Next Article in Journal
Estimating Sensory Properties with Near-Infrared Spectroscopy: A Tool for Quality Control and Breeding of ‘Calçots’ (Allium cepa L.)
Previous Article in Journal
Enhancing Cation Exchange Capacity of Weathered Soils Using Biochar: Feedstock, Pyrolysis Conditions and Addition Rate
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 10
Issue 6
10.3390/agronomy10060825
agronomy-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

Antifungal and Phytotoxic Activities of Essential Oils: In Vitro Assays and Their Potential Use in Crop Protection

by
Imad El-Alam
1,2,3,†,
Robin Raveau
1,†,
Joël Fontaine
1,
Anthony Verdin
1,
Frédéric Laruelle
1,
Sophie Fourmentin
1,
Ramez Chahine
2,3,
Hassane Makhlouf
2,4 and
Anissa Lounès-Hadj Sahraoui
1,*
1
University of Littoral Côte d’Opale, Unit of Environmental Chemistry and Interactions with Living Organisms, UR 4492–UCEIV, SFR Condorcet FR CNRS 3417, F-62228 Calais CEDEX, France
2
Oxidative Stress and Antioxidants Group, Doctoral School of Sciences and Technologies, Lebanese University, Hadath B.P. 1500, Lebanon
3
Faculty of Public Health, Sagesse University, Furn-El-Chebak, Beirut P.O. Box 50501, Lebanon
4
Georesources, Geosciences and Environment Laboratory, Faculty of Sciences, Lebanese University, Fanar Matn, Beirut P.O. Box 90656, Lebanon
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to the present work.
Agronomy 2020, 10(6), 825; https://doi.org/10.3390/agronomy10060825
Submission received: 2 May 2020 / Revised: 27 May 2020 / Accepted: 8 June 2020 / Published: 11 June 2020
(This article belongs to the Section Pest and Disease Management)
Browse Figures
Versions Notes

Abstract

:
(1) Background: The use of natural products based on essential oils (EO) is nowadays arousing great interest as an alternative method to control plant pathogens and weeds. However, EO possess low bioavailability and are highly volatile, and their encapsulation in hydroxypropyl-ß-cyclodextrin (HP-β-CD) could be a means to enhance their stability and maintain their bioactivity. Thus, the current study aims at investigating, in the presence and the absence of HP-β-CD, the antifungal and phytotoxic activities of nine EO, distilled from plant species belonging to Alliaceae, Apiaceae, and Cupressaceae families, with considerations for their chemical composition. (2) Methods: EO antifungal activity was assessed by direct contact and volatility assays against Fusarium culmorum, a major phytopathogenic fungi, while phytotoxic effects were evaluated against lettuce (Lactuca sativa L.) and rye-grass (Lolium perenne L.), by seedling’s emergence and growth assays. (3) Results: These EO inhibit fungal growth in both direct contact and volatility assays, with half-maximal inhibitory concentrations (IC50) ranging from 0.01 to 4.2 g L−1, and from 0.08 up to 25.6 g L−1, respectively. Concerning phytotoxicity, these EO have shown great potential in inhibiting lettuce (IC50 ranging from 0.0008 up to 0.3 g L−1) and rye-grass (IC50 ranging from 0.01 to 0.8 g L−1) seedlings’ emergence and growth. However, the EO encapsulation in HP-β-CD has not shown a significant improvement in EO biological properties in our experimental conditions. (4) Conclusion: All tested EO present antifungal and phytotoxic activities, with diverse efficacy regarding their chemical composition, whilst no increase of their biological effects was observed with HP-β-CD.

Graphical Abstract

1. Introduction

In recent years, there has been a clear tendency towards the utilization of alternative methods in agriculture for pest and disease control, food preservation, and weed control that are less damaging to the environment and human health. Among these methods, the use of essential oils (EO) is arousing great interest for their bactericidal, virucidal, fungicidal, antiparasitical and insecticidal applications [1,2]. EO are indeed complex oily volatile liquids characterized by a strong odor and synthetized by all aromatic plant organs (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and root) as secondary metabolites and stored in secretory cells, cavities, canals, epidermic cells or glandular trichomes. They can contain 20 to 60 active compounds, responsible for their biological activities [3].
Both in the fields and during postharvest storage, cereals, fruits, and vegetables are highly susceptible to fungal spoilage. Food safety from spoilers and pathogens is an important worldwide public health concern, and microbial contamination is a vital factor not only causing food deterioration and shelf life reduction, but also resulting in economic losses and diseases [4,5]. Furthermore, weeds are another major biological constraint in agriculture. Indeed, these plants compete with crops for natural resources and space and result in reduced crop yields and productivity. The control of both fungal diseases and weeds is currently achieved through various methods, with a predominance of synthetic pesticides. However, the inappropriate or overuse of pesticides is known to result in environmental issues (groundwater and soil contamination, presence of toxic residues in agricultural products), noxious effects on human health, as well as leading to the development of herbicide resistance in weeds [6,7,8]. So as to overcome these issues, EO-based products may represent a natural alternative to replace synthetic fungicides and herbicides and stand as potential bio-pesticides [3,9]. Nevertheless, EO have low aqueous solubility and bioavailability, which could be challenging for biological application. This drawback could be overcome by the use of cyclodextrins (CD), which are cyclic oligosaccharides possessing a hydrophilic surface and a hydrophobic cavity that allows the encapsulation of hydrophobic guests in aqueous solutions [10,11]. Hydroxypropyl-ß-cyclodextrin (HP-β-CD-degree of substitution 5.6), a β-cyclodextrin derivative, is cited in the Food and Drug Administration’s list of Inactive Pharmaceutical Ingredients [12]. In that matter, encapsulation in CD may be of interest to formulate oily substances into solid dosage, or to replace organic solvents or surfactants in liquid formulations [10], to avoid degradation and obtain a better persistence of EO’s effects in time [13,14]. Notably, the encapsulation in CD of EO coming from clove (Eugenia caryophyllata L. Merr. and Perry) and Mexican oregano (Lippia berlandieri Schauer) was of interest in enhancing the antifungal activity against Fusarium oxysporum [15]. Similarly, encapsulated cinnamon leaf (Cinnamon verum J. Pres) and oregano (Origanum vulgare L.) EO [16], as well as EO derivatives coming from cinnamon and clove, displayed higher efficiency against Botrytis sp. [17]. On another note, the inclusion of EO distilled from peppermint (Mentha × piperita L.), caraway (Carum carvi L.) and calamus (Acorus calamus L.) in CD exerted phytotoxic properties against maize (Zea mays L.), barnyard grass (Echinochloa crus-galli (L.) P. Beauv.) and lambsquarters (Chenopodium album L.) [18].
Among fungal phytopathogens, Fusarium genus is wildly studied not only for causing plant diseases such as Fusarium head blight and Fusarium crown rot–two major diseases occurring on wheat and responsible for considerable yield loss [19,20]–but also for producing highly toxic secondary metabolites, mycotoxins, that lead to the contamination of the grain harvested from infected ears and cause potential risk to human and animal health [21,22,23]. Most of the time, EO antimicrobial effects are evaluated by direct contact, with EO directly incorporated in the culture medium. Even so, several references have shown an interest of using volatility assays, where the accuracy of such assays has been demonstrated as more reliable and the efficiency of smaller compounds such as monoterpenes was greater [24,25,26]. In addition, the reliability of direct contact assays is sometimes compromised by the low solubility in agar media of EO compounds or by the addition of solvents [27].
Allium species such as garlic (Allium sativum L.), onion (Allium cepa L.), chive (Allium schoenoprasum L.), leek (Allium porum L.), and shallot (Allium ascalonicium L.) are cultivated and consumed in many Asian and European countries and known for their medicinal properties, especially from their organosulfur compounds [28]. In the same way, Apiaceae such as chervil (Anthriscus cerefolium L.) and angelica (Angelica archangelica L.) are used for flavoring and in folk medicine, while thuja (Thuya plicata atrovirens L.), a Cupressaceae species, is mainly used for cosmetic applications [29,30]. However, despite food and cosmetics applications, little attention has been paid to the biological properties of these EO regarding their potential in crop protection and scarce are the studies targeting phytopathogenic species such as Fusarium culmorum. Moreover, to the best of our knowledge, despite the potential benefits of encapsulation, the biological activities of these EO encapsulated in HP-β-CD have not previously been investigated.
In this way, the present study aims to evaluate in vitro antifungal and phytotoxic activities of the nine EO distilled from different plant species belonging to Alliaceae, Apiaceae, and Cupressaceae families, with concerns for the potential of CD encapsulation.

2. Materials and Methods

2.1. Essential Oils

Essential oils tested in the current study were kindly provided by Ferrant PHE (France), an EO producer company [31]. The purchase of the raw material and the distillation procedure were carried out by the EO producer. Flowering aerial parts of chervil (A. cerefolium) as well as bulbs of chive (A. schoenoprasum), garlic (A. sativum), leek (A. porum), onion (A. cepa) and shallots (A. ascalonicium), harvested at fruit maturity, were used as fresh material, and were purchased by the EO producer from food companies and farmers in the north of France (Hauts-de-France region). Prior to distillation, plant material was mechanically shredded into small pieces by blades, to facilitate the distillation process [28]. Distillation occurred through a water-steam entrainment process, on a Clevenger-type apparatus (3-h cycle, saturated water steam-0.3 bar).
Roots or seeds of angelica (A. archangelica) grown by the producer and harvested at flowering stage, were distilled through a similar process (steam-distillation unit, 14 m3 tank-saturated water steam, 0.3 bar), during a 3-h cycle, until no more EO was recovered. Thuja (T. plicata atrovirens) crushed twigs and fresh leaves were also distilled through the same process. No specific solvent was added in the hydrodistillation process.

2.2. Determination of the EO Chemical Composition

Previously diluted in ethyl acetate (ratio 1:200 (v/v)), EO were analyzed by gas chromatography (using FID as a detector) and by gas chromatography–mass spectrometry (Shimadzu QP 2010 Ultra). Similar operating conditions were employed as follows: the system was operated using helium as a carrier gas at a constant linear velocity (60 cm/s) and 0.2 μL of the sample was injected in a split mode (split ratio 1:10, injector at 260 °C) in a Zebron ZB-5MS (5% phenyl-arylene/95% dimethylpolysiloxane-10 m length, 0.10 mm inner diameter, 0.10 μm phase thickness) fused silica capillary column (Phenomenex). The column temperature was linearly programmed from 60 °C (held for 2 min) to 280 °C (held for 1 min) at a rate of 40 °C/min. The transfer line was heated at 280 °C. Mass spectra were acquired in the electronic impact mode (EI at 70 eV) in an m/z range of 35–350. The identification of the compounds was performed by comparing their retention indices and mass spectra with those found in the literature [32] and supplemented by those listed in the NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) and Wiley 275 computer libraries. The Kovats indices were determined from the retention times after co-injection with n-alkanes. Relative percentages of oil constituents were calculated from the GC peak areas. Analyses were led in triplicates.

2.3. Determination of the Retention Capacity of EO by HP-β-CD

The retention capacity of HP-β-CD was quantified by static headspace-gas chromatography (SH-GC), by comparing the sum of the chromatographic peak areas of EO components in the presence of CD with the blank experiments. This analysis was performed according to [10]. EO were added to 10 mL of either water or aqueous CD solutions, placed in 22 mL headspace glass vials. Vials were then sealed, using silicone septa and aluminum foil, and thermostated at 25 ± 0.1 °C. After the establishment of the equilibrium between aqueous and gaseous phases (30 min), 1 mL of vapor present in the upper part of the vial, above the solution, was withdrawn from the vial by using a gas-tight syringe and injected directly into the chromatographic column via a transfer line (250 °C). All measurements were conducted with a headspace autosampler (Agilent, Les Ulis, France) and an Autosystem XL (Perkin Elmer, Courtaboeuf, France), equipped with a FID detector, using a DB624 column (6% cyanopropylphenyl/94% dimethyl-polysiloxane, bonded and crosslinked phase) (length (30 m), diameter (0.53 mm) and phase thickness (3 µm)). Temperature conditions were as follows: initial temperature of 50 °C for 2 min, increased to 200 °C at a constant rate of 5 °C/min. The total runtime for a sample was 32 min, with nitrogen used as a carrier gas. The percentage of retention (r) of EO by HP-β-CD was determined by SH-GC at 25 °C for a 10 mM CD solution and expressed as follows:
r (%) = (1 − (ΣACD/ΣA0)) × 100
where ΣA0 and ΣACD stand for the sum of peak areas of each EO component in the absence and the presence of CD, respectively.
For each EO, in the presence or absence of HP-β-CD, measurements were done in triplicates.

2.4. Biological Activities of EO

2.4.1. EO Antifungal Activity

Antifungal activities of the different EO were tested against F. culmorum by using two in vitro complementary methods: direct contact (mycelial radial growth inhibition assay) and volatility assays in a completely randomized experimental design.
Direct contact assay: antifungal activity by direct contact was assessed according to [33,34], followed with slight modifications. Mycelial discs of F. culmorum (0.9 cm), from the edge of a 7-day-old culture fungal colony, were cut and placed at the center of a 9 cm Petri dish containing potato dextrose agar (PDA) culture medium (40 g L−1). EO were mixed with PDA medium at 50 °C in order to obtain final concentrations ranging from 0.02 to 2.5% of EO in the medium. These latter were previously sterilized by filtration and prepared in 1% ethanolic solution. Direct contact assay was performed in the absence and the presence of HP-β-CD, at a final concentration of 10 mM, in the medium. Analyses were led in triplicates. Samples were incubated for 7 days at 20 °C, and subsequently, the radius of mycelium growth was measured. The inhibition rate (IR) was calculated following the formula:
IR (%) = (X0 − Xi)/X0 × 100
where X0 = average diameter of the fungal colony in control; Xi = average diameter of the fungal colony in treatment.
Aqueous solutions of ethanol 1% (v/v) or HP-β-CD (10 mM) were tested as negative controls. A commercial fungicide, Aviator XPro, was simultaneously evaluated as a positive one. Graphical interpolation analysis was used to calculate the half-maximal inhibitory concentration (IC50) value.
Volatility assay: antifungal activity by volatility was assessed according to [35], followed with minor modifications. Mycelial discs of F. culmorum (0.9 cm), from the edge of a 7-day-old culture fungal colony, were cut and placed at the center of a 5.5 cm Petri dish culture medium plate with PDA. This plate was stuck with PDA into a 9 cm Petri dish that contained EO solutions. These latter were previously sterilized by filtration and prepared in both ethanolic solution and HP-β-CD (10 mM). EO concentrations used ranged from 0.01 to 5%. Analyses were led in triplicates. Samples were incubated for 5 days at 20 °C, and subsequently, the radius of mycelium growth was measured. The inhibition rate (IR) was calculated using Equation (2).
Aqueous solutions of ethanol 1% (v/v) or HP-β-CD (10 mM) were tested as negative controls. A commercial fungicide, Aviator XPro (Stolz, Wailly-Beaucamp, France), was simultaneously evaluated as a positive one. Graphical interpolation analysis was used to calculate the half-maximal inhibitory concentration (IC50)value.

2.4.2. EO Phytotoxicity Assessment

Lolium perenne L. and Lactuca sativa L., one monocotyledon and one dicotyledon plant, respectively, commonly used in phytotoxicity assays and listed in the Organisation for Economic Co-operation and Development (OECD) guidelines (2003) for the assessment of chemicals, were used for an in vitro bioassay. The effects of the different EO were evaluated on seedlings’ emergence and growth of the two plant species in a completely randomized experimental design, by using an adapted in vitro method [36,37,38].
EO were first mixed with agar medium at 50 °C in order to obtain final concentrations ranging from 0.005 to 0.5% of EO in the medium. Seed samples were then placed on agar medium, in sealed Petri dishes and incubated for 8 days on a day/night cycle, with a 16-h photoperiod at 20 °C and an obscurity period of 8 h at 16 °C. After the incubation period, germination rates were evaluated by counting germinated seeds and root elongation was assessed through an imaging software (ImageJ), by measuring root length [38,39]. These in vitro assays were performed in the absence and the presence of HP-β-CD, at a final concentration of 10 mM, in an agar medium. Analyses were led in triplicates. Aqueous ethanolic and HP-β-CD solutions (10 mM) were tested as negative controls, whereas Glyphosate (isopropylamine salt) was used as a positive control.
Graphical interpolation analysis was used to calculate the half-maximal inhibitory concentration (IC50) value, regarding both germination and root elongation parameters.

2.5. Statistical Analysis

Statistical analyses were performed using XLSTAT 2018.1.1 (Adinsoft, Paris, France) software and R 3.6.1 (R core Team, Vienna, Austria, 2019) [40]. Shapiro–Wilk and Bartlett tests were used prior to any statistical analysis to verify the normality of the distribution and the homoscedasticity, respectively. Non-normal data were “square-root” or “log10” transformed. Statistical significance between retention percentages has been analyzed using one-way ANOVA, complemented with a post-hoc Tukey-HSD test. For both antifungal and phytotoxicity assays, the IC50 resulted from non-linear regression analyses from triplicate trials and was expressed with mean values and standard deviations (mean ± SD). Statistical significance of the results was evaluated by two-way ANOVA (Analysis of Variance), with the absence or presence of CD and the “EO donor plant” as fixed factors, complemented with a post-hoc paired comparison (Tukey’s HSD–honestly significant difference). Moreover, a principal component statistical analysis (PCA) based on the Pearson correlation matrix, has been carried out using XLSTAT, to identify the possibly existing correlations between the different EO and to investigate the separation between EO samples according to their main chemical constituents. Prior to the PCA, the variables were standardized for a normalized PCA. The PCA was then carried out on centered and reduced GC-MS data from all tested EO, using identified compounds with relative abundance higher than 0.1%.

3. Results

3.1. Determination of the EO Chemical Composition

The EO chemical composition revealed that EO from thuja and angelica (root and seed) contained mostly terpenes. Chervil EO was particularly rich in eugenol (52%) and estragole (42%). Garlic, leek, shallots, onion, and chive EO all mostly contained organosulfur compounds. On the one hand, garlic EO was dominated by allyl polysulfides, while, on the other hand, leek, shallots, onion and chive EO were characterized by propyl polysulfides (Table 1).
The PCA analysis results displayed a high correlation coefficient corresponding to EO having a highly similar chemical composition and vice versa. Our results have shown that chive, leek, onion and shallot EO are strongly correlated (correlation coefficient ranging from 0.814 to 0.982, data not shown). Garlic EO, which also consists of organosulfur compounds, is not correlated to the previously mentioned EO (correlation coefficients ranging from 0.066 to 0.095). Angelica seed and root EO are also significantly positively correlated, but with a lesser correlation coefficient (0.52). Finally, thuja and chervil EO are not correlated to any other EO (correlation coefficients respectively ranging from 0.021 to 0.059, and from 0.032 to 0.064).
These conclusions are also visible on the correlation circles obtained with the PCA analysis (Figure 1). The projection has been carried out on three different axes, due to the fact that the first two principal components, F1 and F2, represent 59% of the initial variability of the data. In order to avoid misinterpretation, the two additional projections were executed. The results have shown that chive, leek, onion, and shallot EO are strongly correlated, whatever the projection. Angelica seed and root EO are correlated, when projected on F1 and F2 axes (Figure 1A), but this observation is not visible on other correlation circles (Figure 1B, C).

3.2. Determination of the Retention Capacity of EO by HP-β-CD

All the EO evaluated in this study were able to be encapsulated in HP-β-CD. EO retention percentages by HP-β-CD are listed in Table 2.
Among the retention percentages obtained, angelica seed’s EO is the one with the lowest value. All the other EO have retention percentages ranging from 43 to 80%, with the highest retention value obtained for garlic EO. Essential oils are more efficiently retained by HP-β-CD when the retention percentage obtained is higher.

3.3. Biological Activities of EO

3.3.1. EO Antifungal Activity

The results presented in Table 3 have shown that all the tested EO presented antifungal properties against the phytopathogenic agent, F. culmorum, by both direct contact and volatility assays.
Direct contact assay: IC50 obtained by direct contact varied from 0.01 to 4.2 g L−1 for shallot EO and angelica root EO, respectively. Shallot EO has demonstrated a higher potential in terms of antifungal activity by direct contact assay, by presenting a significantly lower IC50 in comparison with the other EO. The IC50 are in the following order: IC50 Shallot EO < IC50 Onion EO < IC50 A. seed EO < IC50 Chive EO < IC50 Leek EO < IC50 Garlic EO < IC50 Chervil EO < IC50 Thuja EO < IC50 A. root EO. On the other hand, no significant differences were observed between the IC50 obtained in the presence and the absence of HP-β-CD. In the presence of HP-β-CD, shallot EO presented the lowest IC50 (0.03 g L−1) and angelica root EO the highest (5.8 g L−1, Table 3). In comparison with the positive control, the obtained IC50 for the tested EO are about 100 to 1000 times higher.
Volatility assay: IC50 obtained by volatility assay ranged from 0.08 g L−1 for shallot EO to 25.6 g L−1 for leek EO. Once again, shallot EO has shown a greater potential antifungal activity by volatility assay in comparison with the other EO tested. The IC50 increase in the following order: IC50 Shallot EO < IC50 A. seed EO <IC50 A. root EO < IC50 Onion EO < IC50 Chervil EO < IC50 Thuja EO < IC50 Chive EO < IC50 Garlic EO < IC50 Leek EO. In the same way, HP-β-CD did not show any beneficial effect on obtained IC50. IC50 obtained by volatility assay, in the presence of HP-β-CD, ranged from 0.09 to 7.3 g L−1 for shallots and angelica root EO, respectively. As for the raw EO, IC50 obtained for the shallot EO, which is significantly lower, depicts a greater potential in terms of antifungal activity (Table 3). As the positive control is intended for use as a contact fungicide, the determination of an IC50 regarding volatility effects was not possible.

3.3.2. EO Phytotoxicity Assessment

Various responses from the two plant species to the EO have been highlighted. Nevertheless, all tested EO have shown a phytotoxic activity, either by inhibiting seed germination or by affecting root elongation.
Seedlings’ emergence assay: Regarding seedlings’ emergence, most of the tested EO exerted a significant inhibitory effect (except for angelica root EO and thuja EO in the presence of HP-β-CD) on lettuce germination by suppressing 100% of the germination, at least at the highest tested concentration. In the same way, a significant germinative inhibitory effect of the tested EO has also been demonstrated on rye-grass, except for garlic and angelica root EO (data not shown). Indeed, angelica seed and onion EO have shown great potential in inhibiting lettuce seed germination, with a significant effect at the lowest EO concentration tested. In terms of IC50, among the nine tested EO, only thuja and angelica root EO have shown a significantly lower anti-germinative potential (Table 4). The IC50 increase in the following order: IC50 A. seed EO < IC50 Leek EO < IC50 Shallot EO < IC50 Onion EO < IC50 Garlic EO < IC50 Chervil EO < IC50 Chive EO < IC50 Thuja EO < IC50 A. root EO.
In the presence of HP-β-CD, no significant improvement has been demonstrated in our experimental conditions, with even significantly higher effects in the absence of HP-β-CD for angelica seed, leek, garlic, chervil, and thuja EO. Due to their limited efficiency at the tested concentrations, the IC50 has not been calculated for thuja and angelica seed EO, as it would have resulted in particularly inaccurate and seemingly high values. In comparison with the positive control, the IC50 are in the same range for the most efficient EO and more than 100 times higher for angelica root EO.
On the other hand, all the EO containing organo-sulfur compounds (garlic, chive, leek, onion, and shallot EO), as well as chervil, thuja and angelica seed EO, exerted an inhibitory effect on rye-grass seedlings’ emergence, suppressing 70 to 100% of the germinative capacity at the highest tested concentration (data not shown). Angelica root EO, on the contrary, has shown a relatively limited anti-germinative capacity. In the presence of HP-β-CD, no significant improvement in terms of anti-germinative activity has been demonstrated for any of the tested EO (Table 4). As with lettuce, the IC50 has not been calculated for angelica root and garlic EO. In comparison with the positive control, the tested EO displayed significantly higher IC50 values (more than 10 times higher). The IC50 are in the following order: IC50 A.seed EO < IC50 Shallot EO < IC50 Chervil EO < IC50 Onion EO < IC50 Thuja EO < IC50 Garlic EO < IC50 Leek EO < IC50 Chive EO < IC50 A.root EO.
Seedlings’ growth assay: IC50 obtained for lettuce root elongation assessment varied from 0.0008 to 0.3 g L−1 for angelica seed and thuja EO, respectively. Obtained IC50 are listed in Table 5, where angelica seed, chervil, and leek EO displayed significantly lower IC50 values (ranging from 0.0008 to 0.02 g L−1). A significant improvement has been obtained in the presence of HP-β-CD for shallot EO only (Table 5). In the presence of HP-β-CD, shallot EO presented the lowest IC50 (0.02 g L−1) and angelica root EO the highest (3.2 g L−1). IC50 were increased as follows (Table 5): IC50 Shallot EO ≤ IC50 Garlic EO ≤ IC50 Onion EO ≤ IC50 A. seed EO ≤ IC50 Chive EO ≤ IC50 Leek EO ≤ IC50 Chervil EO ≤ IC50 Thuja EO ≤ IC50 A. root EO.
On rye-grass, the IC50 expressed ranged from 0.01 to 0.8 g L−1 for angelica seed and thuja EO, respectively (Table 5).
No significant improvement has been demonstrated in the presence of HP-β-CD, whatever the EO. Furthermore, angelica seed EO, in the presence of HP-β-CD, presented the lowest IC50 (0.06 g L−1) and angelica root EO the highest (3.6 g L−1). The IC50 are in the following order: IC50 A. seed EO ≤ IC50 Shallot EO ≤ IC50 Onion EO ≤ IC50 Chervil EO ≤ IC50 Chive EO ≤ IC50 Leek EO ≤ IC50 A. root EO ≤ IC50 Garlic EO < IC50 Thuja EO.
In comparison with the positive control, the tested EO displayed higher IC50 values for all the tested EO, for both lettuce and rye-grass assays, displaying IC50 values up to more than 1000 times higher for thuja EO.

4. Discussion

The use of biocontrol products and especially natural substances including essential oils (EO), in agricultural systems, is arousing great interest. They are indeed bio-sourced products, regarded as more ecological, and brought forward recently as alternative tools to replace synthetic pesticides, which show greater environmental and human health risks.
The results presented herein, based on in vitro antifungal activity against F. culmorum, by both direct contact and volatility assays, have shown that all tested EO are able to inhibit the growth of this phytopathogenic fungus, with higher efficiency for the direct contact method. Indeed, the inhibitory concentrations obtained by direct contact assay were significantly lower in comparison with those obtained with the volatility assay: the half-maximal inhibitory concentrations (IC50) obtained by volatility assay ranged from 1.2 up to 23–fold higher than those obtained by direct contact assay. However, the efficiency of this kind of inhibition was strongly related to the type of EO used.
The antifungal activity of common synthetic antifungal agents has previously been reported. Among others, demethylation inhibitor (DMI), quinone outside inhibitor (QOI) fungicides [41], pimaricin [42] and tetraconazole [43] have been investigated for their fungicidal properties against Fusarium graminearum and Fusarium spp. The most efficient fungicide evaluated was pimaricin, with an IC50 ranging from 2.5 up to 5 µg L−1, whereas the IC50 reported for tetraconazole was 16 µg L−1 and the ones obtained with DMI and QOI were 0.01 mg L−1 and 0.2 to 1.3 mg L−1, respectively. The fungicide evaluated in the present study displayed an IC50 value similar to the highest ones reported in the literature. Altogether, synthetic pesticides’ inhibitory effects occurred at lower concentrations compared to EO. However, if the gap appears to be consistent, EO have shown themselves efficient, with lower noxious effects on both environment and human health and the occurrence of resistance phenomena has, to our knowledge, not been reported so far with the use of EO. Indeed, the use of EO distilled from Mentha arvensis L., Mentha spicata L., Juniperus mexicana Spreng, Citrus sinensis L., Persicaria odorata Lour., Piper nigrum L., Canarium commune L., Cinnamomum zeylanicum L., Boswellia carterii Birdw., Cymbopogon flexuosus L., Litsea cubeba Lour., Artemisia herba-alba Asso, Cistus ladaniferus L., Copaifera tree, Ferula galbaniflua Boiss. et Buhse., Citrus aurantium L. and Schinus terebinthifolius Raddi has been homologated and has shown promising results in agriculture, as reviewed in [3].
Onion and garlic EO were previously studied for their antifungal activity toward Aspergillus niger, Aspergillus terreus, Penicillum cyclopium, Fusarium oxysporum, Candida albicans, Candida tropicalis, Rhodotorula glutinis, Monascus purpureus [4,44,45,46]. However, scarce are the studies that evaluate the antifungal activities of the tested EO by both direct contact and volatility assays, against F. culmorum.
Besides antifungal properties, the tested EO revealed phytotoxic effects, being able to inhibit the seedlings’ emergence of either one or both plant species tested in this in vitro bioassay, lettuce (L. sativa) and rye-grass (L. perenne). Our results have shown promising activities of the EO, especially the one distilled from angelica seeds, presenting the lowest IC50 values (0.0008 and 0.01 g L−1, respectively, on lettuce and rye-grass), notably due to the presence of a high amount of monoterpene compounds, such as beta-phellandrene [47,48,49]. The IC50 obtained ranged from 0.0008 to 0.3 g L−1, respectively, for angelica seed and angelica root EO used on lettuce, whereas the IC50 expressed on rye-grass ranged from 0.01 to 0.8 g L−1 for angelica seed and thuja EO. In terms of seedlings’ emergence inhibition, most of the EO tested in this study have demonstrated an inhibitory effect, especially on L. sativa, with lower EO concentrations resulting in inhibition of seeds’ germination. Overall, angelica seed EO has shown great potential in terms of inhibiting the growth of the studied plants but has been demonstrated as less efficient, especially on ryegrass, in terms of anti-germinative activity. By comparing our results with those obtained for glyphosate, used as a systemic herbicide in previous studies, and evaluated as a positive control in the present work, EO have demonstrated real potential. Indeed, previous studies have reported glyphosate’s IC50 ranging from 23–46.2 mg L−1 [50] and 15.3 mg L−1 [51], regarding ryegrass growth inhibition, whereas the IC50 reported on lettuce ranged from 8.9 mg L−1 [52] to 20 mg L−1 [53]. The obtained IC50 for glyphosate in the present work are similar to those previously reported. Thus, the most efficient EO reported in terms of in vitro phytotoxic effects (especially angelica seed EO) exerted effects similar to those of glyphosate.
Moreover, L. sativa has shown a higher sensibility to EO (in both evaluation methods, herbicidal or anti-germinative activities) compared to L. perenne, which is in line with the literature, reporting a higher sensitivity of lettuce to chemicals and environmental pollutants than many other plants [37,51,52,54,55], possibly because of the plant metabolism. Although there are frequent mentions of this sensitivity to chemicals [56,57,58,59,60], there is, to our knowledge, no clear evidence of the mechanisms and biological pathways involved. Besides, the EO tested in this study have, to the best of our knowledge, not previously been reported for their herbicidal properties, in terms of either seedlings’ emergence or growth inhibition.
As reported above, all EO were efficient regarding all of the biological activities assessed. Nevertheless, some EO, such as those coming from shallot, onion and angelica seed, presented high efficiency for both antifungal and phytotoxicity assays, since lower concentrations were required to obtain a total inhibitory effect. This may be imputed to the difference in the nature and the abundance of the major active principles of EO.
Indeed, the EO tested in this study may be classified into three different groups, regarding their chemical compositions. Angelica (root and seed) and thuja EO are mostly composed of terpene compounds, chervil EO is rich in phenylpropanoids whereas chive, garlic, leek, onion, and shallot EO contain organosulfur compounds. The balance between the active principles of EO among the same subgroup might be the cause of the wide scale of efficiency rates that have been spotlighted in this study. Leek and onion EO, rich in organosulfur compounds, respectively contain mainly dipropyl disulfide (50.4 and 35.5%), dipropyl trisulfide (20.1 and 17.6%), dipropyl tetrasulfide (3.8 and 11.0%), methyl propyl trisulfide (6.4 and 9.1%), bis(1-methyl ethyl)disulfide (4.5 and 6.7%), for instance.
These findings are in line with the literature, as it has been demonstrated that the major principles of EO, belonging to different classes of organic compounds, such as terpenes, flavonoids and isoflavonoids, coumarins, phenolic compounds, and organic acids, are biologically active and that some of them are likely to act synergistically to exert a stronger biological activity [61,62,63]. Additionally, one should note that, besides major compounds, the contribution of the compounds present in smaller amounts should not be neglected regarding biological effects. In fact, previous studies reported that the efficiency of the EO was in most cases greater than those of the major compounds studied separately [64,65]. In particular, EO may play a role in inhibiting fungal cell wall formation, disturbing cell membrane and fungal mitochondria function, as well as inhibiting cell proliferation, DNA, RNA, and protein synthesis [66,67]. These mechanisms of action are principally due to their major active principles such as terpenes (carvone, limonene), and phenolic compounds (eugenol, apiol) [66].
However, concerning the herbicidal assay, it is surprising to find EO from the same subgroup (terpene-containing compounds) in terms of composition, showing very different rates of efficiency, presenting either the lowest or the highest IC50 values. This might be attributed to the relative abundance of major compounds. Indeed, the main differences between angelica seed and root EO reside in the balance between major compounds, especially in mono-terpenes: Beta-phellandrene (resp. 67.2 and 16.5%), alpha-phellandrene (resp. 1.6 and 8.9%), alpha-pinene (resp. 11.4 and 21.4%), limonene (resp. not detected and 7.49%) and cis-beta-ocimene (resp. 4.92 and 6.36%). In addition, the results obtained from the PCA have shown a positive correlation between angelica root and seed EO, showing that the chemical compositions are similar, but the correlation factor is close to 0.5, meaning that the correlation is not optimal. Altogether, we found that EO from shallot, chive, leek and onion were closely related in terms of composition, which, most of the time, is also the case concerning their biological activities. On the contrary, EO from angelica seed and root have very different efficiency rates, despite their seemingly close compositions.
On another note, EO encapsulation in HP-β-CD did not provide a significant difference in the IC50 of EO in all the tested biological activities with respect to free-HP-β-CD treatment. In the presence of HP-β-CD, the IC50 of antifungal activity by direct contact assay varied from 0.03 g L−1 for shallot EO to 5.8 g L−1 for angelica root EO, while IC50 ranged from 0.09 to 7.3 g L−1 for shallot EO and angelica root EO, respectively, by volatility assay.
In the same way, the IC50 of herbicidal activity in the presence of HP-β-CD were not significantly altered, with shallots and angelica root EO presenting the lowest IC50 (16.6 mg L−1; 62 mg L−1 on lettuce and rye-grass, respectively) and angelica root EO the highest (3.2 g L−1; 3.6 g L−1 on lettuce and rye-grass, respectively).
In the presence of HP-β-CD, whatever the biological property assessed, no significant effect of HP-β-CD has been demonstrated. This raises the issue of EO retention by HP-β-CD. It is well known that HP-β-CD can form inclusion complexes with EO components. This phenomenon was highlighted by the decrease in the peak areas of EO in the presence of HP-β-CD. This effect has already been described in previous studies [11,68,69]. For the EO listed above, retention percentage values have shown that EO were efficiently retained by HP-β-CD (except for angelica seed’s EO, presenting a relatively low retention percentage). These results suggest that HP-ß-CD could efficiently retain EO and that way avoid EO evaporation and degradation. Additionally, our results have not demonstrated that a high retention percentage significantly reduces the EO activity and vice versa, in the case of volatility assay. Finally, HP-β-CD might be considered as an efficient material to encapsulate EO and improve the release efficiency and persistence in time of volatile compounds, enlarging the applications of EO from an agricultural perspective.

5. Conclusions

In conclusion, altogether our findings have shown that EO, especially those distilled from shallot, onion, and angelica seed, possessed both interesting antifungal and phytotoxic activities. This may be attributable to the nature of the chemical compounds, their richness, and their relative abundance. One should note that even minor compounds may contribute to the efficiency of the EO. In particular, these EO presented a great potential to control F. culmorum growth by both direct contact and volatility assays. They have also shown phytotoxic effects against mono- and dicotyledon species, displaying inhibitory effects in the same range as common herbicides. Such in vitro tests give an indication of the potential role of EO used as biopesticides to control phytopathogen infection. Afterwards, it would be interesting to carry out in planta assays, using individual or combined EO, to validate EO-based biopesticides’ efficiency. Although this efficacy was strongly related to the EO used and its composition, all EO tested in the current study could potentially be used as natural and eco-friendly pesticides. Additionally, it should be noted, that in our experimental conditions, the encapsulation of the tested EO did not significantly improve the EO efficiency, yet reduced EO volatility. Further analyses should be conducted in glasshouse or field conditions to evaluate the efficiency of the controlled release of EO.

Author Contributions

Conceptualization-methodology-validation, I.E.-A., R.R., J.F., A.V., F.L., S.F., R.C., H.M., A.L.-H.S.; writing—original draft preparation, I.E.-A., R.R.; writing—review and editing, I.E.-A., R.R., J.F., A.V., F.L., S.F., R.C., H.M., A.L.-H.S.; supervision A.L.-H.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by l’Agence De l’Environnement et de la Maîtrise de l’Energie (ADEME, Angers, France). This work has also been carried out in the framework of the Alibiotech project which is financed by the European Union, the French State and the French Region of Hauts-de-France.

Acknowledgments

The authors wish to thank the “Université du Littoral Côte d’Opale” and the “Pôle Métropolitain de la Côte d’Opale” for providing financial support for R. Raveau’s Ph.D thesis, P. Ferrant from Ferrant PHE for having kindly provided the EO and N. Facon for her technical help.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hanif, M.A.; Nisar, S.; Khan, G.S.; Mushtaq, Z.; Zubair, M. Essential Oils. In Essential Oil Research; Springer: Cham, Switzerland, 2019; pp. 3–17. [Google Scholar]
  2. Mittal, R.P.; Rana, A.; Jaitak, V. Essential Oils: An Impending Substitute of Synthetic Antimicrobial Agents to Overcome Antimicrobial Resistance. Curr. Drug Targets 2019, 20, 605–624. [Google Scholar] [CrossRef] [PubMed]
  3. Raveau, R.; Fontaine, J.; Lounès-Hadj Sahraoui, A. Essential Oils as Potential Alternative Biocontrol Products against Plant Pathogens and Weeds: A Review. Foods 2020, 9, 365. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Ye, C.L.; Dai, D.H.; Hu, W.L. Antimicrobial and antioxidant activities of the essential oil from onion (Allium cepa L.). Food Control 2013, 30, 48–53. [Google Scholar] [CrossRef]
  5. da Cruz Cabral, L.; Pinto, V.F.; Patriarca, A. Application of plant derived compounds to control fungal spoilage and mycotoxin production in foods. Int. J. Food Microbiol. 2013, 166, 1–14. [Google Scholar] [CrossRef]
  6. Jallow, M.F.A.; Awadh, D.G.; Albaho, M.S.; Devi, V.Y.; Thomas, B.M. Science of the Total Environment Pesticide risk behaviors and factors in fl uencing pesticide use among farmers in Kuwait. Sci. Total Environ. 2017, 574, 490–498. [Google Scholar] [CrossRef]
  7. Lamichhane, J.R.; Dachbrodt-Saaydeh, S.; Kudsk, P.; Messéan, A. Toward a Reduced Reliance on Conventional Pesticides in European Agriculture. Plant Dis. 2016, 100, 10–24. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Kordali, S.; Cakir, A.; Akcin, T.A.; Mete, E.; Akcin, A.; Aydin, T.; Kilic, H. Antifungal and herbicidal properties of essential oils and n-hexane extracts of Achillea gypsicola Hub-Mor. and Achillea biebersteinii Afan. (Asteraceae). Ind. Crops Prod. 2009, 29, 562–570. [Google Scholar] [CrossRef]
  9. Pavela, R.; Benelli, G. Essential Oils as Ecofriendly Biopesticides? Challenges and Constraints. Trends Plant Sci. 2016, 21, 1000–1007. [Google Scholar] [CrossRef]
  10. Kfoury, M.; Auezova, L.; Greige-Gerges, H.; Fourmentin, S. Promising applications of cyclodextrins in food: Improvement of essential oils retention, controlled release and antiradical activity. Carbohydr. Polym. 2015, 131, 264–272. [Google Scholar] [CrossRef] [PubMed]
  11. Fourmentin, S.; Ciobanu, A.; Landy, D.; Wenz, G. Space filling of β-cyclodextrin and β-cyclodextrin derivatives by volatile hydrophobic guests. Beilstein J. Org. Chem. 2013, 9, 1185–1191. [Google Scholar] [CrossRef]
  12. Kurkov, S.V.; Loftsson, T. Cyclodextrins. Int. J. Pharm. 2013, 453, 167–180. [Google Scholar] [CrossRef]
  13. Del Toro-Sánchez, C.L.; Ayala-Zavala, J.F.; Machi, L.; Santacruz, H.; Villegas-Ochoa, M.A.; Alvarez-Parrilla, E.; González-Aguilar, G.A. Controlled release of antifungal volatiles of thyme essential oil from β-cyclodextrin capsules. J. Incl. Phenom. Macrocycl. Chem. 2010, 67, 431–441. [Google Scholar] [CrossRef]
  14. Munhuweyi, K.; Caleb, O.J.; Lennox, C.L.; van Reenen, A.J.; Opara, U.L. In vitro and in vivo antifungal activity of chitosan-essential oils against pomegranate fruit pathogens. Postharvest Biol. Technol. 2017, 129, 9–22. [Google Scholar] [CrossRef]
  15. Estrada-cano, C.; Antonieta, M.; Castro, A.; Muñoz-castellanos, L.; García-triana, N.A.A. Antifungal Activity of Microcapsulated Clove (Eugenia caryophyllata) and Mexican Oregano (Lippia berlandieri) Essential Oils against Fusarium oxysporum. J. Microb. Biochem. Technol. 2017, 9, 567–571. [Google Scholar] [CrossRef] [Green Version]
  16. Munhuweyi, K.; Caleb, O.J.; van Reenen, A.J.; Opara, U.L. Physical and antifungal properties of β-cyclodextrin microcapsules and nanofibre films containing cinnamon and oregano essential oils. LWT Food Sci. Technol. 2018, 87, 413–422. [Google Scholar] [CrossRef]
  17. Herrera, A.; Rodríguez, F.J.; Bruna, J.E.; Abarca, R.L.; Galotto, M.J.; Guarda, A.; Mascayano, C.; Sandoval-yáñez, C.; Padula, M.; Ramos, F.; et al. Antifungal and physicochemical properties of inclusion complexes based on β -cyclodextrin and essential oil derivatives. Food Res. Int. 2019, 121, 127–135. [Google Scholar] [CrossRef] [PubMed]
  18. Synowiec, A.; Smęda, A.; Adamiec, J.; Kalemba, D. The effect of microencapsulated essential oils on the initial growth of maize (Zea mays) and common weeds (Echinochloa crus-galli and Chenopodium album). Prog. Plant Prot. 2016, 56, 372–378. [Google Scholar] [CrossRef] [Green Version]
  19. Matny, O.N. Fusarium head blight and crown rot on wheat & barley: Losses and health risks. Adv. Plants Agric. Res. 2015, 2, 2–7. [Google Scholar]
  20. Wilson, W.; Dahl, B.; Nganje, W. Economic costs of fusarium head blight, scab and deoxynivalenol. World Mycotoxin J. 2018, 11, 291–302. [Google Scholar] [CrossRef]
  21. Ji, F.; He, D.; Olaniran, A.O.; Mokoena, M.P.; Xu, J.; Shi, J. Occurrence, toxicity, production and detection of Fusarium mycotoxin: A review. Food Prod Process Nutr. 2019, 1, 6. [Google Scholar] [CrossRef]
  22. Wagacha, J.M.; Muthomi, J.W. Fusarium culmorum: Infection process, mechanisms of mycotoxin production and their role in pathogenesis in wheat. Crop Prot. 2007, 26, 877–885. [Google Scholar] [CrossRef]
  23. Rai, A.; Das, M.; Tripathi, A. Occurrence and toxicity of a fusarium mycotoxin, zearalenone. Crit. Rev. Food Sci. Nutr. 2019, 1–20. [Google Scholar] [CrossRef]
  24. Goni, P.; Lopez, P.; Sanchez, C.; Gomez-Lus, R.; Becerril, R.; Nerin, C. Antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential oils. Food Chem. 2009, 116, 982–989. [Google Scholar] [CrossRef]
  25. Nedorostova, L.; Kloucek, P.; Kokoska, L.; Stolcova, M.; Pulkrabek, J. Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria. Food Control 2009, 20, 157–160. [Google Scholar] [CrossRef]
  26. Suhr, K.; Nielsen, P.V. Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi. J. Appl. Microbiol. 2003, 94, 665–674. [Google Scholar] [CrossRef] [PubMed]
  27. Griffin, S.G.; Markham, J.L.; Leach, D.N. An agar dilution method for the determination of the minimum inhibitory concentration of essential oils. J. Essent. Oil Res. 2000, 12, 249–255. [Google Scholar] [CrossRef]
  28. Mnayer, D.; Fabiano-Tixier, A.-S.; Petitcolas, E.; Hamieh, T.; Nehme, N.; Ferrant, C.; Fernandez, X.; Chemat, F. Chemical Composition, Antibacterial and Antioxidant Activities of Six Essentials Oils from the Alliaceae Family. Molecules 2014, 19, 20034–20053. [Google Scholar] [CrossRef] [Green Version]
  29. Smith, J.P.J. Economically Important Plants Arranged Systematically. Available online: http://digitalcommons.humboldt.edu/botany_jps/48 (accessed on 18 March 2020).
  30. Hendawy, S.F.; Hussein, M.S.; El-Gohary, A.E.; Soliman, W.S. Chemical Constituents of Essential Oil in Chervil (Anthriscus cerefolium L. Hoffm.) Cultivated in Different Locations. J. Essent. Oil Bear. Plants 2019, 22, 264–272. [Google Scholar] [CrossRef]
  31. FerrantPHE. Available online: https://www.ferrantphe.com (accessed on 18 March 2020).
  32. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing, Carol Stream: Illinois, IL, USA, 2007. [Google Scholar]
  33. Huang, Y.; Zhao, J.; Zhou, L.; Wang, J.; Gong, Y.; Chen, X.; Guo, Z.; Wang, Q.; Jiang, W. Antifungal activity of the essential oil of illicium verum fruit and its main component trans-anethole. Molecules 2010, 15, 7558–7569. [Google Scholar] [CrossRef]
  34. Quiroga, E.N.; Sampietro, A.R.; Vattuone, M.A. No TitleScreening antifungal activities of selected medicinal plants. J. Ethnopharmacol. 2001, 74, 89–96. [Google Scholar] [CrossRef]
  35. Abarca, R.L.; Rodríguez, F.J.; Guarda, A.; Galotto, M.J.; Bruna, J.E. Characterization of beta-cyclodextrin inclusion complexes containing an essential oil component. Food Chem. 2016, 196, 968–975. [Google Scholar] [CrossRef] [PubMed]
  36. Judd, L.; Jackson, B.; Fonteno, W. Advancements in Root Growth Measurement Technologies and Observation Capabilities for Container-Grown Plants. Plants 2015, 4, 369–392. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Lyu, J.; Park, J.; Kumar Pandey, L.; Choi, S.; Lee, H.; De Saeger, J.; Depuydt, S.; Han, T. Testing the toxicity of metals, phenol, effluents, and receiving waters by root elongation in Lactuca sativa L. Ecotoxicol. Environ. Saf. 2018, 149, 225–232. [Google Scholar] [CrossRef]
  38. Paul, A.L.; Daugherty, C.J.; Bihn, E.A.; Chapman, D.K.; Norwood, K.L.; Ferl, R.J. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in Arabidopsis. Plant Physiol. 2001, 126, 613–621. [Google Scholar] [CrossRef] [Green Version]
  39. Yazdanbakhsh, N.; Fisahn, J. Analysis of Arabidopsis thaliana root growth kinetics with high temporal and spatial resolution. Ann. Bot. 2010, 105, 783–791. [Google Scholar] [CrossRef]
  40. R Core Team. R: A Language and Environment for Statistical Computing; R Core Team: Vienna, Austria, 2019. [Google Scholar]
  41. Avozani, A.; Tonin, R.B.; Reis, E.M.; Camera, J.; Ranzi, C. In vitro sensitivity of Fusarium graminearum isolates to fungicides. Summa Phytopathol. 2014, 40, 231–247. [Google Scholar] [CrossRef] [Green Version]
  42. Kawakami, H.; Inuzuka, H.; Hori, N.; Takahashi, N.; Ishida, K.; Mochizuki, K.; Ohkusu, K.; Muraosa, Y.; Watanabe, A.; Kamei, K. Inhibitory effects of antimicrobial agents against Fusarium species. Med. Mycol. 2015, 53, 603–611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Yörük, E. Tetraconazole leads to alterations in Fusarium graminearum at different molecular levels. Appl. Ecol. Environ. 2018, 16, 6155–6167. [Google Scholar] [CrossRef]
  44. Ankri, S.; Mirelman, D. Antimicrobial properties of allicin from garlic. Microbes Infect. 1999, 1, 125–129. [Google Scholar] [CrossRef]
  45. Benkeblia, N. Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). LWT Food Sci. Technol. 2004, 37, 263–268. [Google Scholar] [CrossRef]
  46. Corzo-Martínez, M.; Corzo, N.; Villamiel, M. Biological properties of onions and garlic. Trends Food Sci. Technol. 2007, 18, 609–625. [Google Scholar] [CrossRef]
  47. 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] [PubMed]
  48. Kotan, R.; Cakir, A.; Dadasoglu, F.; Aydin, T.; Cakmakci, R.; Ozer, H.; Kordali, S.; Mete, E.; Dikbas, N. Antibacterial activities of essential oils and extracts of Turkish Achillea, Satureja and Thymus species against plant pathogenic bacteria. J. Sci. Food Agric. 2010, 90, 145–160. [Google Scholar] [CrossRef]
  49. Amri, I.; Hamrouni, L.; Hanana, M.; Jamoussi, B. Reviews on phytotoxic effects of essential oils and their individual components: News approach for weeds management. Int. J. Appl. Biol. Pharm. Technol. 2013, 4, 96–114. [Google Scholar]
  50. Ghanizadeh, H.; Harrington, K.C.; James, T.K.; Woolley, D.J. Quick tests for detecting glyphosate-resistant Italian and perennial ryegrass. N. Z. J. Agric. Res. 2015, 58, 108–120. [Google Scholar] [CrossRef]
  51. Martin, M.; Ronco, A. Effect of Mixtures of Pesticides Used in the Direct Seeding Technique on Nontarget Plant Seeds. Bull. Environ. Contam. Toxicol. 2006, 77. [Google Scholar] [CrossRef]
  52. Ronco, A.E.; Carriquiriborde, P.; Natale, G.; Martin, M.; Mugni, H.; Bonetto, C. Integrated approach for the assessment of biotech soybean pesticides impact on low order stream ecosystems of the pampasic region. In Ecosystem Ecology Research Trends; Chen, J., Guô, C., Eds.; Nova Science Publishers: New York, NY, USA, 2008; pp. 209–239. [Google Scholar]
  53. Kuang, Y.; Yang, S.X.; Sampietro, D.A.; Zhang, X.F.; Tan, J.; Gao, Q.X.; Liu, H.W.; Ni, Q.X.; Zhang, Y.Z. Phytotoxicity of leaf constituents from bamboo (Shibataea chinensis nakai) on germination and seedling growth of lettuce and cucumber. Allelopath. J. 2017, 40, 133–142. [Google Scholar] [CrossRef]
  54. Charles, J.; Sancey, B.; Morin-Crini, N.; Badot, P.-M.; Degiorgi, F.; Trunfio, G.; Crini, G. Evaluation of the phytotoxicity of polycontaminated industrial effluents using the lettuce plant (Lactuca sativa) as a bioindicator. Ecotoxicol. Environ. Saf. 2011, 74, 2057–2064. [Google Scholar] [CrossRef]
  55. Pennacchio, M.; Jefferson, L.V.; Havens, K. Arabidospis thaliana: A new test species for phytotoxic bioassays. J. Chem. Ecol. 2005, 31, 1877–1885. [Google Scholar] [CrossRef]
  56. Gruda, N.; Rau, B.J.; Wright, R.D. Laboratory bioassay and Greenhouse Evaluation of a Pine Tree substrate used as a container substrate. Eur. J. Hortic. Sci. 2009, 74, 73–78. [Google Scholar]
  57. Marcu, D.; Cristea, V.; Daraban, L. Dose-dependent effects of gamma radiation on lettuce (Lactuca sativa var. capitata) seedlings. Int. J. Radiat. Biol. 2013, 89, 219–223. [Google Scholar] [CrossRef] [PubMed]
  58. Labud, V.; Garcia, C.; Hernandez, T. Effect of hydrocarbon pollution on the microbial properties of a sandy and a clay soil. Chemosphere 2007, 66, 1863–1871. [Google Scholar] [CrossRef] [PubMed]
  59. Santos, S.C.; OLIVEIRA, U.A.; Trindade, L.; ASSIS, M.D.; Campos, J.M.; Salgado, E.G.; Barbosa, S.A. Genotypes selection for plant bioassays using Lactuca sativa L. and Allium cepa L. Pakistan J. Bot. 2017, 49, 2201–2212. [Google Scholar]
  60. Park, J.; Yoon, J.; Depuydt, S.; Oh, J.; Jo, Y.; Kim, K.; Brown, M.; Han, T. The sensitivity of an hydroponic lettuce root elongation bioassay to metals, phenol and wastewaters. Ecotoxicol. Environ. Saf. 2016, 126, 147–153. [Google Scholar] [CrossRef]
  61. Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils—A review. Food Chem. Toxicol. 2008, 46, 446–475. [Google Scholar] [CrossRef]
  62. Christaki, E.; Bonos, E.; Giannenas, I.; Florou-Paneri, P. Aromatic Plants as a Source of Bioactive Compounds. Agriculture 2012, 2, 228–243. [Google Scholar] [CrossRef] [Green Version]
  63. Shaaban, H.A.E.; El-Ghorab, A.H.; Shibamoto, T. Bioactivity of essential oils and their volatile aroma components: Review. J. Essent. Oil Res. 2012, 24, 203–212. [Google Scholar] [CrossRef]
  64. de Araújo Couto, H.G.S.; Blank, A.F.; e Silva, A.M.D.O.; de Lima Nogueira, P.C.; de Fátima Arrigoni-Blank, M.; de Castro Nizio, D.A.; de Oliveira Pinto, J.A. Essential oils of basil chemotypes: Major compounds, binary mixtures, and antioxidant activity. Food Chem. 2019, 293, 446–454. [Google Scholar] [CrossRef]
  65. Ben Kaab, S.; Rebey, I.; Hanafi, M.; Berhal, C.; Fauconnier, M.; De Clerck, C.; Ksouri, R.; Jijakli, H. Rosmarinus officinalis essential oil as an effective antifungal and herbicidal agent. Span. J. Agric. Res. 2019, 17, 1006. [Google Scholar] [CrossRef] [Green Version]
  66. Lagrouh, F.; Dakka, N.; Bakri, Y. The antifungal activity of Moroccan plants and the mechanism of action of secondary metabolites from plants. J. Mycol. Med. 2017, 27, 303–311. [Google Scholar] [CrossRef]
  67. Vicki Caligur, B.S.; McClanahan, C. Antifungals. In Biofiles: Antibiotics for Research Applications; Sigma-Aldrich: St. Louis, MO, USA, 2009; Volume 4, pp. 10–15. [Google Scholar]
  68. Ciobanu, A.; Mallard, I.; Landy, D.; Brabie, G.; Nistor, D.; Fourmentin, S. Retention of aroma compounds from Mentha piperita essential oil by cyclodextrins and crosslinked cyclodextrin polymers. Food Chem. 2013, 138, 291–297. [Google Scholar] [CrossRef] [PubMed]
  69. Kfoury, M.; Auezova, L.; Fourmentin, S.; Greige-Gerges, H. Investigation of monoterpenes complexation with hydroxypropyl-β-cyclodextrin. J. Incl. Phenom. Macrocycl. Chem. 2014, 80, 51–60. [Google Scholar] [CrossRef]
Agronomy 10 00825 g001 550
Figure 1. Correlation circles from the principal component statistical analyses (PCA) on the 9 tested essential oils (EO), with projections on F1 and F2 axes (A), on F1 and F3 axes (B) and on F1 and F4 axes (C).
Figure 1. Correlation circles from the principal component statistical analyses (PCA) on the 9 tested essential oils (EO), with projections on F1 and F2 axes (A), on F1 and F3 axes (B) and on F1 and F4 axes (C).
Agronomy 10 00825 g001
Table
Table 1. Relative chemical composition of the 9 tested EO.
Table 1. Relative chemical composition of the 9 tested EO.
A. Root aA. Seed bChervilChiveGarlicLeekOnionShallotThuja
α-Thujene--------0.45 ± 0.006
Methyl propyl disulfide ---3.5 ± 0.3-3.6 ± 0.022.8 ± 0.096.5 ± 0.1-
α-Pinene21.4 ± 1.111.4 ± 0.2------2.5 ± 0.06
2,5-dimethyl thiophene-------1.7 ± 0.06-
Camphene0.8 ± 0.003-------5.7 ± 0.3
Sabinene5.4 ± 0.051.4 ± 0.01-------
Allyl propyl disulfide-------6.5 ± 0.2-
β-Pinene1.2 ± 0.11.0 ± 0.30.1 ± 0.001-----0.2 ± 0.001
β-Myrcene4.0 ± 0.063.6 ± 0.09------0.8 ± 0.1
α-Phellandrene8.9 ± 0.81.6 ± 0.05-------
δ-3-Carene22.9 ± 1.0-------0.6 ± 0.005
trans-β-Ocimene0.9 ± 0.21.4 ± 0.02------1.8 ± 0.1
D-Limonene7.5 ± 0.6-0.1 ± 0.003-----2.1 ± 0.3
β-Phellandrene16.5 ± 1.367.2 ± 1.6-------
cis-β-Ocimene6.4 ± 0.34.9 ± 0.2-------
γ-Terpinene1.4 ± 0.03-------0.2 ± 0.001
Diallyl disulfide----29.3 ± 1.6----
Terpilonene1.0 ± 0.02--------
Undecane--3.62 ± 0.1------
Dipropyl disulfide---43.4 ± 1.8-50.4 ± 1.935.5 ± 2.323.7 ± 0.9-
3-Thujanone--------71.5 ± 3.2
Disulfide, bis(1-methylethyl)---7.9 ± 0.8-4.5 ± 0.66.7 ± 1.56.8 ± 1.3-
α-Thujone--------10.6 ± 1.0
Allyl methyl disulfide----3.1 ± 0.05----
Methyl propyl trisulfide---8.1 ± 1.1-6.4 ± 0.59.1 ± 0.820.5 ± 1.8-
1,2-Dithiolane----2.9 ± 0.2--1.0 ± 0.1-
Terpinen-4-ol-1.0 ± 0.1------2.9 ± 0.06
Isothujol--------0.2 ± 0
Estragole--42.0 ± 0.3------
Dimethyl tetrasulfide-------1.5 ± 0.03-
1-Propenyl propyl disulfide-----0.4 ± 0.06---
Camphanol acetate0.8 ± 0.07--------
α-limonene diepoxide--0.2 ± 0.04-----0.2 ± 0.005
Oxalic acid--0.3 ± 0.006------
Diallyl trisulfide----24.2 ± 0.9----
Dipropyl trisulfide---25.6 ± 1.3-20.1 ± 0.517.6 ± 0.613.9 ± 0.4-
Allyl n-propyl sulfide-----2.0 ± 0.053.3 ± 0.082.6 ± 0.07-
trans-3,5-Diethyl-1,2,4-trithiolane-----0.8 ± 0.0042.3 ± 0.05--
α-terpinyl acetate-0.6 ± 0.09------0.4 ± 0
1,2,4-Trithiolane, 3,5-diethyl------2.1 ± 0.022.7 ± 0.062.4 ± 0.03-
Allyl-propyl trisulfide----0.2 ± 0----
Copaene0.9 ± 0.052.0 ± 0.080.1 ± 0------
Allyl-methyl trisulfide----2.13 ± 0.06-0.8 ± 0.07--
Methyl 2-propenyl disulfide----1.5 ± 0.04----
Methyl isoeugenol--1.1 ± 0.1------
Methyl isopropyl disulfide---0.9 ± 0.05-0.6 ± 0.083.4 ± 0.16.1 ± 0.3-
Methyleugenol--51.7 ± 1.8------
cis-1-Propenyl propyl trisulfide-----0.4 ± 0.2---
Caryophyllene--0.2 ± 00.7 ± 0.05-0.6 ± 0.1---
β-Germacrene-0.8 ± 0.04-------
δ-Germacrene-0.6 ± 0.03-------
Humulene-1.8 ± 0.2-------
β-Copaene-0.7 ± 0.1-------
Hexadecane--0.4 ± 0.03------
Diallyl tetrasulfide----22.3 ± 0.5----
Dipropyl tetrasulfide---5.8 ± 0.8-3.8 ± 0.111.0 ± 0.65.8 ± 0.3-
Methyl propyl tetrasulfide---0.8 ± 0.2-----
1-Propyl-2-(4-thiohept-2-en-5-yl) disulfide---1.8 ± 0.090.9 ± 0.11.2 ± 0.10.94 ± 0.06--
1-(1-Propenyl)-2-(4-thiohept-5-yl) disulfide---0.5 ± 0.06-0.4 ± 0.041.2 ± 0.03--
8-Ethyl-4,5,6,7,9-pentathiadecane-----0.3 ± 0.01---
4-Methyl-1,2,3,5,6-pentathiepane----0.9 ± 0.07----
2,4-Dimethyl-5,6-dithia-2,7-nonadienal---1.1 ± 0.03-0.7 ± 0.11.2 ± 0.051.0 ± 0.06-
6-Ethyl-4,5,7,8-tetrathianonane-----1.5 ± 0.031.7 ± 0.04--
Diallyl sulfide----9.9 ± 0.5----
Phytol--0.2 ± 0------
(Z)-1-propenyl propyl tetrasulfide-----0.3 ± 0---
Cyclooctasulfur----2.7 ± 0.3----
Values are presented as percentages and depicted as mean ± standard deviation. a A. Root: angelica root EO. b A. Seed: angelica seed EO.: not detected.
Table
Table 2. EO’s percentages of retention in the presence of HP-β-CD.
Table 2. EO’s percentages of retention in the presence of HP-β-CD.
A. Root AA. Seed BChervilChiveGarlicLeekOnionShallotThuja
Retention percentage (%)61 ± 1.9 b27 ± 3.4 e46 ± 2.1 cd54 ± 1.7 c78 ± 4.2 a62 ± 5.7 b71 ± 3.6 a43 ± 2.8 d60 ± 1.8 b
Values are depicted as mean ± standard deviation. A: A. Root: angelica root EO. B: A. Seed: angelica seed EO.
Table
Table 3. IC50 of antifungal activity of EO obtained by both direct contact assay and volatility assay.
Table 3. IC50 of antifungal activity of EO obtained by both direct contact assay and volatility assay.
A. RootA. SeedChervilChiveGarlicLeekOnionShallotThujaPositive Control
Direct Contact assayIC50 (g L−1) without HP-β-CD4.2 ± 0.1 b0.3 ± 0.003 ij3.1 ± 0.04 d0.9 ± 0.007 g1.6 ± 0.1 f1.2 ± 0.03 g0.1 ± 0.002 jkl0.01 ± 0.0003 l3.6 ± 0.02 c0.0010 ± 0.0006 l
IC50 (g L−1) with HP-β-CD5.8 ± 0.1 a0.2 ± 0.001 ij1.9 ± 0.001 e0.3 ± 0.009 ij0.4 ± 0.01 hi0.6 ± 0.001 h0.2 ± 0.005 jk0.03 ± 0.0003 kl3.7 ± 0.02 c
Volatility asssayIC50 (g L−1) without HP-β-CD2.2 ± 0.01 ij’0.8 ± 0.01 k’5.5 ± 0.02 f’16.9 ± 0.1 c’22.6 ± 0.5 b’25.6 ± 0.4 a’2.3 ± 0.06 i’0.08 ± 0.0005 k’9.5 ± 0.08 d’NC
IC50 (g L−1) with HP-β-CD7.3 ± 0.01 e’0.7 ± 0.01 k’6.7 ± 0.02 e’2.4 ± 0.05 i’3.2 ± 0.1 h’4.4 ± 0.1 g’1.4 ± 0.04 j’0.09 ± 0.0002 k’3.6 ± 0.02 gh’
Values are depicted as mean ± standard deviation; IC50: inhibitory concentration; NC: not calculable; different lowercase letters indicate significant difference using two-way ANOVA, between results obtained in the presence or absence of HP-β-CD (p < 0.05) and displayed without and with apostrophe for direct contact and volatility assays, respectively. Results from direct contact and volatility assays have been analyzed separately. A. Root: angelica root EO. A. Seed: angelica seed EO.
Table
Table 4. IC50 of EO’s phytotoxic activity (inhibition of seedling’s emergence) obtained by contact assay, against lettuce and rye-grass.
Table 4. IC50 of EO’s phytotoxic activity (inhibition of seedling’s emergence) obtained by contact assay, against lettuce and rye-grass.
A. RootA. SeedChervilChiveGarlicLeekOnionShallotThujaPositive Control
LettuceIC50 (g L−1) without HP-β-CD4.9 ± 0.9 c0.01 ± 0.002 g0.3 ± 0.05 g0.9 ± 0.1 f0.3 ± 0.05 g0.1 ± 0.04 g0.2 ± 0.04 g0.2 ± 0.04 g2.3 ± 0.4 d0.01 ± 0.006 g
IC50 (g L−1) with HP-β-CD1.1 ± 0.3 fNC a1.7 ± 0.4 e0.8 ± 0.2 f5.4 ± 0.7 b1.6 ± 0.5 e0.083 ± 0.02 f0.1 ± 0.03 gNC a
Rye-grassIC50 (g L−1) without HP-β-CDNC a’0.6 ± 0.07 h’1.7 ± 0.3 ef’3.9 ± 0.5 c’2.9 ± 0.5 d’3.7 ± 0.5 c’1.7 ± 0.3 ef’0.7 ± 0.09 h’2.1 ± 0.5 e’0.04 ± 0.01 i’
IC50 (g L−1) with HP-β-CDNC a’0.5 ± 0.07 h’1.4 ± 0.3 fg’1.2 ± 0.3 g’NC a’1.8 ± 0.3 ef’0.4 ± 0.6 h’1.3 ± 0.3 fg’4.8 ± 0.9 b’
Values are depicted as mean ± standard deviation; IC50: inhibitory concentration; NC: not calculable; different letters indicate significant difference using two-way ANOVA, between results obtained in the presence or absence of HP-β-CD (p < 0.05). Results for lettuce and rye-grass have been analyzed separately and statistical letters are displayed, respectively, without or with an apostrophe. A. Root: angelica root EO. A. Seed: angelica seed EO.
Table
Table 5. IC50 of EO’s phytotoxic activity (inhibition of seedling’s growth) obtained by contact assay, against lettuce and rye-grass.
Table 5. IC50 of EO’s phytotoxic activity (inhibition of seedling’s growth) obtained by contact assay, against lettuce and rye-grass.
A. RootA. SeedChervilChiveGarlicLeekOnionShallotThujaPositive Control
LettuceIC50 (g L−1) without HP-β-CD0.2 ± 0.04 cd0.0008 ± 0.0004 e0.02 ± 0.003 e0.1 ± 0.04 de0.2 ± 0.06 cd0.03 ± 0.004 e0.2 ± 0.04 cd0.2 ± 0.04 cd0.3 ± 0.05 c0.0001 ± 0.0002 e
IC50 (g L−1) with HP-β-CD3.2 ± 0.9 a0.04 ± 0.008 e0.3 ± 0.1 c0.08 ± 0.04 e0.03 ± 0.007 e0.08 ± 0.05 e0.03 ± 0.02 e0.02 ± 0.007 e0.8 ± 0.1 b
Rye-grassIC50 (g L−1) without HP-β-CD0.5 ± 0.06 e’0.01 ± 0.003 h’0.3 ± 0.06 de’0.06 ± 0.02 gh’0.3 ± 0.05 f’0.2 ± 0.04 fg’0.2 ± 0.03 fg’0.2 ± 0.04 fg’0.8 ± 0.1 d’0.001 ± 0.0006 h’
IC50 (g L−1) with HP-β-CD1.3 ± 0.4 c’0.06 ± 0.03 gh’0.09 ± 0.03 gh’0.1 ± 0.04 gh’3.2 ± 0.5 b’0.1 ± 0.04 gh’0.08 ± 0.03 gh’0.07 ± 0.02 gh’3.6 ± 0.8 a’
Values are depicted as mean ± standard deviation; IC50: inhibitory concentration; NC: not calculable; different letters indicate significant difference using two-way ANOVA, between results obtained in the presence or absence of HP-β-CD (p < 0.05). Results for lettuce and rye-grass have been analyzed separately and statistical letters are displayed, respectively, without or with an apostrophe. A. Root: angelica root EO. A. Seed: angelica seed EO.

Share and Cite

MDPI and ACS Style

El-Alam, I.; Raveau, R.; Fontaine, J.; Verdin, A.; Laruelle, F.; Fourmentin, S.; Chahine, R.; Makhlouf, H.; Lounès-Hadj Sahraoui, A. Antifungal and Phytotoxic Activities of Essential Oils: In Vitro Assays and Their Potential Use in Crop Protection. Agronomy 2020, 10, 825. https://doi.org/10.3390/agronomy10060825

AMA Style

El-Alam I, Raveau R, Fontaine J, Verdin A, Laruelle F, Fourmentin S, Chahine R, Makhlouf H, Lounès-Hadj Sahraoui A. Antifungal and Phytotoxic Activities of Essential Oils: In Vitro Assays and Their Potential Use in Crop Protection. Agronomy. 2020; 10(6):825. https://doi.org/10.3390/agronomy10060825

Chicago/Turabian Style

El-Alam, Imad, Robin Raveau, Joël Fontaine, Anthony Verdin, Frédéric Laruelle, Sophie Fourmentin, Ramez Chahine, Hassane Makhlouf, and Anissa Lounès-Hadj Sahraoui. 2020. "Antifungal and Phytotoxic Activities of Essential Oils: In Vitro Assays and Their Potential Use in Crop Protection" Agronomy 10, no. 6: 825. https://doi.org/10.3390/agronomy10060825

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