Evaluation of Essential Oils from Lamiaceae and Myrtaceae Families: Antifungal, Antioxidant, and Chemical Characterization for Multifunctional Purposes

1. Introduction

Phytopathogenic fungi represent a major global agricultural threat, causing substantial yield losses and economic hardships for farmers worldwide [1]. These pathogens affect a wide variety of crops, causing diseases that manifest as wilting, rotting, leaf spots, and other symptoms [2,3]. As these infections spread, they can lead to decreased crop quality and quantity [4]. Yield losses occur due to reduced crop growth, premature ripening, and compromised nutritional value, all of which directly impact farm income and food security [5]. Moreover, farmers often resort to chemical fungicides to combat these fungal diseases, which further add to production costs [6]. In the absence of effective control measures, the cumulative economic losses from fungal infections are staggering, highlighting the critical need for sustainable and eco-friendly strategies to manage these agricultural threats [7].

Essential oils (EOs) are rich in secondary metabolites—compounds produced by plants with diverse biological functions, including defense against pests and pathogens [8]. In recent years, EOs have gained attention as powerful alternatives in the battle against antibiotic, herbicide, and fungicide resistance [9]. These natural compounds contain a diverse array of bioactive constituents with antimicrobial properties, offering a sustainable solution to combat the growing problem of resistance in agriculture and healthcare [10]. EOs can play a pivotal role in eco-friendly agriculture by reducing reliance on synthetic chemicals that contribute to environmental degradation [11]. Their ability to target a broad spectrum of pathogens, while being less prone to resistance development, makes them a promising tool to address the pressing need for sustainable pest and disease management practices that safeguard both crop yields and the planet’s health [12].

Beyond their aromatic qualities, EOs extracted from plants of the Lamiaceae family (Lavandula dentata, Salvia rosmarinus and Thymus vulgaris) have also been recognized for their diverse biological activities [13]. They exhibit potent antioxidant properties, helping to combat oxidative stress and protect cells from damage [14]. Additionally, these EOs have demonstrated strong antibacterial and antifungal actions, making them valuable in the fight against various microbial infections [15]. The EOs extracted from plants of the Lamiaceae family continue to be subjects of scientific interest and practical applications, offering a wealth of benefits to both human health and sustainable agriculture [16].

The Eucalyptus genus, a diverse and globally recognized group of flowering trees, is primarily native to Australia, where it thrives in a wide range of habitats [17]. However, these iconic evergreen plants have also spread to other parts of the world and are cultivated in various regions, including South America, Africa, and Southeast Asia [18]. One of the most remarkable features of the different species from Eucalyptus genus are their EOs, which are mainly derived from the leaves, being responsible for a distinct, invigorating aroma [19]. Eucalyptus EO is renowned for its numerous biological activities, making it a valuable resource in the field of natural medicine and industry [20]. It exhibits potent antioxidant properties, which can help combat free radicals and oxidative stress [21].

The present study aimed to explore the chemical composition of EOs extracted from the aerial parts of Lavandula dentata, Salvia rosmarinus, and Thymus vulgaris, all belonging to the Lamiaceae family, as well as the leaves of seven Eucalyptus species from the Myrtaceae family: Eucalyptus camaldulensis, Eucalyptus cinerea, Eucalyptus grandis, Eucalyptus lehmannii, Eucalyptus leucoxylon, Eucalyptus saligna, and Eucalyptus sideroxylon. Additionally, the study investigated the antifungal activity of these EOs against six phytopathogenic fungal strains: Fusarium culmorum, F. oxysporum, F. proliferatum, Phoma sp., Rhizoctonia solani, and Sclerotinia sclerotiorum. The antioxidant potential of the EOs was assessed using DPPH and ABTS assays.

2. Results

2.1. Essential Oil Yields and Chemical Composition

The EO yields of the selected species from the Lamiaceae and Myrtaceae families exhibited significant variation, as indicated by statistical analysis (p < 0.05) highlighting the variation in oil productivity. For the Lamiaceae family, Lavandula dentata yielded the highest EO content (3.6%) followed by Thymus vulgaris EO (2.7%) and Salvia rosmarinus (0.66%). In the Myrtaceae family, the EO yields ranged from 0.18% (Eucalyptus grandis) to 1.94% (Eucalyptus camaldulensis) (Table 1).

The chemical analysis of the ten EOs allowed the identification of more than 100 compounds, accounting for from 97.23 to 99.92% of the total EOs and distributed acroos five classes of terpene and non-terpene derivatives (Table 1). All EOs showed a specific richness in oxygenated monoterpenes (39.95 to 88.31%) and monoterpenes hydrocarbons (7.02 to 53.65%). A total of 85 compounds were identified in Lavandula dentata EO, representing 99.18% of the entire EO constituents. Oxygenated monoterpenes (72.67%) were the major portion in this oil with 1.8 cineole (61.8%), β-pinene (12.52%) and α-pinene (4.35%) being the dominant compounds. In Salvia rosmarinus EO, 56 chemical components were characterized which accounted for 99.79% of the total oil. Oxygenated monoterpenes (54.52%) and monoterpenes hydrocarbons (36.31%) were the two main subclasses in this oil. The prevalent constituents were 1.8 cineole (40.75%) followed by α-pinene (14.42%) and camphor (7.87%). The chemical analysis of the Thymus vulgaris EO showed 30 compounds representing 99.2% of the total oil. Oxygenated monoterpenes (74.84%) were the primary constituents of this oil. The major components detected in Thymus vulgaris EO were thymol (72.37%), γ-terpinene (7.87%) and p-cymene (7.11%). The chemical analysis of the EOs extracted from the leaves of the seven Tunisian Eucalyptus species allowed the identification of 35 compounds from E. camaldulensis, 33 from E. cinerea, 51 from both E. grandis and E. lehmannii, 55 from E. leucoxylon, 64 from E. saligna and 53 from E. sideroxylon, representing 99.86%, 99.92%, 98.57%, 99.71%, 99.29%, 97.23% and 99.86% of the entire EO constituents respectively. The predominant EO’s constituents for Eucalyptus species (E. camaldulensis, E. cinerea, E. grandis and E. sideroxylon) were 1.8 cineole (39.62 to 86.26%) as oxygenated monoterpenes, followed by α-pinene (4.38 to 31.96%) and p-cymene (1.86 to 12.82%) as monoterpenes hydrocarbons. Camphor (15.2%) was also identified in E. lehmannii in addition to 1.8 cineole (60.74%) and α-pinene (12.69%). The key constituents of E. leucoxylon were 1.8 cineole (62.95%) followed by p-cymene (10.84%) and α-pinene (8.05%). The the main compound in E. saligna EO (29.37%) was p-cymene followed by 1,8- cineole (24.36%) and α-pinene (18.2%).

2.2. Antioxidant Activity

The antioxidant potential of the ten EOs were evaluated by DPPH and ABTS assays, and the results are presented in Table 2.

In both tests, DPPH and ABTS, Thymus vulgaris EO revealed the strongest antioxidant activity with an IC₅₀ of 3.06 ±0.04 and 1.5 ±0.08 µg mL^-1 respectively among the tested species. These values were higher than those obtained for the commonly used antioxidant standard Trolox. Then, also a high antioxidant activity was exhibited by E. saligna (IC₅₀ of 18.5 ±1 and 5 ±0.9 µg mL^-1) and Salvia rosmarinus (IC₅₀ of 42.32 ±3.06 and 7 ±0.2 µg mL^-1) EOs. The EOs from E. camaldulensis, E. grandis and E. leucoxylon were also active with IC₅₀ values ranging from 100.1 ±6.2 to 190.7 ±2.6 µg mL^-1 in DPPH and from 10.8 ±0.8 to 24.3 ±4.1 µg mL^-1 in ABTS assay, respectively. Moreover, E. lehmannii and E. sideroxylon revealed moderate antioxidant activities. No significant differences between these EOs were observed in the DPPH assay. Conversely, these differences were statistically significant in the ABTS assay. Finally, the least active was the EO of Lavandula dentata showing IC₅₀ values of 765.26 ±141.05 and 102.5 ±1.7 µg mL^-1 in DPPH and ABTS assays respectively.

2.3. Antifungal Activity

The effects of increasing concentrations of the ten tested EOs on mycelium growth of the different fungal strains are summarized in Table 3. Most of the EOs inhibited the growth of the tested fungal strains in a dose-dependent manner.

The highest inhibitory activity was exhibited by the Thymus vulgaris EO, with total inhibition of mycelium growth at 2 µL mL^-1 for all fungal strains. Thymus vulgaris EO has shown a fungicidal effect for all the tested strains with MFC values ranging between 2 and 12 µL mL^-1 while it was fungistatic against Phoma sp. Salvia rosmarinus EO totally inhibited the growth of all fungal strains at concentrations ranging from 4 to 8 µL mL^-1. However, it was only fungicidal for both R. solani and S. sclerotiorum at 8 and 10 µL mL^-1 respectively. Lavandula dentata EO showed moderate antifungal activity against the strains tested. The MIC values ranged between 8 and 12 µL mL^-1, however these applied doses were not lethal for all tested strains. Among Eucalyptus species, E. saligna, even at the lowest concentration (2 µL mL^-1), exerted a total growth inhibition and fungicidal effect on S. sclerotiorum strain while it showed a strong antifungal activity with inhibition percentages higher than 70% for all the others strains at the same concentration. Total inhibition was recorded for all strains when E. camaldulensis EO was applied at concentrations of 4 µL mL^-1 or higher with S. sclerotiorum, R. solani and F. culmorum being the most sensitive strains, as indicated by their MFC ranging between 4 and 12 µL mL^-1. Similarly, E. grandis EO completely inhibited the growth of all strains at concentrations ≥4 µL mL^-1, except for F. oxysporum, which showed an inhibition percentage of 80% at the highest concentration. Among the strains, S. sclerotiorum and R. solani exhibited the highest sensitivity to E. grandis EO, with MFC of 6 and 10 µL mL^-1 respectively. This indicates that both E. camaldulensis and E. grandis exert significant antifungal activity, with some variability in their effectiveness against different fungal species. More than 50% inhibition of growth was observed in all fungal strains when E. leucoxylon EO was used at the lowest concentration of 2 µL mL^-1. Complete inhibition was achieved for all strains, with MIC ranging from 4 to 12 µL mL^-1, except for F. oxysporum, which showed 76% inhibition at the highest dose of 12 µL mL^-1. E. leucoxylon EO also exhibited fungicidal effect against S. sclerotiorum and F. culmorum, with MFC of 4 µL mL^-1 and 12 µL mL^-1 respectively. Likewise, E. cinerea EO totally inhibited all strains growth at concentrations ranging between 4 and 12 µL mL^-1 except for F. oxysporum. Both S. sclerotiorum and F. culmorum are the most sensitive strains with MFC of 6 and 12 µL mL^-1 respectively. E. sideroxylon EO at the lowest concentration (2 µL mL^-1) inhibited the growth of all tested strains by approximately 60% or more, except for S. sclerotiorum which was totally inhibited at this concentration. Meanwhile, Phoma sp. and R. solani were fully inhibited at higher concentrations of 6 µL mL^-1 and 10 µL mL^-1, respectively. E. sideroxylon demonstrated a fungistatic effect only on S. sclerotiorum strain with MFC equal to 4 µL mL^-1. E. lehmannii EO exhibited effectiveness against both S. sclorotiurom and R. solani strains with MIC values of 6 and 8 µL mL^-1 and MFC values of 6 and 10 µL mL^-1 respectively.

3. Discussion

Yield variation in EO production is a common challenge that arises due to several factors [22]. It is mainly influenced by the plant species or genotype, seasonal changes, and environmental conditions such as climate, soil, and geography [23,24,25,26]. The harvest stage, plant part used, and extraction method also play important roles in determining EO yield [27,28].

In this study, Lavandula dentata produced the highest EO content (3.6%), which aligns with the findings of El Abdali et al. [29], who reported a content of 3.46%. This yield is higher compared to Lavandula dentata studied in Tunisia (1.76%), Algeria (1.18%), Morocco (0.79 %) and Argentina 0.8% [30,31,32,33].

Golkar et al. [34] reported an EO yield of 0.08% for Thymus vulgaris. Higher yield (0.3%) was obtained by de Oliveira et al. [35]. According to Pavela et al. [36], the EO content in Thymus vulgaris plant parts ranged from 0.3% to 4%. The present study’s findings fall within this range, with an EO yield of 2.7%. Similarly, Dinu et al. [37] recorded an EO yield of 2.5%.

Bayar and Akşit [38] reported an EO yield of 1% for Salvia rosmarinus. Çınar et al. [39] investigated how EO content and quality characteristics of rosemary vary based on location and harvest time, emphasizing the importance of choosing the appropriate site and harvest period for optimal oil yield. Their findings indicated that rosemary oil content ranged from 0.96 to 2.02 %. Rathore et al. [40] found that the EO profile of rosemary in the western Himalayan region was significantly influenced by both the harvesting season and genetic variability among accessions. Higher EO content was recorded during the autumn season (0.87%) compared to summer (0.68%) and the rainy season (0.48%). In line with these findings, the present study recorded a 0.66% EO yield for Salvia rosmarinus.

The yields of EOs from various Eucalyptus species can vary significantly, with reported percentages ranging from 0.1% to 7.3%, highlighting the wide diversity in oil content among different plants [41,42]. For example, the EO yield of Eucalyptus camaldulensis from Thailand has been documented to range between 1.07% and 2.23%, depending on the specific clones tested [43]. Similar yields have been observed in other regions, with values of 1.2% in Turkey [44] and 1.9% in Pakistan [45]. The EO yield of Eucalyptus camaldulensis in the present study (1.94%) is consistent with previously reported data. The EO yield of Eucalyptus cinerea in the present report was found to be 1.45%. In contrast, E. cinerea from Brazil, when extracted from both leaves and stems, produced a significantly higher yield of 5.4% [46]. In Italy, the same species yielded 2.56% when only the leaves were used [47]. Tum et al. [48] studied the impact of varying extraction times, finding that the yield of Eucalyptus grandis ranged from 0.5 to 0.7% which differs from our findings (0.18%). Khedhri et al. [49] found that Eucalyptus lehmannii from a Tunisian arboretum yielded 1.91%, which is higher than the yield observed in our study (1.45%). A similar observation was made for Eucalyptus leucoxylon, as Sebei et al. [50] reported a yield of 1.61%, which exceeds the yield found in our study (1.17%). Ayed et al. [51] reported that the EOs extracted from eight Eucalyptus species growing in Tunisia had yields ranging from 0.12 to 1.32%, with Eucalyptus saligna yielding 0.64%. Similarly, our study recorded a yield of 0.53%. Additionally, Amri et al. [52] reported a yield of 1.3% for Eucalyptus sideroxylon growing in Tunisia, which is consistent with our findings, as we also observed a yield of 1.33%.

Regarding the chemical composition of EOs, the primary constituent in all the studied species was 1.8-cineole (an oxygenated monoterpene) except in Eucalyptus saligna and Thymus vulgaris, where p-cymene and thymol were the dominant components respectively. Our results on chemical profiling of the Thymus vulgaris EO are consistent with those reported by de Oliveira et al. [35] concluding that the main constituents were thymol (40%), p-cymene (19.3%) and γ-terpinene (17.3%). Similar results were detected in Thymus vulgaris EO from Brasil, showing thymol (45.95%), p-cymene (25.11%), and γ-terpinene (8.95%) as major components [53]. Tardugno et al. [54] characterized three Thymus vulgaris EOs obtained from plants growing in Italy. The main constituents were thymol (35.84–41.15%), p-cymene (17.5 -21.73%), c-terpinene (15.06 - 18.42%), linalool (2.55 - 5.37%) and carvacrol (1.45 - 1.7%). Additionally, Iranian Thymus vulgaris EO was mainly composed of thymol (22.1%) followed by p-cymene (21.31%), carvacrol (13.02%), carvacrol acetate (6.72%) and linalool (5.58%) [55]. The major compounds identified within the Romanian Thymus vulgaris EO were thymol (55.44%), m-cymene (11.88%), γ-terpinene (5.74%) and o-cymen-5-ol (5.14%) [37]. However, Thymus vulgaris EO collected from Morocco showed a different chemical composition with carvacrol (59.70 %), thymol (16.5 %), and γ-terpinene (11.11 %) as main constituents [56].

The 1,8-cineole (63%) was the major compound of Lavandula dentata from Brazil [57], which aligns with our findings (61.8%). In Tunisia, Lavandula dentata EO extracted by Dammak et al. [31] and Touati et al. [58] were rich in 1,8-cineole (35 and 33.54 % respectively). In contrast, Dridi et al. [59] identified β-eudesmol (21.17 %), while Msaada et al. [60] reported linalool (47.3 %) as the predominant component. The 1,8-cineole was also observed to be the major constituent of Lavandula dentata cultivated in Algeria (48%), Morocco (32%), Spain (67%) and Argentina (34.33%) [32,61]. Rathore et al. [40] reported that Salvia rosmarinus EO primarily contained 1.8-cineole (45.4 - 48.1%), α-pinene (10.9 - 14%) and camphor (5.4 – 15.8%), emphasizing the variability in major EO constituents across different plant accessions. These findings are in line with the present study, where Salvia rosmarinus EO contained mainly 1.8 cineole (40.75%), α-pinene (14.42%) and camphor (7.87%). Additionally, Micić et al. [62] revealed that the most abundant compounds in Salvia rosmarinus EO originating from Serbia and Russia were α-pinene (23 and 17.76%), 1.8 cineole (17.79 and 23.4%) and camphor (14.39 and 17.17%) respectively. Similarly, Bayar and Akşit [38] found that the major components of Turkish Salvia rosmarinus EO were camphor (21.25%), 1,8-cineole (13.85%) and borneol (11.64%).

Eucalyptus EOs exhibited significant chemical variability across different geographical origins as evidenced by various studies with 1,8 cineole being the major constituent which is in line with our findings [63,64]. In Brazil, a total of 5 components were identified in Eucalyptus camaldulensis containing primarily 1,8-cineole (76.93%), β-pinene (11.49%), and α-pinene (7.15%) [65]. In another Brazilian study, the same plant species yielded EO from leaves with different chemical profiles, with 1,8-cineole (41.61%), α-terpineol (19.87%), and α-pinene (15.81%) as the predominant components [66]. However, when using the aerial parts of E. camaldulensis in Egypt, the main constituents shifted to spathulenol (20.84%), p-cymene (15.16%), and 1,8-cineole (12.01%) [67]. In Pakistan, the leaves of E. camaldulensis had a distinctive profile, with linalool (17%), 1,8-cineole (16%) and p-cymene (12.2%) being the major constituents [45]. These variations extend to other regions like Morocco, Syria, Turkey and Thailand showing p-cymene and γ-terpinene as the major components [43,44,68,69,70,71,72,73]. EO extracted from the leaves and stems of E. cinerea from Brazil, contained mainly 1,8-cineole (55.24%), α-terpinyl acetate (21.64%) and α-pinene (7.94%) [46]. In Italy, the same species yielded 1,8-cineole (67.7%), α-pinene (7.3%) and α-terpinyl acetate (5.2%) when only the leaves were used [47]. Similarly, Sebei et al. [50] reported that the major constituents of E. lehmannii growing in Tunisia were 1,8-cineole (49.07%) α -pinene (26.35%) and α-terpinyl acetate (5.64%) while E. leucoxylon was characterized by the dominance of 1,8-cineole (77.76%) α -pinene (5.85%) and trans-pinocarveol (3.23%). Caetano et al. [66] reported that E. grandis EO main components were 1,8-cineole (37.43%), α-pinene (36.35%), and α-terpineol (8.71%) as the predominant components. Ayed et al. [51] characterized E. saligna EO extracted from Tunisia identifying 1,8-cineole (20.36%), p-cymene (15.27%) and isoborneol (10.54%) as the primary compounds. In contrast, the present study found that p-cymene was the principal component in E. saligna EO (29.37%), followed by 1.8-cineole (24.36%) and α-pinene (18.2%). Similarly, Amri et al. [52] analyzed the E. sideroxylon EO growing in Tunisia, with the main compounds were 1,8-cineole (65.4%), globulol (7.4%) and aromadendrene (2.1%). These findings differ from those observed in the current study, where the dominant compound was 1,8-cineole (86.26%), followed by α-pinene (4.38%) and p-cymene (1.86%), highlighting a distinct chemical composition between the two studies.

Based on the EO composition, different chemotypes of Thymus vulgaris can be distinguished from each other, with thymol being one of the dominant chemotypes [74]. Thymus vulgaris is well-known to have a great antioxidant and antimicrobial ability owing to its chemical composition rich in phenolic compounds [74,75]. Previous studies concluded that the high ability to scavenge free radicals in Thymus vulgaris EO was related to its thymol content [34,53]. Findings of the present study indicated a highly significant antioxidant capacity of Thymus vulgaris EO with IC₅₀ of 3.06 ±0.04 and 1.5 ±0.08 µg mL^-1 outperforming standard Trolox (IC₅₀ of 22.26 ±1.2 and 33.73 ±0.08 µg mL^-1) in DPPH and ABTS assays respectively. These results showed a higher antiradical capacity of the Thymus vulgaris EO when compared to those of Chahboun et al. [56] reporting IC₅₀ values of 5.94 ±0.22 and 3.03 ±0.17 μg mL^-1 in DPPH and ABTS tests respectively. Pilozo et al. [76] demonstrated the antioxidant profile of Thymus vulgaris with a radical neutralizing potential (DPPH) of IC₅₀=1.11 ±0.019 mg mL^-1 and ferric ion reducing power of 93.05 ±0.52 mg equivalent Trolox/g. According to Golkar et al. [34], the biosynthetic pathway of thymol continues with p-cymene as an intermediate. P-Cymene, as a strong antioxidant component, was found in the present study to constitute 29.37% of E. saligna EO, which contributed to its significant antioxidant activity. However, compared to thymol and p-Cymene, 1,8-cineole exhibited lesser antioxidant potential. The findings of this study were compared to previously published results, all of which suggest that 1,8-cineole, the primary constituent found in the EOs of Lavandula dentata, Salvia rosmarinus, and some Eucalyptus species, demonstrated a moderate antioxidant potential [29,77]. In the present study, the antioxidant activity of Salvia rosmarinus EO (42.32 ±3.06 μg mL^-1 DPPH assay) is within the range reported for the EOs from five different sites in Palestine, where IC₅₀ values ranged from 10.23 ±0.11 to 158.48 ± 0.87 µg mL^-1 [78]. Additionally, Eid et al. [79] reported a strong antioxidant activity for Salvia rosmarinus EO, with IC₅₀ value of 22.38 ±0.7 μg mL^-1. The results obtained by Dammak et al. [31] showed that Salvia rosmarinus EO exhibited better antioxidant activity (IC₅₀= 11.12 µg mL^-1) than Lavandula dentata (IC₅₀= 14.03 µg mL^-1) using DPPH test which are consistent with our findings. El Abdali et al. [29] found that Lavandula dentata EO was able to scavenge the free radical DPPH with IC₅₀ value of 12.95 µg mL^-1. Dridi et al. [59] assessed the antioxidant activity Lavandula dentata using three methods, with IC₅₀ values of 113.29, 53.029 and 43.20 µg mL^-1, respectively for DPPH, ABTS and reducing power test. Several studies reported moderate antioxidant activities for Eucalyptus sp. EOs, with some differences depending on the species [80,81,82]. Limam et al. [83] found that EOs extracted from thirteen Tunisian species of the Eucalyptus genus (globulus, maidenii, astringens, camaldulensis, lehmannii, melliodora, erythrocorys, gomphocornuta, gomphocephala, oxidantes, microcarpa, paniculata, angulosa) exhibited a moderate antioxidant activity at concentration of 10 µg mL^-1, with inhibition percentages of free radical DPPH ranging from 10.75 ±1.05 to 52.69 ±4.59%. Sadraoui Ajmi et al. [84] revealed that Eucalyptus cinerea EO collected from Tunisia, exhibited a similar DPPH radical scavenging activity with an IC₅₀ value of 161.59 µg mL^-1 compared to our results (IC₅₀ = 200.5 ±4.1 µg mL^-1). Kouki et al. [81] concluded that EOs from three Tunisian Eucalyptus species (E. oleosa, E. pimpiniana, E. polyanthemos) exhibited moderate antioxidant activities. Significant differences between the EOs were observed in the DPPH assay. E. oleosa was the most active, with an IC₅₀ value of 92.179 µg mL^-1, followed by E. pimpiniana (IC₅₀= 163.593 µg mL^-1) and E. polyanthemos (IC₅₀=359.688 mg mL^-1). EOs from four species of Eucalyptus plantation in northern Thailand (E. camaldulensis, E. citriodora, E. deglupta and E. urophylla) were compared for their antioxidant activities using the DPPH method [73]. The oils exhibited antioxidant activity which varied significatively within plant species. E. citriodora oil rich in citronellal (68.43%) produced the best result (IC₅₀= 2.37 µg mL^-1) followed by E. deglupta (IC₅₀= 10.1 µg mL^-1), E. urophylla (IC₅₀= 19.95 µg mL^-1) and E. camaldulensis oil (IC₅₀ = 25.47 µg mL^-1) with 1,8-cineole as major component.

Several reports have documented the antifungal effect of different species of Thymus against various microorganisms with thymol being mainly responsible for its antifungal activity [85,86,87]. In the present study Thymus vulgaris EO has shown a fungicidal effect for all the tested strains with MIC of 2 µL mL^-1 and MFC varying between 2 and 12 µL mL^-1. While most strains were completely inhibited, Phoma sp. showed fungistatic response, suggesting species-specific sensitivity. In conformity with our findings, the antifungal potential of different thyme species (Thymus convolutes, Thymus pectinatus and Thymus vulgaris) has been demonstrated against plant pathogens including F. oxysporum f. sp radicis-Iycopersici, Phytophthora infestans, and R. solani [88]. The EOs of Thymus vulgaris and Thymus pectinatus completely inhibited the growth of all the fungal strains at a dose of 4 μL per Petri dish. Conversely, other thymus species, such as Thymus convolutes EO showed lower antifungal efficacy, with only partial inhibition of F. oxysporum f. sp radicis-lycopersici and no effect on R. solani and Phytophthora infestans suggesting that antifungal efficacy is influenced by the chemical profile of each thymus species. The study performed by Fonseca-Guerra et al. [89] demonstrated that Thymus vulgaris EO completely inhibited the in vitro growth of F. culmorum, F. equiseti, F. graminearum and F. oxysporum isolates originating from Chenopodium quinoa crops at higher concentrations (MIC value of 10 µL mL^-1) than those required in our study,underscoring the variability in fungal sensitivity depending on isolate origin and experimental conditions. While Bounar et al. [90] demonstrated strong inhibition of four Fusarium species (F. culmorum, F. equiseti, F. avenaceum, and F. moniliforme) causing rot in potato tubers at very low concentrations (0.156-0.313 µL mL^-1), Divband et al. [91] reported higher MIC and MFC values (up to 30 mg mL^-1) against 20 wild-type strains of F. oxysporum isolates. Moghaddam and Mehdizadeh [92] reported higher doses of Thymus vulgaris EO (800 μL L^-1) to affect F. oxysporum and Drechslera spicifera, and even greater concentrations (1600 μL L^-1) were required to inhibit Macrophomina phaseolina. A comparative antifungal study showed that, among several EOs tested, only Origanum vulgare, Syzygium aromaticum and Thymus vulgaris, achieved total inhibitory effect at 500 μg mL^-1 concentration. The in vivo efficacy of Thymus vulgaris EO (MIC of 500 ppm) was demonstrated by Pilozo et al. [76] reporting a significant reduction in postharvest losses caused by Lasiodiplodia theobromae with analogous performance to those of synthetic fungicide, increasing the shelf life of bananas and their commercial value.

Many plants, particularly those belonging to the Lamiaceae family are known for their antimicrobial activity, especially their EOs [62]. In our study, Salvia rosmarinus totally inhibited the growth of all fungal strains at concentrations ranging from 4 to 8 µL mL^-1. It was fungicidal for both R. solani and S. sclerotiorum at 8 and 10 µL mL^-1 respectively. These results are consistent with those of (ElYacoubi et al., 2024) [94], who showed that Salvia rosmarinus EO achieved complete inhibition of a broad-spectrum of phytopathogenic fungi (including F. culmorum, F. oxysporum, F. poae, and Helminthosporium sativum) when applied at the highest tested concentration (1/100 v/v). Ben Kaab et al. [95] found that Salvia rosmarinus EO significantly inhibited the spore germination of F. culmorum (85.99%), F. oxysporum (100%) and Penicillium italicum (95.40%) at slightly higher concentration (6 µL mL^-1) than that used in our study. Similarly, Hussein et al. [96] confirmed that Salvia rosmarinus EO was active against six major ginseng pathogens: Alternaria panax, Botrytis cinerea, Cylindrocarpon destructans, F. oxysporum, S. nivalis and S. sclerotiorum, with MIC ranging from 0.1 to 0.5 % (v/v).

The EOs of the seven studied Eucalyptus species resulted in antifungal activity for all six phytopathogenic fungal strains in a dose-dependent manner. The results of the present study are consistent with those of Ayed et al. [51] who evaluated eight Eucalyptus sp. (E. angulosa, E. cladocalyx, E. diversicolor, E. microcoryx, E. ovata, E. resinifera, E. saligna and E. sargentii) against four Fusarium strains (F. culmorum, F. oxysporum subsp. solani, F. oxysporum f. sp. matthioli and F. redolens) [51]. F. oxysporum and F. redolens exhibited higher sensitivity to most of the EOs tested with complete mycelium growth inhibition at doses between 1 and 6 µL mL^-1. E. cladocalyx was the most effective EO when compared to the others, totally inhibiting mycelial growth of all fungal species studied with MIC ranging from 1 to 3 µL mL^-1. Amri et al. [52] obtained similar results with Tunisian Eucalyptus species (E. citriodora, E. falcata and E. sideroxylon) at a dose of 4 µL mL^-1, particularly against Fusarium species, which appeared more susceptible than Bipolaris sorokiniana. E. citriodora EOs demonstrated the most potent antifungal activity, completely inhibiting all strains except B. sorokiniana while the other two oils produced partial inhibition. Kouki et al. [81] revealed that E. oleosa EO showed the strongest inhibitory activity among three Tunisian Eucalyptus species (E. oleosa, E. pimpiniana and E. polyanthemos), fully inhibiting fungal growth at 6 µL mL^-1. In another study supporting these results, E. camaldulensis EO confirmed its fungicidal property against Fusarium species that affect maize with MICs and MFCs in the range of 7 to 10 µL mL^-1 [71]. Caetano et al. [66] tested Eucalyptus EOs (E. citriodora, E. camaldulensis, E. grandis and E. microcorys) in vivo against the agent of coffee leaf rust (Hemileia vastatrix), reporting good antifungal activity for most species, except E. microcorys. Similarly, Eucalyptus staigeriana, Eucalyptus globulus and Cinnamomum camphora EOs were effective for both in vitro and in vivo assays against Alternaria solani causing early blight disease in tomato [97]. Umereweneza et al. [98] found that E. melliodora EO was the most effective among the tested species, inhibiting the growth of food spoilage fungi and aflatoxin-producing Aspergillus species with MICs varying from 3.3 to 8.1 mg mL^-1 and completele inhibition of aflatoxins production at 6 and 7 µL mL^-1.

4. Materials and Methods

4.1. Plant Material

Plant samples were randomly collected during summer 2023 from different Tunisian regions. Aerial parts of Lavandula dentata, Salvia rosmarinus and Thymus vulgaris (Lamiaceae) and leaves from seven Eucalyptus species (Myrtaceae): Eucalyptus camaldulensis, Eucalyptus cinerea, Eucalyptus grandis, Eucalyptus lehmannii, Eucalyptus leucoxylon, Eucalyptus saligna and Eucalyptus sideroxylon were harvested, then were stored in a glass greenhouse for drying for 5 days (Table 4).

4.2. Essential Oil Extraction

The EOs were obtained by hydro-distillation of 100g of plant material for 3 hours using a Clevenger type apparatus (SAF Wärmetechnik LabHEAT^® KM-ME, 1000 mL, SAF GmbH, Hamm, Germany). The extracted oils were dried over anhydrous sodium sulfate and stored in amber glass vials at 4°C until use.

4.3. GC-FID and GC-MS Analysis

Chemical composition of EOs was determined using Gas Chromatography coupled with Flame Ionization Detection (GC-FID) and Mass Spectrometry (GC-MS).

GC-FID analyses were assessed on an HP6890 (II) gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID), by using an apolar HP-5 capillary column [30 m × 0.32 mm (i.d), 0.25 µm film thickness] (Agilent Technologies). Oil diluted in hexane was injected with a split ratio of 1:50 and a flow percentage of 1.2 mL min^-1. The oven temperature program was: 40°C for 1 min; 40-260°C at a rate of 5°C min^-1; 260°C isothermally for 4 min. The temperature of the injector and the detector were maintained at 250 and 300°C, respectively.

GC–MS analyses were performed on HP 6890 N gas chromatograph coupled to an HP 5975 mass spectrometer (Agilent Technologies). The separation of volatile compounds was assessed using an HP-5MS capillary column (60 m×0.25 mm; 0.25 mm) (Agilent Technologies). The temperature of the oven ramped from 40 to 280°C at a rhythm of 5°C min^-1. Helium was used as a carrier gas at a flow speed of 1.2 mL min^-1. Scan mass range was 50-550 m/z at a sampling speed of 1 scan s^-1.

A standard dilution of a C₆–C₂₅ n-alkanes series was prepared to calculate retention indices (Ri). The EO compounds were identified by comparison of their relative Ri and mass spectra with those from corresponding data (Wiley 275 L library) and/or reported in the literature [99]. The percentage of each compound was obtained from the electronic integration of its relative FID peak area without including a correction factor.

4.4. Antioxidant Activity Assays

The method of Hamdeni et al. [100] was used for the evaluation of the free radical scavenging activity (RSA) of EOs. Briefly, 50 µL of various dilutions of EOs were mixed with 200 µL of 0.1 mM methanolic 2,2-Diphenyl-1-picrylhydrazyl (DPPH) solution (Sigma-Aldrich, St. Louis, MO, USA). The mixture was then shaken vigorously and allowed to stand at room temperature for 30 min in the dark. The decrease in absorbance at 517 nm was measured spectrophotometrically vs. DPPH standard solutions. The RSA of the oil expressed as % inhibition of DPPH was calculated using the formula: % inhibition = [(A₀ - A_s)/A₀] × 100, where A₀ and A_s are the absorbance values of the control and that of the sample, respectively. The concentration (µg mL^-1) which allowed to 50% inhibition (IC₅₀) was calculated from the graph of RSA percentage against oil concentration. All results were reported as means ± standard deviation of three measurements.

The antioxidant activity was estimated by using the 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) (Sigma-Aldrich) based on the reduction of ABTS^•+ radicals by antioxidants present in the EOs. ABTS radical cation (ABTS^•+) was produced by mixing a 7 mM ABTS solution with 2.45 mM K₂S₂O₈ in a ratio 1:1. The mixture was allowed to stand in the dark at room temperature for 12h before use. For the assay, the ABTS^•+ solution was diluted in methanol to an absorbance of 0.7 (±0.02) at 735 nm. Two hundred µL of ABTS^•+ solution were mixed with 25 μL of the corresponding dilution of EOs. The absorbance reading was taken 30 min after initial mixing [101]. All the analyses were performed by triplicate and results were expressed as inhibition percentage of the radical cation ABTS⁺ using the following formula: % inhibition ABTS = [(A₀ - A_s)/A₀] × 100, where A₀ and A_s are the absorbance values of the control and that of the sample, respectively.

4.5. Antifungal Activity

The antifungal effects of EOs were tested in vitro by the direct contact method on agar. The phytopathogenic fungal strains F. culmorum, F. oxysporum, F. proliferatum, Phoma sp., R. solani and S. sclerotiorum were provided by the Plant Protection Laboratory of the National Agriculture Research Institute of Tunisia (INRAT, Tunisia).

EOs were solubilized in a Tween 20 solution (0.1%, v/v) (Sigma-Aldrich) and later they were incorporated at increasing concentrations in Potato Dextrose Agar (PDA) medium (0, 2, 4, 6, 8, 10 and 12 µL mL^-1). Six mm agar plugs of each fungal strain were deposited in the center of PDA plates [81]. Negative and positive controls were carried out and each test was repeated 3 times. Incubation was performed at 24°C for 5 days. The inhibition percentage of fungal growth was determined by the following formula: Inhibition of fungal growth (I %) = [(D – Di)/ D × 100] where D and Di are the diameters of mycelial growth in control and treatment.

Minimum inhibitory concentration (MIC) is defined as the lowest dose at which there is complete inhibition of fungal growth. To establish the minimum fungicidal concentrations (MFC), the inhibited fungal disks were inoculated into PDA plates without EO and their growth was observed. After 3 days of incubation, MFC was obtained as the lowest MIC at which no growth observed in the plates after culturing [51].

4.6. Data Analysis

All of the experiments were carried out in three replicates, with the results represented as mean ± standard deviation. Statistical analyses were performed with STATISTICA software 10. The one-way analysis of variance (ANOVA) was performed to identify the effect of each treatment. The Fisher’s least significant difference test at the 5% threshold was used to compare means.

5. Conclusions

The EOs extracted from various plant species, including Lavandula dentata, Salvia rosmarinus, Thymus vulgaris and seven Eucalyptus species, have shown significant promise as natural alternatives to synthetic fungicides. The chemical analysis of these EOs highlighted their rich composition of oxygenated monoterpenes and monoterpene hydrocarbons, which are key contributors to their antifungal and antioxidant properties. Among the Lamiaceae species, Thymus vulgaris EO stood out for its potent antifungal activity, exhibiting fungicidal effects against all tested fungal strains. Furthermore, Thymus vulgaris EO demonstrated the highest antioxidant potential, outperforming the common antioxidant standard, Trolox. The Eucalyptus species also presented varying levels of antifungal activity, with notable potential for controlling important phytopathogenic strains affecting crops. These findings support the hypothesis that EOs from both Lamiaceae and Myrtaceae families can serve as eco-friendly alternatives to synthetic chemicals against a broad spectrum of phytopathogenic fungi. However, differences in MIC and MFC values reported across studies highlights the influence of EO chemical composition, fungal species, inoculum densities and methodological variabilities. These disparities emphasize the need for further standardization of in vitro assays and chemical characterization of bioactive compounds to optimize their use in sustainable crop protection strategies. Additionally, further research is necessary to evaluate the field efficacy, stability, and economic feasibility of these EOs in real-world agricultural settings. Exploring their combination with other biological control agents and studying their potential for synergistic effects could also provide valuable insights for developing more robust, environmentally friendly pest management systems. Overall, this study contributes to the growing body of evidence supporting the role of EOs in multifunctional landscapes and their potential as effective tools in the fight against phytopathogenic fungi.