1. INTRODUCTION:

Eucalyptus species are also valued for their essential oils¹, which have diverse biological and industrial applications².

In Morocco, Eucalyptus was introduced in the early 20th century, mainly for ecological restoration, erosion control, and to address wood shortages. Species such as E. camaldulensis and E. grandis have been widely adopted due to their fast growth, drought resistance, and ecological adaptability to Mediterranean climates. These species are central to afforestation and agroforestry strategies in the region³.

Eucalyptus essential oils, rich in terpenes such as 1,8-cineole and α-pinene, exhibit notable antimicrobial properties. Their composition varies with genetic and environmental factors, supporting the classification of species into distinct chemotypes suited to targeted applications⁴.

However, EO composition is highly variable, affected by genetic diversity, environmental conditions, and harvest techniques. This variability has prompted the classification of Eucalyptus into chemotypes—genetic variants within a species defined by specific chemical profiles. Chemotype identification has become crucial for selecting elite lines suited for targeted bioapplications⁵.

In recent years, hybridization has emerged as a potent tool to enhance both biomass and EO quality. Interspecific hybrids often show heterosis surpassing their parents in growth, resistance, and metabolic traits. These hybrids can yield novel chemotypes with superior biological activity or unique terpene combinations, as shown in clones like E. grandis × E. urophylla, which exhibit enhanced antibacterial potency^6,7.

In Morocco, controlled hybridization programs were initiated to optimize essential oil profiles, notably involving E. camaldulensis and E. grandis, which show vigorous growth and promising EO yields; however, the chemical composition and therapeutic potential of selected hybrids remain underexplored, noted for its vigorous growth and EO yield. Yet, its chemical composition and therapeutic potential remain underexplored⁸.

Given the urgent demand for natural antimicrobials and sustainable crop protection agents, the search for bioactive Eucalyptus chemotypes is both scientifically and economically justified⁹.

Objectives of the Study:

The present study aims to perform a comprehensive comparative analysis of hybrid clone 2414 and its parental species (Eucalyptus camaldulensis and Eucalyptus grandis) in terms of essential oil yield, chemical composition, and antimicrobial efficacy. Specifically, the study pursues the following objectives:

1. To quantify and compare the essential oil yields obtained from the leaves of the hybrid and both parental species using standardized hydrodistillation techniques.

2. To characterize the chemical profiles of the essential oils through gas chromatography–mass spectrometry (GC–MS) and identify dominant terpenoid compounds and chemotypic patterns.

3. To evaluate the antimicrobial activity of the oils against selected Gram-positive and Gram-negative bacterial strains, as well as a wood-decaying fungal species, using broth microdilution assays.

4. To explore potential correlations between oil composition and biological activity, with an emphasis on the role of major compounds such as 1,8-cineole, α-pinene, and p-cymene.

Through this multi-parameter approach, the study seeks to highlight the potential of controlled hybridization as a tool for developing high-value Eucalyptus genotypes suited for both silvicultural productivity and the sustainable production of bioactive essential oils.

2. MATERIALS AND METHODS

2.1 Plant Material and Sampling Protocol:

Sampling was conducted in Sidi Slimane province (northwestern Morocco), an area suitable for Eucalyptus cultivation.⁸.

Sampling was performed in the morning on healthy mature leaves to preserve oil integrity. Only healthy, mature, and undamaged leaves were selected from each tree, avoiding those affected by disease, herbivory, or senescence. The collected material was immediately placed in aerated paper bags and transported to the laboratory in insulated

Essential oil extraction:

Essential oils were extracted from 200 g of fresh leaves by hydrodistillation in a Clevenger-type apparatus for 3 h (European Pharmacopoeia, 10th ed.). Oils were dried over anhydrous sodium sulfate and stored at 4 °C until analysis¹⁰.

The essential oil yield (Y%) was calculated relative to the dry weight (DW) of the plant material using the following formula:

w Mass of EO extracted (g)

Yield (% --- ) = ------------------------------------- x 100

w Mass of dried plant materials (g)

To obtain dry weights, 20 g of fresh leaves were oven-dried at 60 °C for 48 hours prior to yield determination. All measurements were conducted in triplicate, and results are expressed as mean ± standard deviation¹¹.

2.2 Gas Chromatography–Mass Spectrometry (GC–MS) Analysis:

GC–MS analyses were performed using a HP 6890/5973 system with a HP-5MS capillary column¹².

GC–MS Operating Conditions:

Oven program: 50–250 °C at 4 °C/min; carrier gas: nitrogen; injection: 1 μL, split mode (1:50); EI ionization at 70 eV.

Compound Identification and Quantification:

Identification of EO constituents was achieved by comparing:

1. Retention indices (RI), calculated relative to a homologous series of n-alkanes (C7–C30) injected under identical conditions.

2. Mass spectral data, interpreted against reference spectra from the NIST (National Institute of Standards and Technology) and Wiley libraries;

3. Published spectral data and chromatographic behavior from peer-reviewed literature.

Quantitative data (expressed as relative percentage) were obtained from peak area normalization without correction factors, assuming a consistent detector response across monoterpenes and sesquiterpenes. Only compounds with a match quality above the confidence threshold (>85%) were retained for interpretation.

2.3 Antimicrobial activity:

2.3.1 Bacterial strains:

Bacterial (E. coli, S. aureus, M. luteus, B. subtilis) and fungal strains (C. puteana, G. trabeum, T. versicolor, R. placenta) were tested using broth microdilution assays. MIC values were determined after incubation under standard conditions.¹³.

2.3.2 Antimicrobial assay:

Previous formulations such as essential oil-based lozenges containing eucalyptus have demonstrated notable antimicrobial efficacy, particularly against Staphylococcus aureus¹⁴.

The minimum inhibitory concentrations (MICs) of the EOs were determined using a modified broth microdilution method, following the protocol described by Remmal et al. (1993), with minor adaptations to suit essential oil testing.

To ensure homogeneous dispersion of the hydrophobic oils, each EO was first emulsified in a 0.2% (w/v) agar solution.

Serial volumetric dilutions of each EO were prepared to obtain the following final concentrations (v/v):

1/100, 1/250, 1/500, 1/1000, 1/2000, 1/3000, and 1/5000.

For each concentration:

· 13.5 mL of sterile TSA (Tryptic Soy Agar) for bacterial assays, or PDA for fungal assays, was dispensed into sterile test tubes.

· Once the media cooled to approximately 45 °C, 1.5 mL of the corresponding EO dilution was added under aseptic conditions and mixed thoroughly.

· The mixture was then poured into sterile Petri dishes to solidify¹⁵.

Inoculation procedures:

· Bacterial strains were streaked across the surface using a sterile calibrated platinum loop to ensure consistent inoculum density.

· Fungal strains were inoculated by placing a 1 cm² agar plug (excised from the actively growing margin of a 7-day-old culture) onto the center of each plate.

All tests were performed in triplicate to ensure reproducibility and statistical reliability.

Controls included:

· A negative control (medium + emulsifier without EO) to monitor any intrinsic antimicrobial effect of the dispersant;

· A blank control (uninoculated plates) to ensure sterility of media and reagents.

Microbial growth was monitored visually and, where applicable, quantified to determine the lowest EO concentration inhibiting visible growth, which was recorded as the MIC¹⁶.

Similar testing methodologies have been employed in developing essential oil-based topical formulations, including liquid herbal hand washes with proven antiseptic properties¹⁷.

3. RESULTS AND DISCUSSION:

3.1 The essential oil yield:

The essential oil (EO) yield obtained from the three Eucalyptus taxa showed marked differences (Table 1, Figure 1). The hybrid clone 2414 exhibited the highest yield at 6.93 ± 0.07% (w/w, dry basis), significantly surpassing both parental species: E. grandis (2.67 ± 0.03%) and E. camaldulensis (2.37 ± 0.13%). These differences were statistically significant (p < 0.05), as confirmed by ANOVA followed by Tukey’s HSD test.

Table 1. Essential-oil yield (% w/w, dry basis) of the three Eucalyptus taxa (mean ± SD, n = 3).

Taxon	Yield (% w/w) ± SD	Statistical Group
*E. camaldulensis*	2.37 ± 0.13	b
*E. grandis*	2.67 ± 0.03	b
Hybrid 2414	6.93 ± 0.07	a

The hybridization between E. camaldulensis and E. grandis resulted in a threefold increase in EO yield, highlighting the hybrid's potential for commercial exploitation.

Figure 1. Essential-oil yield (% w/w, dry basis) for E. camaldulensis, E. grandis, and Hybrid 2414 (mean ± SD, n = 3). Different letters indicate significant differences (Tukey’s HSD, p < 0.05).

3.2 Chemical composition:

The chemical composition of the essential oils was analyzed by GC–MS, revealing significant variations between taxa (Table 2, Figure 2). The hybrid oil was primarily composed of 1,8cineole (47.61%) and α-pinene (41.10%), while E. camaldulensis was rich in 1,8-cineole (42.30%), and E. grandis in α-pinene (43.73%).

Table 2. Major constituents (>1%) of the essential oils (% of TIC, total ion current).

Compound	*E. camaldulensis*	*E. grandis*	Hybrid 2414
1,8-cineole	42.30	36.78	47.61
α-pinene	11.40	43.73	41.10
γ-terpinene	10.73	0.07	1.76
β-pinene	7.78	4.49	0.20
p-cymene	3.21	8.02	3.76
α-terpineol	2.15	1.02	2.58

Figure 2. Relative chemical profile of major constituents in essential oils from E. camaldulensis, E. grandis, and Hybrid 2414 ; values as % of TIC.

The hybrid exhibited a balanced profile with high levels of both α-pinene and 1,8-cineole, suggesting additive or synergistic inheritance. Additionally, several minor compounds exclusive to the hybrid (e.g., β-cubebene, dehydroaromadendrene) were detected, indicating metabolic novelty due to hybridization.

3.3 Antimicrobial activity result:

The antimicrobial effectiveness of the EOs was assessed through MIC assays against four bacterial strains and four wood-decaying fungi (Table 3, Figure 3).

Table 3. Minimal inhibitory concentrations (MIC, v/v; mean of three replicates) for each essential oil against bacterial and fungal strains.

Microorganism	*E. camaldulensis*	*E. grandis*	Hybrid 2414
E. coli ATCC 8739	1/250	1/100	1/100
S. aureus ATCC 6538	1/250	1/100	1/100
M. luteus ATCC 9341	1/250	1/100	1/100
B. subtilis ATCC 6633	1/250	1/250	1/100
Coniophora puteana	1/250	1/250	1/1 000

The hybrid oil demonstrated strong antibacterial activity, equivalent to or better than the parental oils. Notably, it exhibited the lowest MIC (1/1000) against C. puteana, suggesting superior antifungal efficacy (figure 3 and 4). This may reflect the synergistic action of its major and minor terpenes, including p-cymene and α-terpineol.

Figure 3. Minimal inhibitory concentration (MIC, v/v) of essential oils against test strains (mean ± SD, n = 3).

Figure 4. Antifungal activity of Hybrid 2414 essential oil against wood-decaying fungi (representative plates; assays performed in triplicate).

4. DISCUSSION:

Hybridization in plants can enhance essential oil yield and bioactivity through heterosis, as shown in several studies on Eucalyptus. Our findings confirm this trend: hybrid clone 2414 produced nearly three times more oil than its parental species, highlighting its potential for commercial exploitation. Similar increases in yield have been reported in other interspecific hybrids of this genus^18,19.

The chemical profile of clone 2414 was distinct, characterized by the co-dominance of 1,8-cineole and α-pinene, whereas each parent was dominated by one of these terpenes. This balanced chemotype suggests an additive or synergistic inheritance pattern. The presence of exclusive minor compounds such as β-cubebene and dehydroaromadendrene further indicates novel metabolic interactions resulting from hybridization. Such chemical novelty has also been reported in other hybrid systems and may underlie unique bioactivities^20,21.

From a biological perspective, the hybrid oil showed stronger antimicrobial activity than either parent, particularly against Coniophora puteana, where the MIC reached 1/1000²². This superior antifungal efficacy can be attributed to synergistic interactions among major and minor terpenes. Both 1,8-cineole and α-pinene are well documented for their antimicrobial actions, and their combined presence may enhance potency²³. Minor compounds such as p-cymene and α-terpineol likely contribute by increasing membrane permeability and potentiating the effect of the dominant terpenes²⁴.

These results support the idea that hybridization can generate new chemotypes with optimized biological properties, offering a valuable tool for developing natural antimicrobial agents²⁵. Previous reports on Eucalyptus hybrids also observed enhanced antimicrobial efficacy, reinforcing our observations²⁶.

Recent reports corroborate these trends, highlighting that selected Eucalyptus hybrids combine enhanced yields with distinct chemotypes and improved bioactivity, thereby expanding industrial applicability^20,21,26,27.

While promising, some considerations remain before practical application. Further studies are required to clarify the mode of action, including biofilm inhibition and time-kill assays. Toxicity and environmental safety should also be assessed through in vitro and in vivo assays to ensure safe pharmaceutical or agro-industrial applications. Seasonal and environmental variability in yield and composition must be monitored to identify optimal harvesting periods. Finally²⁸, genetic and transcriptomic analyses could help elucidate the molecular basis of these enhanced traits and guide future breeding programs^29,30.

Overall, our findings demonstrate that clone 2414 combines high yield with enhanced antimicrobial properties, providing evidence that targeted hybridization is a promising approach for producing elite Eucalyptus genotypes with commercial and therapeutic potential^27,31.

5. CONCLUSION:

This study substantiates the value of hybridization in Eucalyptus for producing essential oils with superior yield and enhanced antimicrobial efficacy. Clone 2414’s unique chemotype, characterized by high concentrations of both 1,8-cineole and α-pinene alongside novel minor metabolites, confers broad-spectrum bioactivity surpassing that of its parental species. These findings not only contribute to our understanding of plant secondary metabolism in hybrids but also hold practical implications for sustainable bioproduct development, natural preservative formulation, and integrated pest management. Further multidisciplinary investigations are recommended to fully harness the potential of such hybrids in industrial and pharmacological contexts.

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Received on 07.08.2025 Revised on 05.11.2025

Accepted on 29.12.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):27-32.

DOI: 10.52711/0974-360X.2026.00004

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.