Chemical Composition and Phytotoxic and Antibiofilm Activity of the Essential Oils of Eucalyptus bicostata, E. gigantea, E. intertexta, E. obliqua, E. pauciflora and E. tereticornis

Eucalyptus species are characterized by their richness in essential oils (EOs) with a great diversity of biological activities. This study reports the chemical composition and the phytotoxic and antibiofilm activities of the EOs of six Eucalyptus species growing in Tunisia: E. bicostata, E. gigantea, E. intertexta, E. obliqua, E. pauciflora and E. tereticornis. Four EOs were rich above all in oxygenated monoterpenes (25.3–91.4%), with eucalyptol as the main constituent. However, in the EOs of E. pauciflora and E. tereticornis, sesquiterpene hydrocarbons (28.8–54.0%) were the main class of constituents; piperitone was the main constituent of both EOs. The phytotoxicity of the EOs was tested against germination and radicle elongation of the weeds Sinapis arvensis and Lolium multiflorum and the crop Raphanus sativus, resulting in the different inhibition of seed germination and radicle elongation depending on both chemical composition and the seed tested, with remarkable phytotoxicity towards S. arvensis and R. sativus. Furthermore, almost all EOs showed antibacterial potential, resulting in significant inhibition of bacterial biofilm formation and the metabolism of Gram-positive (Staphylococcus aureus subsp. aureus and Listeria monocytogenes) and Gram-negative (Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli) bacterial strains, in addition to acting on mature biofilms. The EOs were inhibitory against all bacterial strains tested and usually reluctant to undergo the action of conventional antibiotics. Therefore, these EOs may be considered for applications both as herbicides and in food and health fields.


Introduction
Eucalyptus is a genus of Myrtaceae, native to Australia and including about 900 species. The generic name is a word made up of the Greek terms "ευ," which means "true," and "καλψπτo," which means to cover, referring to the calyx and corolla that form a coating that covers the flower until flowering. At the end of the 17th century, some of its species also began to be planted in Europe until they became widespread throughout the world over the centuries. The plants of this genus have many industrial uses, ranging from flexible and resistant wood for construction, to pulp for paper making, to honey obtained from flowers to rubber that flows along the bark. During the 1950s, 117 species of Eucalyptus were introduced in Tunisia, mainly intended to produce timber and fight against soil erosion [1].
Results are reported as the mean ± SD of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control (inhibition = 0) according to two-way ANOVA followed by Tukey's multiple comparisons test at the significance level of p < 0.05. Table 5 shows the minimal inhibitory concentration of the six EOs necessary to impede the growth of the five pathogenic bacteria used as tester strains. Figure 1 shows typical bacterial biofilms of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus, formed in the 96-well microplates following staining with crystal violet. The capacity of the six EOs to fight bacterial adhesion and the process leading to mature bacterial biofilms is reported in Table 6. Table 7 reports the capacity of the EOs to work on the metabolism of the sessile cells, which can direct the bacterial cells to increase their virulence. Table 5. MIC (µL/mL) of the six Eucalyptus EOs necessary to inhibit the growth of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus. Tetracycline (µg/mL) was used as a positive control.

EO
A. baumannii E. coli L. monocytogenes P. aeruginosa S. aureus The experiments were performed in triplicate and reported as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.0001 vs. tetracycline) according to two-way ANOVA followed by Dunnet's multiple comparison test at the significance level of p < 0.05.     The experiments were performed in triplicate, and results were reported as the mean ± SD of three experiments. *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 vs. control (inhibition = 0) according to two-way ANOVA followed by Dunnet's multiple comparisons test at the significance level of p < 0.05. The experiments were performed in triplicate, and results were reported as the mean ± SD of three experiments. **: p < 0.01, ****: p < 0.0001 vs. control (inhibition = 0) according to two-way ANOVA followed by Dunnet's multiple comparisons test at the significance level of p < 0.05.
Except in a few cases and at the lowest concentration tested, all EOs proved capable of inhibiting biofilm formation by the five pathogenic bacterial strains, with inhibition rates as high as 85.12% (E. gigantea EO vs. A. baumannii) at the highest concentration used. This confirmed the biofilm inhibitory action observed for other Eucalyptus EOs, such as E. gunnii Hook. f., which demonstrated an effective inhibitory activity against some of the same strains used in our experiments, such as S. aureus and E. coli, albeit with greater inhibitory vigor, given the smaller amount of EO required to limit the bacterial biofilm. The results of the inhibitory activity on biofilm formation appeared attractive; those obtained concerning the mature biofilms, when the EOs were in contact with the bacterial strains after 24 h from the beginning of their growth, were still more interesting. A mature biofilm leads the bacteria to modify their morphological, metabolic, and physiological characteristics that generally determine their increased virulence. In our experiments, the tested EOs proved potentially helpful in limiting biofilm formation and acting against mature biofilms. In some cases, their inhibitory efficacy proved even more vigorous. For example, the EO of E. bicostata inhibited A. baumannii biofilm formation by 28.74% when tested at the lower concentration (10 µL/mL). Similar efficacy was also observed in the case of S. aureus and L. monocytogenes, against which E. bicostata EO practically showed similar efficacy on the mature biofilm. The action of the E. bicostata EO was powerful, indeed more decisive on the mature biofilm of P. aeruginosa (85.06% inhibition), compared to the inhibitory efficacy exerted by the same EO on the bacterial adhesion when at the lowest concentration tested, it was ineffective and, at 20 µL/mL, gave 58.74% inhibition. Similarly, the EO of E. pauciflora, which inhibited the P. aeruginosa biofilm formation by only 13.79%, proved to be much more effective on mature biofilms (47.97 and 56.20% inhibition at 10 and 20 µL/mL, respectively). Likewise, the EO of E. obliqua was more effective on the mature biofilm of S. aureus (77.38 and 83. 63% inhibition at 10 and 20 µL/mL, respectively) than on the adhesive process performed by this strain. Furthermore, although in the tests with the other strains, the inhibitory efficacy of the EOs was less pronounced, in each case, it was never insignificant, with the sole exception of the EO of E. intertexta, which was ineffective only against L monocytogenes at the lowest concentration tested. Through the MTT test, we also evaluated the effect that the two EO concentrations exerted on the sessile cell metabolism of the five bacterial strains with an upstream impact after adding the EO at time zero and on the mature biofilm. In the case of the MTT assay performed ab origine, the action of the EOs was, with some exceptions, mainly on bacterial metabolism. This was evidenced by the percent inhibition exhibited by the EOs of E. bicostata, E. gigantea, E. obliqua, and E. pauciflora. Some EOs, such as the EO of E. gigantea, had an intense inhibitory action on the metabolism of all pathogenic strains, with percentages of inhibition never less than 69.79% (10 µL/mL vs. E. coli). They went as high as 83.74% (vs. A. baumannii). The EOs of E. bicostata and E. intertexta, which failed to inhibit the metabolism of sessile E. coli cells, were the least effective. In other cases, some EOs, ineffective at the lowest concentrations, proved capable of acting on bacterial metabolism when tested at the highest concentration. Thus, in some cases, the inhibitory effect exerted by the EOs on the mature biofilm did not essentially translate to the metabolism of sessile cells. For example, the EO of E. gigantea was completely ineffective vs. L. monocytogenes and P. aeruginosa, and the EOs of E. obliqua and E. pauciflora did not act against L. monocytogenes. However, especially in the case of E. pauciflora, which also inhibited the mature biofilm of L. monocytogenes effectively, the inhibitory action did not translate to a step on the cellular metabolism but acted on its other characteristics, as amply demonstrated in the literature.

Discussion
As shown in Table 8, the EO yields varied significantly between the species examined, from 0.03% for E. tereticornis to 3.11% for E. obliqua. These data agree with the yields in EOs found in this genus and with the considerable variability of percent composition found in the literature [15][16][17] 6,[15][16][17]. Instead, the EOs from E. pauciflora and E. tereticornis showed a much lower amount of eucalyptol. This agrees with the literature where these species are characterized by very low quantities of eucalyptol (up to a maximum of 20%) [4,15,16,[18][19][20][21][22][23][24][25]. Figure 2 shows the main constituents of the EOs. As shown in Table 8, the EO yields varied significantly between the species examined, from 0.03% for E. tereticornis to 3.11% for E. obliqua. These data agree with the yields in EOs found in this genus and with the considerable variability of percent composition found in the literature [15][16][17] 6,[15][16][17]. Instead, the EOs from E. pauciflora and E. tereticornis showed a much lower amount of eucalyptol. This agrees with the literature where these species are characterized by very low quantities of eucalyptol (up to a maximum of 20%) [4,15,16,[18][19][20][21][22][23][24][25]. Figure 2 shows the main constituents of the EOs.  The composition of the EO of E. bicostata largely agrees with the literature on EOs obtained from plants of Tunisian origin [3,16], which report eucalyptol as the main component (in our sample in higher amounts), and the presence of α-pinene and transpinocarveol. Limonene and carvacrol are not present in our sample, while viridiflorol is present in a greater quantity, and globulol is present in smaller amounts. The composition The composition of the EO of E. bicostata largely agrees with the literature on EOs obtained from plants of Tunisian origin [3,16], which report eucalyptol as the main component (in our sample in higher amounts), and the presence of α-pinene and trans-pinocarveol. Limonene and carvacrol are not present in our sample, while viridiflorol is present in a greater quantity, and globulol is present in smaller amounts. The composition of E. gigantea agrees with the studies of Elaissi and coworkers [1, 15,16] on EOs obtained from Tunisian plants regarding the presence and percentage of eucalyptol but differs for the absence of limonene and p-cymene and the higher amount of spatulenol. The composition of the EO of E. intertexta agrees with data reported in the literature [25][26][27][28], where the main components were eucalyptol and p-cymene. However, in our sample, a greater number of components and considerable amounts of trans-pinocarveol and spatulenol were registered. Yong and coworkers [17] described the composition of an EO of E. obliqua of Australian origin. Our data agree with this study regarding the components and their percentages. The studies about the composition of the EO of E. pauciflora are inconsistent with our results.
However, the common feature is the very low amount of eucalyptol [15,16,21,[29][30][31]. The data regarding the composition of the EO of E. tereticornis disagree in part with those found in the literature [1,4,10,15,16,18,[20][21][22][23][24]. However, the common feature was the low amount of eucalyptol and the significant presence of p-cymene and spathulenol. Our sample lacks limonene, cryptone, and caryophyllene oxide, previously reported as the main components. However, high amounts of sesquiterpenes are characteristic of our sample. To the best of our knowledge, the existence of chemotypes in the Eucalyptus genus has not yet been hypothesized. On the other hand, environmental conditions can significantly influence the composition of essential oils. The data collected may contribute to further studies that can investigate the diversity of chemical traits within the genus.
All the EOs have been shown to have a certain phytotoxic activity, which, however, is very variable according to the species and the seed considered: the most active EO in preventing the germination of R. sativus was the one obtained from E. pauciflora; the EO from E. tereticornis, on the other hand, is the most active in preventing the germination of S. arvensis and L. multiflorum. As for the inhibition of the radical elongation of R. sativus, the most active EO was the one obtained from E. intertexta; the EO of E. teritecornis was the most active in the inhibition of the radical elongation of S. arvensis, and the EO of E. gigantea was found to be the most active inhibitor of the radical elongation of L. multiflorum. In general, the phytotoxic activity was high towards seeds of R. sativus and S. arvensis and low towards L. multiflorum. For E. bicostata, E. gigantea, and E. intertexta. The phytotoxicity can be attributed to their high eucalyptol content [32]. However, the EOs of E. gigantea and E. intertexta showed a greater variety of compounds that can contribute together with eucalyptol to determine the total phytotoxic activity [33,34]. Different from the case of the EOs of E. pauciflora and mostly of E. tereticornis, with low amounts of eucalyptol, the most active on all three seeds. Therefore, their phytotoxicity was probably due to the synergism between the constituents [24,35]. The phytotoxic properties of the Eucalyptus genus are well recognized and reported [2,9], and the phytotoxic and allelopathic activities of some Eucalyptus species are well known and attributed to the EOs, in particular to monoterpenes such as eucalyptol and limonene [36], capable of acting in various ways, for example, destroying the chlorophyll reserves, interfering with cellular respiration processes [10] or decreasing the water reserves of the seeds [11]. Monoterpenes have been widely reported for their phytotoxic properties for a long time [37][38][39]. Sesquiterpenes have also been reported for their allelopathic properties [40]. In the examined EOs, the presence of components (both monoterpenes and sesquiterpenes) previously described as phytotoxic substances was registered [41,42]. Of importance seems the prevalence of eucalyptol in four of the analyzed EOs. This component is known for its phytotoxic properties [43,44], and it was proposed as a volatile inhibitor influencing the vegetation composition from the earliest studies on allelopathic interactions in Salvia leucophylla Greene populations [45]. Some mechanisms of action have been suggested [46], and eucalyptol has been proposed both for direct use as a bio-herbicide and as a lead compound for herbicide synthesis [47]. However, the EOs of E. pauciflora and E. tereticornis showed phytotoxicity even with low amounts of eucalyptol. Their activity can be, therefore, attributable to other components such as pcymene, terpinen-4-ol, piperitone β-vetivenene, γ-patchoulene, αand β-eudesmol, which are reported in the literature for their phytotoxic activity [10,24,38,48]. The antimicrobial activity of Eucalyptus EOs is well known [6,8,11,16,21]. The antibiofilm activity of the EOs studied is related to their composition: this property may be related to the presence of large amounts of eucalyptol, a compound with antibacterial effectiveness against several bacteria, which can act ab origine, limiting the initial steps of the biofilm formation [49], and, probably, also to a synergistic effect exerted by other constituents. In fact, the EOs with different compositions also showed the same effects: for example, L. monocytogenes, in which sessile metabolism in the mature biofilm was uninfluenced by the EOs of E. pauciflora and E. bicostata, although the two EOs contained 85.5% and 2.0% of eucalyptol, respectively. In any case, the EOs were inhibitory against Gram-positive and Gram-negative bacteria, usually reluctant to undergo the action of conventional antibiotics and can constitute natural products of interest to both the pharmaceutical and food sectors.

Plant Material
Leaves of the six Eucalyptus species were harvested from different Tunisian arboretums (Table 8). For each species, five samples from more than five different trees were collected and mixed for homogenization. The leaves were stored in a dry place for fifteen days. Specimens were identified at the National Institute of Research in Rural Engineering, Waters and Forests (INRGREF), Rue El Menzah, Tunis, Tunisia.

Extraction of the Essential Oils
One hundred grams of dried leaves of each species were submitted to hydrodistillation (500 mL of water) for 4 h using a Clevenger-type apparatus according to the method reported in the European Pharmacopoeia [50]. The EOs were solubilized in n-hexane, dried in an N 2 atmosphere, and stored in amber vials in the refrigerator at 4 • C. Table 8 reports the data relating to the origin of plant material, with information about the collection site and the EO yields.

Analysis of the Essential Oils
The composition of the essential oils was examined by GC and GC-MS. GC analyses were performed using a Perkin-Elmer Sigma 115 gas chromatograph equipped with a flame ionization detector (FID) and a non-polar HP-5 MS capillary column of fused silica (30 m × 0.25 mm; 0.25 µm film thickness). The operating conditions were: injector and detector temperatures, 250 • C and 290 • C, respectively. The analysis was conducted on a scheduled basis: 5 min isothermally at 40 • C; subsequently, the temperature was increased by 2 • C/min until 270 • C, and finally, it was kept in an isothermal state for 20 min. The analysis was also performed on an HP Innowax column (50 m × 0.25 mm; 0.25 µm film thickness) using helium as a carrier gas (1.0 mL/min). GC-MS analysis was carried out through an Agilent 6850 Ser. II Apparatus equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm; 0.25 µm film thickness) and connected to an Agilent Mass Selective Detector (MSD 5973) with an ionization voltage of 70 V and an ion multiplier energy of 2000 V. The mass spectra were scanned in the range of 40-500 amu, with five scans/s. The chromatographic conditions were as reported above; transfer line temperature was 295 • C. Most of the components were identified by comparing their Kovats indices (Ki) with those of the literature [51][52][53] and by a careful analysis of the mass spectra compared to those of pure compounds available in our laboratory or to those present in the NIST 02 and Wiley 257 mass libraries [54]. The Kovats indices were determined with a homologous series of n-alkanes (C 10 -C 35 ) under the same operating conditions. For some components, the identification was confirmed through co-injection with standard compounds. The analyses were carried out in triplicate.

Phytotoxic Activity
To study the phytotoxic effects of the EOs on the seeds of Raphanus sativus L., Sinapis arvenis L., and Lolium multiflorum Lam., a bioassay based on germination and consequent radicle growth was used [55]. The seeds of R. sativus were purchased from Blumen srl, Piacenza, Italy; the seeds of L. multiflorum were purchased from the "Fratelli Ingegnoli" plant nursery, Milan, Italy, while the seeds of S. arvensis were collected from wild populations in Tunisia. The seeds were surface-sterilized with 95% ethanol for 15 s and sown in Petri dishes (Ø = 90 mm) containing five layers of Whatman filter paper impregnated with distilled water (7 mL, control) or a solution of the tested EO (7 mL). The EOs, solubilized in water:acetone (99.5:0.5), were tested at different doses of 1000, 500, 250, and 125 µg/mL using as controls water and a solution of water/acetone 99.5/0.5. Controls carried out with this solution did not differ from the control with water alone. A climatic chamber for growth was used, equipped with adjustable lighting, temperature, and humidity system. The germination conditions were 20 ± 1 • C with a natural photoperiod. The germination process was observed directly in the Petri dishes. A seed was considered germinated when root protrusion was evident [56]. After 120 h (fifth day), the effects on germination (the number of germinated seeds) and radicle elongation (measured in cm) were determined. Each determination was repeated 3 times using Petri dishes, each containing 10 seeds. The data were expressed as mean ± SD for germination and radicle elongation.

Minimal Inhibitory Concentration (MIC)
The MICs of the EOs were evaluated in flat-bottomed 96-well microtiter plates, which were incubated at 37 • C (35 • C for A. baumannii) for 24 h. The value of the MIC was revealed through the color change occurring from dark purple to colorless [57]. Tetracycline (µg/mL) was used as a positive control.

Biofilm Inhibitory Activity
The ability of the EOs to affect bacterial adhesion was investigated following the method of Fratianni and coworkers [58] with flat-bottomed 96-well microtiter plates. Before the test, the bacterial cultures were adjusted to 0.5 McFarland with fresh culture broth. Then, 10 µL of the bacterial cultures and 10 or 20 µL/mL of the EOs were placed in each well, and the wells were filled with different volumes of Luria-Bertani broth to reach a final volume of 250 µL/well. Plates were covered with parafilm tape to avoid evaporation and incubated for 48 h at 37 • C (35 • C for A. baumannii). After the removal of the planktonic cells, sessile cells were washed twice with a sterile physiological solution, which was removed. The plates were left for 10 min under a laminar flow hood. Two hundred µL of methyl alcohol were added to each well to fix the sessile cells and removed after 15 min. Each plate was left to let the dryness of the samples. Two hundred µL of 2% w/v crystal violet solution/well was used for 20 min to stain the sessile cells. Plates were washed with a sterile physiological solution and left to dry. The bound dye's release was obtained by adding 200 µL of glacial acetic acid 20% w/v. The absorbance was assessed 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 samples, inhibition rate of 0%). Triplicate tests were performed, taking into account the average results for reproducibility.

Activity on Mature Bacterial Biofilm
The overnight bacterial cultures were adjusted to 0.5 McFarland with fresh Luria-Bertani culture broth, and 10 µL were added to flat-bottomed 96-well microtiter plates to have a final volume of 250 µL/well. Next, microplates were covered with parafilm tape to avoid evaporation and incubated at 37 • C (35 • C for A. baumannii). After 24 h of bacterial growth, planktonic cells were removed, and the EOs (10 or 20 µL/mL) and Luria-Bertani broth were added to have a final volume of 250 µL/well. After 24 h of incubation, the sequential steps of the experiment, including the calculation of the percent value of inhibition compared with the untreated bacteria, were performed as previously described.

Effects of EOs on Cell Metabolic Activity within the Biofilm
The effect on the metabolic activity of the bacterial cells of two concentrations (10 or 20 µL/mL) of the EOs, which were added at the beginning of the bacterial growth and after 24 h of incubation, was investigated through the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric method [59]. After a 48 h incubation period, planktonic cells were discarded; 150 µL of sterile physiological solution and 30 µL of 0.3% MTT (Sigma, Milano, Italy) were added. Microplates were kept at 37 • C (35 • C for A. baumannii). After two h, the MTT solution was removed, and two washing steps were performed with 200 µL of sterile physiological solution. Then, 200 µL of dimethyl sulfoxide (DMSO, Sigma, Milano, Italy) was added to allow the dissolution of the formazan crystals, measured after 2 h at 570 nm (Cary Varian, Milano, Italy).

Statistical Analysis
Results were expressed as mean ± standard deviation (SD) of three independent experiments and analyzed by one-way analysis of variance (ANOVA) by GraphPad Prism 6.0 (Software Inc, San Diego, CA, USA). Results were considered significant for p < 0.05.

Conclusions
The data collected in this study on the chemical composition of the EOs of the studied species help to shed light on the complex phytochemistry of EOs of the Eucalyptus genus, even if grown outside its habitat. Moreover, the phytotoxic activity of the studied EOs can be exploited to obtain selective herbicides on target species. EOs in agriculture have many advantages: they come from plant organisms already present in nature and therefore are characterized by high eco-compatibility. Their use is considered a safe strategy in crop management systems in the context of the circular economy and respect for the environment. EOs are a valid alternative to control pathogens, agricultural pests, and weeds, thus avoiding the indiscriminate use of agrochemicals that negatively influence the environment and human health. The activity of the studied EOs against the pathogenic bacteria tested could be considered of noticeable significance in different fields of application. In recent years, the increase in some infections has been linked to the expansion of the presence in several environments of the strains used in our experiments. Such bacteria have developed a more robust evolutionary drug resistance due to different factors, including inappropriate use of conventional drugs if not required or indispensable. Thus, the interest in natural alternatives to prevent biofilm formation and fight mature biofilms, which are more challenging to eradicate, augmented the research to identify natural agents as alternatives to conventional sanitizers to control biofilm development by acting on bacteria metabolism and/or other bacterial cell parameters. Under such a point the view, the six EOs demonstrated an important role in fighting the bacterial biofilm both at the beginning of the biofilm formation process and at the mature stage, which is the most ideal situation for bacteria, which, protected by the biofilm niches, become more protected and less sensitive to the action of conventional drugs. Finally, a suggestive working hypothesis could orient future research toward a possible link between phytotoxic and antibacterial activities.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.