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This study evaluated the antifungal effects of various volatile organic compounds (VOCs) against two common pathogens: Fusarium culmorum and Cochliobolus sativus. Among the various VOCs, methyl propanoate (MP) and methyl prop-2-enoate (MA) exhibited remarkable antifungal effects under different experimental conditions (direct or indirect contact) and at different concentrations (500–1000 μM). In addition, the type of antifungal effect (fungistatic or fungicidal) appeared to be strongly correlated with the VOC concentrations. Additional tests revealed that both molecules increased membrane permeability of pathogenic spores, which resulted in a decreased efflux of K+ ions into the intracellular medium.
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International Journal of
Environmental Research
and Public Health
Article
Antifungal Properties of Two Volatile Organic
Compounds on Barley Pathogens and Introduction to
Their Mechanism of Action
Amine Kaddes 1, *, Marie-Laure Fauconnier 2, Khaled Sassi 3, Bouzid Nasraoui 4and
M. Haïssam Jijakli 1
1Integrated and Urban Plant Pathology Laboratory, Gembloux Agro-Bio Tech (GxABT), University of Liège,
5030 Gembloux, Belgium
2Laboratory of Chemistry of Natural Molecules, Gembloux Agro-Bio Tech (GxABT), University of Liège,
5030 Gembloux, Belgium
3Department of Agronomy and Plant Biotechnology, National Agronomic Institute of Tunisia,
University of Carthage, Tunis 1082, Tunisia
4RL/Biogressors and Integrated Protection in Agriculture, National Agronomic Institute of Tunisia,
University of Carthage, Tunis 1082, Tunisia
*Correspondence: amine.kaddes@doct.uliege.be; Tel.: +32-81-622-433
Received: 13 May 2019; Accepted: 8 August 2019; Published: 10 August 2019


Abstract:
This study evaluated the antifungal eects of various volatile organic compounds (VOCs)
against two common pathogens: Fusarium culmorum and Cochliobolus sativus. Among the various VOCs,
methyl propanoate (MP) and methyl prop-2-enoate (MA) exhibited remarkable antifungal eects
under dierent experimental conditions (direct or indirect contact) and at dierent concentrations
(500–1000
µ
M). In addition, the type of antifungal eect (fungistatic or fungicidal) appeared to be
strongly correlated with the VOC concentrations. Additional tests revealed that both molecules
increased membrane permeability of pathogenic spores, which resulted in a decreased eux of K
+
ions into the intracellular medium.
Keywords:
methyl prop-2-enoate; methyl propanoate; biocontrol; volatile organic compound; mode
of action
1. Introduction
Fusarium culmorum and Cochliobolus sativus are known to cause root rot in dierent cereal varieties,
especially wheat and barley. The incidence and severity of cereal contamination by these fungi have
increased worldwide and resulted in serious yield losses. Damage caused by the contamination of
sensitive barley cultivars by Fusarium culmorum and Cochliobolus sativus can raise up to 16–33% [
1
].
The damage induced by these two pathogens includes brown discoloration of the roots, coleoptiles,
and sub-crown internodes of the host plants and mycotoxins contamination. Following the global
ecologically sustainable approach to agricultural production, the use of chemical pesticides for cereal
protection has grown more and more limited. Dierent eco-friendly techniques have been developed
with a view to assessing alternative sustainable methods for cereal disease control [
2
5
]. In this context,
many studies highlight the benefits of non-host crop techniques in managing foot and root rot [
2
,
6
,
7
].
This approach has attracted great attention and has been applied in dierent countries such as the
United Kingdom and Australia [
2
,
4
,
5
,
8
,
9
]. At the same time, the development of biocontrol methods
has also been suggested as an alternative method to fight against cereal pathogens. Globally, biocontrol
methods use living organisms or natural substances produced by these organisms, such as pheromones
and plant extracts, to inhibit pathogen growth [
10
]. Since the 1980s, volatile organic compounds
Int. J. Environ. Res. Public Health 2019,16, 2866; doi:10.3390/ijerph16162866 www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2019,16, 2866 2 of 14
(VOCs) produced by dierent endophytic fungi have attracted special interest due to their particular
antifungal potential [
11
,
12
]. Fiers et al. [
13
] studied the eect of a spectrum of VOCs on the interaction
between barley and the two main agents of root rot: C. sativus and F. culmorum. VOCs inhibited the
growth of the pathogenic fungi. Interestingly, two VOCs, methyl prop-2-enoate (MA) and methyl
propionate (MP), exhibited significant antifungal activities. These molecules inhibited fungal growth
by more than 81% for both fungi [
14
]. Despite the unique ability of methyl prop-2-enoate (MA) and
methyl propionate (MP) to inhibit a large spectrum of pathogens, their mechanisms of action still
remains poorly studied. This prompted us to undertake the present study.
The main objectives of this work were (i) to evaluate the direct and indirect inhibitory eects
of methyl prop-2-enoate (MA) and methyl propanoate (MP) on the growth of the two fungal strains
C. sativus and F. culmorum, and (ii) to start deciphering the way these two molecules inhibit mycelial
growth and spore germination.
2. Materials and Methods
2.1. Fungal Strains
F. culmorum (MUCL 28166) and C. sativus (MUCL 46854) strains were provided by the Belgian
Co-ordinated Collection of Microorganisms (BCCM-MUCL) (Louvain-la-Neuve, Belgium). The
strains were grown on PDA (Merck KGaA, Darmstadt, Germany) at 23
C and were subjected to
16 hL:8 hD photoperiod.
2.2. Evaluation of the Eect of Five VOCs in Gas Phase on F. culmorum and C. sativus Growth Without
Direct Contact
The antifungal activities of five VOCs—methyl propanoate, methyl prop-2-enoate, isobutylformate,
p-cymene, and longifolene (Sigma-Aldrich)—were evaluated against the two pathogens F. culmorum
and C. sativus. Dierent commercial solutions of VOCs (Sigma-Aldrich) were prepared to a final
concentration of 500
µ
M. PDA (3.9%; w/v) and WA (1% agar (Difco, Grenoble, France); w/v) media
were prepared and inoculated with 10-day-old cultures of F. culmorum and 3-week-old active C. sativus
cultures. Each VOC was placed on a filter paper in a small 2 cm Petri dish with a lid in the middle. Thus,
there was no direct contact between the pathogen and the VOCs, gases phase could be easily spread on
the square-shaped petri dish (Greiner, Belgium). The petri dishes were placed in a growth chamber
under LED light (94 mmol photons/m2/s) with a 16 hL:8 hD photoperiod for 10 days (Figure S1).
The radial growth (RG) of the fungi was determined by measuring two perpendicular diameters
with a graduated ruler and averaging the values every 24 h until T =240 h. A total of 15 Petri dishes
was used for each VOC and each fungal strain. Each assay was independently replicated three times.
Petri dishes were randomly placed in the culture chamber. The growth inhibition rate was calculated
as follows [14]:
Growth inhibition rate =(RG control RG treated sample)/(RG control) ×100 (1)
Statistical analyses were performed using Minitab 17 [15] (Minitab Inc., State College, PA, USA).
The growth rate of every fungus in the presence of each VOC was determined by an analysis of
variance (p<0.05) (AV1) using one-way ANOVA, followed by Tukey and Dunnett multiple comparison
test (p<0.05).
2.3. Evaluation of the Eect of Methylprop-2-Enoate and Methylpropanoate on F. culmorum and C. sativus
Growth in Direct Contact
The eect of methylprop-2-enoate and methylpropanoate on F. culmorum and C. sativus growth in
direct contact was determined according to methods described by Kaddes et al. [
14
], using PDA as the
culture medium.
Int. J. Environ. Res. Public Health 2019,16, 2866 3 of 14
2.4. Evaluation of the Fungicidal/Fungistatic Effect of the Most Efficient VOCs on C. sativus and F. culmorum Growth
To evaluate the fungicidal/fungistatic eects of methylprop-2-enoate and methylpropanoate, both
molecules were supplemented on PDA medium at two dierent concentrations (500 and 1000
µ
M) and
poured in a 600 mL cell culture flask where a 70 mm disk of peripheric culture of each fungal strain
had been placed. Three flasks were prepared for each concentration and were incubated at 23
C and
under a 16 hL:8 hD photoperiod for 10 days. The negative controls were VOC-free. After 10 days,
when mycelium growth was not observed, the flask was opened, and the mycelial disk was transferred
to another PDA Petri dish. All petri dishes were maintained at 23
C and under the same photoperiod
for 10 days to evaluate the eect of the VOCs. The experiment was performed in triplicate, and 3 flasks
per compound and per concentration were analyzed in each replicate.
2.5. Evaluation of the Release of K+Ions in the Extracellular Medium
The amount of K
+
ions released in the extra-cellular medium was used as an indicator of the eect
of MA and MP on the integrity of the fungal spore membranes. Five milliliters of spore suspensions at a
concentration of 105 conidia/mL were prepared for each fungus in PDB medium. Methyl prop-2-enoate
and methyl propanoate were added to obtain the following concentrations: 100, 500, and 1000
µ
M.
A Quantofix
®
kit of colored strips (Macherey-Nagel) was used to measure the release of K
+
ions. The
strips were used according to the protocol provided by the manufacturer (Appendix A). For each
fungus, measurements were made every hour for 5 h.
2.6. Data Analysis
All experiments were performed in triplicate. Data were analyzed using analysis of variance
(
p<0.05
). Tukey and Dunnett multiple comparison test (p<0.05) was performed using the Minitab
16.2.2 software [16].
3. Results
3.1. Evaluation of the Eect of Five VOCs on F. culmorum and C. sativus Growth
In the first part of the study, we evaluated the antifungal capacity of five VOCs (methyl propanoate,
methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene) against the two pathogenic strains
F. culmorum and C. sativus. According to Fiers et al. [
13
], all five VOCs emitted de novo substances b
infected barley roots.
The experiments were performed according to the direct contact protocol using WA as a culture
medium. The VOC concentration (500
µ
M) was chosen on the basis of previous results reported by
Kaddes et al. [
14
]. As can be seen in Figure 1A,B, the growth rate of both pathogens closely depended
on the type of VOC added to the culture medium. Methylprop-2-enoate and methylpropanoate
exhibited the highest antifungal potential against both pathogens. The growth rates of F. culmorum and
C. sativus after application of methylprop-2-enoate for 240 h were estimated to be only 3% and 2%,
respectively. The same behavior was also observed after application of methyl propanoate. In contrast,
isobutylformate, longifolene, and p-cymene exhibited low antifungal activities against both fungal
strains. Based on this first set of experiments, only methylprop-2-enoate and methyl propanoate were
retained for further analyses.
Int. J. Environ. Res. Public Health 2019,16, 2866 4 of 14
Int. J. Environ. Res. Public Health 2019, 16, x 4 of 15
Figure 1. Growth of F. culmorum (A) and C. sativus (B) in the presence of volatile organic compounds
(VOCs) at 500 μM in gas phase. (**): Significant result in comparison with the control, according to
Dunnett’s test (p < 0.05). (***): Highly significant result in comparison with the control, according to
Dunnett’s test (p < 0.05). ns: Non-significant result in comparison with the control, according to
Dunnett’s test (p < 0.05).
3.2. Evaluation of the Inhibitory Effect of the Most Efficient VOCs on Fungal Growth in Direct and Indirect
Contact
We evaluated the antifungal effect of methyl prop-2-enoate and methyl propanoate against F.
culmorum and C. sativus in direct and indirect contact. Different experiments were performed on PDA
medium or WA supplemented with 500 μM of each VOC before being inoculated with the
appropriate fungal strain.
Figure 1.
Growth of F. culmorum (
A
) and C. sativus (
B
) in the presence of volatile organic compounds
(VOCs) at 500
µ
M in gas phase. (**): Significant result in comparison with the control, according to
Dunnett’s test (p<0.05). (***): Highly significant result in comparison with the control, according
to Dunnett’s test (p<0.05). ns: Non-significant result in comparison with the control, according to
Dunnett’s test (p<0.05).
3.2. Evaluation of the Inhibitory Effect of the Most Efficient VOCs on Fungal Growth in Direct and Indirect Contact
We evaluated the antifungal eect of methyl prop-2-enoate and methyl propanoate against
F. culmorum and C. sativus in direct and indirect contact. Dierent experiments were performed on PDA
medium or WA supplemented with 500
µ
M of each VOC before being inoculated with the appropriate
fungal strain.
Int. J. Environ. Res. Public Health 2019,16, 2866 5 of 14
3.2.1. Eect of Methyl Prop-2-Enoate and Methyl Propanoate in Direct Contact
Figure 2A,B presents the time course of the growth of the two fungal strains in PDA medium
supplemented with VOCs or not. In the control medium, the growth diameter of the fungal
strains increased and reached 1.24 cm and 12.54 cm for F. culmorum and C. sativus after 240 h of
incubation, respectively. Despite this dierence, supplementation of the culture medium with VOCs
drastically inhibited mycelial growth. The growth rate of F. culmorum was estimated to be only 4%
after application of methyl propionate or methyl prop-2-enotate. The percentage of inhibition of
C. sativus mycelial growth ranged between 88.6% and 100% after application of methylpropionate
and methylprop-2-enoate, respectively. Taking these results into account, we evaluated the antifungal
potential of the two VOCs against F. culmorum and C. sativus in dierent experimental conditions (type
of culture medium and type of contact).
Int. J. Environ. Res. Public Health 2019, 16, x 5 of 15
3.2.1. Effect of
Methyl Prop-2-Enoate and Methyl Propanoate in Direct Contact
Figure 2A,B presents the time course of the growth of the two fungal strains in PDA medium
supplemented with VOCs or not. In the control medium, the growth diameter of the fungal strains
increased and reached 1.24 cm and 12.54 cm for F. culmorum and C. sativus after 240 h of incubation,
respectively. Despite this difference, supplementation of the culture medium with VOCs drastically
inhibited mycelial growth. The growth rate of F. culmorum was estimated to be only 4% after
application of methyl propionate or methyl prop-2-enotate. The percentage of inhibition of C. sativus
mycelial growth ranged between 88.6% and 100% after application of methylpropionate and
methylprop-2-enoate, respectively. Taking these results into account, we evaluated the antifungal
potential of the two VOCs against F. culmorum and C. sativus in different experimental conditions
(type of culture medium and type of contact).
Figure 2. Evolution of F. culmorum (A) and C. sativus (B) mycelial growth in direct contact with methyl
prop-2-enoate and methyl propanoate at 500 μM on PDA medium. (***): Highly significant result in
comparison with the control, according to Dunnett’s test (p < 0.05).
A comparative study was conducted to evaluate the effects of the culture medium on the
antifungal capacity of methylprop-2-enoate and prop-2-enotae. As shown by Figure 3A,B a
comparison of F. culmorum and C. sativus mycelial growth in direct contact with the VOCs at 500 μM
on water agar and PDA medium. The results of mycelial growth on WA medium were provided from
the study by Kaddes et al. [14]. The results highlighted similar antifungal effects of methyl
propanoate against F. culmorum on both culture media. Nevertheless, significant differences were
observed with methylprop-2-enoate: C. sativus growth in its presence significantly depended on the
Figure 2.
Evolution of F. culmorum (
A
) and C. sativus (
B
) mycelial growth in direct contact with methyl
prop-2-enoate and methyl propanoate at 500
µ
M on PDA medium. (***): Highly significant result in
comparison with the control, according to Dunnett’s test (p<0.05).
A comparative study was conducted to evaluate the eects of the culture medium on the
antifungal capacity of methylprop-2-enoate and prop-2-enotae. As shown by Figure 3A,B a comparison
of F. culmorum and C. sativus mycelial growth in direct contact with the VOCs at 500
µ
M on water
agar and PDA medium. The results of mycelial growth on WA medium were provided from the
Int. J. Environ. Res. Public Health 2019,16, 2866 6 of 14
study by Kaddes et al. [
14
]. The results highlighted similar antifungal eects of methyl propanoate
against F. culmorum on both culture media. Nevertheless, significant dierences were observed with
methylprop-2-enoate: C. sativus growth in its presence significantly depended on the culture medium.
By contrast, no significant dierence was observed under methyl prop-2-enoate treatment.
Int. J. Environ. Res. Public Health 2019, 16, x 6 of 15
Figure 3. Comparison of F. culmorum (A) and C. sativus (B) mycelial growth in direct contact with the
VOCs at 500 μM on water agar and PDA medium. Identical lower-case letters indicate non
significantly different results according to Tukey’s test (p < 0.05).
3.2.2. Effect of Methyl Prop-2-Enoate and Methyl Propanoate in Indirect Contact
In the second part of the study, we evaluated the antifungal capacity of methyl prop-2-enoate
and methyl propanoate on C. sativus and F. culmorum in indirect contact using PDA as a culture
medium (Figure 4A,B). The results highlighted a strong inhibitory effect of both VOCs against C.
sativus and F. culmorum. The percentages of mycelial growth inhibition after application of methyl
propanoate reached 95% and 98% for C. sativus and F. culmorum, respectively. Methyl prop-2-enoate
inhibited 99% of growth in both pathogens.
Figure 3.
Comparison of F. culmorum (
A
) and C. sativus (
B
) mycelial growth in direct contact with the
VOCs at 500
µ
M on water agar and PDA medium. Identical lower-case letters indicate non significantly
dierent results according to Tukey’s test (p<0.05).
3.2.2. Eect of Methyl Prop-2-Enoate and Methyl Propanoate in Indirect Contact
In the second part of the study, we evaluated the antifungal capacity of methyl prop-2-enoate and
methyl propanoate on C. sativus and F. culmorum in indirect contact using PDA as a culture medium
(Figure 4A,B). The results highlighted a strong inhibitory eect of both VOCs against C. sativus and
F. culmorum. The percentages of mycelial growth inhibition after application of methyl propanoate
reached 95% and 98% for C. sativus and F. culmorum, respectively. Methyl prop-2-enoate inhibited 99%
of growth in both pathogens.
Int. J. Environ. Res. Public Health 2019,16, 2866 7 of 14
Int. J. Environ. Res. Public Health 2019, 16, x 7 of 15
Figure 4. Evolution of F. culmorum (A) and C. sativus (B) mycelial growth in gas phase with MA and
MP at 500 μM on PDA medium. (***): highly significant result in comparison with the control
according to Dunnett’s test (p < 0.05).
We then compared the indirect inhibitory effects of the two VOCs on PDA medium and WA
medium. The antifungal effects of both molecules were completely independent of the media
composition (Figure 5).
Figure 4.
Evolution of F. culmorum (
A
) and C. sativus (
B
) mycelial growth in gas phase with MA and MP
at 500
µ
M on PDA medium. (***): highly significant result in comparison with the control according to
Dunnett’s test (p<0.05).
We then compared the indirect inhibitory eects of the two VOCs on PDA medium and WA
medium. The antifungal eects of both molecules were completely independent of the media
composition (Figure 5).
Int. J. Environ. Res. Public Health 2019,16, 2866 8 of 14
Int. J. Environ. Res. Public Health 2019, 16, x 8 of 15
Figure 5. Comparison of F. culmorum (A) and C. sativus (B) mycelial growth in gas phase with VOCs
at 500 μM on water agar medium and PDA medium. Identical lower-case letters indicate non
significantly different results according to Tukey’s test (p < 0.05).
3.3. Effect of the VOC Concentrations on VOC Antifungal Ability
We conducted a new set of direct/indirect experiments to evaluate the effect of the VOC
concentration of the antifungal capacity of methyl prop-2-enoate and methyl propanoate. The final
concentration of each molecule was adjusted to 500–1000 μM in PDA/WA medium. Table 1
summarizes the results of the fungistatic/fungicide effect of each molecule at different concentrations.
The mycelial growth of both pathogens was inhibited in the presence of methyl prop-2-enoate at 500
and 1000 μM. However, the mycelial growth of C. sativus was inhibited only in the presence of methyl
propanoate at 1000 μM.
Implants [A1] that were treated with methyl prop-2-enoate and methyl propanoate at
concentrations of 500 and 1000 μM during the first part of the test were transferred to PDA medium.
In MP-treated cultures, mycelium of pathogens was observed in vials. This suggests that fungistatic
effect of MP was weak. In contrast mycelial growth of both pathogens was completely inhibited when
cultures were treated with MA. This suggests that MA exerts powerful fungicide effects.
Figure 5.
Comparison of F. culmorum (
A
) and C. sativus (
B
) mycelial growth in gas phase with VOCs at
500
µ
M on water agar medium and PDA medium. Identical lower-case letters indicate non significantly
dierent results according to Tukey’s test (p<0.05).
3.3. Eect of the VOC Concentrations on VOC Antifungal Ability
We conducted a new set of direct/indirect experiments to evaluate the eect of the VOC
concentration of the antifungal capacity of methyl prop-2-enoate and methyl propanoate. The final
concentration of each molecule was adjusted to 500–1000
µ
M in PDA/WA medium. Table 1summarizes
the results of the fungistatic/fungicide eect of each molecule at dierent concentrations. The mycelial
growth of both pathogens was inhibited in the presence of methyl prop-2-enoate at 500 and 1000
µ
M.
However, the mycelial growth of C. sativus was inhibited only in the presence of methyl propanoate at
1000 µM.
Implants [A1] that were treated with methyl prop-2-enoate and methyl propanoate at
concentrations of 500 and 1000
µ
M during the first part of the test were transferred to PDA medium.
In MP-treated cultures, mycelium of pathogens was observed in vials. This suggests that fungistatic
eect of MP was weak. In contrast mycelial growth of both pathogens was completely inhibited when
cultures were treated with MA. This suggests that MA exerts powerful fungicide eects.
Int. J. Environ. Res. Public Health 2019,16, 2866 9 of 14
Table 1.
Fungicidal/fungistatic eect of MA and MP on F. culmorum and C. sativus at dierent
concentrations. First part of the results (A), second part of the results (B).
A
VOCs Fusarium culmorum Cochliobolus sativus
MP MA MP MA
500 µM
- – –
- – –
- – –
1000 µM
- – –
- — –
- — –
Controls
+ + + +
+ + + +
+ + + +
B
VOCs Fusarium culmorum Cochliobolus sativus
MA MP MP MA
500 µM
-+ + -
-+ + -
-+ + -
1000 µM
-+ + -
-+ + -
-+ + -
Int. J. Environ. Res. Public Health 2019, 16, x 9 of 15
Table 1. Fungicidal/fungistatic effect of MA and MP on F. culmorum and C. sativus at different
concentrations. First part of the results (A), second part of the results (B).
A
VOCs
Fusarium culmorum Cochliobolus sativus
MP MA MP MA
500 µM
- -- -- --
- -- -- --
- -- -- --
1000 µM
- -- -- --
- --- -- --
- --- -- --
Controls
+ + + +
+ + + +
+ + + +
B
VOCs Fusarium culmorum Cochliobolus sativus
MA MP MP MA
500 µM
- + + -
- + + -
- + + -
1000 µM
- + + -
- + + -
- + + -
- Means “Absence of mycelium of the pathogen in vials”
+ Means “Presence of mycelium of the pathogen in vials”
3.4. Evaluation of the Release of K+ Ions into the Extracellular Medium
The above-mentioned results confirm the antifungal effects of methyl prop-2-enoate and methyl
propanoate. Therefore, we tried to understand the mode of action of these molecules through a new
set of experiments. The amount of K+ ions released into the extra-cellular medium was used as an
indicator of the effect of MA and MP on the membrane of fungal spores.
Methyl prop-2-enoate and methyl propanoate had an effect on F. culmorum and C. sativus conidia
that was related to the emission of K+ ions into the extracellular medium (Tables 2 and 3, Figures S4
and S5).
After 3 h of incubation, the quantity of potassium released during treatment with methyl prop-
2-enoate and methyl propanoate at 500 μM reached 700 mg/L. This quantity increased up to 1000
mg/L after 5 h of incubation with methyl prop-2-enoate at 1000 μM. Similar results were observed
with C. sativus treated with either molecule.
Means “Absence of mycelium of the pathogen in vials”;
Int. J. Environ. Res. Public Health 2019, 16, x 9 of 15
Table 1. Fungicidal/fungistatic effect of MA and MP on F. culmorum and C. sativus at different
concentrations. First part of the results (A), second part of the results (B).
A
VOCs
Fusarium culmorum Cochliobolus sativus
MP MA MP MA
500 µM
- -- -- --
- -- -- --
- -- -- --
1000 µM
- -- -- --
- --- -- --
- --- -- --
Controls
+ + + +
+ + + +
+ + + +
B
VOCs Fusarium culmorum Cochliobolus sativus
MA MP MP MA
500 µM
- + + -
- + + -
- + + -
1000 µM
- + + -
- + + -
- + + -
- Means “Absence of mycelium of the pathogen in vials”
+ Means “Presence of mycelium of the pathogen in vials”
3.4. Evaluation of the Release of K+ Ions into the Extracellular Medium
The above-mentioned results confirm the antifungal effects of methyl prop-2-enoate and methyl
propanoate. Therefore, we tried to understand the mode of action of these molecules through a new
set of experiments. The amount of K+ ions released into the extra-cellular medium was used as an
indicator of the effect of MA and MP on the membrane of fungal spores.
Methyl prop-2-enoate and methyl propanoate had an effect on F. culmorum and C. sativus conidia
that was related to the emission of K+ ions into the extracellular medium (Tables 2 and 3, Figures S4
and S5).
After 3 h of incubation, the quantity of potassium released during treatment with methyl prop-
2-enoate and methyl propanoate at 500 μM reached 700 mg/L. This quantity increased up to 1000
mg/L after 5 h of incubation with methyl prop-2-enoate at 1000 μM. Similar results were observed
with C. sativus treated with either molecule.
Means “Presence
of mycelium of the pathogen in vials”.
3.4. Evaluation of the Release of K+Ions into the Extracellular Medium
The above-mentioned results confirm the antifungal eects of methyl prop-2-enoate and methyl
propanoate. Therefore, we tried to understand the mode of action of these molecules through a new
set of experiments. The amount of K
+
ions released into the extra-cellular medium was used as an
indicator of the eect of MA and MP on the membrane of fungal spores.
Methyl prop-2-enoate and methyl propanoate had an eect on F. culmorum and C. sativus conidia
that was related to the emission of K
+
ions into the extracellular medium (Tables 2and 3, Figures S4
and S5).
Table 2.
Amount of K
+
ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to dierent methyl prop-2-enoate and methyl propionate treatments for
dierent incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 µM 400 400 400 400 400 400
MA 500 µM 400 400 400 700 700 700
MA 1000
µ
M
400 400 400 700 700 1000
MP 100 µM 400 400 400 400 400 400
MP 500 µM 400 400 400 400 400 700
MP 1000 µM 400 400 400 700 700 700
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
400 mg/L K
+
present in the extracellular medium of a spore suspension of Fusarium culmorum.
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
700 mg/L K
+
present in the extracellular medium of a spore suspension of Fusarium culmorum.
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
1000 mg/L
K+present in the extracellular medium of a spore suspension of Fusarium culmorum.
Int. J. Environ. Res. Public Health 2019,16, 2866 10 of 14
Table 3.
Amount of K
+
ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to dierent methyl prop-2-enoate and methyl propanoate treatments for
dierent incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 µM 400 400 400 400 400 700
MA 500 µM 400 400 400 400 1000 1000
MA 1000
µ
M
400 400 400 400 1000 1000
MP 100 µM 400 400 400 400 400 700
MP 500 µM 400 400 400 400 700 1000
MP 1000 µM 400 400 400 400 700 1000
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
400 mg/L K
+
present in the extracellular medium of a spore suspension of Cochliobolus sativus.
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
700 mg/L K
+
present in the extracellular medium of a spore suspension of Cochliobolus sativus.
Int. J. Environ. Res. Public Health 2019, 16, x 10 of 15
Table 2. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Fusarium culmorum subjected to different methyl prop-2-enoate and methyl propionate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 400
MA 500 μM 400 400 400 700 700 700
MA 1000 μM 400 400 400 700 700 1000
MP 100 μM 400 400 400 400 400 400
MP 500 μM 400 400 400 400 400 700
MP 1000 μM 400 400 400 700 700 700
ent in the extracellular medium of a spore suspension of Fusarium
culmorum.
Fusarium culmorum. 1000 mg/L K+ present in the extracellular medium of a spore suspension
of Fusarium culmorum.
Table 3. Amount of K+ ions (mg/L) present in the extracellular medium of a spore suspension of
Cochliobolus sativus subjected to different methyl prop-2-enoate and methyl propanoate treatments for
different incubation times.
Concentration 0 h 1 h 2 h 3 h 4 h 5 h
Control 400 400 400 400 400 400
MA 100 μM 400 400 400 400 400 700
MA 500 μM 400 400 400 400 1000 1000
MA 1000 μM 400 400 400 400 1000 1000
MP 100 μM 400 400 400 400 400 700
MP 500 μM 400 400 400 400 700 1000
MP 1000 μM 400 400 400 400 700 1000
sativus.
resent in the extracellular medium of a spore suspension of
Cochliobolus sativus. 1000 mg/L K+ present in the extracellular medium of a spore
suspension of Cochliobolus sativus.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on
the two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters
(methyl prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct
and indirect contact. In contrast, the antifungal effects of isobutylformate, p-cymene, and longifolene
were negligible. These results are in accordance with those of Kaddes et al. [14], who reported that p-
cymene exhibited a slight inhibitory effect when used in direct contact. This could be explained by
its low molar mass, which makes it less volatile as compared to methyl prop-2-enoate and methyl
propanoate. Moreover, our results confirm those of Kaddes et al. [14], who noted the highest
percentages of pathogen growth inhibition under methyl prop-2-enoate and methyl propanoate
treatment. According to these authors, methyl prop-2-enoate and methyl propanoate treatment at 500
μM resulted in up to 81% growth inhibition for F. culmorum and C. sativus, while p-cymene had a
lower effect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory effects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal effect
1000 mg/L
K+present in the extracellular medium of a spore suspension of Cochliobolus sativus.
After 3 h of incubation, the quantity of potassium released during treatment with methyl
prop-2-enoate and methyl propanoate at 500
µ
M reached 700 mg/L. This quantity increased up to
1000 mg/L after 5 h of incubation with methyl prop-2-enoate at 1000
µ
M. Similar results were observed
with C. sativus treated with either molecule.
4. Discussion
The first part of our study focused on the evaluation of the antifungal capacity of five VOCs,
namely methyl propanoate, methyl prop-2-enoate, isobutylformate, p-cymene, and longifolene, on the
two pathogenic fungi F. culmorum and C. sativus. Among the five VOCs, two organic esters (methyl
prop-2-enoate and methyl propanoate) exhibited a particular antifungal potential in direct and indirect
contact. In contrast, the antifungal eects of isobutylformate, p-cymene, and longifolene were negligible.
These results are in accordance with those of Kaddes et al. [
14
], who reported that p-cymene exhibited
a slight inhibitory eect when used in direct contact. This could be explained by its low molar mass,
which makes it less volatile as compared to methyl prop-2-enoate and methyl propanoate. Moreover,
our results confirm those of Kaddes et al. [
14
], who noted the highest percentages of pathogen growth
inhibition under methyl prop-2-enoate and methyl propanoate treatment. According to these authors,
methyl prop-2-enoate and methyl propanoate treatment at 500
µ
M resulted in up to 81% growth
inhibition for F. culmorum and C. sativus, while p-cymene had a lower eect (73%) on both fungi [14].
In indirect contact, methyl prop-2-enoate had similar inhibitory eects on both fungal strains
grown on PDA medium or WA medium. The same results were observed when the antifungal eect
of methyl propanoate was evaluated against C. sativus. By contrast, the antifungal eect of methyl
propanoate against F. culmorum was strongly dependent on the medium composition.
Furthermore, there was no significant dierence between the inhibitory eect of methyl
prop-2-enoate and methyl propanoate on F. culmorum in direct contact on PDA or WA. However, this
was not the case for C. sativus, which developed better in the presence of methyl propanoate. This
could be explained by the fact that C. sativus was resistant to methyl propanoate on PDA because
environmental conditions were more favorable for its growth. This resistance could be related to the
structure of its conidia, whose generally thick cell wall facilitates survival [1719].
Methyl prop-2-enoate and methyl propanoate had a significant eect on C. sativus, especially
on WA medium. This can be linked to the physiology of the fungus, as unfavorable environmental
conditions can considerably aect C. sativus development. Neched [
20
] reported that the oxidative
action of oxygen or humidified ozone generated oxidative stress and induced a clear decrease of the
contamination level or even an absence of C. sativus on barley seeds. Poor environmental conditions
therefore seem to make C. sativus more sensitive to VOCs.
Int. J. Environ. Res. Public Health 2019,16, 2866 11 of 14
Our results also show that methyl prop-2-enoate and methyl propanoate had a fungicidal or
fungistatic eect against both pathogenic fungal strains in PDA medium. At a concentration of 500
µ
M,
methyl prop-2-enoate had a fungicidal eect. A Combretum racemosum natural extract used
in vitro
at 2 g/L had a fungicidal eect on the mycelial growth of three telluric fungal pathogens of tomato
crops in Ivory Coast(Zirihi et al. [
21
]). In comparison with that study, methyl prop-2-enoate had an
important antifungal eect at lower concentrations (500–1000 µM).
The antifungal activity of MP and MA could be related to a cell disruption phenomenon. Both
VOCs induced an increase in the membrane permeability of pathogenic spores and a decrease of the
eux of K+ions into the intracellular medium [21]. To compensate for this imbalance, the activity of
the proton pumps has to increase to ensure the eux of H
+
ions into the intracellular medium and
maintain electrical charges on either side of the membrane at equilibrium [
22
24
]. This could induce a
drastic change of the pH in the intracellular medium and inhibit fungal growth [
18
,
19
]. Many studies
have highlighted the ability of nonanoic acid molecules and strobilurin to disturb the pH gradient
between the intracellular medium and the extracellular media [
25
28
]. To our knowledge, this is the
first study that has focused on the mode of action of these molecules on these two pathogens.
5. Conclusions
In the last few years, VOCs, a complex mixture of volatile compounds, have attracted great
attention due to their antimicrobial potential. Their ability to be used as antifungal agents to fight
against numerous pathogens has been considered as an attractive biocontrol strategy in agriculture. In
the present study, we noticed the strong eect of two organic esters (methyl prop-2-enoate and methyl
propanoate) against the two pathogens Fusarium culmorum and Cochliobolus sativus. We highlighted
the ability of methyl prop-2-enoate to inhibit F. culmorum growth by more than 99% and 97% in direct
contact phase and in gas phase, respectively. In the same way, methyl propanoate showed remarkable
inhibitory activity against F. culmorum. As for C. sativus, its proliferation was totally inhibited by
both organic esters in direct and indirect contact. In a second step, we attempted to understand
the mechanism of action of methyl propanoate and methyl prop-2-enoate. After contact between
fungal spores and the VOCs, an imbalance of the distribution of K
+
ions between the intracellular
medium and the extracellular medium was observed. This result suggests that both VOCs aect the
integrity of the fungal membrane and disturb the pH gradient between the intracellular medium and
the extracellular medium. However, further work is needed to confirm this hypothesis. In order to
promote the use of methyl prop-2-enoate and methyl propanoate in agriculture, the development of
cost-eective preparations is highly required. Alginate beads were recently used as vectors to ensure a
slow diusion of volatile molecules in the soil to fight against parasitoids and aphid predators [
12
,
29
].
The same strategy could be adopted to coat the antifungal molecules methyl prop-2-enoate and methyl
propanoate and ensure their diusion in the soil.
Supplementary Materials:
The following are available online at http://www.mdpi.com/1660- 4601/16/16/2866/s1,
Figure S1: Growth test of C. sativus or F. culmorum in the gas phase with a VOC on water agar or PDA media.,
Figure S2: Growth test of F. culmorum in direct contact with methyl prop-2-enoate on PDA media., Figure S3:
Growth test of F. culmorum in direct contact with methyl propanoate on PDA media., Figure S4: Evaluation of
the quantity of K+ions released by VOCs treated or non-treated, by conidia of F. culmorum after 3 h, Figure S5:
Evaluation of the quantity of K+ions released by VOCs treated or non-treated, by conidia of C. sativus after 4 h.
Author Contributions:
Conceptualization, A.K. and Y.Y.; methodology, A.K.; software, A.K.; validation,
M.H.J., N.B., S.K. and M.L.F.; formal analysis, A.K.; investigation, A.K.; resources, A.K.; data curation, A.K.;
writing—original draft preparation, A.K.; writing—review and editing, A.K; visualization, A.K.; supervision,
M.H.J. and N.B.; project administration, M.H.J..; funding acquisition, M.H.J.
Funding: This research received no external funding.
Acknowledgments:
The statistical analyses benefited from advice provided by Yves Brostaux (Gembloux Agro-Bio
Tech, University of Liège).
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Environ. Res. Public Health 2019,16, 2866 12 of 14
Appendix A
Int. J. Environ. Res. Public Health 2019, 16, x 13 of 15
Appendix A
Int. J. Environ. Res. Public Health 2019,16, 2866 13 of 14
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