Comprehensive Study on the Potential of Domesticated Clones of Rosemary (Salvia rosmarinus Spenn.): Implications for Large-Scale Production and Waste Recovery in the Development of Plant-Based Agrochemicals

Abstract

Rosemary is a versatile Mediterranean shrub valued for its culinary and medicinal uses, also finding applications as a food additive (E-392). This study explores the potential of rosemary for large-scale cultivation as well as the valorization of its distillation residue, which constitutes more than 95% of the total biomass. Rich in bioactive compounds, this solid waste represents a valuable opportunity to develop renewable plant-based products. This study monitored the agronomic adaptations of cultivated clones of rosemary and evaluated the essential oil and phenolic content. This study also evaluated the biological potential of the ethanolic extracts from the distilled residue as an antifungal, antioxidant, chelator, and biostimulant in model tests. Interestingly, the extracts showed substantial phenolic content, exhibiting strong antifungal activity, antioxidant capacity, and efficient metal chelation. Furthermore, all extracts also demonstrated promising biostimulant effects on rooting. Among the clones evaluated, Pina de Ebro stood out especially for its balanced adaptability, high essential oil yield, and outstanding phenolic content, along with uniform biological capacities among individual plants and plots. Therefore, this study highlights the potential of utilizing the entire rosemary plant, enhancing the overall profitability of the crop and meeting the growing demand for eco-friendly and renewable resources in the market.

1. Introduction

Rosemary (Salvia rosmarinus Spenn., syn. Rosmarinus officinalis L.) is a woody shrub that can grow up to 1.8 m in height. It is typically erect and is one of the most widely distributed aromatic plants in the Mediterranean basin due to its adaptability to a wide variety of substrates and diverse habitats [1]. This species is well-known for its diverse traditional applications in food preparations and its numerous medicinal properties. So far, it is the only plant extract regulated as an antioxidant by the European Commission in food preservation (E-392; [2]). Additionally, numerous studies have proven its other biological capacities, such as being antimicrobial [3,4], anti-inflammatory [4], and antiproliferative to cancer cells [5], having allelopathic properties [6] and phytostimulant activity [7], as well as its use as a bioplaguicide [8,9]. These bioactivities have been observed in different herbal preparations, including essential oils [10,11,12] and plant extracts using solvents such as hydroalcoholic mixtures [5,13], glycerol [14], glycol derivatives [4], etc.

The primary marketable product of rosemary is likely the dried plant, while the essential oil (EO) is extensively used in various food, pharmacological, and cosmetic applications. Except for certain phenological stages and bioclimatic areas as shown by Jordán et al. [15], wild rosemary plants rarely yield EO levels above 2% [10,16,17]. Consequently, the distilled biomass often exceeds 95–98% of the total dry plant, representing an undervalued waste. However, these residues presents numerous opportunities for valorization, due to their content of compounds with significant biological activities [7,12,15,18,19]. By extracting and incorporating these bioactive components into new natural formulations, high-value products can be developed and marketed alongside EOs, thereby maximizing resource utilization and significantly enhancing overall crop profitability.

Ortiz de Elguea-Culebras et al. [20] reviewed how appropriate solvents and extraction techniques, such as Soxhlet extraction with ethanol, can yield valuable extracts from distilled rosemary biomass, amounting to 10–15% recovery [21]. Notably, phenolic compounds stand out for their diverse biological functions and substantial abundance, frequently exceeding 10% in rosemary residue extracts [7]. Considering a typical rosemary biomass production of 0.4–0.5 kg./plant [22], this suggests a potential release of 5–10 g of phenolic compounds per plant from distillation residues. Furthermore, optimizing processes with sustainable solvents, with minimal environmental and health risks, can enhance the concentration of bioactive metabolites in rosemary extracts [23].

In our evaluation of the potential of domesticated rosemary clones for (1) large-scale cultivation and (2) the development of agrochemicals from distillation by-products, we meticulously chose five Salvia rosmarinus clones with different essential oil profiles. The selection of the most profitable candidate aligned with these objectives prioritized two key aspects—(i) agronomic perspective + evaluation of essential oils and (ii) phenolic content + biological potential of waste extracts—while guaranteeing uniformity between plants and plots.

2. Material and Methods

2.1. Chemicals and Reagents

Gallic acid, Folin-Ciocalteu’s phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide (III), trichloroacetic acid, iron (III) and iron (II) chlorides, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (FerroZine™ Iron Reagent), copper(II) sulphate, and pyrocatechol violet were acquired from Merck KGaA (Darmstadt, Germany). All solvents were of analytical grade.

2.2. Vegetal Material Collection

Five domesticated clones of Salvia rosmarinus Spenn. (syn. Rosmarinus officinalis L.) were specifically chosen for this study due to their previously identified yield and variability in essential oils [24]. From each clone, 30 cuttings were randomly selected in April 2018, rooted in a greenhouse, and planted in three plots (or repetitions) with ten individuals (N = 150; 5 clones × 3 plots × 10 individuals) at the Albaladejito Agroforestry Research Center (CIAF-IRIAF; 40.0673, −2.1963). The trial was conducted under rainfed conditions with minimal intervention, limited to weed control. To ensure a uniform rosemary crop, dead or non-viable individuals (bad appearance) were monitored annually and replaced with ramets from the original clones, using identical rooting techniques. This process continued until a 90% survival rate of living individuals was achieved. After reaching this condition, four individuals from each plot and clone (N = 60; 5 clones × 3 plots × 4 individuals) were randomly harvested simultaneously. Adaptability (%): number of live plants/total planted ramets. Fresh Plant (kg/plant): Weight per plant after harvesting. Dry Plant (kg/plant): Weight per plant after shade drying at room temperature. Moisture content (%): [(Fresh Plant − Dry Plant)/Fresh Plant] × 100.

2.3. Vegetal Material Extraction and Characterization

Fifty grams of leaves, separated from the woody part of the dried rosemary plant, were subjected to hydrodistillation in a Clevenger apparatus for 2.5 h after the first condensed drop. Following this, the EOs were collected and dried using anhydrous sodium sulfate. Additionally, the distilled biomass was collected, shade dried at room temperature and then subjected to a 12 h Soxhlet extraction with 96% ethanol. The ethanol extract (EE) was obtained after rotary evaporation to remove the solvent followed by final drying in an oven for 48 h at 45 °C. Both the EOs and EEs were stored under refrigeration (4 °C) until further analysis. Essential oil yield (%v/w): Volume of EO (mL) per 100 g of dry leaves. Ethanolic extract yield (%w/w): Weight of EE (g) per 100 g of distilled leaves.

2.4. Chemical Characterization of the EOs (GC)

The essential oils (EOs) were chemically characterized using gas chromatography (GC) in a Varian 400-GC system equipped with a VF-5MS column (60 m × 0.25 mm × 0.25 μm film thickness, cross-linked phenyl-methylsiloxane) from Agilent Technologies, Inc. (Santa Clara, CA, USA). The analytical procedure and the identification and quantification of the chemical constituents were conducted following the indications of Melero-Bravo et al. [24].

2.5. Phenolic Characterization of the EEs

2.5.1. Total Phenol Content (TPC)

The total phenol content (TPC) of the S. rosmarinus EEs was determined using the Folin-Ciocalteu reagent. Briefly, 5 µL of the EEs at a concentration of 1 mg/mL was incorporated in quadruplicate into 96-well microplates along with 155 µL of water, 10 µL of Folin-Ciocalteu reagent, and, after 5 min, 30 µL of 20% Na₂CO₃. The plates were incubated at 40 °C for 30 min and then scanned on an EPOCH 2 microplate reader (Agilent Technologies, Inc., Santa Clara, CA, USA) at 765 nm. Gallic acid was employed as a standard for the calibration curve (1–6 µg/mg; y = 0.059x − 0.001; R² = 0.996), and the results were expressed in grams of equivalent gallic acid (GAE) per 100 g of S. rosmarinus EE (g GAE/100 g EE).

2.5.2. Phenolic Profile (HPLC)

The phenolic profiles of the ethanolic extracts (EEs) were analyzed using a Shimadzu HPLC (Shimadzu Co., Tokyo, Japan) equipped with a column Zorbax SB-C18 (5 μm, 4.6 × 250 mm; Agilent Technologies, Inc., Santa Clara, CA, USA) and a diode array detector (UV-Vis) SPD-M20A (Shimadzu Co., Tokyo, Japan). The methodology used for this evaluation matched that reported in Jordán et al. [15]. Phenolic compounds were identified using commercial standards when possible [25] and with reported data on S. rosmarinus [7,19,26,27]. Quantification was achieved through calibration curves employing commercial standards with molecular similarity.

2.6. Evaluation of the Potential of EEs for Agrochemical Development

2.6.1. Antifungal Capacity

The antifungal capacity of rosemary EEs was evaluated on the test model fungus Aspergillus flavus [28]. Approximately 10–15 mL of potato dextrose agar (PDA) was evenly distributed in sterile 55 mm Ø Petri plates. Subsequently, 475 µL of S. rosmarinusi EE at an initial concentration of 5 mg/mL or 96% ethanol (control) was added in triplicate to the plate surface (=100 µg EE/cm²). After solvent evaporation, a 0.6 cm-diameter well was made in the center of each plate and was filled with 10 µL of the inoculum with an approximate concentration of 1 × 10⁴ CFU/mL, which was obtained through a regression test correlating the optical density of the medium (OD; λ = 530 nm) with the logarithmic number of conidia growth (y = 1.335x + 5.922, R² = 0.943). The plates were then incubated in darkness at a controlled temperature of 26 ± 2 °C and scanned every 24 h for a total of 7 days. Growth areas (mm²) were determined by means of the Image J Version 1.37r, 2010 software (National Institutes of Health, Bethesda, MD, USA). Inhibition capacities (%) were estimated through a potential regression test between growth areas and time (y = axb) as [(C − T)/C × 100], where C and T represented the growth halos of the control (ethanol) and EEs, respectively. Furthermore, the EEs underwent a dose–response test to statistically derive the logarithmic expression between concentrations (6.25–100 µg/cm²) and growth inhibition. This allowed for the mathematical estimation of concentrations that inhibited 50% (IC50) and 90% (IC90) of fungal growth and the minimum inhibitory concentration (MIC) value.

2.6.2. Antioxidant Potential

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Radical Scavenging Activity (DPPH): In 96-well microplates, 50 µL of each S. rosmarinus EE at a concentration ranging from 10 to 200 µg/mL was mixed with 200 µL of 0.005% DPPH radical [29]. After incubating the plates for 1 h at room temperature, absorbance was recorded at 517 nm. The inhibition capacity (%) was estimated as [(C − T)/C × 100], with C being the absorbance of the negative control (water) and T that of each EE. The analyses were carried out twice with two repetitions each, and the dose inhibiting 50% of the DPPH radical (IC₅₀) was calculated from the linear regression equation between concentration and inhibition.

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Ferric Reducing Antioxidant Power (FRAP): Similarly, 50 µL of EE (10–200 µg/mL) was mixed in 96-well plates with 50 µL of 0.2 M sodium phosphate buffer pH 6.6 and50 µL of 1% potassium ferricyanide (III) [29]. After incubating at 50 °C for 20 min and cooling, 100 µL of 5% trichloroacetic acid and 10 µL of 0.1% iron (III) chloride were added. Following another 10 min incubation at 50 °C, the plates were measured at 700 nm. The linear regression equation between concentration and absorbance was obtained from duplicate analyses with 2 replicates for each EE, determining the dose required to achieve 0.5 absorbance units (AU0.5).

2.6.3. Chelating Agent Properties

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Fe²⁺ Chelating Agent Capacity (Fe²⁺-CA): In 96-well plates, 50 µL of EE at a concentration between 100 and 500 µg/mL was mixed with 200 µL of 0.1 M sodium acetate buffer pH 4.9 and 10 µL of 1 mM iron (II) chloride [29]. The mixture was incubated for 1 h at room temperature, and the absorbance of the medium was measured at 562 nm. Additionally, 20 μL of 5 mM ferrozine was added, and the absorbance was immediately recorded at the same wavelength. Chelating activity (%) was calculated as [(C − ΔT)/C × 100], where C represents the absorbance of water, and ΔT was the absorbance before and after the incorporation of ferrozine. The analyses were carried out twice with 2 replicates each, and, from the linear regression equation between concentration and inhibition, the dose necessary to chelate 50% of the Fe²⁺ ions (IC50) was estimated.

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Cu²⁺ Chelating Agent Capacity (Cu²⁺-CA): 50 µL of the EEs (100–500 µg/mL) were mixed with 200 µL of 0.05 M sodium acetate buffer pH 6.0 and 10 µL of 5.5 mM copper (II) sulphate in 96-well plates [29]. The mixture was incubated for 1 h at room temperature. After measuring the absorbance at 632 nm, 10 µL of 2 mM pyrocatechol violet was added, and the absorbance was immediately recorded. Chelating activity (%) was calculated as [(C − ΔT)/C × 100], where C was the absorbance of water, and ΔT was the absorbance before and after the incorporation of pyrocatechol violet. The analyses were performed in duplicate with 2 replicates each, and the dose necessary to chelate 50% of the Cu²⁺ ions (IC50) was estimated from the linear regression equation between concentration and inhibition.

2.6.4. Biostimulant Effects

The growth-promoting effect of S. rosmarinus EEs was evaluated using model seeds of Lactuca sativa var. Carrascoy (dicotyledonous) and Lolium perenne var. certified (2n) (monocotyledonous) provided by Semillas Fitó (Spain) [29]. In brief, 2.5 cm-diameter paper filters were placed in 12-well plates, and 40 µL of each S. rosmarinus EE at a concentration of 5 mg/mL or ethanol was applied. Following solvent evaporation, 10 seeds of each species (L. perenne seeds were pre-hydrated for 24 h) were incorporated in quadruplicate along with 700 µL of distilled water. The plates were then sealed in plastic bags and incubated at a temperature of 22 °C with a photoperiod of 16:8 (L:D). Germination progress was monitored every 24 h for 7 days for L. sativa or 10 days for L. perenne. Subsequently, 25 seeds were randomly obtained for each extract, and the roots (L. sativa) and roots/leaves (L. perenne) were measured using Image J.

2.7. Statistical Analysis

In order to assess statistical differences in the adaptative abilities, essential oil yield and composition, phenolic recovery and characterization, and biological capacities of the S. rosmarinus EEs, the HSD Tukey multirange post-hoc test was conducted via one-way ANOVA using the SPSS Statistics Version 25 (IBM Corp., Armonk, NY, USA). To select the clone demonstrating the best balance considering uniform results among plants and plots, the homogeneity grade (HG) was determined from the coefficient of variation using the following formula: [1 − (SD/$\overline{\mathrm{X}}$) × 100]. Here, SD and $\overline{\mathrm{X}}$ represent the standard deviation and average values for the 4 individuals analyzed from each clone and plot (N = 60). Three homogeneity grades were established as follows: *** > 90%, ** > 80%, and * > 70%. Principal component analysis (PCA) was applied to the compositions of S. rosmarinus (EOs and EEs), with the two main factors being represented in a 2D scatterplot. Furthermore, a Pearson’s bivariate correlation test was conducted to establish connections between biological activities and phenolic compounds.

3. Results

3.1. Agronomic Perspective

On average, the implantation success of the trial in the first year was around 50% (

Table 1. Dry biomass, extraction yield, and extract and phenolic recovery of S. rosmarinusi. Results represent the mean values of 5 clones × 3 plots × 4 individuals ± SD (mean min. plot–mean max. plot).

		Almorox	Lorca	Moratalla	Lliria	Pina de Ebro	S. rosmarinus
Adaptability (%)	2018	46.67 ± 47.3 (10.00–100.00)	23.33 ± 40.4 (0.00–70.00)	93.33 ± 5.8 (90.00–100.00) ***	46.67 ± 20.8 (30.00–70.00)	53.33 ± 23.1 (40.00–80.00)	52.67 ± 35.3 (0.00–100.00)
	2019	63.33 ± 35.1 (30.00–100.00)	50.00 ± 10.0 (40.00–60.00) **	90.00 ± 17.3 (70.00–100.00) **	83.33 ± 15.3 (70.00–100.00) **	63.33 ± 25.2 (40.00–90.00)	70.00 ± 24.2 (30.00–100.00)
	2020	100.00 ± 0.0 (100.00–100.00) ***	100.00 ± 0.0 (100.00–100.00) ***	93.33 ± 11.5 (80.00–100.00) **	90.00 ± 17.3 (70.00–100.00) **	93.33 ± 5.8 (90.00–100.00) ***	95.33 ± 9.2 (70.00–100.00) ***
Fresh Plant (kg/plant) 1		0.55 ± 0.4 b (0.27–1.04)	0.29 ± 0.2 b (0.08–0.46)	0.46 ± 0.2 b (0.42–0.48)	0.66 ± 0.2 b (0.52–0.78) *	1.15 ± 0.5 a (0.83–1.37)	0.61 ± 0.4 (0.08–1.37)
Moisture (%)		60.20 ± 13.0 (58.09–61.50) **	60.29 ± 5.1 (56.84–66.87) ***	60.21 ± 4.0 (58.96–62.05) ***	61.90 ± 3.0 (60.26–63.21) ***	61.56 ± 3.2 (60.90–61.94) ***	60.72 ± 7.0 (56.84–66.87) **
Dry Plant (kg/plant) 1		0.22 ± 0.2 b (0.12–0.41)	0.13 ± 0.1 b (0.03–0.19)	0.18 ± 0.1 b (0.16–0.20)	0.25 ± 0.1 b (0.19–0.30) *	0.44 ± 0.2 a (0.32–0.52)	0.24 ± 0.2 (0.03–0.52)
Essential Oil (EO) 2		4.26 ± 0.4 ab (4.03–4.55) ***	3.86 ± 0.5 b (3.65–4.03) **	3.16 ± 0.8 c (2.73–3.46) **	3.63 ± 0.6 bc (3.13–4.28) **	4.71 ± 0.6 a (4.36–4.96) **	3.95 ± 0.7 (2.73–4.96) **
Ethanolic Extract (EE) 3		25.73 ± 1.7 ab (25.30–26.29) ***	23.56 ± 2.9 bc (20.16–25.89) **	23.07 ± 0.9 c (22.69–23.69) ***	25.45 ± 2.2 b (23.66–27.39) ***	27.80 ± 0.8 a (27.58–28.06) ***	25.06 ± 2.5 (20.16–28.06) ***
Total Phenol Content (TPC) 4		9.31 ± 1.5 (8.60–10.69) **	9.68 ± 2.9 (7.74–12.58) *	8.43 ± 1.5 (7.47–9.37) **	10.33 ± 2.3 (9.79–11.52) *	10.40 ± 2.3 (9.24–11.26) *	9.58 ± 2.2 (7.47–11.52) *

¹ Weight (kg) per plant; ² mL EO/100 g dry leaves (%); ³ g EE/100 g distilled leaves; ⁴ g GAE/100 g EE (GAE: gallic acid equivalents); homogeneity grade (%): *** > 90%; ** > 80%; * > 70%; different letters in the same row indicate statistically significant differences between the different rosemary clones (Tukey’s test; p < 0.05).

), increasing to over 90% by the third year of cultivation (2020). Among the clones, Moratalla demonstrated full adaptation after the initial implantation, achieving a 93% success rate. This high success rate not only makes it attractive from an agronomic perspective, but could also present potential benefits for forestry replanting. In contrast, Lorca presented a success rate of 23%. Regarding biomass, the trial averaged a fresh weight of 0.6 kg/plant with over 60% humidity, reaching a dry biomass value of 0.24 kg/plant. Moisture content was consistent across clones, but fresh and dry biomass varied from 0.29 to 1.15 kg/plant and from 0.13 to 0.44 kg/plant, respectively. Pina de Ebro had the highest values, with fresh and dry biomass nearly double the average, being the only clone with fresh biomass higher than 1 kg/plant. In contrast, Lorca exhibited the lowest biomass production at 0.13 kg/plant (ranging from 0.03 to 0.19 kg/plant.

3.2. Chemical Characterization of the EOs

Rosemary leaves exhibited an average essential oil (EO) yield of 4% (Table 1), with statistically significant differences among clones. Pina de Ebro had the highest yield (4.71%), while Moratalla had the lowest EO yield (3.2%). Regarding their chemical compositions (

Table 2. Chemical analysis of essential oils (EOs) from S. rosmarinus. Results represent the mean values of 5 clones × 3 plots × 4 individuals ± SD (mean min. plot–mean max. plot).

Compound	Almorox	Lorca	Moratalla	Lliria	Pina de Ebro	S. rosmarinus
α-pinene	9.30 ± 0.7 d (8.96–9.49) ***	11.58 ± 1.2 c (11.01–12.09) ***	12.11 ± 0.6 c (11.90–12.36) ***	20.33 ± 1.9 a (19.22–21.52) ***	14.55 ± 0.9 b (13.74–14.98) ***	13.08 ± 3.7 (8.96–21.52) *
Camphene	5.01 ± 0.3 d (4.90–5.03) ***	9.91 ± 1.2 a (9.31–10.80) **	7.72 ± 0.4 b (7.60–7.82) ***	8.22 ± 0.4 b (7.94–8.40) ***	5.92 ± 0.1 c (5.91–5.93) ***	7.31 ± 1.9 (4.90–10.80) *
β-pinene + Myrcene 1	37.93 ± 1.4 b (37.25–38.91) ***	7.57 ± 0.5 d (7.15–8.08) ***	7.01 ± 0.6 d (6.68–7.46) ***	25.16 ± 3.7 c (22.41–26.60) **	43.46 ± 1.0 a (43.07–43.75) ***	24.12 ± 15.7 (6.68–43.75)
Limonene	3.47 ± 0.2 b (3.42–3.52) ***	3.30 ± 0.2 bc (3.14–3.42) ***	3.09 ± 0.2 c (3.00–3.19) ***	3.39 ± 0.4 b (3.04–3.59) **	3.82 ± 0.1 a (3.75–3.90) ***	3.42 ± 0.3 (3.00–3.90) ***
1,8-cineole	13.14 ± 1.0 c (12.25–13.91) ***	22.13 ± 2.8 a (20.84–24.08) **	18.70 ± 0.8 b (18.39–18.90) ***	7.22 ± 0.6 d (6.96–7.95) ***	4.65 ± 0.3 e (4.35–4.87) ***	13.67 ± 6.8 (4.35–24.05)
γ-terpinene	1.83 ± 0.1 a (1.75–1.88) ***	1.41 ± 0.2 b (1.35–1.48) **	0.93 ± 0.1 d (0.93–0.94) ***	1.11 ± 0.2 c (0.97–1.17) **	1.04 ± 0.1 cd (0.99–1.09) ***	1.29 ± 0.4 (0.93–1.88) *
p-cymene	0.69 ± 0.2 a (0.62–0.77) *	0.69 ± 0.2 a (0.54–0.77) *	0.32 ± 0.1 b (0.27–0.39) *	0.57 ± 0.2 a (0.49–0.70)	0.75 ± 0.1 a (0.72–0.80) **	0.61 ± 0.2 (0.27–0.80)
Camphor	9.93 ± 1.7 d (9.21–10.72) **	20.17 ± 3.4 b (19.45–21.74) **	29.82 ± 1.8 a (28.37–30.80) ***	17.24 ± 2.9 c (16.08–20.49) **	12.44 ± 0.5 d (12.35–12.61) ***	17.85 ± 7.5 (9.21–30.80)
Linalool	2.67 ± 0.4 a (2.33–3.00) **	1.07 ± 0.2 c (1.00–1.12) **	1.59 ± 0.1 b (1.53–1.63) ***	0.79 ± 0.1 d (0.69–0.87) **	1.53 ± 0.1 b (1.48–1.57) ***	1.58 ± 0.7 (0.69–3.00)
Bornyl acetate	0.64 ± 0.1 d (0.57–0.67) ***	3.58 ± 0.7 b (3.29–4.17) **	2.45 ± 0.2 c (2.30–2.55) ***	4.63 ± 0.8 a (3.86–5.10) **	0.65 ± 0.1 d (0.62–0.67) ***	2.25 ± 1.6 (0.57–5.10)
Borneol	0.80 ± 0.7 b (0.59–1.21)	2.47 ± 0.3 a (2.29–2.66) **	2.13 ± 0.1 a (2.07–2.22) ***	0.72 ± 0.1 b (0.69–0.79) **	0.40 ± 0.0 b (0.39–0.41) ***	1.35 ± 0.9 (0.39–2.66)
Terpinen 4-ol	1.36 ± 0.1 a (1.28–1.44) ***	1.43 ± 0.3 a (1.27–1.54) **	0.67 ± 0.0 b (0.65–0.68) ***	0.64 ± 0.1 b (0.60–0.68) ***	0.81 ± 0.0 b (0.79–0.83) ***	1.01 ± 0.4 (0.60–1.54)
α-terpineol	1.63 ± 0.1 b (1.58–1.66) ***	2.03 ± 0.2 a (1.88–2.15) ***	2.08 ± 0.1 a (2.07–2.10) **	1.28 ± 0.1 c (1.25–1.31) ***	1.00 ± 0.1 d (0.97–1.03) ***	1.63 ± 0.4 (0.97–2.15) *
Verbenone	3.13 ± 0.5 a (2.82–3.32) **	2.98 ± 1.0 a (2.39–3.36)	1.73 ± 0.2 b (1.62–1.93) **	0.08 ± 0.0 c (0.06–0.10)	2.21 ± 0.5 b (2.00–2.38) *	2.17 ± 1.2 (0.06–3.36)
t-caryophyllene	0.97 ± 0.4 b (0.77–1.29)	0.36 ± 0.2 c (0.31–0.44)	1.44 ± 0.2 a (1.33–1.57) ***	0.96 ± 0.2 b (0.65–1.07) *	0.92 ± 0.2 b (0.89–0.96) **	0.92 ± 0.4 (0.31–1.57)
TOTAL	92.49 ± 0.6 ab (92.22–92.94)	90.68 ± 0.6 c (90.65–90.73)	91.78 ± 1.1 bc (91.11–92.72)	92.33 ± 3.1 bc (91.15–93.31)	94.16 ± 1.0 a (93.43–94.64)	92.26 ± 1.8 (90.65–94.64)

¹ Co-elution; homogeneity grade (%): *** > 90%; ** > 80%; * > 70%; different letters in the same row indicate statistically significant differences between the different rosemary clones (Tukey’s test; p < 0.05).

), sixteen main compounds were detected in the EOs of rosemary leaves of the clones of the trial, representing quantitatively over 92% of the EO. They were especially noted for β-pinene + myrcene (24.1%), camphor (17.9%), 1,8-cineole (13.7%), α-pinene (13.1%), and camphene (7.3%). However, some components of the EO showed statistical differences (p < 0.05) among clones, distinguishing them from each other. In this regard, Almorox presented elevate concentrations of verbenone (3.1%) and linalool (2.7%); Lorca was rich in 1,8-cineole (22.1%), camphene (9.9%), verbenone (3%), borneol (2.5%), and α-terpineol (2.0%), and Moratalla showed a high content of camphor (29.8%), borneol (2.1%), and α-terpineol (2.1%); Lliria oils were notable for containing α-pinene (20.3%) and bornyl acetate (4.6%); and the EOs of Pina de Ebro stood out for β-pinene + myrcene (43.5%) and limonene (3.8%). The principal component analysis (PCA) identified two main clusters: Cluster 1, consisting of Lorca and Moratalla, and Cluster 2, comprising Almorox and Pina de Ebro, with Lliria displaying intermediate characteristics (Figure 1). The Spanish standard UNE 84306 [30] “Essential oil of rosemary (Rosmarinus officinalis L.) from Spain” establishes a chromatographic profile that identifies a range of values for 14 representative compounds. Generally, the Moratalla clone would be the one that best fits the standard (10 compounds), while at the opposite end would be Almorox (5 compounds). If studied in more detail, and in relation to the standard, it should be noted that the Almorox, Lliria, and Pina de Ebro clones have a very high content of β-pinene + myrcene; Lliria and Pina de Ebro have a low content of 1,8-cineole; and Lliria is the only clone whose α-pinene content is within the standard.

3.3. Phenolic Characterization of the EEs

The extraction yield (Table 1) of rosemary dry residue showed an average value of 25%, ranging from 23% (Moratalla) to 28% (Pina de Ebro). These data provide valuable insights to estimate the actual extract production of each clone based on the weight of the dry solid residue of each plant. Consequently, S. rosmarinus presented an average of 61 g EE/plant, with Pina de Ebro doubling that amount at 117 g EE/plant, estimating at nearly 140 g EE/plant in one plot. Contrarily, Lorca presented the poorest yield (31 g EE/plant) due to the limited adaptability of the plant in one plot, as previously noted. As depicted in Table 1, S. rosmarinus EEs reported a phenol content of approximately 10% (or g GAE/100 g EE). Despite no statistical differences being discerned among clones, Lliria and Pina de Ebro displayed the best results, especially one of the plots of Pina de Ebro, which exceeded 11%. Contrarily, Moratalla presented the lowest content at 8.4%. In this regard and considering plant weight, the results indicated an average TPC value of 6 g TPC/plant, with Pina de Ebro doubling this value at 12 g TPC/plant, achieving a maximum value close to 15 g TPC/plant in one of the plots. The phenolic profile of the EEs was determined by HPLC (

Table 3. Phenolic profile (mg/g EE) of the ethanolic extracts from distilled plants of S. rosmarinus. Results represent the mean values of 5 clones × 3 plots × 4 clones ± SD (mean min. plot–mean max. plot).

Compound	Almorox	Lorca	Moratalla	Lliria	Pina de Ebro	S. rosmarinus	Ref.
Gallocatechin	1.66 ± 0.5 a (1.41–1.95) *	0.53 ± 0.1 c (0.49–0.56) **	0.63 ± 0.2 c (0.46–0.80)	1.11 ± 0.4 b (0.81–1.36)	1.80 ± 0.5 a (1.57–1.95) *	1.14 ± 0.6 (0.46–1.95)	[26]
6-hydroxyluteolin 7-glucoside	7.68 ± 1.4 a (7.33–8.31) ***	1.21 ± 0.4 b (1.12–1.37)	1.90 ± 0.3 b (1.64–2.18) **	7.22 ± 2.2 a (6.22–8.76) *	6.32 ± 2.2 a (4.10–8.31)	4.71 ± 3.1 (1.12–8.76)	[19]
Hesperidin	1.80 ± 0.6 a (1.70–1.95)	1.15 ± 0.4 b (0.76–1.56)	1.13 ± 0.3 b (0.91–1.32) *	1.54 ± 0.2 ab (1.48–1.61) **	1.76 ± 0.5 a (1.50–1.95) *	1.47 ± 0.5 (0.76–1.95)	Stnd.
Homoplantaginin	2.11 ± 1.3 (1.55–2.96)	1.62 ± 1.1 (1.25–2.31)	1.81 ± 0.7 (1.25–2.38)	1.25 ± 0.9 (1.15–1.55)	1.89 ± 0.9 (1.69–2.08)	1.76 ± 1.0 (1.15–2.96)	[19]
Rosmarinic acid	5.33 ± 2.1 (4.01–6.48)	6.28 ± 3.1 (3.16–8.03)	5.54 ± 2.0 (4.28–8.05)	6.42 ± 2.7 (5.09–8.18)	6.18 ± 1.8 (5.33–6.94) *	5.92 ± 2.3 (3.16–8.18)	Stnd.
Salvianolic acid A	2.02 ± 0.8 (1.56–2.37)	3.28 ± 1.4 (1.89–4.40)	3.18 ± 1.2 (2.15–4.04)	2.86 ± 1.6 (2.64–3.07)	2.78 ± 1.4 (2.14–3.55)	2.82 ± 1.3 (1.56–4.40)	[27]
Cirsimaritin	1.82 ± 0.2 bc (1.72–1.96) **	2.15 ± 0.3 a (1.96–2.38) **	1.45 ± 0.1 d (1.37–1.50) ***	2.07 ± 0.2 ab (1.88–2.17) ***	1.81 ± 0.2 a (1.72–1.88) ***	1.85 ± 0.3 (1.37–2.38) **	[19]
Unidentified flavanone	3.25 ± 0.5 a (2.83–3.53) **	1.91 ± 0.2 b (1.85–1.96) **	1.17 ± 0.2 c (1.09–1.28) **	1.93 ± 0.4 b (1.87–1.99) **	3.43 ± 0.9 a (3.22–4.20) *	2.34 ± 1.0 (1.09–4.20)	-
Rosmanol	2.75 ± 1.7 (2.28–3.63)	3.35 ± 2.6 (1.76–6.09)	2.40 ± 0.6 (1.89–2.71) *	4.55 ± 6.5 (2.04–7.60)	2.31 ± 0.8 (2.16–2.55) *	3.01 ± 3.0 (1.76–7.60)	Stnd.
Epirosmanol	2.04 ± 1.3 (1.41–3.25)	1.60 ± 0.9 (1.05–2.58)	1.49 ± 0.5 (1.20–1.81)	2.80 ± 1.5 (2.00–3.65)	2.21 ± 1.3 (1.22–3.75)	1.98 ± 1.2 (1.05–3.75)	[19]
Genkwanin	1.56 ± 0.3 bc (1.28–1.76) *	2.73 ± 0.4 a (2.60–2.87) **	1.16 ± 0.1 c (1.10–1.26) **	1.86 ± 0.4 b (1.65–1.94) *	1.15 ± 0.5 c (0.87–1.34)	1.69 ± 0.7 (0.87–2.87)	Stnd.
Methoxhycarnosol	4.90 ± 1.7 ab (3.60–6.26)	1.18 ± 0.4 c (0.87–1.51)	0.68 ± 0.2 c (0.58–0.78) *	6.21 ± 1.8 a (4.96–6.96) *	4.23 ± 1.5 b (2.98–4.85)	3.28 ± 2.5 (0.58–6.98)	[26]
Salvigenin	1.31 ± 0.3 a (1.03–1.49) *	1.26 ± 0.3 a (1.10–1.55) *	0.71 ± 0.1 b (0.66–0.76) **	1.20 ± 0.3 a (1.05–1.36) *	1.23 ± 0.2 a (1.03–1.42) **	1.14 ± 0.3 (0.66–1.55) *	Stnd.
Carnosol	46.83 ± 11.6 (41.44–52.51) *	52.90 ± 9.8 (45.60–58.93) **	47.39 ± 9.8 (37.46–54.31) *	47.18 ± 14.6 (41.53–59.21)	53.90 ± 14.5 (46.53–59.24) *	49.70 ± 12.0 (37.46–59.24) *	Stnd.
Rosmadial	8.09 ± 5.0 (6.03–10.84)	5.81 ± 2.7 (3.66–8.21)	4.76 ± 1.4 (3.99–5.48) *	4.76 ± 3.7 (2.62–6.03)	5.33 ± 3.9 (3.93–7.40)	5.81 ± 3.7 (2.62–10.84)	[19]
4′-Methoxytectochrysin	1.41 ± 0.7 ab (1.09–1.93)	1.00 ± 0.6 bc (0.77–1.18)	0.68 ± 0.3 c (0.66–0.70)	1.76 ± 0.5 a (1.26–2.07)	1.21 ± 0.6 abc (0.94–1.56)	1.18 ± 0.6 (0.66–2.07)	[19]
Carnosol isomer	1.26 ± 1.0 (0.85–1.91)	0.98 ± 0.6 (0.71–1.25)	0.73 ± 0.2 (0.56–0.83)	1.62 ± 2.8 (0.38–2.72)	1.45 ± 0.8 (1.01–1.95)	1.18 ± 1.3 (0.38–2.72)	Stnd.
Rosmaridiphenol	4.69 ± 2.9 (4.02–5.91)	5.11 ± 1.5 (4.61–6.11) *	6.07 ± 1.2 (5.03–6.89) **	4.91 ± 2.4 (4.14–7.14)	6.91 ± 3.2 (5.91–8.11)	5.55 ± 2.4 (4.02–8.11)	[7]
Carnosic acid	64.00 ± 20.7 b (49.61–78.26)	94.52 ± 47.4 ab (35.99–127.33)	85.80 ± 18.0 ab (78.16–92.14) *	75.18 ± 31.1 ab (59.10–96.50)	105.69 ± 24.0 a (78.26–122.76) *	85.20 ± 32.6 (35.99–127.33)	Stnd.
Methyl carnosate	7.55 ± 2.8 bc (6.64–8.59)	7.69 ± 2.4 bc (7.52–8.01)	9.86 ± 1.9 b (8.49–10.97) **	14.64 ± 1.5 a (14.32–15.78) **	6.29 ± 3.0 c (4.72–8.59)	8.97 ± 3.6 (4.72–15.78)	[7]
NI	8.78 ± 1.3 a (8.16–9.99) **	3.88 ± 2.8 c (2.13–7.13)	4.22 ± 0.8 c (3.88–4.49) **	4.97 ± 0.7 bc (4.83–5.34) **	6.82 ± 2.8 ab (4.89–9.99)	5.76 ± 2.7 (2.13–9.99)	-
Salviol	7.23 ± 0.8 a (6.47–8.00) **	3.46 ± 1.3 c (2.75–4.22)	7.17 ± 1.3 a (6.57–7.98) **	5.26 ± 0.6 b (4.68–5.72) **	5.56 ± 2.1 b (4.13–8.00)	5.77 ± 1.9 (2.75–8.00)	[26]
TOTAL	188.08 ± 20.5 b (176.38–204.96) **	203.60 ± 40.3 ab (156.82–230.47) **	189.92 ± 18.0 b (186.58–194.29) ***	201.31 ± 35.4 ab (187.36–234.87) **	230.21 ± 32.0 a (204.96–247.56) ***	206.78 ± 36.3 (156.82–247.56) **

Stnd.: Identification using commercial standards; Homogeneity grade (%): *** > 90%; ** > 80%; * > 70%; different letters in the same row indicate statistically significant differences between the different rosemary clones (Tukey’s test; p < 0.05).

). The results revealed a total of 207 mg/g EE for the rosemary extracts, ranging from 188 mg/g EE (Almorox) to 230 mg/g EE (Pina de Ebro). Variations in metabolite levels were detected among clones, with notable differences being observed for 6-hydroxyluteolin 7-glucoside, rosmadial, rosmaridiphenol, carnosic acid, methyl carnosate, and salviol. Specifically, Almorox exhibited elevated levels of 6-hydroxyluteolin 7-glucoside, methoxhycarnosol, rosmadial, and salviol; Lorca was rich in rosmadial and carnosic acid; Moratalla displayed high levels of rosmadial, rosmaridiphenol, and salviol; Lliria showed an elevated presence of methoxycarnosol and methyl carnosate; finally, Pina de Ebro stood out for its content of 6-hydroxyluteolin 7-glucoside, rosmaridiphenol, and carnosic acid. The principal component analysis (PCA) identified two main clusters: Cluster 1, consisting of Lorca and Moratalla, and Cluster 2, comprising Almorox and Pina de Ebro; whereas Lliria presented intermediate characteristics (Figure 2).

3.4. Biological Potential of EEs for Agrochemical Development

S. rosmarinus EEs clones revealed inhibition effects against the tested mold (

Table 4. Biological capacities of the ethanolic extracts from distilled plants of S. rosmarinusi. Results represent the mean values of 5 clones × 3 plots × 4 clones ± SD (mean min. plot–mean max. plot).

		Almorox	Lorca	Moratalla	Lliria	Pina de Ebro	S. rosmarinus
Antifungal Capacity 1	A. flavus (EC50) 1	25.91 ± 9.9 (14.30–35.33)	40.03 ± 39.3 (24.52–69.91)	29.46 ± 9.2 (21.78–35.77) *	27.78 ± 11.7 (23.44–32.20)	23.82 ± 8.9 (19.45–26.39)	29.59 ± 20.3 (14.30–69.91)
	A. flavus (EC90) 1	146.14 ± 32.0 (139.18–153.98) *	293.36 ± 387.4 (124.11–570.88)	157.61 ± 54.4 (148.44–165.95)	151.28 ± 56.2 (108.63–187.67)	103.01 ± 28.8 (79.67–120.13) *	172.50 ± 189.3 (79.67–570.88)
	A. flavus (MIC) 1	232.30 ± 68.1 (219.96–251.77) *	489.14 ± 691.8 (184.85–974.66)	245.54 ± 106.5 (236.15–259.29)	234.49 ± 91.9 (164.70–309.42)	149.35 ± 40.8 (113.40–176.84) *	274.23 ± 338.3 (113.40–974.66)
Antioxidant capacity	DPPH (IC50) 2	25.75 ± 6.1 (22.51–28.66) *	24.49 ± 14.0 (16.66–38.14)	26.12 ± 8.5 (22.95–29.45)	23.29 ± 5.4 (20.46–26.85) *	17.73 ± 3.4 (16.22–18.51) *	23.59 ± 8.8 (16.22–38.14)
	FRAP (AU0.5) 3	60.59 ± 15.8 b (58.54–63.77) *	52.13 ± 18.7 ab (42.25–70.98)	54.59 ± 9.7 ab (53.03–55.93) **	51.22 ± 13.3 ab (41.78–58.51) *	42.20 ± 8.1 a (37.78–47.15) **	52.37 ± 14.6 (37.78–70.98) *
Chelating capacity	Fe2+ CA (IC50) 2	282.65 ± 62.7 ab (257.07–300.96) *	276.31 ± 47.0 ab (252.05–311.67) **	366.36 ± 151.3 b (328.97–388.74)	252.01 ± 57.9 a (229.66–294.55) *	220.62 ± 51.0 a (149.72–249.27) *	282.12 ± 96.0 (149.72–388.74)
	Cu2+ CA (IC50) 2	339.85 ± 72.6 ab (313.03–371.60) *	335.79 ± 104.8 ab (258.79–413.65)	355.84 ± 59.3 b (329.14–405.12) **	339.03 ± 50.7 ab (324.39–358.50) **	267.04 ± 55.6 a (260.35–271.46) *	327.97 ± 76.6 (258.79–413.65) *
Biostimulant effects	Ls Germ. 24 h 4	94.17 ± 6.2 (92.50–98.13) ***	92.29 ± 6.8 (89.38–93.75) ***	95.21 ± 4.1 (93.75–96.25) ***	98.06 ± 2.1 (96.25–100.00) ***	95.68 ± 4.8 (94.38–98.33) ***	94.91 ± 5.3 (89.38–99.38) ***
	Ls Germ. 48 h 4	98.96 ± 2.9 (96.88–100.00) ***	98.75 ± 2.9 (96.25–100.00) ***	99.17 ± 1.2 (98.75–99.38) ***	99.44 ± 1.1 (98.75–100.00) ***	99.55 ± 1.5 (98.75–100.00) ***	99.15 ± 2.1 (96.25–100.00) ***
	Ls Growth Roots 5	4.22 ± 0.6 (3.97–4.43) **	4.54 ± 0.7 (4.14–4.97) **	3.91 ± 0.5 (3.57–4.15) **	4.54 ± 0.5 (4.04–4.80) ***	4.21 ± 0.4 (3.90–4.45) ***	4.27 ± 0.6 (3.57–4.97) **
	Lp Germ. 72 h 4	81.88 ± 6.5 ab (76.88–85.00) ***	80.91 ± 5.0 ab (77.50–85.00) ***	83.54 ± 10.2 a (78.13–93.75) **	74.44 ± 8.5 b (67.50–76.67) **	75.00 ± 5.8 ab (72.50–79.38) ***	79.55 ± 8.0 (67.50–93.75) ***
	Lp Germ. 144 h 4	94.79 ± 2.7 (93.13–95.63) ***	94.09 ± 3.9 (91.88–96.88) ***	94.38 ± 4.9 (89.38–98.13) ***	93.61 ± 2.8 (93.33–93.75) ***	94.55 ± 4.4 (92.50–96.67) ***	94.29 ± 3.7 (89.38–98.13) ***
	Lp Germ. 192 h 4	96.88 ± 2.7 (95.63–98.75) ***	97.04 ± 1.0 (95.83–97.50) ***	94.38 ± 4.9 (92.50–99.38) ***	96.67 ± 2.5 (95.00–97.50) ***	96.14 ± 3.0 (94.38–97.50) ***	96.61 ± 2.8 (92.50–99.38) ***
	Lp Germ. 240 h 4	97.08 ± 2.7 (96.5–98.75) ***	97.50 ± 1.6 (96.25–98.75) ***	94.38 ± 4.9 (93.75–99.38) ***	97.22 ± 2.3 (96.67–98.13) ***	96.82 ± 2.5 (95.63–98.33) ***	97.14 ± 2.4 (93.75–99.38) ***
	Lp Growth Roots 5	6.52 ± 0.8 (6.00–7.02) **	6.40 ± 0.6 (6.08–6.81) ***	6.64 ± 0.6 (6.39–6.86) ***	6.33 ± 0.3 (6.18–6.48) ***	6.41 ± 0.5 (5.96–6.68) ***	6.47 ± 0.6 (5.96–7.02) ***
	Lp Growth Leaves 5	4.73 ± 0.3 (4.57–4.86) ***	4.54 ± 0.2 (4.51–4.58) ***	4.61 ± 0.3 (4.60–4.63) ***	4.59 ± 0.2 (4.53–4.63) ***	4.60 ± 0.2 (4.39–4.77) ***	4.62 ± 0.2 (4.39–4.86) ***

¹ Concentration (µg EE/cm²) that inhibits fungal growth by 50%, 90%, or the minimum inhibitory concentration. ² Concentration (μg EE/mL) required to reduce/chelate 50% of radicals/cations. ³ Concentrations (μg EE/mL) required to yield 0.5 absorbance units; ⁴ % seed germination (out of 40 seeds); ⁵ seedlings lengths at 7 (L. sativa) and 10 days (L. perenne) with control values of 2.9 ± 0.2 (L. sativa roots) and 5.06 ± 0.4 (L. perenne roots) and 4.60 ± 0.2 (L. perenne leaves); homogeneity grade (%): *** > 90%; ** > 80%; * > 70%; different letters indicate statistical differences (Tukey’s test; p < 0.05) among clones.

), presenting EC₅₀ and EC₉₀ values of 30 and 173 µg EE/cm². The MIC (minimum inhibitory concentration) value was statistically estimated at 274 µg EE/cm². In terms of clones, Pina de Ebro exhibited better antifungal activity than the average value observed for S. rosmarinusi EEs, with an estimated MIC value of 149 µg EE/cm². The antifungal activity of rosemary EEs is statistically correlated to its higher content of phenols (0.505; p < 0.01), especially concerning the unidentified flavanone (0.412; p < 0.01) and carnosic acid (0.465; p < 0.01). In contrast, correlation tests showed biostimulation for the phenols rosmarinic acid (0.345; p < 0.05) and carnosol isomer (0.287; p < 0.05). S. rosmarinus EEs presented a robust antioxidant potential with IC50 and AU0.5 values around 24 and 52 µg EE/mL for the in vitro DPPH and FRAP assays, respectively (Table 4). Despite no statistical differences being observed for DPPH values, Pina de Ebro demonstrated superior antioxidant capacities in the FRAP test, with the plots ranging from 37.8 to 47.2 µg EE/mL. This represents a notable improvement in the antioxidant capacities compared to the mean value obtained for S. rosmarinusi. Contrarily, the Almorox clone was the sample that presented the lowest antioxidant capacities. These findings directly correlate with the major TPC observed in the analyzed samples (p < 0.01); where statistical correlations reported the highest antioxidant capacities for the acids rosmarinic, salvianolic A and carnosic (p < 0.01). Additionally, S. rosmarinusi EEs showed moderate chelating properties for Fe²⁺ and Cu²⁺ cations, with IC50 values exceeding 200 µg EE/mL (Table 4). Among the clones, differences were observed (p < 0.05), and Pina de Ebro exhibited the best results, exhibiting better effects than the average values detected for S. rosmarinusi, especially regarding the Cu²⁺ cation. In contrast, Moratalla showed the poorest capacities. The Pearson’s test revealed a direct correlation between phenolic compounds and chelating capacities, especially for Cu²⁺ (0.564; p < 0.01), mainly influenced by the carnosic acid (0.571; p < 0.01) and the unidentified flavanone (0.367; p < 0.01). In contrast, Fe²⁺ chelating effects were mainly affected by the unidentified flavanone (0.387; p < 0.01) and salvigenin (0.381; p < 0.01). The results indicated that all S. rosmarinusi EEs enhanced rooting in Lactuca sativa and Lolium perenne seeds (Table 4). L. sativa roots increased on average from 2.9 to 4.3 cm and from 5.1 to 6.5 cm in the roots of L. perenne, whereas no increase was detected in L. perenne leaves. Regarding the clones, no statistical differences were observed among clones. The root length of L. sativa showed a certain correlation (0.301; p < 0.05) with the TPC, suggesting the role of phenols as rooting phytoestimulants, especially for cirsimaritin (0.308; p < 0.05) and salvigenin (0.286; p < 0.05), and showing certain phytotoxicity for the salviol (0.340, p < 0.05). On the contrary, the role of phenols in the rooting effects of L. perenne is not clear, so further analysis should elucidate the nature of the components responsible for this bioactivity in both seeds.

4. Discussion

From an agronomic perspective, the trial was conducted under rainfed conditions with minimal cultural practices, mainly limited to weed control. Therefore, incorporating irrigation and enhancing soil nutrient content, as demonstrated by La Bella et al. [31] could enhance implantation success ratios. Our findings for Pina de Ebro align with those of Flamini et al. [22], who reported comparable dry biomass values for rosemary (0.4–0.5 kg/plant). Overall, this small-scale trial (N = 150 plants) yielded an estimated 2100–2400 kg/ha of dry biomass, which could potentially reach up to 3600–4200 kg/ha if this trial had been carried out only with individuals from Pina de Ebro clone. Additionally, this clone also presented major surviving individuals during the storm Filomena (January 2021), a climatic episode characterized by extreme snowy and cold conditions (−1.5 °C) compared to the average temperatures in January 2019 (2.6 °C) and 2020 (2.2 °C) [32]. In contrast, the Lorca and Moratalla clones were most affected by this abnormal climatic event. This multi-year assessment, considering potential future climate change scenarios, also provides valuable insights for the selection of rosemary clones for forest plantations. Consequently, Pina de Ebro exhibits desirable aspects such as adaptability, tolerance to extreme cold temperatures, and high dry biomass production, making it a favorable selection.

This small-scale experiment (N = 150 plants) had an average EO yield of around 4%, which surpasses values reported in previous studies for this species [15,17]. The chemical analysis of the rosemary oils indicated that the five major components (α-pinene, camphene, β-pinene + myrcene, 1,8-cineole, and camphor) constituted around 75% of the total oil constituents. Considering these principal compounds for the selection and cultivation of specific chemotypes, Lorca should be selected for its high levels of camphene and 1,8-cineole, Moratalla for its elevated camphor content, Lliria for its enriched α-pinene concentrations, and Pina de Ebro and Almorox for their outstanding β-pinene + myrcene contents. The chemical compositions of these clones are very similar to those obtained in their populations of origin cultivated in previous trials in the same area [24]. The PCA test identified two main clusters of rosemary clones: one consisting of those from the southeast of Spain (Lorca and Moratalla) and another from the center (Almorox and Pina de Ebro). Essential oils from Lliria, located in the middle east of the country, were categorized separately. This suggest that the geographical origin of clones present a strong influence on the production of different oil metabolites, as also reported for the EOs of S. lavandulifolia [33]. Although some of these EOs do not fit the UNE 84306 standard and will probably be discarded by market agents for perfumery or cosmetics, they can be useful in other applications. For example, myrcene-rich EOs have been reported to have high antioxidant activity and can be used as natural antioxidants for food preservation [34]. In addition, it is important to highlight that more than 95% of the total dry weight of the rosemary plant was released as distilled residue. Consequently, cultivation of rosemary with these clones could potentially generate over 2000 kg/ha of solid waste, which could exceed 4000 kg/ha for the most productive clone (Pina de Ebro). This underscores the substantial resource recovery potential of this agro-industrial side-stream, offering avenues for sustainable utilization and value addition.

The findings regarding the TPC of the rosemary EEs are consistent with the previous studies of Oreopoulou et al. [35], Sánchez-Vioque et al. [7], and Dorman et al. [36], who reported TPC values ranging from 4 to 15% for similar leaves’ extracts. Considering clones, the Pina de Ebro clone emerged as the most promising for phenolic recovery, while also demonstrating uniformity among plants and plots. Among the predominant compounds detected, carnosic acid, carnosol, methyl carnosate, rosmaridiphenol, rosmarinic acid, rosmadial, salviol, and 6-hydroxyluteolin 7-glucoside were the most prevalent, as reported in previous studies [7,19,26]. The PCA test also revealed that plants from the southeast of Spain (Moratalla and Lorca) and the center of Spain (Almorox and Pina de Ebro) formed distinct clusters. Meanwhile, Lliria, located in the middle east of Spain, was positioned between Clusters 1 and 2. This distribution suggests that phenol production can also be influenced by the climatic conditions of the geographical origin of each clone. Additionally, considering that the dry biomass of Pina de Ebro was nearly double that that of the other clones, the potential for extracting certain desirable compounds from rosemary, especially carnosic acid or carnosol, is significantly higher.

The findings of this study shed light on the multifaceted potential of rosemary waste extracts in addressing various challenges encountered for plant health and agricultural practices. Fungal diseases represent a significant threat, stemming from a wide array of fungi capable of attacking various plant organs, ranging from roots to leaves and fruits. Of particular concern is the impact of plant pathogens like Aspergillus flavus, which commonly affects crops such as corn, peanuts, and cotton; these are also notorious for their production of aflatoxins, which are potent natural carcinogenic mycotoxins. Notably, the antifungal efficacy observed in this study for the rosemary EEs offers promising avenues for disease management. Addressing this, Jordán et al. [37] highlighted the potential enhancement of the antibacterial activity of rosemary extracts depending on the carnosol:carnosic acid ratio. Moreover, the presence of antioxidant compounds in rosemary EEs presents an opportunity to protect plants from oxidative damage within their cells while potentially enhancing their resilience to different stress factors. Notably, phenolic compounds have demonstrated capacity to absorb UV radiation, with the possible mitigation of UV-induced damage, contributing to overall plant health and vitality. Prior research has also underscored the antioxidant potential of solid distilled by-product extracts of rosemary plants [7,18,35,36]. Additionally, the moderate chelating capacities observed for the EEs on iron and copper cations also offers natural solutions to one of the greatest challenges in plant nutrition, namely the low solubility of certain essential nutrients, such as iron (Fe²⁺), manganese (Mn²⁺), copper (Cu²⁺), or zinc (Zn²⁺), in calcareous soils. To address this situation, the agrochemical sector develops chelates and chemical complexes (fertilizers) to increase the bioavailability of metal cations, making them more absorbable in plant uptake. From these findings, rosemary EEs emerge as a possible alternative to synthetic chelating molecules. Similarly, comparable chelating capacities to those observed in this study have been reported by Sánchez-Vioque et al. [7]. Finally, the observed rooting capacities of model seeds in response to rosemary extracts confirmed their potential in promoting root growth. This is crucial for achieving higher yields with improved nutritional quality, while reducing reliance on synthetic agrochemicals. Lactuca sativa (lettuce) is a dicotyledonous plant known for its strong germination capabilities, often sprouting in less than 24 h. In contrast, Lolium perenne (English ryegrass) is a monocotyledonous plant commonly used in model assays due to its rapid root and leaf growth. Previous studies have also demonstrated biostimulant capacities for similar rosemary extracts [7]. In contrast, others have associated phytotoxic effects to rosemary due to the carnosic acid content [38]. Therefore, strategies aimed at mitigating such effects, such as pretreatments to remove carnosic acid [14], offer opportunities to increase the biostimulant potential of rosemary extracts, enhancing their overall efficacy in sustainable agricultural practices.

5. Conclusions

This study investigated the potential of domesticated rosemary clones for large-scale cultivation and the valorization of the resulting distillation by-products for the development of plant-based agrochemicals. Among the clones studied, Pina de Ebro consistently outperformed others in several key aspects. This clone exhibited agronomic adaptation (despite low initial implantation), characterized by increased biomass production, along with a strong tolerance to extreme cold temperatures. This clone also produced the highest content of essential oils, standing out as a chemotype rich in α-pinene (≈15%) and β-pinene + myrcene (≈44%), while also containing a significant amount of camphor (≈12%). In addition, Pina de Ebro also presented the greatest potential for phenol recovery from distilled waste, containing high concentrations of some of the most desirable phenolic compounds of this species (carnosol and carnosic acid). Furthermore, statistical analysis demonstrated that the chemical composition (terpene and phenolic compounds) of rosemary clones from the southeast and center of Spain form distinct clusters, with Lliria from the middle east positioned between these clusters. These findings suggest that plant metabolites are influenced by climatic conditions. In terms of biological applications, all rosemary extracts showed strong antifungal activity and important antioxidant and chelating properties, with Pina de Ebro extract also demonstrating the greatest efficacy. Furthermore, the extracts promoted rooting effects, with Lliria and Pina de Ebro showing the most consistent results. The findings confirmed the great potential to valorize the solid waste from rosemary distillation, supporting its commercial exploitation.

In summary, Pina de Ebro is undoubtedly emerging as the most promising clone destined for large-scale cultivation and the development of agrochemicals due to its balanced agronomic behavior, essential oil, phenolic content, and biological potential, with notable uniformity between plants and plots. However, more research is needed to optimize the growing practices of Pina de Ebro plants, such as determining the best collecting stages, irrigation conditions, fertilizer addition, planting density, harvest periods, and other agronomic factors that can further improve the biosynthesis of the desired metabolites. In addition, multi-environmental trials should also be carried out to evaluate the influence of soil-climatic conditions and estimate large-scale productivity for optimal crop profitability.