry weight (DW); dry matter (DM); chlorophyll a (Chl a); chlorophyll b (Chl b); total chlorophylls (Total Chl); total chlorophylls (Total Chl); carotenoids (Car); nitrogen (N); phosphorus (P); potassium (K); sodium (Na); 2,2-diphenyl-1-picrylhydrazyl (DPPH); ferric reducing antioxidant power (FRAP); 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); hydrogen peroxide (H₂O₂); malondialdehyde (MDA).

exhibits the results yielded by the two-way ANOVA analysis on the main impacts of the fertilization application and foliar application, as well as their interaction, on S cypria and O. dubium plants. In the case of S cypria, the different fertilization schemes affected the chlorophyll a:chlorophyll b ratio at p < 0.05; plant fresh weight, dry matter content, macronutrient content (N, P, K, and Na), total phenolics content, antioxidant capacity (DPPH, FRAP, and ABTS), flavonoids, and stress indicators (MDA and H₂O₂) at p < 0.001. The different foliar applications mainly affected dry matter content, ABTS, and flavonoids at p < 0.05; FRAP at p < 0.01; and the macronutrient content, total phenolics, DPPH, and stress indicators at p < 0.001. Finally, their interaction mainly affected the plants’ fresh weight at p < 0.05; plants’ macronutrient content, total phenolics, antioxidant capacity, flavonoids, and stress indicators at p < 0.001.

In the case of O. dubium, the different fertilization applications affected the total phenolics and DPPH at p < 0.05; chlorophyll a, the chlorophyll a to chlorophyll b ratio, the carotenoids to total chlorophylls ratio FRAP at p < 0.01; SPAD, dry matter content, macronutrient content (N, P, K, and Na), carotenoids, ABTS, flavonoids, and stress indicators (MDA, H₂O₂) at p < 0.001. The different foliar applications mainly affected dry matter content and chlorophyll b at p < 0.05; total chlorophylls at p < 0.01; macronutrient content, total phenolics, the antioxidant capacity (assayed by DPPH, FRAP, and ABTS), flavonoids, and stress indicators at p < 0.001. Finally, their interaction mainly affected the plants’ fresh weight at p < 0.05; FRAP at p < 0.01; chlorophylls (a, b, and total), macronutrient content, total phenolics, the antioxidant capacity (DPPH and ABTS), flavonoids, and stress indicators at p < 0.001.

3.3. Plant Biomass, Photochemistry, and Mineral Content

The impacts of fertilization and foliar application of Fe and Zn on S. cypria plants are shown in

Table 2. Effect of different fertilization applications [No fertilization (NoFert), Organic fertilization (OrFert), Conventional fertilization (CoFert)] and foliar applications (water-H₂O, iron-Fe, zinc-Zn) on Sideritis cypria and Origanum dubium plant biomass fresh weight (FW; g plant⁻¹), dry weight (DW; g plant⁻¹), and dry matter content (DM; %).

	Fertilization	Foliar	Plant FW	Plant DW	Plant DM
Sideritis cypria	NoFert	H2O	193.37 ± 13.84 cd	83.54 ± 7.64 d	43.60 ± 3.89 cd
		Fe	260.43 ± 36.26 abc	111.08 ± 12.58 bcd	43.59 ± 1.83 cd
		Zn	305.58 ± 21.65 ab	150.09 ± 8.30 a	49.78 ± 2.66 bc
	OrFert	H2O	243.12 ± 23.29 bc	121.30 ± 11.56 abc	50.06 ± 1.26 bc
		Fe	234.87 ± 22.56 bcd	126.81 ± 10.40 ab	54.48 ± 1.57 ab
		Zn	335.65 ± 37.20 a	140.88 ± 12.56 ab	42.58 ± 1.54 d
	CoFert	H2O	254.80 ± 26.39 abc	111.34 ± 7.00 bcd	44.83 ± 2.68 cd
		Fe	224.48 ± 32.52 bcd	109.50 ± 15.18 bcd	48.73 ± 2.02 bcd
		Zn	155.45 ± 15.36 d	88.63 ± 8.40c	57.12 ± 1.25 a
	Fertilization	Foliar	Plant FW	Plant DW	Plant DM
Origanum dubium	NoFert	H2O	164.90 ± 31.92 c	66.03 ± 13.42	40.49 ± 2.48 ab
		Fe	209.62 ± 29.20 bc	84.20 ± 10.19	40.60 ± 0.92 ab
		Zn	172.43 ± 16.10 c	69.42 ± 6.21	40.32 ± 0.83 ab
	OrFert	H2O	140.42 ± 15.20 c	61.98 ± 6.81	42.73 ± 1.34 a
		Fe	141.53 ± 19.16 c	58.82 ± 10.11	40.73 ± 1.54 ab
		Zn	234.68 ± 40.20 bc	74.09 ± 11.39	33.74 ± 3.69 b
	CoFert	H2O	350.77 ± 34.28 a	93.14 ± 14.24	25.73 ± 1.73 c
		Fe	283.75 ± 24.20 ab	70.95 ± 10.38	24.09 ± 2.34 c
		Zn	287.35 ± 40.72 ab	70.06 ± 16.53	22.18 ± 3.18 c

Significant differences (p < 0.05) among applications are indicated by different letters. Values are means (±SE) of six replicates for each treatment.

. The highest fresh weight was recorded for plants grown with OrFert and foliar Zn, and the lowest for plants grown with NoFert when sprayed with H₂O. Interestingly, in NoFert, the foliar application of both Fe and Zn increased the plants biomass, while in OrFert, only the application of Zn increased the plants biomass, compared to the foliar application of H₂O. In contrast, in CoFert, a significant reduction in fresh weight was evidenced after Zn spraying, compared to foliar application of H₂O. Furthermore, the highest and lowest dry weights were found in the NoFert treatment, with the Zn spraying and without spraying, respectively. Finally, the use of OrFert and CoFert, in combination with Zn spraying, resulted in the lowest and highest dry matter content, respectively.

In the case of O. dubium plants, the highest fresh weight was marked in plants grown with CoFert and foliar applications of H₂O, while no significant changes were evidenced with the foliar application of Fe and Zn in the same fertilization application (Table 2). Furthermore, a significant decrease was evidenced in the dry matter content of plants grown with CoFert, regardless of foliar application, in comparison to the rest of the treatments. Finally, the plants’ dry weight was unchanged by the tested applications.

The photochemistry and therefore the photosynthetic attributes in plants were affected by the treatments examined. In S. cypria leaves, SPAD was significantly increased with the use of NoFert with Fe spraying and CoFert with Zn spraying, compared to the use of OrFert with Zn spraying (

Table 3. Effect of different fertilization applications [No fertilization (NoFert), Organic fertilization (OrFert), Conventional fertilization (CoFert)] and foliar applications (water-H₂O, iron-Fe, zinc-Zn) on Sideritis cypria and Origanum dubium SPAD value, chlorophyll content (Chlorophyll a: Chl a; Chlorophyll b: Chl b; Total chlorophylls: Total chls; mg g⁻¹ FW), carotenoid content (Car: mg g⁻¹ FW), chlorophyll a to chlorophyll b and carotenoids to Total chlorophylls ratios.

	Fertilization	Foliar	SPAD	Chl a	Chl b	Total Chls	Car	Chl a/Chl b	Car/Total Chl
Sideritis cypria	NoFert	H2O	56.10 ± 1.65 ab	0.76 ± 0.04 a	0.38 ± 0.03 ab	1.13 ± 0.07 a	0.118 ± 0.003	2.05 ± 0.07 ab	0.105 ± 0.005
		Fe	67.45 ± 5.21 a	0.70 ± 0.03 ab	0.35 ± 0.02 ab	1.04 ± 0.05 ab	0.115 ± 0.005	2.04 ± 0.05 ab	0.108 ± 0.003
		Zn	49.35 ± 3.69 b	0.81 ± 0.03 a	0.42 ± 0.03 a	1.23 ± 0.05 a	0.120 ± 0.007	1.93 ± 0.09 b	0.098 ± 0.006
	OrFert	H2O	57.98 ± 5.56 ab	0.71 ± 0.04 ab	0.35 ± 0.03 ab	1.06 ± 0.07 ab	0.110 ± 0.009	2.02 ± 0.04 ab	0.105 ± 0.005
		Fe	56.75 ± 2.33 ab	0.72 ± 0.05 ab	0.37 ± 0.05 ab	1.10 ± 0.10 ab	0.110 ± 0.004	1.98 ± 0.12 b	0.103 ± 0.009
		Zn	47.30 ± 3.79 b	0.74 ± 0.02 ab	0.38 ± 0.01 ab	1.12 ± 0.03 a	0.110 ± 0.007	1.96 ± 0.03 b	0.103 ± 0.006
	CoFert	H2O	53.68 ± 3.35 ab	0.72 ± 0.02 ab	0.34 ± 0.01 ab	1.06 ± 0.03 ab	0.115 ± 0.003	2.11 ± 0.02 ab	0.110 ± 0.004
		Fe	57.45 ± 6.83 ab	0.62 ± 0.04 b	0.28 ± 0.02 b	0.89 ± 0.06 b	0.103 ± 0.005	2.22 ± 0.03 a	0.115 ± 0.003
		Zn	66.73 ± 3.76 a	0.71 ± 0.06 ab	0.35 ± 0.05 ab	1.07 ± 0.10 ab	0.115 ± 0.003	2.06 ± 0.12 ab	0.113 ± 0.013
	Fertilization	Foliar	SPAD	Chl a	Chl b	Total chls	Car	Chl a/Chl b	Car/Total chl
Origanum dubium	NoFert	H2O	36.93 ± 2.96	0.76 ± 0.02 de	0.46 ± 0.01 cd	1.22 ± 0.03 de	0.10 ± 0.00 abc	1.63 ± 0.03 bc	0.08 ± 0.00 bcd
		Fe	40.55 ± 2.49	0.84 ± 0.02 ab	0.61 ± 0.03 a	1.45 ± 0.04 a	0.06 ± 0.01 d	1.39 ± 0.07 d	0.04 ± 0.01 e
		Zn	40.23 ± 3.96	0.69 ± 0.01 f	0.37 ± 0.01 e	1.06 ± 0.02 f	0.09 ± 0.00 bc	1.84 ± 0.03 a	0.08 ± 0.00 abc
	OrFert	H2O	36.95 ± 3.60	0.78 ± 0.03 cde	0.49 ± 0.04 bcd	1.26 ± 0.06 cd	0.09 ± 0.01 c	1.63 ± 0.08 bc	0.07 ± 0.01 cd
		Fe	43.95 ± 3.21	0.80 ± 0.01 bcd	0.42 ± 0.01 de	1.21 ± 0.02 de	0.10 ± 0.00 abc	1.91 ± 0.02 a	0.08 ± 0.00 abc
		Zn	41.28 ± 2.24	0.81 ± 0.01 bc	0.53 ± 0.03 bc	1.34 ± 0.04 bc	0.09 ± 0.01 c	1.54 ± 0.06 cd	0.06 ± 0.01 d
	CoFert	H2O	37.78 ± 3.10	0.75 ± 0.01 e	0.38 ± 0.01 e	1.13 ± 0.02 ef	0.11 ± 0.00 ab	1.98 ± 0.04 a	0.10 ± 0.00 a
		Fe	38.93 ± 3.22	0.80 ± 0.01 bcde	0.45 ± 0.02 de	1.24 ± 0.03 cd	0.11 ± 0.00 a	1.80 ± 0.09 ab	0.09 ± 0.00 ab
		Zn	44.45 ± 2.45	0.86 ± 0.00 a	0.54 ± 0.03 b	1.40 ± 0.02 ab	0.09 ± 0.01 abc	1.62 ± 0.09 bc	0.07 ± 0.01 cd

Significant differences (p < 0.05) among applications are indicated by different letters. Values are means (±SE) of six replicates for each treatment.

). Furthermore, chlorophyll a was significantly increased with the application of NoFert without foliar sprays and with foliar Zn. Chlorophyll b was significantly increased with the application of NoFert with foliar Zn. Total chlorophylls were significantly increased with the use of NoFert without foliar sprays and with Zn spraying, as well as OrFert with Zn spraying, compared to the lowest values of chlorophylls (a, b, and total) achieved by CoFert with foliar Fe. Furthermore, the chlorophyll a to chlorophyll b ratio was significantly increased with the use of CoFert with Fe spraying, compared to NoFert with foliar Zn and OrFert with foliar Fe and Zn.

In the case of O. dubium leaves, chlorophyll a was the highest with the use of CoFert and Zn spraying (Table 3). Interestingly, while the treatment of NoFert and Fe spraying did not differ significantly from the previously mentioned treatment (CoFert + Zn), the treatment of NoFert and Zn spraying produced plants with the lowest chlorophyll a content. Similarly, the NoFert and Zn spraying treatment resulted in the lowest total chlorophyll content, while in the same treatment, the CoFert with foliar application of H₂O resulted in the lowest chlorophyll b content. In contrast, the application of NoFert and Fe spraying produced plants with the highest content of chlorophyll b and total chlorophylls, while the same treatment recorded the lowest content of carotenoids, as well as the lowest ratios of chlorophyll a to chlorophyll b and carotenoids to chlorophylls. Regarding carotenoids, the highest content was recorded in plants grown with the CoFert and Fe spraying treatment. The highest chlorophyll a to chlorophyll b ratios were recorded for NoFert and Zn spraying, OrFert and Fe spraying, and CoFert and foliar application of H₂O. Additionally, the highest carotenoids to chlorophylls ratio was recorded for the CoFert and foliar application of H₂O.

Results regarding the macronutrient content of S. cypria plant leaves are presented in Figure 1, where fluctuations were recorded for the examined fertilization and foliar applications. Particularly, plants treated with CoFert and Zn spraying recorded the highest content of N, P, K, and Na (16.56, 20.72, 3.92, and 1.98 g kg⁻¹, respectively) (Figure 1(A1–A4)), while plants treated with NoFert and Zn spraying recorded the lowest content of N, showing a decrease of 33.9% over the former treatment. Additionally, the use of NoFert with foliar application of H₂O resulted in the lowest P and K contents in leaves (Figure 1(A2,A3)), showing a decrease of 36.4% and 67.3%, compared to the CoFert and Zn spraying treatment, respectively. Finally, the lowest overall Na was evidenced in plants treated with NoFert and foliar Fe, noting a 77.3% decrease compared to plants treated with CoFert and Zn spraying (Figure 1(A4)).

In the case of the O. dubium, at the NoFert treatments, the foliar application of H₂O resulted in the lowest content of N, while a significant increase was observed with the foliar application of both Fe and Zn over the rest of the treatments (Figure 1(B1)). Similarly, the Fe spraying increased the N content of plants grown with OrFert, an effect that was also exhibited in both P and K contents (Figure 1(B2,B3)). For K particularly, the highest and lowest K content was found in plants grown with OrFert, with Fe spraying and without foliar applications, respectively. Finally, the lowest Na content was marked for plants treated with NoFert, with Fe or without foliar applications, while a significant increase was evidenced with Zn spraying over the abovementioned treatments (Figure 1(B4)). Similarly, foliar applications in the OrFert treatment increased the content of Na over foliar applications of H₂O, with the OrFert and Zn treatment recording the highest Na content.

Figure 2 presents the results regarding total phenolics, flavonoids, and antioxidant capacity of S. cypria (Figure 2(A1–A5)) and O. dubium (Figure 2(B1–B5)) plants grown under varying fertilization and foliar applications. In the case of S. cypria, total phenolics were the highest when plants were grown with CoFert, using foliar application of H₂O or of Fe, while the use of Zn spraying caused a significant decrease over the previously mentioned treatments (Figure 2(A1)). Furthermore, total phenolics were the lowest when plants were grown with NoFert, using foliar application of H₂O, while Fe and Zn spraying increased total phenolics. The plants that received CoFert and Fe spraying had the highest flavonoids content, while the ones that received OrFert and Fe spraying had the lowest flavonoids content (Figure 2(A2)). The highest DPPH was recorded in plants treated with CoFert with foliar application of H₂O (Figure 2(A3)), the highest FRAP was recorded in plants treated with CoFert and Fe spraying (Figure 2(A4)), while the highest ABTS was recorded in both abovementioned treatments (Figure 2(A5)). In contrast, the antioxidant capacity (assayed by FRAP and ABTS) was the lowest in plants treated with OrFert and Fe spraying (Figure 2(A4,A5)).

In the case of the O. dubium, total phenolics were increased with foliar application of H₂O under all fertilizer applications, while in the case of NoFert, an increase was observed with Zn spraying (Figure 2(B1)). In the case of NoFert, the application of Fe decreased the content of total phenolics, while in the case of OrFert and CoFert, both Fe and Zn alleviated the production of total phenolics. Flavonoids followed the trend of the type of fertilization applied, as the highest contents were observed with no fertilizer application, while a decrease was found with the use of OrFert and NoFert (Figure 2(B2)). The antioxidant capacity (after DPPH and FRAP assays) of O. dubium plants increased without the foliar applications under all fertilizer applications, while a significant decrease was noted with the Fe and Zn spraying (Figure 2(B3,B4)). Furthermore, the highest ABTS content was observed in plants grown with NoFert or OrFert, treated with H₂O foliar applications and foliar Zn, and plants grown with CoFert, without foliar applications (Figure 2(B5)). Generally, the lowest antioxidant capacity (after DPPH, FRAP, and ABTS assays) was evidenced in plants treated with CoFert and Zn spraying.

The stress response of S. cypria plants, as assayed by MDA content and H₂O₂ production, was presented in Figure 3(A1,A2). Plants that received conventional fertilization, in conjunction with Fe spraying, had the highest content of MDA and H₂O₂. Furthermore, in the case of MDA, the lowest content was observed in plants grown with OrFert and supplied with Fe and Zn spraying (Figure 3(A1)). Finally, the lowest H₂O₂ was observed in plants treated with NoFert and Fe spraying, exhibiting a significant decrease compared to the other foliar treatments of NoFert (Figure 3(A2)).

Regarding the effects of the examined applications on the stress response of O. dubium plants, the highest lipid peroxidation, through the MDA content, was found in plants grown using no fertilization and either foliar application of Fe or Zn, while lower values were observed without foliar applications (Figure 3(B1)). A significant decrease in MDA was found in plants treated with both OrFert and CoFert. In the case of OrFert, MDA increased with the Fe and Zn spraying, compared to H₂O spraying, while in the case of CoFert, MDA was decreased with the Fe spraying, compared to Zn and H₂O spraying. The accumulation of H₂O₂ was also influenced by the treatments examined (Figure 3(B2)). The highest H₂O₂ content was observed in plants grown with OrFert without foliar application. The Fe or Zn spraying at OrFert and CoFert applications caused a significant decrease in H₂O₂ when compared to foliar application of H₂O.

In S. cypria, the WUE tended to increase only after Zn spraying for both the NoFert and OrFert treatments, while the opposite results were evidenced after Zn spraying at the CoFert treatment (Figure 4(A1)). The NUE after fertilization was obviously increased after OrFert and CoFert applications, while NUE was decreased after Zn spraying at the CoFert treatment compared to the H₂O or Fe spraying at the same treatment (i.e., CoFert) (Figure 4(A2)). The foliar efficacy, after comparing the Fe or Zn spraying with the H₂O spraying, revealed a similar trend to the WUE, as the foliar efficacy was increased after Zn spraying at the NoFert and OrFert treatments and decreased after Zn spraying at the CoFert treatment (Figure 4(A3)).

In the case of O. dubium, WUE tended to increase at OrFert but decreased at CoFert treatments after Zn spraying, whereas Fe spraying did not have any effects (Figure 4(B1)). The NUE was negatively affected by the Fe and Zn spraying only at the CoFert treatment (Figure 4(B2)).

The foliar application efficacy in S. cypria was evidenced with the Zn spraying at the NoFert and OrFert treatments but not in the case of the CoFert treatment, where the foliar efficacy decreased compared to the H₂O or Fe spraying (Figure 4(A3)). In the case of O. dubium, the foliar efficacy was not evidenced, as foliar application seems to not alter the fresh biomass produced (Figure 4(B3)).

4. Discussion

Sustainability and preservation of biodiversity are the guiding principles of organic farming and the utilization of organic fertilizers [4,13]. Since MAPs and other underutilized crops are adapted to both abiotic and biotic stressors and require minimal inputs, they are well-suited for integration into organic farming systems [20,57]. S. cypria and O. dubium are two promising MAPs that were used in the current research to investigate and encourage the adoption of sustainable farming techniques. Maintaining a proper balance of inputs is essential in the context of ecologically sound and organic agriculture, given that fertilization has a critical role in crop development and quality and ultimately in crop output [17,58]. Application rates and fertilizer type determine how organic fertilization and the combination of chemical and organic fertilizers affect crop development and soil fertility. Organic fertilizer application rates are generally determined by crop N requirements and the predicted organic fertilizer N supply rates; however, P and K content are not considered in general. Optimal fertilization techniques are crucial in boosting the plant growth and quality-related characteristics of MAPs [59]. On the other hand, conventional fertilization might raise the MAP yield but not necessarily mirror advances in the quality and safety of the consumed/used products, because the level of bioactive substances and, thus, the plants nutritional value are decreased by mineral fertilizers [58].

Prior studies reported that conventional fertilization resulted in similar Sideritis perfoliata yield compared to organic fertilization [9], which is in agreement with the S. cypria yield for the present study. However, O. dubium had 2.5 times higher yield in conventional fertilization compared to the organic scheme, indicating the various responses of plant metabolism to fertilization and different plants mineral needs. Such variation in mineral needs is important at a farm level in order to optimize the crop selection based on mineral needs and soil fertility. Zinc spraying increased S. cypria fresh weight in the NoFert and OrFert treatments, but Fe spraying did not have such effects.

According to other studies, organic medicinal plants had a much higher dry matter content and a lower water content than conventionally produced plants [58], which aligns only with the findings of the O. dubium plants, since S. cypria plants had the same dry matter content in all fertilized or non-fertilized plants. Previous reports indicated similar dry matter content in organically vs. conventionally grown peppermint (Mentha piperita L.) and rosemary (R. officinalis L.) [58]. Moreover, irrigation is also an important cultivation practice to be considered, as the MAPs under drought stress activate metabolic processes towards water stress, with reduced cell multiplication and expansion, which negatively affects plant growth with decreased yield, increased dry matter content, and the activation of various antioxidant mechanisms towards the water shortage challenge [9,60]. Interestingly, Zn spraying exerted fertilizer-specific effects on S. cypria dry matter content, decreasing it under the OrFert treatment but increasing it under the CoFert treatment. The observed variation in plant responses as a function of base fertilization is related to the differential availability of nutrients, particularly N, which has a significant role in the efficacy of utilization of exogenously applied Zn [61]. In addition, the dry matter content of O. dubium plants with OrFert application was negatively impacted by the Zn spraying, compared to H₂O spraying. Similarly, Adamczyk-Szabela and Wolf [62] demonstrated that Zn supplementation had a differential impact on the dry biomass. It was mentioned that dry biomass was decreased with Zn supplementation in common nettle (Urtica dioica L.), increased in borage (Borago officinalis L.), or unaffected in basil (Ocimum basilicum L.) and peppermint (M. piperita L.) [62].

Fertilization constitutes a critical factor in the synthesis of chlorophylls by supplying key macronutrients such as N. Variations in the availability and mineralization of nutrients between organic and conventional fertilization can significantly affect the production of chlorophylls, thus influencing photosynthesis and overall plant development [54]. Chrysargyris et al. [18] observed that organic fertilization decreased the relative chlorophyll content of spearmint (M. spicata L.) leaves, as indicated by lower SPAD values, compared to conventional fertilization. In contrast, Hajisolomou et al. [54] reported that Portulaca oleracea L. and Sonchus oleraceus L. plants subjected to organic fertilization had SPAD values and chlorophyll contents comparable to those observed under conventional fertilization. Similarly, in the current study, the fertilization type did not significantly affect SPAD or total chlorophyll content in the leaves of S. cypria or O. dubium. However, plant genotype responses depend on variable soil fertility [63]. For instance, in O. dubium, the interaction of fertilization and foliar applications influenced the content of chlorophylls. More specifically, total chlorophyll contents were significantly elevated under NoFert with Fe spraying and CoFert with Zn spraying. This may be related to the critical roles of Fe and Zn in the biosynthesis of chlorophyll, as Fe is involved in the formation of chlorophyll precursors, while Zn influences enzyme activities, the metabolism of photosynthetic proteins, and N uptake [64,65,66]. In addition, N fertilization has a synergistic effect on Zn accumulation, especially when Zn supply is not limiting, thus influencing chlorophyll content [67]. In accordance with these results, Fe and Zn spraying have been associated with greater chlorophyll accumulation in pepper (Capsicum annum L.) and wheat (Triticum aestivum L.), respectively [64,68].

The mineral content of MAPs is a significant factor influencing their medicinal efficacy and nutritional status [45]. For optimal growth, plants require an adequate supply of nutrients, tailored to their specific needs and developmental stage. Organic farming generally consists of lower nutrient addition with organic fertilizers compared to conventional ones, resulting in a decreased mineral content in soils [9]. In addition, nutrient availability is harder to predict, as it depends on the mineralization of organic matter, which is influenced by factors such as temperature, available microorganisms, pH, and soil moisture [48]. Furthermore, to enhance the nutritional value of MAPs, agronomic biofortification applications have been gaining interest [57]. In the current study, both examined plants exhibited varying responses to basic fertilization and foliar applications. Nitrogen content in S. cypria leaves was increased by Zn spraying under both CoFert and OrFert, likely due to the positive effect of Zn supply on N absorption. However, this effect is contingent upon N availability and form [67]. Zn is an important component of enzymes that regulate key metabolic processes in plants, while also having a critical role in auxin and protein synthesis that contribute to enhanced N uptake [69]. This is consistent with previous research. Tzortzakis et al. [31] reported that under sufficient N levels in hydroponically grown S. cypria, Zn spraying mediated N uptake. In addition, Alinejad Elahshah et al. [70] demonstrated that Zn spraying significantly enhanced N absorption by strawberry (Fragaria × ananassa Duch.) plants. In O. dubium, however, N content was significantly increased by both Fe and Zn spraying, but only under NoFert, suggesting that under nutrient limitation, foliar micronutrients boost N assimilation. Phosphorus content in S. cypria was also improved under Zn spraying with CoFert, possibly due to Zn stimulating the activation of P-solubilizing enzymes such as phosphatase and phytase [71]. In O. dubium, Fe spraying was linked to increased P acquisition, particularly under OrFert and CoFert. Similarly, Neofytou et al. [72] reported elevated P accumulation in S. cypria leaves after application of Fe spraying under increased P availability. Potassium uptake was enhanced with Zn spraying under fertilized S. cypria treatments. This result aligns with the findings of Alinejad Elahshah et al. [70], as Zn spraying led to increased K uptake in strawberry (F. × ananassa Duch.) plant leaves. Interestingly, O. dubium was also found to have increased K levels under Zn spraying, especially under CoFert, while Fe spraying had a more pronounced effect on leaf K content, being in accordance with previous reports in peppermint (M. piperita L.) [73].

The evaluated species exhibited distinct variations in their phenolic profile and antioxidant capacity in response to the applied fertilization schemes and/or the foliar applications. Total phenols, flavonoids, and antioxidant capacity of S. cypria were triggered by CoFert following foliar with H₂O or Fe, underscoring the critical role of Fe in this species in its metabolic and physiological processes. For instance, Fe spraying to S. cypria plants induced non-enzymatic antioxidant mechanisms and flavonoid contents, contributing to enhanced antioxidant capacity [33]. This effect was limited to CoFert, where nutrient availability, particularly N, was more immediate and sufficient, especially during periods of rapid plant growth. In contrast, organic fertilization, which releases nutrients more gradually, may not adequately meet the demands of the plants [74]. This may have limited the plants’ metabolic response to Fe spraying, leading to decreased phenolics, flavonoids, and antioxidant capacity of S. cypria. Nonetheless, Fe and Zn spraying modulated the production of phenolic compounds and antioxidants, causing a reduction in lipid peroxidation [72]. Although organic fertilization is often associated with enhanced produce quality, particularly in terms of phytochemical richness, this is not consistently observed [74]. For instance, Lv et al. [25] found no changes on total phenols but only on individual phenolic components on peppermint (M. piperita L.) and cinnamon (Cinnamomum verum L.) that were grown in organic and conventional farms. In contrast, organically grown S. perfoliata [9], R. officinallis, and Melissa officinalis [58] revealed higher total phenolics than the conventional one.

In the case of the O. dubium, total phenolics, flavonoids, and antioxidants were stimulated with the foliar application of H₂O, while both Fe and Zn applications resulted in plants with decreased levels of phenolics, flavonoids, and antioxidants, especially under fertilization schemes (CoFert and OrFert). In contrast, Fe and Zn nanoparticle applications in M. officinalis L. reportedly improved biochemical attributes, including total phenolic and flavonoid content [75]. This discrepancy may be related to the increased availability of nutrients, and especially N, as it influences nitrogen-containing metabolites, including free amino acids, proteins, and alkaloids. This in turn causes a decline in phenolics, flavonoids, and vitamin C content [58]. In addition, the carbon:nitrogen (C:N) ratio influences the synthesis of key plant-defense components [76]. In contrast, N shortage stimulates the production of defense components [18]. This is caused by the reallocation of available carbon, with plants diversifying and increasing secondary metabolism, which includes phenolics, flavonoids, and vitamin C increases [77].

Application of organic fertilizers has been associated with increased stress tolerance by improving water and nutrient uptake, antioxidant activity, and gene expression [78]. In addition, Fe and Zn have direct roles in mitigating oxidative damage by activating several antioxidant enzymes, which decrease reactive oxygen species (ROS) [79]. Zinc is involved in several physiological processes that influence plants’ stress response, including the protection and maintenance of the cell membrane’s stability and being a cofactor of enzymes such as superoxide dismutase, peroxidase, and catalase [79,80]. For instance, Zn had a significant effect on the resistance of rice plants to low temperatures, influencing the activity of antioxidant enzymes and subsequently decreasing the plants’ MDA content [81]. Likewise, Fe is associated with antioxidant enzymes, being present in the active site of enzymes involved in ROS scavenging and thus facilitating tolerance to various abiotic stressors [65,82]. For S. cypria, the lowest MDA content was observed in plants grown with OrFert and treated with Fe and Zn spraying. This indicates the influence of organic fertilization in conjunction with foliar micronutrient applications in attenuating oxidative damage markers and enhancing antioxidant activities [83]. In the case of O. dubium, the highest lipid peroxidation was observed under the NoFert treatment, while a mediation effect was observed following fertilization. In general, nutrient deprivation and deficiencies affect metabolic processes, which potentially result in a stress response [84]. Interestingly, Fe and Zn spraying modulated the production of H₂O₂, especially in fertilized treatments. This is attributable to the enhancement of antioxidant enzyme activities that scavenge ROS, thus mitigating oxidative stress [85].

The current study demonstrated that Zn spraying tended to increase WUE of S. cypria plants under NoFert and OrFert treatments and of O. dubium plants under OrFert. Zinc has been found to have an influence on WUE by increasing osmolytes, improving stomatal conductance, and maintaining membrane integrity, supporting these findings [86]. In contrast, the opposite occurred under CoFert for both S. cypria and O. dubium. This may be related to nutrient imbalances, particularly the elevated uptake and accumulation of P observed in S. cypria, which could have interfered with Zn utilization [87]. In addition, both plant species exhibited increased NUE under CoFert application. Unlike organic fertilizers, where nutrient release depends on mineralization, conventional fertilizers immediately supply nutrients under high plant demand. The result indicates that the steady but delayed nutrient supply of organic amendments may not have been fully realized within the duration of the experiment [88]. In contrast, under CoFert, decreases occurred when Zn spraying was applied, mainly due to the decrease in biomass production.

5. Conclusions

This study examined the use of organic and conventional fertilizers in the field cultivation of Sideritis cypria and Origanum dubium, in conjunction with foliar applications of iron (Fe) and zinc (Zn). In terms of yield, S. cypria presented minute differences among organic fertilization (OrFert) and conventional fertilization (CoFert), but also with no fertilization. In contrast, O. dubium revealed a 2.5 times increase in fresh weight under conventional fertilization relative to organic fertilization, indicating a species-specific response to fertilization method. In terms of foliar applications, Zn produced fertilizer-specific effects on S. cypria dry matter content, related to the availability of nutrients, particularly N, which influences the efficacy of Zn. In contrast, the dry matter content of O. dubium under OrFert application was decreased under Zn spraying, demonstrating application responses that are specific to each species. S. cypria showed distinct phenolic and antioxidant responses to fertilization and foliar treatments, mostly benefiting from conventional fertilization and Fe spraying, while organic fertilization showed variable effects, likely due to slower nutrient release. In contrast, the lowest malondialdehyde content was observed in S. cypria grown with OrFert and treated with Fe and Zn spraying, with their combination indicating an amelioration effect. In O. dubium, the phenolics content, antioxidant activity, and hydrogen peroxide production were decreased by Fe and Zn spraying under adequate nutrition. Finally, Zn spraying resulted in improvements of water use efficiency on both species, contingent on fertilization application, while conventional cultivation led to an improvement on nutrient use efficiency, provided by the immediate supply of nutrients. In conclusion, the utilization of organic fertilizers in S. cypria and O. dubium cultivation, although inducing variations in terms of leaf chlorophyll content, exhibited adequate fertilizer efficiency. Additionally, Fe and Zn spraying had a significant role in the antioxidant capacity and mineral uptake of the examined species. However, appropriate fertilization management is needed to fully utilize the benefits of foliar applications, especially in the context of organic practices.