A New Lavender (Lavandula multifida L.) Ecotype from Arid Tunisia, with Differential Essential Oil Composition and Higher Antimicrobial Potential

The lavender Lavandula multifida L., a medicinal plant grown in arid regions of Tunisia, was recently considered an endangered species; thus, its habitats regressed to some difficult zones in terms of access, such as the watershed of Oued Agareb in central-eastern Tunisia. This species was recorded only in deep and narrow shady Wadi of the watershed and benefited from protection against overgrazing, erosion and sunlight. L. multifida was rarely observed in an open area, such as a plateau or large-bed valley. The plant’s metabolism is linked to its response to environmental conditions, which is of particular interest to understanding the components of the considered population of L. multifida. Consequently, biochemical and antimicrobial analyses have been evaluated. Using gas chromatography-mass spectrometry (GC-MS) analysis reveals that among the 58 compounds identified in L. multifida essential oil extracted from aboveground plant tissues, camphor was the major component (15.68%), followed by 1,8-cineole (14.14%) and alpha-pinene (13.82%). Moreover, it has been observed that Escherichia coli was more susceptible than Staphylococcus aureus to the antimicrobial properties of L. multifida essential oil, while in the case of camphor, S. aureus was more susceptible than E. coli. The protected population of L. multifida exhibits a distinctive vegetative development and growth cycle, resulting in specific secondary metabolites and distinguished antimicrobial activity.


Introduction
The family of Lamiaceae has been recognized for a long time as a source of many medicinal and aromatic species. The essential oil extracted from Lamiaceae species is proved by many studies to be of a high quality, and it was used by multiple industries, such as pharmaceutic and cosmetic, but especially in traditional medicine in many rural communities [1][2][3]. The commerce of plants and essential oils has increased considerably in both developed and developing countries [4]. In Lamiaceae species, the majority of the utilized material is derived from wild populations that have frequently been developed in disturbed environments [5][6][7]. Classical selection of medicinal plants (traditional selection) and biotechnological approaches for plant improvement (using biotechnological methods, such as tissue and organ culture, genetic transformation) were restricted to some renowned taxa (e.g., Mentha spp., Lavandula spp., Salvia spp., Ocimum spp., Origanum spp.). However, a severe decline and enormous loss, linked to both natural populations and many species belonging to the Lamiaceae family, have been reported during the last decade. portance in traditional medicine and the pharmaceutic and cosmetic bio-industry, similar to most of the Lamiaceae species, L. multifida was over-exploited, leading to its becoming endangered [32,33]. Due to ongoing anthropogenic stresses, most L. multifida populations were fragmented and represented by scattered individuals in some protected locations [5]. Habitat fragmentation enhances population divergence and contributes to genetic diversity loss [30], while the maintenance of genetic variety is critical for population evolution and adaptability to environmental change [34]. As a result, understanding the current status of these endangered species (distribution area, regeneration capability, floral biology, genetic diversity) is critical to ensure their sensible usage.
The investigation of morphological heterogeneity, ecological adversity and population structure offers critical information for developing restorative initiatives and long-term conservation and improvement of the species [35,36]. The synthesis and accumulation of primary and secondary metabolites varies intensely amongst specimens of the same plant species cultivated under various environmental conditions and habitats [37][38][39]. Secondary metabolites that are stimulated by environmental factors (for example, water and salt stress in arid and semi-arid regions) act as a chemical interaction between the plant and its surroundings [38]. Plants and their environments interact largely through secondary metabolite production, which performs biological tasks, such as a flexible adaptive response to their habitats [40,41]. Therefore, the investigation of such discrepancies is extremely valuable for the chemical characterisation of individual plants of the same species collected from different habitats, especially in considering the different geographical origins of plant material [42,43]. These processes (genetic differences within a plant species) include either long-term acclimation or local adaptation, as well as seasonal variations in phenology, environmental fluctuations linked to biotic and abiotic factors and geographical disparities affecting diverse populations [44].
The aim of this research was the study of the morphological traits and ecological response of Lavandula multifida L. grown in the watershed of Agareb, one of the restricted areas of this species in mid-eastern Tunisia. Furthermore, secondary metabolites were analysed using GC-MS. Moreover, antibacterial properties were evaluated against two resistant strain bacteria, i.e., Escherichia coli and Staphylococcus aureus.

The Study Site
This study was carried out along 8 zones covering 29 stations in the Agareb region, which is considered a representative portion of the washout of Agareb ( Figure 1). The Agareb region belongs to the Sahel of Sfax, located between latitudes 34 • 40 -34 • 50 and longitudes 10 • 25 -10 • 45 . It is bounded from west to east by the plateaux of Jouaouda, Araba, El Gouna and Traka and the plains of Sfax, Ouled Ameur and Soualah.
The watershed of Oued Agareb, located on the southern coast of Sfax and subjected to an irregular climate, is characterized by shallow topography, a soft structure and a dense river network. These factors make the watershed fragile, thus triggering various forms of soil degradation. Washouts and deep wadi are the fundamental erosion structures that characterise this region. As narrow and deep structures, washouts can hold many sensitive species, such as L. multifida, that cannot resist the climatic severity outside the river area.
The climate of the study zone is characterised by an annual mean temperature ranging from 2 to 24 • C, with mean annual precipitation of 196 mm. During the study period, the mean air temperature of the cold season was 12.1 ± 0.5 • C and that of the hot season 26.4 ± 1.4 • C; the rainfall varied from 7 mm during the dry period to 75 mm in autumn and 65 mm in winter. The climate traits are characteristic of a Mediterranean Lower Arid climate with temperate winter. The soil is an alkaline sandy loam, with gypsum outcrops and crumbly caliches at a depth of 10-25 cm. The topography is characterised by hills traversed by many valleys and gully erosion. The flora is composed of a remaining degraded steppe of Alfa grass Stipa tenacissima L., hosting a sparse perennial vegetation mainly composed and Retama raetam (Forssk.) Webb.
Washouts of Agareb have special geomorphology characterized by a network of many valleys. The studied zone is located in the Sahel Sfax, the eastern Tunisian platform. The Agareb region has a monotonous topography showing mounds with a curvature of a large radius, separated by wide valleys, plateaus and plains, generally subsiding and occupied by sebkhas (i.e., smooth flat plains sometimes occupied after a rain by a shallow lake). The altitudes rarely exceed 200 m. In this area, the highest altitudes reach 141 m at Hamadet El Houch and 183 m at Ksar El Merdjine. The eight study areas are represented by wadis and their edges, as well as the plateaus located in proximity. The depth and width of the wadi beds are the fundamental differences between the different sites.

Plant Material and Morphological Traits
Lavandula multifida L. that belongs to the Lamiaceae family-known by the vernacular name fern leaf lavender and Halhal, as well as wild Khezama, a local name-is an herbaceous perennial, highly aromatic, ever-blooming plant native to Southern Italy, Spain and Northern Africa (Morocco, Algeria, Tunisia and Libya) and frequently connected with open, disturbed regions and settlement.
The specimens were collected from eight sites along the studied washouts during the period 2019-2020. Dr. Khalil Mseddi (Botanic section) from the department of Biology, Faculty of Science, University of Sfax, has identified the plant at the species level. A voucher specimen (AGT 001) was deposited in the herbarium of the Department of Biology, University of Sfax, Tunisia.
The geographic coordinates were estimated by GPS. Ten morphological characteristics, i.e., root length (cm), number of root ramifications, root diameter (cm), stem length Washouts of Agareb have special geomorphology characterized by a network of many valleys. The studied zone is located in the Sahel Sfax, the eastern Tunisian platform. The Agareb region has a monotonous topography showing mounds with a curvature of a large radius, separated by wide valleys, plateaus and plains, generally subsiding and occupied by sebkhas (i.e., smooth flat plains sometimes occupied after a rain by a shallow lake). The altitudes rarely exceed 200 m. In this area, the highest altitudes reach 141 m at Hamadet El Houch and 183 m at Ksar El Merdjine. The eight study areas are represented by wadis and their edges, as well as the plateaus located in proximity. The depth and width of the wadi beds are the fundamental differences between the different sites.

Plant Material and Morphological Traits
Lavandula multifida L. that belongs to the Lamiaceae family-known by the vernacular name fern leaf lavender and Halhal, as well as wild Khezama, a local name-is an herbaceous perennial, highly aromatic, ever-blooming plant native to Southern Italy, Spain and Northern Africa (Morocco, Algeria, Tunisia and Libya) and frequently connected with open, disturbed regions and settlement.
The specimens were collected from eight sites along the studied washouts during the period 2019-2020. Dr. Khalil Mseddi (Botanic section) from the department of Biology, Faculty of Science, University of Sfax, has identified the plant at the species level. A voucher specimen (AGT 001) was deposited in the herbarium of the Department of Biology, University of Sfax, Tunisia.
The geographic coordinates were estimated by GPS. Ten morphological characteristics, i.e., root length (cm), number of root ramifications, root diameter (cm), stem length (cm), number of stem ramifications, leaf number per stem, spike length (cm), number of flowers per spike and flower length (cm), were measured in the collected samples.

Extraction of Essential Oil
Essential oils, also called volatile odoriferous oils, are aromatic oily liquids extracted from different parts of plants, such as the leaves, peels, barks, flowers, buds and seeds. They can be extracted from plant materials by several methods, including steam distillation, Life 2023, 13, 103 5 of 16 a method that has been widely used, especially for commercial-scale production. The composition of each essential oil depends not only on the family, but also on the part of the plant from which it is extracted, and sometimes on the soil where the plant grows, or even on the time of the harvest. Therefore, gas chromatography is necessary to characterize an essential oil.
Extraction of essential oil from the collected plant material was performed, as previously described by Baj et al. [45]. Briefly, 50 g of dried aerial parts of L. multifida collected from deep wadi in semi-arid Tunisia was placed into a round flask, and 400 mL of distilled water was added. The Hydro distillation was performed by a Clevenger apparatus (Glassco) for 3 h, according to the above-mentioned method. Extracted essential oils were collected in vials and stored at 4 • C until GC-MS analysis.

Chemical Analysis
The analysis was carried out using a gas chromatograph-mass spectrometer, GC-MS QP 2018, Shimadzu, equipped with a split-splitless injector and a ZB-5MS capillary column (30 m × 0.25 mm; 0.25 µm film).

Chromatographic Conditions
The column's initial temperature was 45 • C, which was held for 2 min before ramping up to 280 • C at a rate of 5 • C per minute and then remaining steady for 5 min. The injector's temperature was 265 • C. Helium (the carrier gas) flowed at a rate of 1 mL/min. The following conditions were applied for acquiring mass spectra: (instrument current) ionization voltage, 70 eV; ion source, 200 • C; filament emission current, 60 mA. Automatic essential oil injections were performed at a 1:15 split ratio, and the injection of the essential oil was at (1 µL,). The range of the mass spectrometric detector, which was in scan mode, was 35 to 550 m/z. By comparing mass spectra and retention indices in a high-quality Shimadzu mass spectral library, essential oils were identified.

Tested Microorganisms
Lavandula multifida L. is a member of the genus of Lavender that belongs to the mint family (Lamiaceae). These aromatic plants produce valuable and essential oils (EOs) in the manufacture of medicines, perfumes, cosmetics and food, as it has antioxidant properties and biological properties against the growth of many microorganisms, especially Candida albicans, Staphylococcus aureus and Escherichia coli. It is also widely used in folk medicine as an antispasm.
More than 95% of urinary tract infections are caused by E. coli, which is the most pathogenic organism. However, many other bacteria can also cause pathogenicity, such as Staphylococcus, Pseudomonas, Proteus, Klebsiella and Neisseria. Antibiotic resistance among E. coli has been recorded internationally, and growing resistance rates among E. coli are of increasing concern in both developed and developing countries.
The various components of plant extracts play an important role as antibacterial agents, as it has been suggested that vegetable oils act through the lipophilic part that interacts with the fatty parts of cell membranes. In recent years, multi-antibiotic resistance has arisen due to the indiscriminate use of antibiotics; hence, this problem requires efforts to find effective treatments by searching for effective materials against the microorganisms that cause these diseases. In addition, synthetic antibiotics are powerful and life-saving medicines. Yet, they do more harm than good when not used correctly; thus, there is also a need to develop alternative antimicrobial medicines to treat infectious diseases from other sources. The antibacterial properties of the essential oils of Lavandula multifida were evaluated against these two resistant bacteria strains, i.e., Escherichia coli and Staphylococcus aureus.

Antimicrobial Activity
The antimicrobial activity of the L. multifida essential oil was tested against two strains of pathogenic bacteria: Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6583. The two microorganisms were derived from the culture collection of the QC department of the State company for the drugs industry and medical Appliance/Samarra-Iraq. The agar well diffusion method was employed for the determination of the antimicrobial activities of the tested diluted essential oil, as described by Oumzil et al. [46] with some modifications. Briefly, the test was performed in sterile Petri dishes containing Muller Hinton agar. A total of 6 wells (6 mm in diameter) were impregnated with 100 µL of oil and were placed on the Petri plates previously inoculated with a microbial suspension. The suspension of bacteria was obtained from 18 h cultures (one microorganism per Petri plate). All Petri plates were incubated at 37 • C for 24 h. The diameters of inhibition zones were measured in millimeters.
For determining the minimum inhibitory concentration (MIC) values of the essential oil and camphor material against the two bacterial strains, we tested 5 serial concentrations, ranging from 5 to 2.5, 1.25, 0.625 and 0.312 µg/mL. This method was done according to the CLSI protocol (CLSI, 2017), with some modifications as previously described by Sakkas et al. [47]. The dilutions of oil were prepared in Mueller-Hinton broth (MHB, Oxoid Ltd., Basingstoke, UK). Tween 20 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a solubilizer at a concentration of 0.5% (v/v). Bacterial inoculum measured at turbidity of 0.5 McFarland (1-1.5 × 108 CFU/mL) was transferred into MHB to obtain a bacterial count of 5 × 105 CFU/mL. After incubation for 24 h at 37 • C, an appropriate amount of 10 µL was added to MHB and incubated for 24 h at 37 • C. The visible method was used for the determination of MIC value. The MIC is defined as the lowest concentration of an antimicrobial agent (EO) that prevents the visible growth of a microorganism in a broth dilution susceptibility test.

Ecological Context and Distribution
Due to rangeland overgrazing and climate severity, L. multifida has become a rare species in the watershed of Oued Agareb. This work shows that the studied population exhibited shade preference strictly related to deep and narrow Wadi, which were the result of channel erosion along gully beds, whereas this species was absent in the open area of the plateau and hill zones (Table 1). This might be attributed to overgrazing and strong winter winds.
However, it was seen as a random individual species only in the protected park of Algonna (created near the study zone), associated with some phanerophytes species, such as Retama reatam that can create a protective micro-environment and habitat and minimize sunlight effect. Hydraulic networks crossed by washouts were the fundamental erosion structures that can be seen in this region, which can support the growth of many sensitive species, such as Lavandula multifida, that cannot resist or tolerate the climatic severity outside the valley.

Communities Associated with L. multifida
The growth of L. multifida can be associated with that of numerous neighbor species (Table 2). However, it was observed that the species Retama raetam, Lycium shawii, Thymelaea hirsuta, Cenchrus ciliaris, Rhus tripartita and Aspargus albus were the nearest. These species can offer protection against both overgrazing and climate severity via their upright stem development and/or spiny leaves and stems.
Some other species can be associated in a distance of one to two meters around L. multifida. These species do not have any direct contact among aerial shoots, but they may communicate with their underground parts (rhizomes and roots). The most common species were Trigonella stellata, Stipa capensis, Rhanterium suaveolens, Lygeum spartum, Artemesia herba-alba, Gymnocarpos decander, Asparagus albus and Hyparrhenia hirta. Species such as Hammada scoparia, Artemisia campestris, Anabasis oropediorum, Ajuga iva, Peganum harmala, Periploca laevigata, Teucrium polium, Allium roseum, Fagonia cretica, Asparagus stipularis and Atractylis serratuloides were also seen in the same habitats as L. multifida.

Phenology and Life Cycle
According to the Raunkiaer classification [48], Lavandula multifida is a Chamaephyte (dwarf shrub) plant, exhibiting stems growing near the ground, and it bears dormant, hibernating buds that grow close to the ground, approximately 25 cm above the soil surface. The advantage of being close to the soil surface is that the buds can be protected from many negative environmental impacts. As a result, the chamaephyte habitat is more widespread in stressed niches, with prolonged periods of drought and severe herbivore grazing pressures.
Current varieties of L. multifida have an autumnal-winter life cycle, which is different than the European variety (spring-summer cycle) [49]. Indeed, this species grows rapidly and with high density just after the first autumnal rain (September-October), reaching maximum development in winter ( Figure 2). L. multifida blossoms between late autumn and winter (i.e., from September to January). The development of fruits and seed dispersal are both completed in the beginning of spring. During the dry summer with elevated temperatures, the plants of L. multifida lose the majority of their leaves and become dry, woody shrubs bearing hidden buds during the unfavorable season.

Morphological Traits
The mean values of the considered parameters were calculated on collected samples from the study zones, and the results are presented in Table 3. The population of L. multifida in the Agareb region typically grows up to 58.7 cm height on straight stems with deeply lobed, lacy, silver-green leaves (Figure 3). It has been published that L. multifida is a perennial species (Missouri Botanical Garden, L. multifida); however, it is actually a subshrub because it develops woody stems over time in areas where it is winter resistant. Its roots can reach 41 cm in length and cross with other roots of neighbor plants, such as Cenchrus ciliaris and Retama reatam.

Morphological Traits
The mean values of the considered parameters were calculated on collected samples from the study zones, and the results are presented in Table 3. The population of L. multifida in the Agareb region typically grows up to 58.7 cm height on straight stems with deeply lobed, lacy, silver-green leaves (Figure 3). It has been published that L. multifida is a perennial species (Missouri Botanical Garden, L. multifida); however, it is actually a subshrub because it develops woody stems over time in areas where it is winter resistant. Its roots can reach 41 cm in length and cross with other roots of neighbor plants, such as Cenchrus ciliaris and Retama reatam.

Minimum Inhibitory Concentration (MIC)
The results of the minimum inhibitory concentration of the L. multifida oil and synthetic camphor are shown in Table 5. A difference in the minimum inhibitory concentration between the extracted lavender oil and camphor was detected, but there was a similarity in their inhibitory concentrations on each one of the two bacteria.

Microbiological Activity
Measurement of the antibacterial activity of L. multifida essential oil against E. coli and S. aureus was made in comparison with the antibacterial activity of camphor as a major compound, in terms of concentration in GC analysis. According to the results ( Table 6; Figure 4)

Microbiological Activity
Measurement of the antibacterial activity of L. multifida essential oil against E. coli and S. aureus was made in comparison with the antibacterial activity of camphor as a major compound, in terms of concentration in GC analysis. According to the results (Table 6;

Discussion
Various studies have reported the genetic diversity [19,20,50] and the biochemical compositions [26,51] of different populations of Lavandula multifida. However, no previous works linking the morphological and phenological traits to the secondary metabolism and the essential oil composition have been reported. The current study shows that the mor-

Discussion
Various studies have reported the genetic diversity [19,20,50] and the biochemical compositions [26,51] of different populations of Lavandula multifida. However, no previous works linking the morphological and phenological traits to the secondary metabolism and the essential oil composition have been reported. The current study shows that the morphological traits and habitat proprieties strongly affect the chemical composition of L. multifida essential oil and its antimicrobial activities.
In the current study, we found that the roots of L. multifida are 40 cm in length and 1 cm in diameter, a structure that may serve as either a drought tolerance or avoidance mechanism. The parameters of the shoots were quite different than those reported by Upson and Jury (2002) [52] for varieties of this species grown in Morocco. In addition, genetic analysis reported the high diversity in L. multifida populations from Tunisia [19,50].
The studied population starts its life cycle at the beginning of autumn with the first rain and rises to a maximum development in the end of the autumn and mid-winter. The blooming period is extended during winter, and fruiting is completed during spring. This life cycle is characteristic of the south Mediterranean population, i.e., North Africa regions [52]. However, in North Mediterranean regions (Spain and Italy), the flowering period begins in spring and is completed in summer [53].
Due to anthropogenic effects on the natural environment, L. multifida populations appear to be diminished and dispersed in North Africa (Morocco, Algeria, Tunisia, Egypt), Southern Spain and Portugal, Sicily, and Southern Italy [54][55][56]. Numerous of the abovementioned studies have reported that biometric variables, such as morphological and reproductive parameters, display very different values, testifying to the high interspecific diversity [52]. Further, the secondary metabolism was related to environmental conditions [57].
The diversity of the composition of the volatile oil obtained from different accessions of L. multifida can be explicated by endogenous (plant varieties, vegetative state, organ tested) and exogenous factors, such as climatic features, soil characteristics and seasons [61].
The biological activity of L. multifida essential oil against gram-negative and grampositive bacteria may be due to the effect of its chemical components, especially linalool and comphor. Our results are consistent with previous investigations, which evaluated the MIC of linalool for L. augustifolia (52.59%), exhibiting biological activity against gram-negative and gram-positive bacteria at a concentration of 1.25 µg/mL [62]. However, Jianu et al. (2013) [63] published the effect of L. augustifolia essential oil with a high content of camphor Life 2023, 13, 103 13 of 16 against S. aureus and E. coli. In another study [64], it was shown that L. hybrida essential oil containing linalool (32.8%) has inhibitory activity against S. aureus, while the opposite holds true for E. coli. Moreover, it has been published that the essential oil of Lavandula stoechas possesses inhibitory activity against both E. coli and S. aureus at MIC limits, which range between 0.16 and11.9 µg/mL [65].
Based on our findings, the essential oil of L. multifida was found to be more active against the used bacterial strains than camphor. As has been reported by Xu et al. (2005) [66], camphor's mode of action on the development of microorganisms involves disruption of the phospholipid bilayer structure, interaction with membrane enzymes and proteins, and acting as a proton exchanger. The higher antibacterial activity of L. multifida essential oil against S. aureus and E. coli in comparison with camphor could be explained by the synergistic action of the components of this essential oil extract, especially the linalool and camphor content.
The extracted lavender oil from aerial plant tissues of L. multifida was biologically effective on gram-negative and gram-positive bacteria because it contained multiple biologically active substances, including camphor, which, in turn, exhibited an inhibitory effect against bacteria. The spread of L. multifida within the biodiversity of naturally growing plants in the valley seems to play a key role in the diversity of the extracted compounds.