Next Article in Journal
The Establishment of an Ex Situ Collection of Primula veris in Bulgaria
Next Article in Special Issue
Essential Oils: Useful Tools in Storage-Pest Management
Previous Article in Journal
Potassium Humate and Plant Growth-Promoting Microbes Jointly Mitigate Water Deficit Stress in Soybean Cultivated in Salt-Affected Soil
Previous Article in Special Issue
An Update on Effectiveness and Practicability of Plant Essential Oils in the Food Industry
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 11
Issue 22
10.3390/plants11223017
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Flavio Polito
1,
Habiba Kouki
2,
Sana Khedhri
3,
Lamia Hamrouni
3,
Yassine Mabrouk
2,
Ismail Amri
2,3,
Filomena Nazzaro
4,
Florinda Fratianni
4 and
Vincenzo De Feo
1,4,*
1
Department of Pharmacy, University of Salerno, Via San Giovanni Paolo II 132, 84084 Fisciano, Italy
2
Laboratory of Biotechnology and Nuclear Technology, National Center of Nuclear Science and Technology, Sidi Thabet, B.P. 72, Ariana 2020, Tunisia
3
Laboratory of Management and Valorization of Forest Resources, National Institute of Researches on Rural Engineering, Water and Forests, P.B. 10, Ariana 2080, Tunisia
4
Institute of Food Science, CNR-ISA, Via Roma 64, 83100 Avellino, Italy
*
Author to whom correspondence should be addressed.
Plants 2022, 11(22), 3017; https://doi.org/10.3390/plants11223017
Submission received: 13 October 2022 / Revised: 3 November 2022 / Accepted: 5 November 2022 / Published: 8 November 2022
(This article belongs to the Special Issue Phytotoxic Activity and Application of Plant Essential Oils)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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 “καλψπτο,” 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].
However, only some species of this genus have been exploited to obtain essential oils (EOs), starting above all from the leaves, the uses of which are mainly in the pharmaceutical and cosmetic fields. These EOs are rich in monoterpenes and sesquiterpenes; other secondary metabolites are macrocarpals, alkaloids, phenols, flavonoids, tannins and phenolaldehydes [2]. The characteristic component of the volatile fractions of most Eucalyptus species is eucalyptol (1,8-cineole), whose content in the EO can reach up to 80–90% of the total [3]. Other main components are spatulenol, p-cymene, viridiflorol, α-phellandrene α-terpineol, limonene and α-pinene [4], along with α-, β- and γ-eudesmol, and piperitone [5]. Over the years, studies have been carried out to evaluate the possible biological activities of the EOs from leaves of Eucalyptus species. Traditional uses are oriented toward the treatment of various infectious diseases, flu, sore throat, cold, respiratory pathologies and painful states [6]. More recent studies have highlighted other biological activities not homogeneously distributed among the hundreds of species belonging to this genus. In particular, antimicrobial properties have been highlighted, mainly due to the presence of monoterpenes such as eucalyptol, α-pinene, β-pinene and limonene [7]. Additionally, phytotoxic and herbicidal activities on weeds and crops have been reported and attributed to EO components that can alter physiological and biochemical processes underlying the germination and elongation of roots [8]. For a long time, studies have been carried out on the allelopathic impact of the cultivation of some Eucalyptus species and of their metabolites (in particular monoterpenes) on both natural and agricultural ecosystems [9,10,11].
A bacterial biofilm constitutes a fairly complex structure made up of microbial cells associated with each other, which adhere to a surface, and are, in a certain sense, kept isolated from the external environment (although they are able to exert an important influence on it) through the formation of a sort of polysaccharide “dome” [12]. In the biomedical field, biofilms are involved in a wide range of diseases, such as joint and orthopedic diseases, and they also characterize a large number of chronic bacterial infections that have always been a major clinical problem, which can still be faced with many difficulties today. Bacterial biofilms are considered a serious hygiene problem in the environment, in human health and in the food industry. In fact, a biofilm makes bacteria much more resistant to disinfectants and to antimicrobial agents [13]. The cells contained in the biofilm are much more difficult to reach, and it becomes very difficult for synthetic drugs to “break” the organization of the biofilm due to how it is structured and composed. For this, there is a need to research and experiment with new types of substances that can make a fundamental contribution to the formation of biofilms and that are able to prevent their formation, persistence or even exacerbation after treatment. A promising way is that which analyzes how other organisms can defend themselves from bacterial colonization. Many plant organisms, for example, are continuously exposed to a wide range of potentially harmful microorganisms that can grow on their surfaces. Therefore, it is useful to study and understand the defense mechanisms that plants exploit to fight the microorganism. Many plant-derived compounds, especially EOs, have demonstrated anti-biofilm properties [14]. The chemical diversity among the countless plant species ensures an enormous reserve of substances that could make a fundamental contribution to the fight against biofilms, as well as creating mixtures of compounds to exploit multiple mechanisms at the same time.
The aim of this work was the study of the chemical composition of the EOs from six species of Eucalyptus grown in Tunisia, E. bicostata Maiden, Blakeley & Simmonds (=E. globulus subsp. bicostata (Maiden, Blakeley & Simmonds) J.B. Kirkp.), E. pauciflora Sieber ex Spreng., E. gigantea Hook f., E. intertexta R. T. Baker, E. obliqua L’Hér., and E. tereticornis Sm., and the valuation of their possible phytotoxic and antibiofilm activities. These species were chosen because they are well-adapted and acclimatized in Tunisia and have been poorly studied, both regarding the chemical composition of the EO and their biological activities.

2. Results

2.1. Composition of the EOs

The composition of the EOs is reported in Table 1 according to the elution order on a 5 HP column. The presence of 102 components distributed among the six species has been detected. The EO of E. bicostata showed the lowest number of components (19), with oxygenated monoterpenes (91.4%) as the main class. The other components are almost uniformly distributed among monoterpene hydrocarbons (1.7%), sesquiterpene hydrocarbons (1.1%), and oxygenated sesquiterpenes (2.5%). The main component was eucalyptol (85.5%); the other most representative components, whose concentration exceeded 1%, were α-pinene, trans-pinocarveol, pinocarvone and viridiflorol. This is the EO with the greatest percentage of eucalyptol among those analyzed. E. gigantea EO showed the presence of 36 components, with oxygenated monoterpenes (68.90%) as the main class. The main component was eucalyptol (59.3%), followed by spathulenol (11.8%) and α-terpineol (4.5%). Other components ranged from 0.1 to 2.0%; among these, α-eudesmol (2.0%), α-pinene (1.6%), allo-ocimene (1.5%), γ-terpinene (1.3%), terpinen-4-ol (1.3%) and α-epi-7-epi-5-eudesmol (1.0%) had a percentage higher than 1.0%. Forty-four components have been identified in the EO of E. intertexta, with a prevalence of oxygenated monoterpenes (75.0%). The main component was eucalyptol (65.9%), followed by spathulenol (8.1%), α-pinene (6.7%) and trans-pinocarveol (4.0%). The other components were present in very low quantities ranging from 0.1% to 1.2%, with cubebol (1,2%), pinocarvone (1,1%) and α-gurjunene (1,1%) with a percentage higher than 1.0%. The EO of E. obliqua revealed the greatest number of components (46), mostly oxygenated monoterpenes (68.6%) and small amounts of sesquiterpene hydrocarbons (0.7%). Eucalyptol was the main component (54.9%), followed by α-pinene (13.2%), spathulenol (3.8%), trans-pinocarveol (3.3%) and dihydrocarveol (3.2%). Other components in percentages higher than 1.0% were β-pinene (1.7%), p-cymene (1.4%), α-terpineol (1.2%) and globulol (1.2%). The EO of E. pauciflora showed the presence of 39 components distributed between monoterpene hydrocarbons (14.0%), oxygenated monoterpenes (25.5%), sesquiterpene hydrocarbons (28.8%), and oxygenated sesquiterpenes (27.1%). The most representative compounds were piperitenone and β-vetivenene (both 8.8%), followed by β-eudesmol (8.1%), p-cymene (7.6%), trans-dauca-4 (11), 7-diene (6.4%), γ-pathcoulene (6.3%) and α-eudesmol (6.3%). Eucalyptol resulted in only 2.0% of the total EO. In the EO of E. tereticornis, 33 components have been identified, primarily oxygenated sesquiterpenes (54.0%), followed by oxygenated monoterpenes (25.3%), oxygenated monoterpenes (11.4%) and monoterpene hydrocarbons (6.1%). The main component was piperitone (19.4%), followed by trans-dauca-4-(11),7-diene (17.9%), and β-vetivenene (17.3%). In this EO, eucalyptol was also present in a small amount (2.4%).

2.2. Phytotoxic Activity

Table 2, Table 3 and Table 4 report the phytotoxic activity of the EOs on R. sativus, S. arvensis and L. multiflorum, respectively. The results show a remarkable phytotoxic effect by the tested EOs, resulting in a dose-response inhibition of both germination and radical elongation. As regards E. bicostata, the inhibition of germination of R. sativus is high but never complete: the highest activity is at 1000 µg/mL (92.3% inhibition), while it is reduced at the other concentrations. However, it totally inhibited the germination of S. arvensis at concentrations of 1000 and 500 µg/mL, whereas at the lower concentrations tested, no appreciable activity was registered. This EO proved ineffective against L. multiflorum. The greatest inhibition on the radical elongation of R. sativus occurs at 1000 µg/mL (77.14%) and is slightly lower at 500 and 250 µg/mL (68.57 and 62.86%, respectively). The inhibitory activity on the elongation of L. multiflorum is instead low, exceeding 50% only at 1000 µg/mL (61.11%). E. gigantea EO showed a similar feature but with lower activity than E. bicostata EO. In fact, at 1000 µg/mL, this EO weakly inhibited the germination of R. sativus and S. arvenis (6.0 and 3.6%, respectively). It is unable to inhibit the germination of L. multiflorum. The inhibition of radical elongation against R. sativus is very low, being appreciable only at concentrations of 1000 (67.74%) and 500 µg/mL (54.84%). On the other hand, a higher inhibition was recorded against S. arvensis where, at all concentrations tested, it exceeds 80% with a maximum of 98.21% at 1000 µg/mL. Against L. multiflorum, the greatest activity occurs at 1000 µg/mL (88.24% inhibition). The E. intertexta EO inhibited the germination of R. sativus and S. arvenis at 1000 µg/mL, but the activity decays at lower concentrations, especially in the case of S. arvensis. This EO was unsatisfactory in inhibiting the germination of L. multiflorum. The inhibition activity of radical elongation was very high towards R. sativus: it resulted in total of 1000 µg/mL and of 80% at 500 µg/mL. The same activity is shown against S. arvensis where, however, there was appreciable activity even at 250 µg/mL (84.84%). Against L. multiflorum, this EO inhibited radical elongation by 88.64% at 1000 µg/mL. E. obliqua EO completely inhibited the germination of S. arvensis at concentrations of 1000 and 500 µg/mL while maintaining an appreciable activity at lower doses (16.7%). On the other hand, its action against R. sativus and L. multiflorum was unsatisfactory at all tested concentrations. The inhibitory activity on the radical elongation of R. sativus was 83.33% at 1000 µg/mL, while it did not reach 50% at other concentrations tested. The activity was instead very high against S. arvensis, where it was complete at 1000 and 500 µg/mL and achieved values of 80% at 250 and 72% at 125 µg/mL. Against L. multiflorum, the greatest activity occurred at 1000 (86.6%) and 500 µg/mL (70% inhibition). E. pauciflora EO showed significant activity against R. sativus, with 100% inhibition at 1000 µg/mL; at lower concentrations, the activity decayed. The activity was low towards S. arvensis and unsatisfactory towards L. multiflorum. The inhibition on radical elongation against R. sativus was total at 1000 µg/mL, while at other concentrations, it was not very noticeable. In the case of S. arvensis, the greatest activity occurred at 1000 µg/mL (81.08%), while the lower inhibition was registered at 500 µg/mL (56.76%). The concentrations of 125 and 250 µg/mL showed moderate activity (62.1 and 72.97%, respectively). In the case of L. multiflorum, the greatest inhibition occurred at 100 µg/mL (85.42%). E. tereticornis EO showed the most significant activity. At 1000 µg/mL, it totally inhibited the germination of all tested seeds, and at 500 µg/mL, it also completely inhibited the germination of S. arvensis. At lower doses, the activity decreased, especially against L. multiflorum. The inhibitory activity on the radical elongation of R. sativus is complete at 100 µg/mL. As regards S. arvensis, the inhibitory activity is complete at 1000 and 500 µg/mL and very high at 250 (93.33%) and 125 µg/mL (86.68%). In the case of L. multiflorum, the inhibitory activity was complete at 1000 µg/mL, but at other concentrations, it was not appreciable.

2.3. Antibacterial and Antibiofilm Activity

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.
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.

3. 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]. The EOs of E. bicostata, E. gigantea, E. intertexta and E. obliqua were characterized by the prevalence of monoterpenes (93.1, 74.7, 82.3, and 87.3%, respectively) with oxygenated monoterpenes as the main class (91.4, 68.9, 75.0, and 68.6%, respectively). Sesquiterpenes predominated in the EOs of E. pauciflora and E. tereticornis (55.9% and 65.4%, respectively), with hydrocarbons accounting for 28.8 and 54.0%, respectively. Eucalyptol was the main component in the EOs from E. bicostata, E. gigantea, E. intertexta and E. obliqua: this agrees with the literature, where this compound is reported as the main component of the EOs of most Eucalyptus species [1,6,15,16,17]. 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 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 p-cymene, terpinen-4-ol, piperitone β-vetivenene, γ-patchoulene, α- and 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.

4. Materials and Methods

4.1. 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.

4.2. 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 N2 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.

4.3. 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 (C10–C35) 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.

4.4. 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.

4.5. Antimicrobial Activity

4.5.1. Microorganisms and Culture Conditions

Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa DSM 50071, Escherichia coli DSM 8579 (Gram-negative), Staphylococcus aureus subsp. aureus Rosebach ATCC 25923 and Listeria monocytogenes ATCC 7644 (Gram-positive bacteria), used as bacterial test strains, were cultured for 18 h at 37 °C in Luria Broth at 80 rpm (Corning LSE, Pisa, Italy). A. baumannii was cultured at 35 °C under the same conditions.

4.5.2. 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.

4.5.3. 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.

4.5.4. 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.

4.5.5. 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)-2,5-diphenyltetrazolium 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).

4.6. 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.

5. 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.

Author Contributions

Conceptualization, I.A., L.H. and V.D.F.; methodology, I.A., F.N. and V.D.F.; analysis, F.P., H.K., S.K., Y.M. and F.F.; investigation, F.P., H.K., S.K., Y.M. and F.F.; resources, I.A., L.H., V.D.F. and F.N.; data curation, I.A., L.H., V.D.F. and F.N; writing—original draft preparation, F.P., I.A. and F.N.; writing—review and editing F.P., I.A., F.F., F.N. and V.D.F.; supervision, I.A., F.N. and V.D.F.; funding acquisition, V.D.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Elaissi, A.; Marzouki, H.; Medini, H.; Larbi Khouja, M.; Farhat, F.; Lynene, F.; Harzallah-Skhiri, F.; Chemli, R. Variation in volatile leaf oils of 13 Eucalyptus species harvested from Souinet Arboreta (Tunisia). Chem. Biodivers. 2010, 7, 909–921. [Google Scholar] [CrossRef] [PubMed]
  2. Dhakad, A.K.; Pandey, V.V.; Beg, S.; Rawat, J.M.; Singh, A. Biological, medicinal and toxicological significance of Eucalyptus leaf essential oil: A review. J. Sci. Food Agric. 2018, 98, 833–848. [Google Scholar] [CrossRef] [PubMed]
  3. Sebei, K.; Sakouhi, F.; Herchi, W.; Khouja, M.L.; Boukhchina, S. Chemical composition and antibacterial activities of seven Eucalyptus species essential oils leaves. Biol. Res. 2015, 48, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Pino, J.A.; Marbot, R.; Quert, R.; García, H. Study of essential oils of Eucalyptus resinifera Smith, E. tereticornis Smith and Corymbia maculata (Hook.) K.D. Hill & L.A.S. Johnson, grown in Cuba. Flavour Fragr. J. 2002, 17, 1–4. [Google Scholar]
  5. Toloza, A.C.; Lucia, A.; Zerba, E.; Masuh, H.; Picollo, M.I. Interspecific hybridization of Eucalyptus as a potential tool to improve the bioactivity of essential oils against permethrin-resistant head lice from Argentina. Bioresour. Technol. 2008, 99, 7341–7347. [Google Scholar] [CrossRef]
  6. Caputo, L.; Smeriglio, A.; Trombetta, D.; Cornara, L.; Trevena, G.; Valussi, M.; Fratianni, F.; De Feo, V.; Nazzaro, F. Chemical composition and biological activities of the essential oils of Leptospermum petersonii and Eucalyptus gunnii. Front. Microbiol. 2020, 11, 409. [Google Scholar] [CrossRef] [Green Version]
  7. Sliti, S.; Ayadi, S.; Kachouri, F.; Khouja, M.A.; Abderrabba, M.; Bouzouita, N. Leaf essential oils chemical composition, antibacterial and antioxidant activities of Eucalyptus camaldulensis and E. rudis from Korbous (Tunisia). J. Mater. Environ. Sci. 2015, 6, 743–748. [Google Scholar]
  8. Danna, C.; Cornara, L.; Smeriglio, A.; Trombetta, D.; Amato, G.; Aicardi, P.; De Martino, L.; De Feo, V.; Caputo, L. Eucalyptus gunnii and Eucalyptus pulverulenta ‘baby blue’essential oils as potential natural herbicides. Molecules 2021, 26, 6749. [Google Scholar] [CrossRef]
  9. Chu, C.; Mortimer, R.E.; Wang, H.; Wang, Y.; Liu, X.; Yu, S. Allelopathic effects of Eucalyptus on native and introduced tree species. For. Ecol. Manag. 2014, 323, 79–84. [Google Scholar] [CrossRef]
  10. Kaur, S.; Pal Singh, H.; Batish, D.; Kumar Kholi, R. Role of monoterpenes in Eucalyptus communities. Curr. Bioact. Compd. 2012, 8, 101–107. [Google Scholar] [CrossRef]
  11. Singh, D.; Kohli, R.K.; Saxena, D.B. Effect of eucalyptus oil on germination and growth of Phaseolus aureus Roxb. Plant Soil 1991, 137, 223–227. [Google Scholar] [CrossRef]
  12. Costerton, J.W.; Cheng, K.J.; Geesey, G.G.; Ladd, T.I.; Nickel, J.C.; Dasgupta, M.; Marrie, T.J. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 1987, 41, 435–464. [Google Scholar] [CrossRef] [PubMed]
  13. Jamal, M.; Ahmad, W.; Andleeb, S.; Jalil, F.; Imran, M.; Nawaz, M.A.; Hussain, T.; Rafiq, M.; Kamil, M.A. Bacterial biofilm and associated infections. J. Chin. Med. Assoc. 2018, 81, 7–11. [Google Scholar] [CrossRef] [PubMed]
  14. Husain, F.M.; Ahmad, I.; Khan, M.S.; Ahmad, E.; Tahseen, Q.; Khan, M.S.; Alshabib, N.A. Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria. Front. Microbiol. 2015, 6, 420. [Google Scholar] [CrossRef] [Green Version]
  15. Elaissi, A.; Rouis, Z.; Salem, N.A.B.; Mabrouk, S.; ben Salem, Y.; Salah, K.B.H.; Aouni, M.; Farhat, F.; Chemli, R.; Harzallah-Skhiri, F.R.; et al. Chemical composition of 8 Eucalyptus species’ essential oils and the evaluation of their antibacterial, antifungal and antiviral activities. BMC Complement. Altern. Med. 2010, 12, 81. [Google Scholar] [CrossRef] [Green Version]
  16. Elaissi, A.; Salah, K.H.; Mabrouk, S.; Larbi, K.M.; Chemli, R.; Harzallah-Skhiri, F. Antibacterial activity and chemical composition of 20 Eucalyptus species’ essential oils. Food Chem. 2011, 129, 1427–1434. [Google Scholar] [CrossRef]
  17. Yong, W.T.L.; Ades, P.K.; Goodger, J.Q.; Bossinger, G.; Runa, F.A.; Sandhu, K.S.; Tibbits, J.F. Using essential oil composition to discriminate between myrtle rust phenotypes in Eucalyptus globulus and Eucalyptus obliqua. Ind. Crops Prod. 2019, 140, 111595. [Google Scholar] [CrossRef]
  18. Jucá, D.M.; da Silva, M.T.B.; Campos Junior, R.; de Lima, F.J.B.; Okoba, W.; Lahlou, S.; Brandt de Oliveira, R.; Aguiar dos Santos, A.; Magalhães, P.J.C. The essential oil of Eucalyptus tereticornis and its constituents, α-and β-pinene, show accelerative properties on rat gastrointestinal transit. Planta Med. 2011, 77, 57–59. [Google Scholar] [CrossRef] [Green Version]
  19. Kaur, S.; Singh, H.P.; Batish, D.R.; Kohli, R.K. Chemical characterization and allelopathic potential of volatile oil of Eucalyptus tereticornis against Amaranthus viridis. J. Plant Interact. 2011, 6, 297–302. [Google Scholar] [CrossRef] [Green Version]
  20. Lima, F.J.; Brito, T.S.; Freire, W.B.; Costa, R.C.; Linhares, M.I.; Sousa, F.C.; Lahlou, S.; Leal-Cardoso, J.H.; Santos, A.A.; Magalhães, P.J. The essential oil of Eucalyptus tereticornis, and its constituents α-and β-pinene potentiate acetylcholine-induced contractions in isolated rat trachea. Fitoterapia 2010, 81, 649–655. [Google Scholar] [CrossRef]
  21. Miguel, M.G.; Gago, C.; Antunes, M.D.; Lagoas, S.; Faleiro, M.L.; Megías, C.; Cortés-Giraldo, I.; Vioque, J.; Figueiredo, A.C. Antibacterial, antioxidant, and antiproliferative activities of Corymbia citriodora and the essential oils of eight Eucalyptus species. Medicines 2018, 5, 61. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Shalinder, K.; Harminder, P.S.; Daizy, R.B.; Ravinder, K.K. Chemical characterization, antioxidant and antifungal activity of essential oil from Eucalyptus tereticornis. J. Med. Plants Res. 2011, 5, 4788–4793. [Google Scholar]
  23. Singh, H.P.; Mittal, S.; Kaur, S.; Batish, D.R.; Kohli, R.K. Characterization and antioxidant activity of essential oils from fresh and decaying leaves of Eucalyptus tereticornis. J. Agric. Food Chem. 2009, 57, 6962–6966. [Google Scholar] [CrossRef] [PubMed]
  24. Vishwakarma, G.S.; Mittal, S. Bioherbicidal potential of essential oil from leaves of Eucalyptus tereticornis against Echinochloa crus-galli L. J. Biopestic. 2014, 7, 47. [Google Scholar]
  25. Assareh, M.H.; Jaimand, K.; Rezaee, M.B. Chemical composition of the essential oils of six Eucalyptus species (Myrtaceae) from south west of Iran. J. Essent. Oil Res. 2007, 19, 8–10. [Google Scholar] [CrossRef]
  26. Safaei-Ghomi, J.; Abbasi-Ahd, A.; Behpour, M.; Batooli, H. Antioxidant activity of the essential oil and metanolic extract of Eucalyptus largiflorens and Eucalyptus intertexta from central Iran. J. Essent. Oil-Bear. Plants 2010, 13, 377–384. [Google Scholar] [CrossRef]
  27. Sefidkon, F.; Assareh, M.H.; Abravesh, Z.; Mirza, M. Chemical composition of the essential oils of five cultivated Eucalyptus species in Iran: E. intertexta, E. platypus, E. leucoxylon, E. sargentii and E. camaldulensis. J. Essent. Oil-Bear. Plants 2006, 9, 245–250. [Google Scholar] [CrossRef]
  28. Sefidkon, F.; Assareh, M.H.; Abravesh, Z.; Kandi, M.N.H. Seasonal variation in the essential oil and 1, 8-cineole content of four Eucalyptus species (E. intertexta, E. platypus, E. leucoxylon and E. camaldulensis). J. Essent. Oil-Bear. Plants 2010, 13, 528–539. [Google Scholar] [CrossRef]
  29. Bignell, C.M.; Dunlop, P.J.; Brophy, J.J. Volatile leaf oils of some south-western and southern Australian species of the genus Eucalyptus. Part XIX. Flavour Fragr. J. 1998, 13, 131–139. [Google Scholar] [CrossRef]
  30. Ghazghazi, H.; Rssghaier, B.; Riguene, H.; Rigane, G.; El Aloiu, M.; Oueslati, M.A.; Ben Salem, R.; Safdi Zouauoi, N.; Naser, Z.; Laarbi Khouja, M. Phytochemical analysis, antioxidant and antimicrobial activities of Eucalyptus essential oil: A comparative study between Eucalyptus marginata L. and Eucalyptus pauciflora L. Rev. Roum. Chim. 2019, 64, 1055–1062. [Google Scholar] [CrossRef]
  31. Li, H.; Madden, J.L.; Potts, B.M. Variation in volatile leaf oils of the Tasmanian Eucalyptus species. Subgenus Monocalyptus. Biochem. Syst. Ecol. 1995, 23, 299–318. [Google Scholar] [CrossRef]
  32. Ibáñez, M.D.; Blázquez, M.A. Phytotoxicity of essential oils on selected weeds: Potential hazard on food crops. Plants 2018, 7, 79. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Vaughn, S.F.; Spencer, G.F. Volatile monoterpenes as potential parent structures for new herbicides. Weed Sci. 1993, 41, 114–119. [Google Scholar] [CrossRef] [Green Version]
  34. Abdelgaleil, S.A.; Abdel-Razeek, N.; Soliman, S.A. Herbicidal activity of three sesquiterpene lactones on wild oat (Avena fatua) and their possible mode of action. Weed Sci. 2009, 57, 6–9. [Google Scholar] [CrossRef]
  35. Kordali, S.; Cakir, A.; Sutay, S. Inhibitory effects of monoterpenes on seed germination and seedling growth. Z. Naturforsch. C 2007, 62, 207–214. [Google Scholar] [CrossRef] [Green Version]
  36. Javaid, A. Herbicidal potential of allelopathic plants and fungi against Parthenium hysterophorus—A review. Allelopathy J. 2010, 25, 331–334. [Google Scholar]
  37. Asplund, R.O. Monoterpenes; relation between structure and inhibition of germination. Phytochemistry 1968, 7, 1995–1997. [Google Scholar] [CrossRef]
  38. De Martino, L.; Mancini, E.; Rolim de Almeida, L.F.; De Feo, V. The antigerminative activity of twenty-seven monoterpenes. Molecules 2010, 15, 6630–6637. [Google Scholar] [CrossRef] [Green Version]
  39. Vokou, D.; Douvli, P.; Blionis, G.J.; Halley, J.M. Effects of monoterpenoids, acting alone or in pairs, on seed germination and subsequent seedling growth. J. Chem. Ecol. 2003, 29, 2281–2301. [Google Scholar] [CrossRef]
  40. Abd-ElGawad, A.M.; El Gendy, A.E.N.G.; Assaeed, A.M.; Al-Rowaily, S.L.; Alharthi, A.S.; Mohamed, T.A.; Nassar, M.I.; Dewir, Y.H.; Elshamy, A.I. Phytotoxic effects of plant essential oils: A systematic review and structure-activity relationship based on chemometric analyses. Plants 2020, 10, 36. [Google Scholar] [CrossRef]
  41. Singh, N.; Singh, H.P.; Batish, D.R.; Kohli, R.K.; Yadav, S.S. Chemical characterization, phytotoxic, and cytotoxic activities of essential oil of Mentha longifolia. Environ. Sci. Pollut. Res. 2020, 27, 13512–13523. [Google Scholar] [CrossRef] [PubMed]
  42. Hamdi, A.; Majouli, K.; Vander Heyden, Y.; Flamini, G.; Marzouk, Z. Phytotoxic activities of essential oils and hydrosols of Haplophyllum tuberculatum. Ind. Crops Prod. 2017, 97, 440–447. [Google Scholar] [CrossRef]
  43. Da Silva, E.R.; Igartuburu, J.M.; Overbeck, G.E.; Goncalves Soares, G.L.; Macias, F.A. Are phytotoxic effects of Eucalyptus saligna (Myrtaceae) essential oil related to its major compounds? Aust. J. Bot. 2021, 69, 174–183. [Google Scholar] [CrossRef]
  44. Zhou, L.; Li, J.; Kong, Q.; Luo, S.; Wang, J.; Feng, S.; Yuan, M.; Chen, T.; Yuan, S.; Ding, C. Chemical composition, antioxidant, antimicrobial, and phytotoxic potential of Eucalyptus grandis × E. urophylla leaves essential oils. Molecules 2021, 26, 1450. [Google Scholar] [CrossRef] [PubMed]
  45. Muller, C.H. The role of chemical inhibitors (allelopathy) in vegetational composition. Bull. Torrey Bot. Club 1966, 93, 332–351. [Google Scholar] [CrossRef]
  46. Romagni, J.G.; Duke, S.O.; Dayan, F.E. Inhibition of plant asparagine synthetase by monoterpene cineoles. Plant Physiol. 2000, 123, 725–732. [Google Scholar] [CrossRef] [Green Version]
  47. Barton, F.M.; Dell, B.; Knight, A.R. Herbicide activity of cineole derivatives. J. Agric. Food Chem. 2010, 58, 10147–10155. [Google Scholar] [CrossRef]
  48. Li, A.; Wu, H.; Feng, Y.; Deng, S.; Hou, A.; Che, F.; Liu, Y.; Geng, Q.; Ni, H.; Wei, Y. A strategy of rapidly screening out herbicidal chemicals from Eucalyptus essential oils. Pest Manag. Sci. 2020, 76, 917–927. [Google Scholar] [CrossRef]
  49. Nazzaro, F.; Fratianni, F.; De Martino, L.; Coppola, R.; De Feo, V. Effect of essential oils on pathogenic bacteria. Pharmaceuticals 2013, 6, 1451–1474. [Google Scholar] [CrossRef]
  50. Council of Europe. European Pharmacopeia, 5th ed.; Council of Europe: Strasbourg Cedex, France, 2004. [Google Scholar]
  51. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Publishing Co.: Carol Stream, IL, USA, 2007. [Google Scholar]
  52. Davies, N.W. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and Carbowax 20M phases. J. Chromatogr. A 1990, 503, 1–24. [Google Scholar] [CrossRef]
  53. Jennings, W.; Shibamoto, T. Qualitative Analysis of Flavour and Fragrance Volatiles by Glass Capillary Gas Chromatography; Academic Press: New York, NY, USA, 1980. [Google Scholar]
  54. McLafferty, F.W. Wiley Registry of Mass Spectral Data, with NIST Spectral Data CD Rom, 7th ed.; John Wiley & Sons: New York, NY, USA, 1998. [Google Scholar]
  55. Smeriglio, A.; Trombetta, D.; Cornara, L.; Valussi, M.; De Feo, V.; Caputo, L. Characterization and phytotoxicity assessment of essential oils from plant byproducts. Molecules 2019, 24, 2941. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Bewley, J.D.; Black, M. Seeds: Physiology of Development and Germination; Plenum Press: New York, NY, USA, 1985. [Google Scholar]
  57. Sarker, S.D.; Nahar, L.; Kumarasamy, Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007, 42, 321–324. [Google Scholar] [CrossRef] [PubMed]
  58. Fratianni, F.; d’Acierno, A.; Ombra, M.N.; Amato, G.; De Feo, V.; Ayala-Zavala, J.F.; Coppola, R.; Nazzaro, F. Fatty acid composition, antioxidant, and in vitro anti-inflammatory activity of five cold-pressed Prunus seed oils, and their anti-biofilm effect against pathogenic bacteria. Front. Nutr. 2021, 8, 775751. [Google Scholar] [CrossRef] [PubMed]
  59. Fratianni, F.; Ombra, M.N.; d’Acierno, A.; Caputo, L.; Amato, G.; De Feo, V.; Coppola, R.; Nazzaro, F. Polyphenols content and in vitro α-glycosidase activity of different Italian monofloral honeys, and their effect on selected pathogenic and probiotic bacteria. Microorganisms 2021, 9, 1694. [Google Scholar] [CrossRef] [PubMed]
Plants 11 03017 g001 550
Figure 1. Typical bacterial biofilm after crystal violet staining and before dissolution with acetic acid 20%. Ab: A. baumannii; Ec: E. coli; Lm: L. monocytogenes; Pa: P. aeruginosa; Sa: S. aureus.
Figure 1. Typical bacterial biofilm after crystal violet staining and before dissolution with acetic acid 20%. Ab: A. baumannii; Ec: E. coli; Lm: L. monocytogenes; Pa: P. aeruginosa; Sa: S. aureus.
Plants 11 03017 g001
Plants 11 03017 g002 550
Figure 2. The main constituents of the EOs.
Figure 2. The main constituents of the EOs.
Plants 11 03017 g002
Table
Table 1. Chemical composition (%) of the EOs.
Table 1. Chemical composition (%) of the EOs.
Compound NameE. bicostataE. giganteaE. intertexaE. obliquaE. paucifloraE. tereticornisKi aKi bIdentification c
1α-Pinene1.7 ± 0.11.6 ± 0.16.7 ± 0.313.2 ± 0.40.2 ± 0.0-86710121,2,3
2Camphenet-0.1 ± 0.00.2 ± 0.0--87610751,2,3
3β-Pinenet0.3 ± 0.00.2 ± 0.11.7 ± 0.20.1 ± 0.1-90211101,2,3
4α-Phellandrene--0.1 ± 0.10.7 ± 0.12.2 ± 0.21.1 ± 0.293011771,2,3
5α-Terpinene-0.1 ± 0.0-0.1 ± 0.00.7 ± 0.20.2± 0.094211701,2,3
6p-Cymene---1.4 0 ± 017.6 ± 0.34.6 ± 0.395212501,2,3
7β-Phellandrene----2.8 ± 0.2-95411891,2,3
8Eucalyptol85.5 ± 0.659.3 ± 0.565.9 ± 0.554.9 ± 0.52.0 ± 0.02.4 ± 0.195812101,2,3
9(E)-β-Ocimene-0.5 ± 0.0----96812421,2,3
10(Z)-β-Ocimene----0.1 ± 0.0-977-1,2,3
11p-Mentha-2,4(8)-diene---0.8 ± 0.0--983-1,2
12γ-Terpinene-1.3 ± 0.1---0.1 ± 0.0-98412211,2,3
13cis-Sabinenehydrate-0.2 ± 0.0----99711151,2
14Terpinolene-0.5 ± 0.10.1 ± 0.00.3 ± 0.00.3 ± 0.00.2 ± 0.0100812671,2,3
156-Camphenone--0.1 ± 0.0---1011-1,2
16Linalool-0.5 ± 0.1----102415061,2,3
17endo-Fenchol----4.4 ± 0.5-1025-1,2
183-Methylbutyl 3-methylbutanoate--0.2 ± 0.00.2 ± 0.0--102912851,2
19exo-Fenchol0.1-0.3 ± 0.00.4 ± 0.0--103115911,2
20trans-p-Mentha-2,8-dien-1-ol---0.1 ± 0.0--104016391,2
21cis-p-Menth-2-en-1-ol----2.9 ± 0.60.2 ± 0.01041-1,2
22α-Campholenal- 0.1 ± 0.00.2 ± 0.0--104314851,2
23allo-Ocimene-1.5 ± 0.00.1 ± 0.20.3 ± 0.0--105113881,2,3
24trans-Pinocarveol2.5± 0.20.5± 0.14.0 ± 0.23.3 ± 0.2-0.1 ± 0.0105716641,2
25cis-β-Terpineol-----0.1 ± 0.01058-1,2
26cis-Verbenol---0.4 ± 0.0--105816651,2
27Camphor--0.1 ± 0.0---105914911,2,3
28Citronellal--0.1 ± 0.0---106314871,2
29Sabina ketone-0.4 ± 0.00.1 ± 0.00.5 ± 0.0--106716511,2
30trans-Pinocamphone--t0.1 ± 0.0--1074-1,2
31Pinocarvone1.6 ± 0.2-1.1 ± 0.10.7 ± 0.0--107715861,2
32Borneol0.2 ± 0.00.4 ± 0.00.3 ± 0.00.6 ± 0.0--108217151,2,3
33p-Mentha-1,5-dien-8-ol--0.2 ± 0.0---108716701,2
34Terpinen-4-ol-1.3 ± 0.10.5 ± 0.0-3.1 ± 021.7 ± 0.2109515901,2,3
35(E)-iso-Citral----0.6 ± 0.00.9 ± 0.01099-1,2
36cis-Pinocarveol--0.3 ± 0.00.2 ± 0.0--1099-1,2
37trans-Isocarveol0.4 ± 0.0-----109918101,2
38cis-Dihydrocarvone---0.8 ± 0.1-0.2 ± 0.01101-1,2
39Dihydrocarveol0.5 ± 0.0-0.2 ± 0.03.2 ± 0.2--1102-1,2
40Cryptone-0.4 ± 0.0 ----110316591,2
41cis-Piperitol----1.1 ± 0.0-110617581,2
42Myrtenol0.2 ± 0.0-0.3 ± 0.0---110717911,2
43α-Terpineol0.1 ± 0.04.5 ± 0.20.9 ± 0.11.2 ± 0.11.1 ± 0.10.3 ± 0.0111016611,2,3
44Safranal---0.3 ± 0.0--111716481,2
45trans-Piperitol----1.5 ± 0.1-112016901,2
46cis-4-Caranone---0.1 ± 0.0--1134-1,2
47cis-Carveol--0.1 ± 0.0---113518481,2
48Verbenone---0.2 ± 0.0--114317261,2
49cis-p-Mentha-1(7),8-dien-2-ol0.3 ± 0.00.2 ± 0.00.3 ± 0.0---114418961,2
50Cuminaldehyde---0.4 ± 0.0--114917531,2
51Carvone---0.2 ± 0.0--115617361,2
52exo-Fenchyl acetate--0.2 ± 0.0---1158-1,2
53Piperitone-0.9 ± 0.1-0.1 ± 0.08.8 ± 0.319.4 ± 0.5116617481,2
54α-Terpinen-7-al-0.4 ± 0.0----119818111,2
55Thymol-0.3 ± 0.0-0.7 ± 0.1--121821721,2,3
56γ-Terpinen-7-al---0.1 ± 0.0--1236-1,2
57δ-Elemene----0.7 ± 0.12.2 ± 0.1123714791,2,3
58trans-Verbenyl acetate---0.2 ± 0.0--1244-1,2
596-camphenol acetate--0.1 ± 0.0---1245-1,2
60p-Menth-1-en-9-ol---0.4 ± 0.0--1252-1,2
61Copaene-0.1 ± 0.0----126514771,2,3
62α-Cubebene-----0.1 ± 0.0127014421,2
63β-Elemene----0.1 ± 0.00.2 ± 0.01290-1,2,3
64β-Longipinene----0.1 ± 0.00.3 ± 0.01298-1,2
65α-Gurjunene-0.5 ± 0.01.1 ± 0.1---130015351,2
66α-Caryophyllene0.1 ± 0.0-----130716171,2
67(Z)-Caryophyllene--0.2 ± 0.0---130816171,2
68Germacrene D---0.2 ± 0.0--132717121,2
69Longifolene----0.8 ± 0.12.6 ± 0.2132815741,2
70Aromadendrene0.8 ± 0.0-0.2± 0.0-0.6 ± 0.02.9 ± 0.3134816311,2
71allo-Aromadendrene0.2 ± 0.0---0.1 ± 0.0-134916601,2
72(E)-Caryophyllene-0.2 ± 0.0----135516121,2
73α-Himachalene----0.3 ± 0.0-1366-1,2
749-epi-(E)-Caryophyllene-0.7--0.4 ± 0.00.8 ± 0.11376-1,2
75cis-β-Guaiene---0.3 ± 0.0--1383-1,2
76γ-Gurjunene-0.1 ± 0.00.4 ± 0.0-1.2 ± 0.12.8 ± 0.21384-1,2
77α-Vetispirene-----0.4 ± 0.01401-1,2
78γ-Amorphene-----0.11408-1,2
79epi-Cubebol-0.8 ± 0.0 -0.2 ± 0.0--142619571,2
80γ-Patchoulene----6.3 ± 0.24.2 ± 0.21438-1,2
81Cubebol--1.2 ± 0.1---1441-1,2
82Viridiflorene-0.7 ± 0.00.1 ± 0.0 0.2 ± 0.00.2 ± 0.01.2 ± 0.11448-1,2
83trans-β-Guaiene--0.5 ± 0.0-2.8 ± 0.31.0 ± 0.01449-1,2
84β-Vetivenene----8.817.31463-1,2
85Viridiflorol1.8--0.4 ± 0.0--146421101,2
86Globulol0.4-0.9 ± 0.01.2 ± 0.0--146621041,2
87Spathulenol-11.8 ± 0.58.1 ± 0.33.8 ± 0.3--146821271,2
88Cubeban-11-ol-0.5 ± 0.00.3 ± 0.00.2 ± 0.0--1476-1,2
89trans-Dauca-4(11),7-diene----6.4 ± 0.117.9 ± 0.31477-1,2
90Guaiol----2.5 ± 0.1-147820941,2
91Rosifoliol--0.5 ± 0.00.5 ± 0.0--1483-1,2
92α-epi-7-epi-5-Eudesmol -1.0 ± 0.0--2.7 ± 0.14.2 ± 0.11485-1,2
93allo-Aromadendreneepoxide--0.2 ± 0.0---1496-1,2
94epi-Cedrol 0.4 ± 0.0--1.5 ± 0.1 2.6 ± 0.11497-1,2
95α-Cadinol--0.2 ± 0.0---150222241,2
96γ-Eudesmol-0.4 ± 0.0-0.5 ± 0.04.7 ± 0.12.0 ± 0.1150821781,2
9714-hydroxy-(Z)-Caryophyllene--0.1 ± 0.0---1513-1,2
98cis-Cadin-4-en-7-ol-0.8 ± 0.0--1.3 ± 0.1-1515-1,2
99β-Eudesmol0.30.8 ± 0.00.4 ± 0.0-8.1 ± 0.52.2 ± 0.1152722481,2
100α-Eudesmol-2.0 ± 0.00.3 ± 0.00.3 ± 0.06.3 ± 0.4-153022471,2
1015-Hydroxy-isobornyl isobutanoate-0.1 ± 0.0-0.1 ± 0.0--1540-1,2
102Vulgarone B-----0.4 ± 0.01543-1,2
Total96.796.097.496.195.596.8
Monoterpene hydrocarbons1.75.87.318.714.06.1
Oxygenated monoterpenes91.468.975.068.625.525.3
Sesquiterpene hydrocarbons1.12.32.50.728.854.0
Oxygenated sesquiterpenes2.518.512.27.127.111.4
a,b The Kovats retention indices determined relative to a series of n-alkanes (C10–C35) on the apolar HP-5 MS and the polar HP Innowax capillary columns, respectively. c Identification method: 1 = comparison of the Kovats retention indices with published data, 2 = comparison of mass spectra with those listed in the NIST 02 and Wiley 275 libraries and with published data, and 3 = co-injection with authentic compounds; t = trace (<0.1%). - = absent.
Table
Table 2. Phytotoxic activity of the EOs on R. sativus.
Table 2. Phytotoxic activity of the EOs on R. sativus.
Germinated Seeds
E. bicostataE. giganteaE. intertextaE. obliquaE.paucifloraE. tereticornis
Control (H2O)8.7 ± 1.29.7 ± 0.68.3 ±0.610.0 ± 0.09.3 ± 0.69.9 ± 3.5
125 µg/mL2.0 ± 1.0 ****5.7 ± 0.6 ****4.0 ± 1.7 *8.3 ± 1.26.7 ± 1.5 ****6.9 ± 1.5
250 µg/mL1.7 ± 2.1 ****4.7 ± 0.6 ****6.3 ± 1.57.7 ± 0.6 *4.7 ± 0.6 ****3.0 ± 1.0
500 µg/mL1.0 ± 1.0 ****1.3 ± 0.6 ****3.0 ± 2.0 ***7.3 ± 1.2 *1.3 ±0.6 ****3.0 ± 1.0
1000 µg/mL0.7 ± 0.6 ****0.7 ± 0.6 ****0.0 ± 0.0 ****6.0 ± 1.0 ****0.0 ± 0.0 ****0.0 ± 0.0 *
Radical Length (cm)
E. bicostataE. giganteaE. intertextaE. obliquaE.paucifloraE. tereticornis
Control (H2O)3.5 ± 0.43.1 ± 0.32.5 ± 1.34.8 ± 0.32.6 ± 0.11.4 ± 1.2
125 µg/mL2.5 ± 1.52.5 ± 0.10.9 ± 0.3 *3.8 ± 0.41.7 ± 0.1 ****1.8 ± 0.7
250 µg/mL1.3 ± 1.4 *2.1 ±0.2 **0.9 ± 0.1 *3.8 ± 0.41.2 ± 0.1 ****1.4 ± 1.3
500 µg/mL1.1 ± 0.9 **1.4 ± 0.3 ****0.5 ± 0.1 **3.3 ± 1.21.3 ± 0.2 ****0.4 ± 0.3
1000 µg/mL0.8 ± 0.7 **1.0 ± 0.9 ****0.0 ± 0.0 ****0.8 ± 0.30.0 ± 0.0 ****0.0 ± 0.0 *
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
Table 3. Phytotoxic activity of the EOs on S. arvensis.
Table 3. Phytotoxic activity of the EOs on S. arvensis.
Germinated Seeds
E. bicostataE. giganteaE. intertextaE. obliquaE.paucifloraE. tereticornis
Control (H2O)10.0 ± 0.09.3 ± 0.69.7 ± 0.610.0 ± 0.09.7 ± 0.69.7 ± 0.6
125 µg/mL8.3 ± 0.68.0 ± 1.09.0 ± 1.01.7 ± 0.6 ****7.7 ± 0.6 **4.7 ± 1.5 ***
250 µg/mL6.0 ± 2.6 *4.3 ± 1.2 ****4.0 ± 0.4 ***1.7 ± 0.6 ****5.0 ± 0.0 ****0.7 ± 0.6 ****
500 µg/mL0.0 ± 0.0 ****1.0 ± 0.0 ****3.0 ± 2.6 ****0.0 ± 0.0 ****1.3 ± 0.6 ****0.0 ± 0.0 ****
1000 µg/mL0.0 ± 0.0 ****0.3 ± 0.6 ****0.0 ± 0.0 ****0.0 ± 0.0 ****1.0 ± 0.0 ****0.0 ± 0.0 ****
Radical Length (cm)
E. bicostataE. giganteaE. intertextaE. obliquaE.paucifloraE. tereticornis
Control (H2O)2.8 ± 0.25.6 ± 0.33.3 ± 0.62.5 ± 0.13.7 ± 0.23.0 ± 0.7
125 µg/mL1.1 ± 0.21.1 ± 0.3 ****2.1 ± 0.70.7 ± 0.2 ****1.4 ± 0.2 ****0.4 ± 0.1 ****
250 µg/mL1.0 ± 0.41.1 ± 0.2 ****0.5 ± 0.2 ****0.5 ± 0.1 ****1.0 ± 0.3 ****0.2 ± 0.1 ****
500 µg/mL0.0 ± 0.0 ***0.5 ± 0.2 ****0.6 ± 0.5 ****0.0 ± 0.0 ****1.6 ± 0.1 ****0.0 ± 0.0 ****
1000 µg/mL0.0 ± 0.0 ***0.1 ± 0.2 ****0.0 ± 0.0 ****0.0 ± 0.0 ****0.7 ± 0.1 ****0.0 ± 0.0 ****
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
Table 4. Phytotoxic activity of the EOs on L. multiflorum.
Table 4. Phytotoxic activity of the EOs on L. multiflorum.
Germinated Seeds
E. bicostataE. giganteaE. intertextaE. obliquaE. paucifloraE. tereticornis
Control (H2O)9.0 ± 1.08.3 ± 0.610.0 ± 0.010.0 ± 0.09.7 ± 0.67.7 ± 0.6
125 µg/mL8.7 ± 0.69.3 ± 0.68.7 ± 0.69.3 ± 0.69.7 ± 0.67.7 ± 1.2
250 µg/mL8.3 ± 1.59.0 ± 0.06.3 ± 1.5 *8.7 ± 0.69.0 ± 0.08.0 ± 1.0
500 µg/mL6.0 ± 1.07.3 ± 0.67.0 ± 0.07.7 ± 0.6 *6.0 ± 1.0 ****5.3 ± 1.5
1000 µg/mL4.7 ± 4.2 *6.7 ± 0.6 *5.0 ± 1.0 **6.0 ± 3.0 ****0.0 ± 0.0 ****0.0 ± 0.0 ****
Radical length (cm)
E. bicostataE. giganteaE. intertextaE. obliquaE. paucifloraE. tereticornis
Control (H2O)3.6 ± 0.45.1 ± 0.54.4 ± 0.13.0 ± 0.34.8 ± 0.23.3 ± 0.6
125 µg/mL3.1 ± 1.02.9 ± 0.3 ****3.3 ± 0.41.6 ± 0.2 ***2.7 ± 0.3 ****2.2 ± 0.6
250 µg/mL2.1 ± 0.82.7 ± 0.3 ****2.8 ± 0.2 *1.3 ± 0.2 ****2.6 ± 0.2 ****2.3 ± 0.2
500 µg/mL2.0 ± 0.11.8 ± 0.1 ****1.8 ± 0.7 ****0.9 ± 0.2 ****1.8 ± 0.2 ****1.2 ± 0.4 ***
1000 µg/mL1.4 ± 1.5 *0.6 ± 0.1 ****0.5 ± 0.1 ****0.4 ± 0.1 ****0.7 ± 0.1 ****0.0 ± 0.0 ****
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
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.
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.
EOA. baumanniiE. coliL. monocytogenesP. aeruginosaS. aureus
E. bicostata30 ± 225 ± 228 ± 2 *30 ± 228 ± 2 ***
E. gigantea25 ± 2 **23 ± 125 ± 2 ***28 ± 2 **25 ± 3 ***
E. intertexta35 ± 242 ± 1 ***25 ± 2 ***28 ± 1 **28 ± 2 ***
E. obliqua33 ± 242 ± 1 ***28 ± 2 *30 ± 228 ± 3 ***
E. pauciflora25 ± 2 **35 ± 3 ***25 ± 2 ***30 ± 235 ± 3
E. teritcornis33 ± 328 ± 125 ± 2 ***28 ± 3 **30 ± 3 ***
Tetracycline31 ± 224 ± 233 ± 134 ± 138 ± 1
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.
Table
Table 6. Percent inhibition of two doses of the EOs on biofilm formation of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus at 0 and 24 h.
Table 6. Percent inhibition of two doses of the EOs on biofilm formation of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus at 0 and 24 h.
Time 0A. baumanniiE. coliL. monocytogenesP. aeruginosaS. aureus
E. bicostata 10 µL/mL0.00 ± 0.000.00 ± 0.0054.34 ± 1.25 ****0.00 ± 0.000.00 ± 0.00
E. bicostata 20 µL/mL28.74 ± 1.8 ****79.61 ± 1.06 ****65.62 ± 0.31 ****58.74 ± 2.75 ****72.55 ± 0.40 ****
E. gigantea 10 µL/mL79.71 ± 0.14 ****79.22 ± 0.06 ****82.67 ± 0.10 ****78.76 ± 0.07 ****77.51 ± 0.11 ****
E. gigantea 20 µL/mL89.34 ± 0.33 ****85.70 ± 0.10 ****85.11 ± 0.16 ****79.69 ± 0.08 ****81.61 ± 0.19 ****
E. intertexta 10 µL/mL5.99 ± 0.37 ****0.00 ± 0.000.00 ± 0.0075.97 * ± 0.17 ****76.20 ± 0.09 ****
E. intertexta 20 µL/mL63.45 ± 0.21 *0.00 ±0.0080.32 ± 0.13 ****74.70 ± 0.35 ****76.47 ± 0.18 ****
E. obliqua 10 µL/mL2.96 ± 0.31 ***0.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.00
E. obliqua 20 µL/mL61.50 ± 0.28 ****0.00 ± 0.0070.02 ± 0.16 ****60.21 ± 0.19 ****70.42 ± 0.30 ****
E. pauciflora 10 µL/mL83.92 ± 0.04 ****20.54 ± 1.04 ****0.00 ± 0.000.00 ± 0.002.02 ± 0.84 ****
E. pauciflora 20 µL/mL86.73 ± 0.02 ****46.93 ± 0.23 ****79.47 ± 1.35 ****13.79 ± 0.83 ****31.08 ± 5.81 ****
E. tereticornis 10 µL/mL5.89 ± 1.46 ****0.00 ± 0.0049.84 ± 1.00 ****74.17 ± 0.34 ****5.46 ± 2.30 ****
E. tereticornis 20 µL/mL58.73 ± 0.42 ****69.57 ± 0.33 ****83.72 ± 0.45 ****70.90 ± 0.48 ****71.80 ± 0.15 ****
Time 24 hA. baumanniiE. coliL. monocytogenesP. aeruginosaS. aureus
E. bicostata 10 µL/mL62.99 ± 0.91 ****34.13 ± 0.71 ****56.63 ± 0.84 ****51.18 ± 0.45 ****46.46 ± 0.79 ****
E. bicostata 20 µL/mL73.24 ± 0.29 ****55.13 ± 1.07 ****62.82 ± 0.80 ****85.06 ± 1.92 ****73.38 ± 0.33 ****
E. gigantea 10 µL/mL40.81 ± 0.66 ****0.00 ± 0.0047.01 ± 0.45 ****66.55 ± 0.84 ****62.91 ± 0.44 ****
E. gigantea 20 µL/mL51.94 ± 1.15 ****28.62 ± 0.20 ****55.44 ± 1.34 ****69.79 ± 0.57 ****64.78 ± 0.43 ****
E. intertexta 10 µL/mL32.44 ± 0.68 ****40.35 ± 1.53 ****67.75 ± 0.5256.20 ± 0.9952.42 ± 0.23 ****
E. intertexta 20 µL/mL27.92 ± 1.01 ****19.04 ± 0.68 ****0.00 ± 0.0036.66 ± 0.77 ****67.30 ± 0.25 ****
E. obliqua 10 µL/mL23.17 ± 0.96 ****39.82 ± 1.14 ****0.00 ± 0.007.94 ± 0.74 ****77.38 ± 0.45 ****
E. obliqua 20 µL/mL34.96 ± 0.68 ****42.56 ± 0.25 ****14.52 ± 0.94 ****29.79 ± 0.55 ****83.63 ± 0.47 ****
E. pauciflora 10 µL/mL9.71 ± 0.89 ****28.60 ± 0.74 ****0.00 ± 0.0047.97 ± 1.01 ****49.93 ± 0.54 ****
E. pauciflora 20 µL/mL32.44 ± 1.21 ****40.35 ± 1.22 ****67.75 ± 0.32 ****56.20 ± 0.68 ***52.42 ± 0.65 ****
E. tereticornis 10 µL/mL10.99 ± 2.218.09 ± 3.84 ****38.86 ± 2.88 ****13.95 ± 1.33 ****0.00 ± 0.00
E. tereticornis 20 µL/mL43.88 ± 1.4533.75 ± 2.06 ****68.62 ± 2.36 ****14.10 ± 1.38 ****24.08 * ± 1.18
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.
Table
Table 7. Percent inhibition of two doses of the EOs on biofilm metabolic activity of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus at 0 and 24 h.
Table 7. Percent inhibition of two doses of the EOs on biofilm metabolic activity of A. baumannii, E. coli, L. monocytogenes, P. aeruginosa, and S. aureus at 0 and 24 h.
Time 0A. baumanniiE. coliL. monocytogenesP. aeruginosaS. aureus
E. bicostata 10 µL/mL58.01 ± 0.95 ****0.00 ± 0.000.00 ± 0.000.00 ± 0.0054.96 ± 0.48 ****
E. bicostata 20 µL/mL60.88 ± 0.70 ****0.00 ± 0.005.15 ± 0.77 **34.26 ± 7.33 ****69.26 ± 1.24 ****
E. gigantea 10 µL/mL83.35 ± 2.78 ****75.29 ± 1.34 ****80.04 ± 1.64 ****79.71 ± 2.04 ****79.03 ± 1.56 ****
E. gigantea 20 µL/mL85.12 ± 2.88 ****78.23 ± 1.66 ****83.22 ± 1.14 ****81.93 ± 2.19 ****81.77 ± 1.16 ****
E. intertexta 10 µL/mL0.00 ± 0.000.00 ± 0.0079.14 ± 2.05 ****75.88 ± 2.59 ****78.51 ± 1.58 ****
E. intertexta 20 µL/mL69.08 ± 0.35 ****0.00 ± 0.0079.48 ± 1.70 ****78.81 ± 1.78 ****80.62 ± 0.84 ****
E. obliqua 10 µL/mL52.84 ± 1.06 ****20.14 ± 1.41 ****20.67 ± 2.98 ****1.06 ± 1.1711.91 ± 1.37 ****
E. obliqua 20 µL/mL57.60 ± 0.98 ****63.66 ± 1.02 ****75.41 ± 1.25 ****74.42 ± 0.33 ****55.43 ± 1.41 ****
E. pauciflora 10 µL/mL41.05 ± 1.10 ****0.00 ± 0.000.00 ± 0.000.00 ± 0.0057.44 ± 0.81 ****
E. pauciflora 20 µL/mL55.18 ± 0.78 ****7.70 ± 1.36 ****44.69 ± 1.82 ****14.36 ± 2.07 ****72.03 ± 1.74 ****
E. tereticornis 10 µL/mL0.00 ± 0.000.00 ± 0.0071.13 ± 0.32 ****0.00 ± 0.000.62 ± 1.68
E. tereticornis 20 µL/mL35.63 ± 0.67 ****62.65 ± 0.40 ****88.95 ± 0.25 ****83.06 ± 1.60 ****85.38 ± 0.18 ****
Time 24 h
E. bicostata 10 µL/mL4.66 ± 3.38 ****33.52 ± 0.39 ****29.90 ± 1.09 ****0.00 ± 0.0030.88 ± 1.52 ****
E. bicostata 20 µL/mL38.75 ± 1.19 ****51.34 ± 0.92 ****54.04 ± 0.88 ****7.08 ± 0.44 ****33.22 ± 0.52 ****
E. gigantea 10 µL/mL0.00 ± 0.0020.59 ± 0.54 ****0.00 ± 0.000.00 ± 0.007.95 ± 0.90 ****
E. gigantea 20 µL/mL0.00 ± 0.0056.68 ± 0.38 ****0.00 ± 0.000.00 ± 0.0021.38 ± 0.64 ****
E. intertexta 10 µL/mL0.00 ± 0.0031.89 ± 1.35 ****0.00 ± 0.0033.95 ± 0.59 ****11.46 ± 0.60 ****
E. intertexta 20 µL/mL12.96 ± 1.72 ****34.32 ± 1.03 ****0.00 ± 0.000.00 ± 0.0018.77 ± 1.13 ****
E. obliqua 10 µL/mL0.00 ± 0.0010.32 ± 0.99 ****0.00 ± 0.000.00 ± 0.0018.43 ± 1.34 ****
E. obliqua 20 µL/mL19.87 ± 1.85 ****27.08 ± 1.03 ****0.00 ± 0.009.73 ± 0.61 ****30.54 ± 1.11 ****
E. pauciflora 10 µL/mL0.00 ± 0.0010.32 ± 0.94 ****0.00 ± 0.000.00 ± 0.0018.43 ± 1.11 ****
E. pauciflora 20 µL/mL88.39 ± 1.07 ****27.08 ± 0.82 ****0.00 ± 0.009.73 ± 0.71 ****30.54 ± 0.81 ****
E. tereticornis 10 µL/mL22.79 ± 1.12 ****0.00 ± 0.0026.68 ± 1.03 ****44.15 ± 0.12 ****25.62 ± 0.35 ****
E. tereticornis 20 µL/mL34.69 ± 0.23 ****65.92 ± 0.87 ****29.26 ± 0.06 ****45.63 ± 0.28 ****64.24 ± 1.15 ****
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.
Table
Table 8. Data on plant material, yields, place of origin and climatic conditions.
Table 8. Data on plant material, yields, place of origin and climatic conditions.
Arboretum
(Governorate)		Harvest PeriodBioclimatic
Conditions		Yield (%)
E. bicostataChoucha
(Bizerte)		March 2021Upper humid1.40
E. giganteaZerniza
(Bizerte)		July 2021Upper humid0.20
E. intertextaDjebel Manasour (Zaghouen)May 2021Upper and middle semi-arid0.55
E. obliquaHenchirNaam
(Siliana)		April 2021Upper and middle semi-arid3.11
E. paucifloraZerniza
(Bizerte)		July 2021Upper humid0.10
E. tereticornisZerniza
(Bizerte)		July 2021Upper humid0.03
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Polito, F.; Kouki, H.; Khedhri, S.; Hamrouni, L.; Mabrouk, Y.; Amri, I.; Nazzaro, F.; Fratianni, F.; De Feo, V. 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. Plants 2022, 11, 3017. https://doi.org/10.3390/plants11223017

AMA Style

Polito F, Kouki H, Khedhri S, Hamrouni L, Mabrouk Y, Amri I, Nazzaro F, Fratianni F, De Feo V. 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. Plants. 2022; 11(22):3017. https://doi.org/10.3390/plants11223017

Chicago/Turabian Style

Polito, Flavio, Habiba Kouki, Sana Khedhri, Lamia Hamrouni, Yassine Mabrouk, Ismail Amri, Filomena Nazzaro, Florinda Fratianni, and Vincenzo De Feo. 2022. "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" Plants 11, no. 22: 3017. https://doi.org/10.3390/plants11223017

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop