Chemistry and Bioactivities of Six Tunisian Eucalyptus Species

The complex taxonomy of Eucalyptus genus, the renewed interest in natural compounds able to combat microbial strains, the overuse of synthetic pesticides, the consequent request for alternative control methods were the reasons for this research. The essential oils (Eos) of Eucalyptus bosistoana, Eucalyptus melliodora, Eucalyptus odorata, Eucalyptus paniculata, Eucalyptus salmonopholia, and Eucalyptus transcontinentalis were analyzed by GC/MS and their potential phytotoxic activity was evaluated against the germination and radicle elongation of Sinapis arvensis, Raphanus sativus and Lolium multiflorum. The antibiofilm activity was assayed against both Gram-positive (Staphylococcus aureus and Listeria monocytogenes) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii) bacteria. Monoterpenoids were the most representative constituents in all EOs and eucalyptol was the dominant component except in E. melliodora EO, in which p-cymene was the most abundant. In phytotoxic assays, the EOs from E. odorata and E. paniculata were the most active against germination and radical elongation of the tested seeds. Finally, the Eucalyptus EOs proved their capacity to effectively inhibit the adhesion process of all five pathogen strains, with percentages often reaching and exceeding 90%. These Eucalytpus EOs could have possible employments in the food, health and agricultural fields.


Introduction
In 1788, the term eucalyptus, which means 'well covered', was coined by the French botanist, Charles Louis L'Héritier de Brutelle, referring to a genus with the operculate nature of the flower which lacks conspicuous petals and sepals [1].
Native to Australia, the genus Eucalyptus (Myrtaceae family) contains about 900 species and subspecies [2]. It has spread worldwide, particularly in Africa, because of its easy adaptability and fast growth [3] and it is now extensively cultivated in many countries for several applications such as cellulose, pulp, gum, essential oils, and honey production, as well as for construction and as an ornamental plant [4].
In order to improve the forest production, great efforts of reforestation based on Eucalyptus L'Hér species were implemented in Tunisia [3], where 117 species have been introduced. They were used as fire wood, for the production of mine wood, and in the fight against erosion [5].
An EO from E. melliodora plants growing in Rwanda was reported by Umereweneza and coworkers [1] with a composition similar to the EO studied in this paper: eucalyptol and α-pinene were the most abundant components together with aromadendrene, not found in the studied sample. Sadeghi et al. [28] found an EO from E. melliodora with a similar composition: the authors reported eucalyptol and α-pinene with percentages of 51.1 and 9.5%, respectively. On the other hand, Eid and coworkers [29] reported a different constitution of an E. melliodora EO, with p-cymene (30.04%) and spathulenol (25.09%) as the predominant components.
The composition of E. salmonopholia essential oil was not previously reported in literature. Elaissi et al. [5,30,31] reported chemical constitutions of an EO from E. odorata quite different from our data, with the ketone cryptone and p-cymene as the main components and a low amount of oxygenated monoterpenes.
However, the same authors [32] reported the composition of an E. paniculata EO with higher amounts (89.8%) of oxygenated components, first of all sesquiterpenes alcohols (τ-cadinol and 7-epi-α-eudesmol), sesquiterpenes oxides (caryophyllene oxide) and monoterpene oxides (eucalyptol). Dorsaf and coworkers [3] inferred the chemical characterization of a Tunisian EO from E. transcontinentalis; also in this case, the results displayed eucalyptol as the main component followed by α-pinene. Elaissi et al. [33] reported the composition of the EOs from several Eucalyptus species harvested in Tunisia, including E. transcontinentalis. This EO was characterized by a high amount of eucalyptol, followed by viridiflorol; in general, the results showed that the EO was rich in oxygenated sequiterpenes.

Phytotoxic Activity
The phytotoxic effects of the EOs, tested on germination and radical elongation of Raphanus sativus L. cv 'Saxa', Sinapis arvenisis L. and Lolium multiflorum Lam, were reported in Table 2. Table 2. Effects of different doses of the EOs on germination (number of germinated seeds) and radical elongation (cm) of Raphanus sativus, Sinapis arvensis and Lolium multiflorum.
The germination and radical elongation of radish were significantly inhibited by all doses of E. bosistoana, E. odorata, and E. paniculata EOs. Other EOs showed significant inhibitory effects on radish germination in the following order: E. transcontinentalis > E.
salmonopholia > E. melliodora; whereas for the significant inhibitory effect of radish radical elongation the order was E. transcontinentalis > E. melliodora > E. salmonopholia.
The EO from E. odorata completely inhibited the germination of S. arvensis, at all doses tested. E. paniculata was the second most active oil; in fact, at the lowest doses tested, the inhibition of germination was 82.8% and the inhibition of radical elongation was 79.3%. The other EOs showed inhibition of germination >50% only at the two highest doses used. The EO of E. salmonopholia was the less active on radical growth of S. arvensis, while the other three oils showed inhibition of radical elongation of more than 50%, at all doses tested.
Only E. odorata and E. paniculata EOs totally inhibited the germination of L. multiflorum, at the highest doses tested. In the same case, E. salmonopholia and E. melliodora EOs seemed the less active EOs, with an inhibition of germination of 20.7 and 24%, respectively, at the highest doses tested. All other EOs (except E. salmonopholia) inhibited in a significant way the radical elongation of the seeds at all doses tested.
Recently, our research group reported the phytotoxicity and eco-compatibility of essential oils from Eucalyptus gunnii Hook. f. and E. pulverulenta Sims 'Baby Blue' cultivated in Italy [4]. The oils were tested on weeds (L. multiflorum Lam. and Portulaca oleracea L.) and crops (Raphanus sativus L., Lactuca sativa L., Lepidium sativum L., Solanum lycopersicum L., Pisum sativum L., and Cucumis sativus L.): both EOs inhibited P. oleracea seed germination, but only E. pulverulenta EO inhibited L. multiflorum radical elongation. Concerning crop species, the investigated EOs showed phytotoxicity in R. sativus.
Specifically, no reports on the phytotoxic activity of these EOs are present in the literature. Several studies reported the potential herbicidal effect of the constituents of Eucalyptus EOs: Kaur et al. [34] studied the role of monoterpenes in Eucalyptus communities, highlighting the phytotoxicity/allelopathic activity of eucalypt EOs and of their monoterpenes constituents against germination and growth of many crops and weeds, as previously reported by many other authors [34][35][36][37][38].
Kordali et al. [39] investigated the inhibitory effects of monoterpenes on seed germination and seedling growth of different seeds and they concluded that, in general, oxygenated monoterpenes, especially alcohols, showed higher phytotoxic effects in comparison with monoterpene hydrocarbons, thus suggesting a potential use as bio-herbicides. In particular, De Martino et al. [40,41] reported that, between oxygenated monoterpenes, alcohols (borneol, citronellol, geraniol, α-terpineol) appeared as the most inhibitory compounds, followed by ketones (carvone, menthone, camphor) and aldehydes against germination of tested seeds. Alcohols and ketones were the most inhibitory mainly on radish radicle growth and monoterpene alcohol derivatives were more phytotoxic than their acetate derivatives [41]. The herbicidal effect could probably be related to the change in several biochemical and physiological processes, influencing the germination of seeds and/or the elongation of radicle hypocotyl [4]. However, as previously reported in the literature, Eucalyptus EOs can also cause damage to some crops, making it essential to assess their selectivity [4]. It is therefore critical to maximize the herbicidal activity of Eucalyptus against weeds but at the same time to minimize the negative impact on crop growth [2].
Zhou and coworkers [42] studied the phytotoxic and antimicrobial activities of some Eucalyptus essential oils, allowing to hypothesize a possible correlation between these effects. In fact, Khamassi et al. [21] suggested that the application of the essential oils on plant seeds during the germination process could generate oxidative stress, provoking a release of malondialdehyde from fatty acids of membrane phospholipids and so an alteration of membrane integrity, with relative leakage of electrolytes and loss of vital membrane functions. He et al. [43] suggested that when a microbial cell was treated with essential oils, the permeability of its membrane changed, producing a damage to a cell integrity and cell physiological functions. Table 3 shows the minimal inhibitory concentration of the Eucalyptus EOs necessary to inhibit the bacterial growth of the five pathogens used in our study. MIC results allowed to evaluate, subsequently, the capacity of the six Eucalyptus EOs to influence the bacterial biofilm and their ability to affect the metabolism of the sessile bacterial cells. Such results could provide more information about the performance of EOs in limiting bacterial virulence which leads to increased bacterial resistance to conventional antibiotics. The results are shown in Tables 4 and 5, respectively.    As reported in Table 3, the MIC values ranged between 26 and 32 µL/mL. Based on such results, two sub-lethal doses (10 and 20 µL/mL) were used in the antibiofilm tests.

Antibiofilm Activity
The EOs proved their capacity to effectively inhibit the adhesion process of all five pathogenic tested strains. The inhibition was almost absolute in many cases, with percentages often reaching and exceeding 90% (Table 4). Except for the E. melliodora EO, which inhibited at 25.44% the adhesion of A. baumannii, the other EOs affected the biofilm formation with a powerful inhibitory action provoked by E. transcontinentalis EO (93.99%) and E. odorata EO (92.59%). Exciting results were also obtained by the Eucalyptus EOs against E. coli. In fact, some of these caused an inhibition even superior to 90% (E. paniculata and E. transcontinentalis EOs). The EOs, often already at a dose of 10 µL/mL, showed inhibitory efficacy vs. the bacterial adhesion of more than 80 percent compared to the control.
The experiments on L. monocytogenes (with percentages of inhibition ranging between 59.39 and 90.41%), P. aeruginosa (with inhibition ranges between 51.21 and 91.65%), and S. au-reus (48.68-90.19%) indicate that all the EOs were capable of blocking, ab origine, the capacity of this strain to form a biofilm, the first step that leads to the increase of virulence. The antibiofilm ability exhibited by the Eucalyptus EOs is of particular meaning. The pathogenic bacteria used in our experimentation play a particular role in the food and clinical fields: the common peculiarity was that they were often resistant to antibiotics, or at least increased their resistance due to the inappropriate use of conventional drugs. A. baumannii is widely considered one of the most threatening pathogens on a global scale, particularly in healthcare institutions, being one of the most difficult microorganisms to treat with conventional antibiotics and disinfectants [44]. The A. baumannii bacteremia could cause a significant increase in mortality with respect to other Gram-negative organisms [45]. It has been one of the "red alert" pathogenic bacteria that critically compromise the benefit of the presently applied antibacterial agents [46]. E. coli O157:H7 presents a legitimate concern for the public health [47], found in several foodstuffs, including milk and derivatives, meat and meat products, fruits and derivatives and green salads [48]. P. aeruginosa is a common Gram-negative able to adapt to adverse environments. It is capable of causing disease in humans, plants, and animals. Its connection with serious illnesses, especially hospitalacquired infections, indicated the difficulty of fighting such microorganism with standard drugs, also due to its capacity to form a biofilm [49]. Studies indicated that L. monocytogenes is the only species of the genus involved in known food-borne outbreaks of listeriosis, causing 10 to 40% mortality in patients [50]. S. aureus is an opportunistic pathogen, cause of nosocomial infections with high morbidity and mortality rates [51]. These aspects are also linked to its capacity to secrete multiple toxins and exoenzymes [52], and also to its ability to form biofilm [53]. These characteristics, together with its high multidrug resistance [54], determine a high degree of S. aureus pathogenicity.
The inhibitory activity exhibited by these EOs confirmed previous studies on these bacteria [21,55,56]. On the other hand, for first time, such powerful inhibitory effects have been noted on a range of Gram-negative and Gram-positive pathogens. This indicates that, while generally the non-specific resistance or sensitivity of the bacteria could be also depending on whether they are Gram-positive or Gram-negative bacteria [57], our results have indeed highlighted a wide potential applicability of these EOs. For example, the E. transcontinentalis EO, already at a dose of 10 µL/mL, caused an inhibition of not less than 79% (vs. E. coli) reaching 91.50% (vs. A. baumanni).
Comparing the ability of the Eucalyptus EOs to inhibit the adhesion process with the ability, in some ways even more important, to act on sessile cells present in the mature biofilm (thus capable of exhibiting even more pronounced virulence and more difficult to eradicate), the inhibitory efficacy was maintained, reaching inhibition percentages as high as 93.63 and 91.14% (E. paniculata EO vs. L. monocytogenes and P. aeruginosa, respectively). In some cases, an increase in inhibitory efficacy was evident: for example, E. melliodora EO increased its inhibitory capacities on mature A. baumannii biofilm from 25.44 to 36.37%. In each case, the inhibition was never less than 28.50%. This confirmed the inhibitory activity of the Eucalyptus EOs on the mature biofilm of different pathogens, including those used here, as indicated by other researches with the EOs of different Eucalyptus species [58][59][60][61].
As indicated in Tables 4 and 5, the inhibitory effect of the Eucalyptus EOs could be ascribable mainly to their influence on the metabolism of the bacterial cell, particularly in the case of the EO of E. odorata. This EO determined an inhibition of the adhesion process of 88.56% and concurrently was capable of affecting the metabolism of the sessile cells at 83.36%. Only when the E. salmonopholia EO was tested against P. aeruginosa, the activity was extremely weak or absent. Therefore, the inhibitory effect of this oil, which produced an inhibition on the immature biofilm at 77.72%, could be due to other mechanisms such as the action on bacterial cells, DNA, bacterial permeabilization system, and others [57]. The effect of the EOs against the sessile cell metabolism within the mature biofilm indicated that often they did not act against the metabolic pathways of the bacterial cells. It happened when E. melliodora EO was added to the mature biofilm of A. baumanni and L. monocytogenes, or when E. odorata EO was administered to the mature biofilm of E. coli, P. aeruginosa, and L. monocytogenes. Again, the biofilm inhibitory action could be triggered by factors other than metabolic ones, also considering the different bacterial physiology in the mature biofilm. With the just mentioned exceptions, the test conducted using MTT showed that the Eucalyptus EOs were capable of acting on sessile cell metabolism even in the mature biofilm, reaching inhibition rates as high as 91.14% (E. paniculata vs. P. aeruginosa), 88.75% (E. paniculata vs. E. coli), and 85.95% (E. paniculata vs. A. baumannii). Furthermore, in all cases when the EOs worked, the inhibition rate never fell below 28.15% (E. odorata vs. L. monocytogenes) and 28.50% (E. melliodora vs. A. baumannii).

Extraction of Essential Oils
The leaves collected in April-May 2021 were reduced to fragments and then subjected to hydro-distillation for 3 h as reported in the European Pharmacopoeia [62]. The EOs were dissolved in n-hexane, dried over anhydrous sodium sulfate and kept under N 2 at 4 • C in the dark until analysis.

Analysis of Essential Oils
Analytical gas chromatography was conducted on a Perkin-Elmer Sigma-115 gas chromatograph accessorized with an FID and a data handling processor. The separation was obtained with a HP-5MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). Column temperature: 40 • C, with 5 min initial hold, and then to 270 • C at 2 • C/min, 270 • C (20 min); splitless injection (1 µL of a 1:1000 n-hexane solution). Injector and detector temperatures were 250 • C and 290 • C, respectively. Analysis was also run by using a fused silica HP Innowax polyethylenglycol capillary column (50 m × 0.20 mm i.d., 0.25 µm film thickness). In both cases, He was employed as carrier gas (1.0 mL/min).
GC-MS analyses were conducted with a Hewlett-Packard 5890 A gas chromatograph linked on line to an HP mass selective detector (MSD 5970HP), equipped with a DB-5 fused-silica column (25 m × 0.25 mm i.d.; 0.33 µm film thickness). Ionization energy voltage 70 eV; electron multiplier energy 2000 V. Gas-chromatographic conditions were those described above; transfer line 295 • C.
Most components were identified by comparing their Kovats retention indices with those reported in the literature [63,64] or with those of authentic compounds available in our laboratory. The Kovats retention indices were calculated on the basis of a homologous series of n-alkanes (C 10 -C 35 ) under the same operating conditions. Further identification was done comparing their mass spectra on both columns with either those present in NIST 02 and Wiley 275 libraries or with the literature [64][65][66], and in a personal library. Components' relative concentrations were calculated by peak area normalization. Response factors were not considered.

Phytotoxic Activity
The phytotoxic activity of the EOs was evaluated on germination and root elongation of radish (Raphanus sativus L. cv 'Saxa'), charlock mustard (Sinapis arvensis L.), and Ital-ian ryegrass (Lolium multiflorum Lam) following the method previously reported [40]. These seeds are histologically well known and they are usually employed in phytotoxic assays for their fast germinability. The seeds were bought from Blumen group srl (Piacenza, Italy); their surface was sterilized in 95% ethanol for 15 s. The seeds were put in Petri dishes (Ø = 90 mm) with five layers of Whatman filter paper, impregnated with distilled water (7 mL, control) or the essential oil solution (7 mL) at different doses. The germination conditions were 20 ± 1 • C with natural photoperiod. The EOs and the pure compounds solubilized in water-acetone mixture (99.5:0.5) were tested at the doses of 1000, 500, 250, 125 µg/mL. No differences between controls performed with water-acetone mixture and controls with water alone were detected. Seed germination was checked directly in Petri dishes every 24 h. A seed was considered germinated when the protrusion of the root became evident [67]. After 120 h, the radicle lengths were measured and expressed in cm. Each determination was replicated three times, using Petri dishes containing 10 seeds each. The results were reported as the mean ± SD for both germination and radicle elongation.

Minimal Inhibitory Concentration (MIC)
The resazurin microtiter-plate assay evaluated the MIC [68]. The tests were performed in flat-bottomed 96-well microtiter plates incubated at 37 • C and 35 • C, depending on the strain, for 24 h. The MIC value was revealed by the color change from dark purple to colorless. Determinations were performed in triplicate and results expressed as the mean ± SD.

Biofilm Inhibitory Activity
The capacity of the EOs to influence the bacterial biofilm formation was evaluated in flat-bottomed 96-well microtiter plates (Falcon ® , VWR International, Milano, Italy) [69]. Before the test, the overnight bacterial cultures were adjusted to 0.5 McFarland with fresh culture broth. Then, in each well 10 µL of the bacterial cultures, 10 or 20 µL/mL of each EO, and Luria-Bertani broth (LB, Sigma Aldrich Italia, Milano, Italy) were brought to a final volume of 250 µL. Microtiter plates were covered with a parafilm tape to preclude the evaporation of material included in the wells and incubated for 48 h at 37 • C (35 • C for A. baumannii). Following the discard of the planktonic cells, sessile cells were lightly washed twice with sterile phosphate buffered saline (PBS), which was discarded, leaving the plates kept for 10 min under a flow laminar hood. Two hundred µL of methanol were included in each well, leaving it to act for 15 min to permit the fixation of the sessile cells. Methanol was discarded, and each plate was left to dry. The adhered cells were stripped by adding 200 µL of 2% w/v crystal violet solution to each well. After 20 min, the staining solution was discarded, and the plates were lightly washed with sterile PBS and left to dry. The bound dye was released by adding 200 µL of glacial acetic acid 20% w/v. The absorbance was measured at 540 nm (Cary Varian, Milano, Italy). The percent value of adhesion was calculated with respect to the control (formed by the cells grown without the presence of the EOs, for which the inhibition rate was assumed as 0%). Triplicate tests were done, taking the average results for reproducibility, and results were expressed as the mean ± SD.

Inhibition of the Bacterial Metabolism
The effect of EOs on the metabolic activity of the sessile bacterial cells was evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method [69], using 96-well microtiter plates. The overnight bacterial cultures were adjusted to 0.5 McFarland, and the plates with 10 or 20 µl/mL of each EO and LB up to 250 µL were prepared as previously described. After 48 h of incubation in total, bacterial suspension representing the planktonic cells was removed, and 150 µL of PBS and 30 µL of 0.3% MTT (Sigma, Milano, Italy) were added, keeping microplates at 37 • C (35 • C for A. baumannii). The MTT solution was removed after 2 h, and the plates were washed twice with 200 µL of sterile physiological solution. Next, 200 µL of DMSO were added, leading to the formazan crystals' dissolution, measured at 570 nm (Cary Varian, Milano, Italy) after 2 h. Triplicate tests were done taking the average results for reproducibility, and results were expressed as the mean ± SD.

Statistical Analysis
All assays were carried out in triplicate. Data of each experiment were expressed as the mean ± SD, and statistically analyzed by two-way ANOVA followed by Dunnett's multiple comparisons test at the confidence level of 0.05 using GraphPad Prism 6.0 software.

Conclusions
The results agree with the previous literature regarding the phytotoxic properties of the Eucalyptus EOs, which could therefore represent an interesting green alternative in the herbicide scenario for use in agriculture. Furthermore, their demonstrated efficacy against the Gram-positive and Gram-negative bacteria used, often resistant to conventional antibiotics, could be related to their ability to decrease bacterial virulence and weaken the mechanisms of bacterial aggression, which is often the cause of the greater difficulty in eradicating the infections they are responsible for. Therefore, the activity exhibited by these Eucalyptus EOs against the pathogens such as P. aeruginosa, S. aureus, E. coli, L. monocytogenes and A. baumannii present in foods, workplaces and hospitals and responsible for several infections could be of considerable importance, both for food and health purposes. Moreover, a suggestive working hypothesis could orient the future research towards a possible link between phytotoxic and antibacterial activities.