Fresh leaves and stems of M. rotundifolia (L.) Huds. were collected during the flowering stage in June 2024 from the experimental station of the National Institute of Agronomic Research of Algeria (INRAA), Touggourt region, Southeast Algeria (Latitude: 33°06′18″ N; Longitude: 6°03′28″ E). Approximately 0.5 kg of fresh aerial parts were harvested for the extraction process. The plant material was taxonomically authenticated by a botanist from INRAA, and a voucher specimen was deposited in the institute’s herbarium for future reference.

Immediately after collection, the plant material was carefully cleaned to remove dust and impurities, then air-dried in the shade under controlled ambient conditions (25 ± 2°C; relative humidity 45–50%) for 10 days to prevent photodegradation and preserve volatile constituents. The dried leaves and stems were then finely ground using a mechanical grinder to obtain a uniform powder. The powdered material was preserved in airtight, light-protected glass containers at 4°C to minimize oxidation and maintain chemical stability until essential oil extraction [17,18].

The essential oil (EO) of M. rotundifolia was obtained by hydrodistillation using a Clevenger-type apparatus. For the extraction, 300 g of the dried and homogenized plant material were mixed with 3 L of distilled water in a round-bottom flask. The mixture was subjected to gentle heating for 3 h at approximately 100°C using a temperature-regulated heating mantle to ensure the optimal release of volatile constituents. Upon completion, the oil layer was carefully separated from the aqueous phase, dried over anhydrous sodium sulfate, and stored in amber glass vials at 4°C to prevent oxidation and photodegradation prior to GC–MS and biological analyses [19]. The extraction yield was calculated according to the formula: Yield (%) = (volume of essential oil (mL)/weight of dry plant material (g)) × 100. The hydrodistillation process lasted for 3 h at approximately 100°C.

The volatile constituents of M. rotundifolia essential oil (MREO) were characterized using gas chromatography–mass spectrometry (GC–MS) to achieve comprehensive identification and quantification of its components. Analyses were carried out on an Agilent 5973 GC–MS system operating with an electron ionization energy of 70 eV. A fused-silica capillary column HP-5MS (30 m × 0.25 mm i.d., 0.25 μm film thickness; 5% phenyl-methylpolysiloxane stationary phase, Agilent Technologies, USA) was employed. To ensure an accurate and representative metabolite profile, three chromatographic runs were performed using capillary columns with different stationary phases, enabling enhanced resolution of complex volatiles.

The temperature program began at 60°C (held for 5 min), followed by a 10°C/min ramp up to 300°C, where it was held isothermally for 10 min. The injector temperature was maintained at 250°C (splitless mode), and the detector at 280°C. Helium served as the carrier gas at a constant flow rate of 1.0 mL/min.

Compound identification was achieved by comparing the obtained mass spectra with those in the NIST 14 and Wiley 9 spectral libraries, as well as by comparing experimental retention indices (RIexp) with literature or theoretical RIs (RIlit) from published compilations and NIST/Wiley databases [19]. Quantification of each compound was expressed as relative peak area percentage using the following equation: Yi% = [Pi/(P1 + P2 +…+ Pn)] × 100 where Y represents the percentage abundance and P represents the individual peak area [20].

Retention indices (RI) were experimentally determined using the Kovats linear retention index method, calculated relative to an n-alkane series (C₈–C₄₀) analyzed under identical chromatographic conditions. The integration of spectral matching and retention index comparison ensured robust compound confirmation and reduced misidentification risk.

Thin-layer chromatography (TLC) was employed as a preliminary qualitative method to assess the chemical composition and homogeneity of M. rotundifolia essential oil prior to GC–MS analysis.

Separations were performed on silica gel G plates (20 × 10 cm) as the stationary phase, using a n-hexane:ethyl acetate solvent system (90:10, v/v) as the mobile phase. For each analysis, 10 μL of the essential oil was precisely spotted on the plate using a calibrated glass capillary. The plates were developed in a saturated chromatographic chamber until the solvent front reached approximately 1 cm from the upper edge, and duplicate plates were prepared for each sample to ensure reproducibility.

After development, the separated compounds were visualized under UV light at 254 nm and 365 nm. Chemical detection reagents including vanillin, sulfuric acid, aluminum chloride (AlCl₃), and Dragendorff reagent were applied to selectively visualize distinct chemical classes, terpenoids, phenolics, and alkaloids, respectively—following standard phytochemical protocols [20].

TLC served as a rapid, cost-effective tool to visualize major chemical classes, assess inter-batch consistency, and guide the optimization of GC parameters. The subsequent GC–MS analysis provided definitive identification and quantification of individual volatile constituents, ensuring comprehensive chemical characterization of the essential oil.

Ten isolates of Fusarium oxysporum f. sp. albedinis were obtained from diseased date palm tissues exhibiting typical symptoms of Bayoud disease. The isolates were preserved in the mycological collection of the Biotechnology and Plant Improvement Division at INRAA. The fungal isolates were cultured on 90-mm Petri dishes containing Potato Dextrose Agar (PDA) and incubated in darkness at 25 ± 2°C for seven days to ensure optimal growth.

Fungal isolates were obtained from symptomatic date palm tissues and identified at the INRAA mycology unit using classical morphological criteria (macroscopic colony characters, microscopic morphology of macro- and micro-conidia and chlamydospores) following standard keys [21]. Pathogenicity of representative isolates was confirmed on susceptible date-palm material.

The antifungal potential of M. rotundifolia essential oil (MREO) against Fusarium oxysporum f. sp. albedinis (Foa), the causal agent of Bayoud disease, was evaluated using the poisoned food technique as previously described by Grover and Moore [20], with slight modifications.

A stock solution of the essential oil was first prepared by dissolving the required volume of MREO in 5% (v/v) Tween 20 aqueous solution, used as an emulsifying agent to enhance oil dispersion in the medium. Potato Dextrose Agar (PDA) medium was prepared and autoclaved at 121°C for 20 min, then cooled to approximately 45°C before adding the essential oil at the desired concentrations.

The final concentrations tested were 0 (control), 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 μL/mL of essential oil. An additional concentration of 1.75 μL/mL was also included to verify total growth inhibition. The essential oil–medium mixture was stirred for 1–2 min to ensure complete homogeneity, and 20 mL of the treated medium was poured into 90-mm sterile Petri dishes.

After solidification, a 5-mm agar disc containing actively growing mycelium of F. oxysporum f. sp. albedinis (from a 7-day-old pure PDA culture) was aseptically placed at the center of each plate. Control plates were prepared with PDA containing 5% Tween 20 but no essential oil.

All treatments were performed in triplicate, and the plates were incubated at 25 ± 2°C in complete darkness for seven (7) days. Following incubation, colony diameters were measured along two perpendicular axes, and the mean values were used to calculate mycelial growth inhibition (MGI) using the formula: M G I   ( % )   =   ( D a   −   D t ) D a   ×   100 where: D_a: Diameter of the control colony (without essential oil), D_t: Diameter of the treated colony (with essential oil) [22,23]. The percentage of inhibition was plotted against essential oil concentration to evaluate the dose–response relationship and determine the concentration producing complete fungal growth inhibition (MIC equivalent).

All experiments were performed in triplicate, and the results are presented as mean values ± standard deviations (SD). To determine the statistical significance of differences among treatments, data were subjected to one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to assess pairwise differences between means. A p-value of less than 0.05 (p < 0.05) was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics software (version 25.0, IBM Corp., Armonk, NY, USA). Prior to analysis, data were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) to validate the assumptions of ANOVA.

The selection of an appropriate solvent system is crucial in thin-layer chromatography (TLC) as it significantly influences the separation and resolution of sample components. In this study, a solvent system comprising hexane (90%) and ethyl acetate (10%) was employed to optimize the separation of various chemical constituents.

The TLC analysis revealed the presence of nine fractions under UV light at 245 nm with retention factor (Rf) values of 0.085, 0.136, 0.203, 0.288, 0.356, 0.424, 0.593, 0.746, and 0.779. Under UV light at 365 nm, only three fractions were detected with Rf values of 0.593, 0.746, and 0.932, as shown in Fig. 1. Following the application of Vanillin/Sulfuric acid reagent, nine distinct spots were observed, corresponding to Rf values of 0.153 (grey-violet), 0.203 (red-violet), 0.271 (blue-violet), 0.322 (green), 0.373 (blue-violet), 0.441 (brown), 0.593 (brown), 0.661 (yellow), and 0.745 (brown). Additionally, the application of AlCl₃ reagent resulted in the formation of three spots with Rf values of 0.625 (blue), 0.786 (yellow), and 0.893 (green), These visualizations are presented in Fig. 2.

TLC serves as a reliable and time-efficient analytical tool for the preliminary identification of compounds in complex mixtures. In our study, we utilised this method as a preliminary analysis tool for examining the essential oil [24]. The utilisation of a specific mixture of solvents, comprising 90% Hexane and 10% Ethyl acetate, proved to be effective in achieving improved separation of various chemicals in the given sample. Moreover, the introduction of several reagents such as iodine, sulfuvaniline, Dragendorff, and AlCl₃ during the TLC (thin-layer chromatography) process significantly enhanced the differentiation between the separated compounds, enabling more precise identification and analysis. The presence of monoterpenes and sesquiterpenes were detected through the appearance of blue-violet, red-violet, grey-blue, or blue spots [25].

The hydrodistillation of Mentha rotundifolia leaves and stems produced a pale-yellow essential oil with a characteristic aromatic odor and an extraction yield of 1.25% (v/w, based on dry weight). This yield is comparable to previously reported values for M. rotundifolia essential oils, which generally range from 0.47% to 4.33%, depending on origin, environmental conditions, and extraction parameters [26,27].

Variations in extraction yield among studies can be attributed to several factors, including geographical location, climatic conditions, harvest season, soil type, plant maturity, and extraction duration or temperature [28]. For instance, Soilhi et al., (2019) reported a yield of (1.1-2.5%) for Tunisian M. rotundifolia, while Hassani, (2020) obtained a higher yield of 3.85% from Moroccan samples, underscoring the influence of regional and environmental variations on essential oil productivity [29,30]. The yield obtained in this study (1.25%) therefore lies within the normal range observed in the literature, confirming that the hydrodistillation conditions employed (3 h at 100°C) were appropriate for efficient volatile oil recovery without degradation of thermolabile constituents.

GC–MS analysis identified 23 volatile compounds (Table 1; Fig. 3), representing more than 99.8% of the total composition. The oil was dominated by piperitenoneoxide (62.53%), followed by trans-piperitone epoxide (9.84%), carvone (4.12%), and 1,8-cineole (3.76%), confirming a chemotype rich in oxygenated monoterpenes characteristic of the Mentha genus [31]. Minor compounds included α-pinene, β-pinene, terpinen-4-ol, and carvone oxide, which, although present in lower concentrations, play important roles in enhancing the bioactivity and antifungal efficacy of the oil.

Oxygenated monoterpenes such as α-pinene, β-pinene, terpinen-4-ol, and carvone oxide are well-documented for their strong antimicrobial and antioxidant properties, acting through cell membrane disruption, enzyme inhibition, and oxidative stress modulation in pathogenic microorganisms. Their synergistic interaction with major compounds like piperitenone oxide likely contributes to the potent antifungal effect of M. rotundifolia essential oil against Fusarium oxysporum f. sp. albedinis.

RI_exp = experimentally measured retention index (this work, C₈–C₄₀ n-alkane series); R_Ilit= literature/library retention index used for confirmation.

The exceptional abundance of piperitenone oxide (62.53%) in Mentha rotundifolia essential oil (MREO) aligns with previous reports describing this compound as a chemical marker of M. rotundifolia chemotypes, particularly those from Algeria, the Mediterranean Basin, and South America [32,33]. Piperitenone oxide is a key oxygenated monoterpene known for its strong antimicrobial and antifungal activity, especially against plant-pathogenic fungi, including several Fusarium species [34]. Its predominance in the present study strongly correlates with the high antifungal efficiency observed against F. oxysporum f. sp. albedinis, the causal agent of Bayoud disease.

The dose-dependent inhibition (7.82–83.41%) and complete suppression (100%) of fungal growth at 1.75 μL/mL confirm that piperitenone oxide plays a dominant role in MREO’s fungicidal activity. This compound’s biological action is primarily attributed to its lipophilic structure, which facilitates penetration into fungal membranes, leading to altered membrane integrity, ion leakage, and inhibition of key metabolic enzymes involved in respiration and cell wall synthesis. These effects collectively impair spore germination and mycelial development, resulting in the observed growth inhibition.

In addition to piperitenone oxide, the presence of other bioactive oxygenated monoterpenes, including α-pinene (1.31%), β-pinene (1.85%), terpinen-4-ol (0.51%), and carvone oxide (4.40%)—significantly enhances the oil’s overall antifungal potential. These minor constituents act synergistically with major compounds, amplifying antimicrobial efficacy through multi-target interactions that disrupt cellular structures, denature fungal proteins, and induce oxidative stress. Such synergy is well documented in complex natural matrices, where the combined effect of several terpenoids exceeds that of individual components.

This synergistic phenomenon has also been observed in other essential oils rich in oxygenated monoterpenes, such as those from Origanum vulgare, Thymus vulgaris, and Lavandula angustifolia, which exhibit potent antifungal activity against Fusarium, Aspergillus, and Candida species [35,36]. Like MREO, these oils contain compounds such as carvacrol, thymol, linalool, and 1,8-cineole, which share similar hydrophobic and reactive oxygen species (ROS)-modulating properties, further supporting the hypothesis that antifungal potency in essential oils is driven by the collective and complementary action of oxygenated monoterpenes.

The correlation between chemical composition and antifungal activity in this study underscores the chemotype-specific efficacy of MREO. The dominance of piperitenone oxide, supported by a suite of synergistic minor constituents, explains the observed differences in sensitivity among F. oxysporum isolates (DL₅₀ values ranging from 0.95 to 1.58 μL/mL). Isolates with lower DL₅₀ values may be more susceptible to oxidative or structural stress induced by the oil’s active terpenoids.

Environmental factors play a crucial role in determining MREO’s chemical composition and biological activity. Variations in climate, soil composition, altitude, and sunlight exposure influence the biosynthesis of secondary metabolites in Mentha species [37]. For instance, samples from coastal or humid regions tend to accumulate higher proportions of oxygenated monoterpenes, whereas plants grown in arid or high-altitude environments often display increased sesquiterpene and ketone contents. Previous studies in Algeria reported piperitenone oxide contents ranging from 23.5% (Miliana) to 38.6% (Rouina) [36], illustrating the strong effect of ecological and edaphic conditions on essential oil profiles.

The detection of sesquiterpenes such as trans-caryophyllene (1.63%), β-cubebene (1.80%), and (+)-epi-bicyclosesquiphellandrene (1.55%) further supports the bioactivity of MREO. These compounds contribute anti-inflammatory and antioxidant properties, with trans-caryophyllene notably acting as a CB2receptor agonist comparable to non-steroidal anti-inflammatory drugs (NSAIDs) [38]. Ketones such as carvone oxide (4.40%) and isophorone (0.77%) also reinforce MREO’s antimicrobial potential by interfering with fungal enzymatic systems and membrane-bound processes [39,40,41,42].

Finally, the mode of antifungal action of MREO can be attributed to membrane disruption and enzyme inhibition mechanisms, as supported by its chemical composition. Oxygenated monoterpenes readily integrate into fungal lipid bilayers, altering permeability and causing leakage of cellular contents, while also inhibiting key enzymatic pathways responsible for ergosterol biosynthesis and cell wall maintenance. This multi-targeted mechanism reduces the likelihood of resistance development and supports the use of MREO as a natural, eco-friendly alternative to synthetic fungicides for managing Bayoud disease.

Collectively, these findings demonstrate that the chemical richness and compositional balance of MREO dominated by piperitenone oxide and supported by synergistic oxygenated monoterpenes—are directly responsible for its potent antifungal, antioxidant, and anti-inflammatory properties. This work thus reinforces M. rotundifolia essential oil as a promising biocontrol agent in sustainable agriculture and as a natural ingredient for pharmaceutical and food preservation applications.

The antifungal activity of Mentha rotundifolia essential oil (MREO) was assessed against ten field isolates of Fusarium oxysporum f. sp. albedinis (Foa) using the poisoned food technique at seven concentrations (0, 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 μL/mL).

The results revealed a strong and dose-dependent inhibition of mycelial growth across all isolates, with inhibition percentages ranging from 7.82% to 83.41% after seven days of incubation (Fig. 4). The antifungal effect intensified progressively with increasing concentrations of the essential oil. At 1.75 μL/mL, complete inhibition (100%) of mycelial growth was achieved in all isolates, confirming the potent fungicidal activity of MREO.

These findings demonstrate that M. rotundifolia essential oil exhibits broad-spectrum antifungal efficacy against F. oxysporum f. sp. albedinis, likely attributed to its high content of bioactive oxygenate monoterpenes such as piperitenone oxide, terpinen-4-ol, α-pinene, and β-pinene, which are known to disrupt fungal cell membranes and interfere with metabolic processes.

Statistical analysis (one-way ANOVA followed by Tukey’s test, p < 0.05) confirmed significant differences in antifungal responses among isolates. The calculated DL₅₀ values (concentration required to inhibit 50% of mycelial growth) ranged from 0.95 to 1.58 μL/mL (Table 2), highlighting variability in isolate sensitivity to the essential oil.

Isolate S20/17 exhibited the lowest DL₅₀ value (0.95 μL/mL), indicating the highest susceptibility, while isolates S24/18, S30/18, and S40 showed higher DL₅₀ values (up to 1.58 μL/mL), reflecting relatively lower sensitivity. These inter-isolate differences may be attributed to genetic variability among F. oxysporum populations, physiological adaptation mechanisms, or differences in cell wall composition and permeability to lipophilic essential oil components.

Overall, the results confirm that M. rotundifolia essential oil exerts strong antifungal activity against F. oxysporum f. sp. albedinis at low concentrations, with complete inhibition achieved at 1.75 μL/mL, demonstrating its potential as a natural, eco-friendly alternative to synthetic fungicides for managing Bayoud disease in date palms.

Numerous studies have investigated the antifungal activity of natural products such as microbial metabolites, plant extracts, and essential oils against Fusarium oxysporum f. sp. albedinis (F.o.a), the causative agent of Bayoud disease in date palms. Despite these efforts, effective biological control strategies remain limited. Essential oils, known for their broad-spectrum antimicrobial properties, present promising alternatives to synthetic fungicides. Previous research has demonstrated varying degrees of inhibitory activity among different essential oils. For instance, essential oils derived from Origanum compactum, Myrtus communis, Thymus satureioides, Lavandula dentata, and Rosmarinus officinalis have shown differential efficacy against F.o.a. Notably, O. compactum exhibited the highest antifungal activity with a minimum inhibitory concentration (MIC) of 2.5 μL/mL, while the other oils had MICs ranging from 10 to 40 μL/mL [43]. Lakhdari et al. reported significant antifungal activity of Cladanthus eriolepis essential oil against F.o.a, with inhibition rates ranging from 35.80% to 86.20% at concentrations of 2000 and 4000 ppm, respectively, suggesting its potential as a natural agent for managing Bayoud disease [44]. Similarly, Tanacetum annuum (Blue Tansy) essential oil demonstrated strong antifungal effects at low concentrations, with an MIC of 3.33 μL/mL [45]. Essential oils from Artemisia herba-alba, Foeniculum vulgare, and Citrus sinensis were also evaluated, with A. herba-alba showing the most potent antifungal activity, having the lowest LC50 value (0.1 μL/mL), although all three oils presented MIC and CMF values above 50 μL/mL [46]. Hassan et al. reported MIC values for Rosmarinus officinalis (0.2 g/L), Salvia officinalis (2.5 g/L), Lavandula dentata (0.6 g/L), and Cymbopogon citratus (0.5 g/L). The lethal concentrations (MLC) for L. dentata and C. citratus were 1.75 g/L and 0.95 g/L, respectively, indicating their strong antifungal potential [47]. These results collectively highlight the inhibitory effects of various essential oils on the mycelial growth of F.o.a. Consistent with these findings, our study confirms the pronounced antifungal activity of Mentha rotundifolia essential oil (OEMR), achieving 100% inhibition at a concentration of just 1.75 μL/mL—a lower dose compared to those reported in other studies. Several authors have examined the antimicrobial properties of essential oils from different Mentha species. MacNair et al. demonstrated that Mentha spicata essential oil exhibits a dose-dependent inhibitory effect, with inhibition rates reaching 90% against Fusarium solani and F. oxysporum, and 100% against Alternaria citri at 20 μL/mL. The IC50 values ranged between 8 and 15 μL/mL [48]. Jeldi et al. also noted antifungal effects of Mentha pulegium extracts against Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Fusarium culmorum [49]. Similarly, Akotowanou et al. evaluated the essential oils of Pimenta racemosa and Mentha piperita against F. oxysporum in tomato, reporting minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 0.75 μL/mL and 1.25 μL/mL for P. racemosa, and 3.75 μL/mL and 5 μL/mL for M. piperita, respectively [50]. The notable antifungal activity of Mentha rotundifolia essential oil is likely attributed to its chemical composition, particularly its richness in oxygenated monoterpenes. These compounds, along with sesquiterpenes containing aromatic rings and phenolic hydroxyl groups, are capable of forming hydrogen bonds with fungal enzymes, thereby interfering with essential biological functions [51,52,53]. Chibane et al. further supported these findings by demonstrating the inhibitory effects of various monoterpenes on mycelial growth in multiple phytopathogenic fungi [54]. Our study shows that M. rotundifolia essential oil exhibits potent antifungal activity against F.o.a isolates, with a notably low DL₅₀ value. GC-MS analysis revealed that this efficacy is largely due to the presence of highly fungitoxic compounds, especially Piperitenone oxide, which was identified as the major constituent. This result is in agreement with previous studies that also identified Piperitenone oxide as a dominant compound in Mentha species [50,55]. Piperitenone oxide is recognized for its strong antimicrobial, antioxidant, and anti-inflammatory activities [56]. The high abundance of this compound in M. rotundifolia essential oil likely plays a crucial role in its therapeutic efficacy, particularly in countering microbial resistance and oxidative stress. Similar findings have been reported for Mentha longifolia and Mentha spicata, reinforcing the important role of Piperitenone oxide across the Mentha genus [57].