Industrial Crops & Products 141 (2019) 111808

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Industrial Crops & Products

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Nano silver-encapsulation of Thymus daenensis and Anethum graveolens T

essential oils enhances antifungal potential against strawberry anthracnose
⁎
Weria Weisanya, , Jahanshir Aminib, Saadi Samadic, Somaieh Hossainib, Shima Yousefia,
Paul C. Struikd
a
Department of Agriculture and Food Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Plant Protection, College of Agriculture, University of Kurdistan, P.O. Box 416, Sanandaj, Iran
c
Laboratory of Asymmetric Synthesis, Department of Chemistry, Faculty of Science, University of Kurdistan, P.O. Box 416, Sanandaj, Iran
d
Centre for Crop Systems Analysis, Plant Sciences, Wageningen University & Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, Netherlands

A R T I C LE I N FO A B S T R A C T

Keywords: Nanotechnology is an eco-friendly strategy in managing plant diseases. In combination with existing practices
Dill nanotechnology can enhance the protection of agricultural products and food. For example, essential oils (EOs)
Essential oil encapsulation of thyme (Thymus daenensis L.) and dill (Anethum graveolens L.) have an antimicrobial potential and this potential
Silver nanoparticles may be enhanced by certain nanoparticle systems. Here we demonstrate that encapsulating EOs of thyme and
Strawberry anthracnose
dill in silver nanoparticles increases their fungicidal activity against Colletotrichum nymphaeae, causing an-
Thyme
thracnose in many horticultural crops. Using GC-MS analysis, we identified p-cymene, thymol, carvacrol and (E)-
caryophyllene as the main EOs of thyme and limonene, cis-dihydrocarvone, cyclohexanon, and carvone as the
main EOs of dill. When the EOs of the two sources were encapsulated in silver nanoparticles, synergistic effects
against C. nymphaeae were observed, resulting in more than 80% inhibition of mycelium growth of C. nym-
phaeae. Moreover, conidia germination was suppressed by nano-encapsulated EOs. We also observed consider-
able morphological changes in the fungal hyphae when nano-encapsulated EOs were applied. Our study de-
monstrates the potential of encapsulated EOs in controlling pathogens that can be very applicable as antifungal
agents.

1. Introduction major components, and their functional groups. Moreover, synergistic

Essential oils (EOs) are unstable, aromatic liquids that naturally
occur in various aromatic plant species. They can be harvested from
such plants using different extraction techniques, including distillation
and cold pressing (Pavela et al., 2019). The EOs from one plant species
can be very diverse: 20–60 (even up to over 100) volatile substances
can be derived; however their concentrations in the plant material vary
greatly.
EOs have a potential use in the ecofriendly management of plant
diseases, because it has been demonstrated that EOs possess diverse
biological activities, including inhibiting the growth of bacteria, fungi
and yeast (Karimi et al., 2016; Bakkali et al., 2008). For example, the
phenolic monoterpenes carvacrol and thymol have been proven to be
powerful, ecofriendly and effective agents with strong antibacterial,
antifungal and insecticidal effects (Yahyazadeh et al., 2008; Tabari
et al., 2017). These effects obviously depend on the type of mixture of
chemical components in the pool of EOs, the chemical nature of the
interactions between some of the major and minor constituents may
occur and such interactions may enhance the antimicrobial effects
(Dorman and Deans, 2000). Effects can be considerable: EOs from dill
(Anethum graveolens L.) impede Penicillium verrucosum growth by 50%
and also significantly inhibit mycelium growth and conidia germination
of Colletotrichum nymphaeae (Karimi et al., 2016).
Anthracnose is caused by Colletotrichum spp. and can be considered
a serious disease in strawberry worldwide (Howard et al., 1992). The
causal agent is recent multi-gene phylogenetic studies have demon-
strated that several Colletotrichum spp. within C. acutatum sensu lato and
C. gloeosporioides sensu lato are involved (Damm et al., 2012; Weir et al.,
2012). In Iran, Colletotrichum nymphaeae (belonging to C. acutatum sensu
lato) is probably the sole causal agent of the anthracnose. In recent
years, anthracnose became a real epidemic in the strawberry fields in
the province of Kurdistan (Karimi et al., 2016, 2019). Designing an
ecofriendly, effective control strategy is therefore economically of great
importance. Use of EOs could be developed into such a strategy,

⁎
Corresponding author.
E-mail address: weria.wisany@gmail.com (W. Weisany).

https://doi.org/10.1016/j.indcrop.2019.111808
Received 2 July 2019; Received in revised form 10 September 2019; Accepted 21 September 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
W. Weisany, et al.
Fig. 1. Scanning electron micrograph (SEM) of silver nanoparticles.
especially if their effectiveness could be enhanced.
Ghosh et al. (2013) showed that there was a strong synergistic effect
of cinnamaldehyde and silver NPs on the development of spores of
Bacillus cereus and Clostridium perfringens. Not all encapsulations are
equally efficient: Zhang et al. (2014) demonstrated that encapsulating
thymol with zein NPs had a larger growth-inhibiting effect on Gram-
positive bacteria than encapsulating thymol with other NPs. En-
capsulating EOs is effective as it keeps a liquid EO in a carrier matrix
(i.e. in the form of a dry powder), thus increasing its shelf life, reducing
its volatility and improving the precision with which the EO is applied.
Encapsulating EOs also helps control accurately the time and place of
their release (Li et al., 2007a,b).
Many different compounds have been used to encapsulate EOs.
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Industrial Crops & Products 141 (2019) 111808
Eugenol encapsulated in polycaprolactone NPs improved the stability of
eugenol during storage by protecting it against light oxidation (Choi
et al., 2009). Duncan et al. (2015) produced various NPs encapsulating
peppermint EOs and cinnamaldehyde. Similarly, Cham Paula et al.
(2016) formulated Lippia sidoides EOs (with large quantities of thymol)
using chitosan-gum NPs.
Silver NPs are effective antimicrobials (Rai et al., 2009, 2016). It has
been shown that they are effective against spores of several fungi that
are plant pathogenic, including Fusarium culmorum (Kasprowicz et al.,
2010) and F. oxysporum (Musarrat et al., 2010) and that they possess a
strong antifungal activity against F. semitectum, Botrytis cinerea, Phoma
glomerata and Ph. herbarum, (Gajbhiye et al., 2009) and C. gloeospor-
ioides (Aguilar-Mendez et al., 2010).
W. Weisany, et al.
Fig. 2. FT-IR spectrum of silver nanoparticles.
Fig. 3. Essential oil release profiles of thyme (A) and dill (B) essential oils en-
capsulated in silver nanoparticles or not encapsulated.
Pure EOs are effective antimicrobial agents. As indicated above,
combining different EOs can create synergistic effects. Such synergistic
effects might also occur between EOs and other antifungal agents, in-
cluding commercial antibiotics, organic acids (Moon et al., 2011) and
NP (Moon et al., 2011; Rai et al., 2009) and silver nanoparticles.
The hypotheses of this experiment are: (1) the combination of EOs
with NPs may facilitate their applicability as antimicrobials (2) nano-
encapsulation of EOs enhances the physical stability of the EOs, pro-
tects them against environmental influences and enhances their
bioactivity (3) nano-encapsulation allows to create synergy between the
antimicrobial effects of NPs with the antimicrobial effect of the EOs.
However, no research has been reported that specifically aimed to as-
sess the synergetic effect of EOs nanoencapsulated with silver NP. In the
present study we aimed to verify whether encapsulation of thyme and
dill EOs with silver NP enhances the overall effectiveness against the
plant pathogen C. nymphaeae.
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Industrial Crops & Products 141 (2019) 111808
2. Material and methods
2.1. Origin of plant material
Dill (Anethum graveolens) and thyme (Thymus daenensis) were grown
at the experimental fields of the Research Centre for Agriculture and
Natural Resources, in the Kurdistan Province of Iran, in 2017. The size
of each plot was 4 m × 5 m. The crops were rainfed, but we supplied
additional water through irrigation, whenever necessary. Sowing dates
of dill and thyme were 13 April 2017. For dill we collected seeds when
they were mature, 15 weeks after sowing the plants; for thyme we
harvested leaves at flowering, 16 weeks after sowing.
2.2. EO release study
Release profiles of EOs of thyme and dill EOs from the nanocapsule
systems were assessed over a period of 10 h. Encapsulated EOs were
redispersed in 3 mL of phosphate buffered saline (PBS) solution (pH
7.4) and 22% ethanol and agitated at 50 rpm and 37 °C. Samples (3 mL)
were withdrawn at frequent intervals. Their absorbance was measured
at the appropriate wavelength, and 3 mL of the sample buffer was re-
placed. The release was quantified as follows:
Release (%) = [Released EO/Total EO] × 100
2.3. Assessing contact fungicide activity
For each of the concentrations, the essential oils extracted from the
leaves of the thyme plants or the seeds of the dill plants were solved
individually in 0.1% (v/v) tween 80 (60 mL) and 3 mL of 0.5% ethanol
(90%). At a temperature of 45 °C, the essential oils were added to 97 mL
of melted potato dextrose agar realizing the following final concentra-
tions: 0 (control), 80, 240, 720 and 1000 ppm of the original EOs. With
a sterilized pipette we plated 20 mL of medium in Petri dishes. Per EO
concentration, three Petri plates were subsequently inoculated by in-
jecting a sterile striker in the conidia formed on acervuli of
Colletotrichum nymphaeae over a distance of 1 mm and then inserting
this striker in the center of the Petri dish. These Petri dishes were then
incubated in the dark at 26 °C for 12 days. Mycelium growth was as-
sessed after 3, 6, 9 and 12 days. To calculate the percentage of growth
inhibition by the essential oils we used the following equation (Soylu
et al., 2006):
2.4. Assessing effects of essential oils on germination of spores
We evaluated the influence of EOs on the percentage germination of
spores of the pathogen. In order to do that, we first diluted the required
amount of EO in a 0.5 mL/L Tween 80 (0.1%) solution. Subsequently,
each vial of diluted EO was added to so-called Falcon tubes (Sigma-
Aldrich, USA) that contained 4.5 mL of potato dextrose broth (PDB) to
achieve the following concentrations of EOs: 0 (control), 80, 240, 720
and 1000 ppm. In addition, by diluting the original stock solution of
essential oils and silver nanoparticles in Falcon tubes containing 4.5 mL
PDB, we prepared the following concentrations of EOs encapsulated in
silver nanoparticles: 0, 52, 145, 410 and 625 ppm. Moreover, we pre-
pared a suspension of conidia with 1 × 106 conidia mL−1 (density
realized on the basis of counts using a homocytometer). Of this sus-
pension of conidia, 200 μL was added to each Falcon tube. Falcon tubes
were subsequently incubated at 25 °C for 10 h, while shaking at a speed
of 120 rpm. We put droplets of the various suspensions on microscope
glass slides and stained them with lacto-phenol-cotton blue; after fixing
them, we covered them by cover glass and assessed the level of ger-
mination of the spores of the pathogen. We observed 100 spores in each
treatment calculated the inhibition of germination in comparison with
the control was calculated and expressed the inhibition as a percentage.
W. Weisany, et al. Industrial Crops & Products 141 (2019) 111808

Fig. 4. Mycelium growth inhibition of Colletotrichum nymphaeae after 3 (A), 6 (B), 9 (C) and 12 (D) days of incubation as affected by 0 (control), 80, 240, 720 and
1000 ppm thyme (T) and dill (D) essential oil. Results are the means of three replications. Means sharing the same letter are not significantly different at the 5%
probability level using the Least Significant Different (LSD) test.

Fig. 5. Mycelium growth inhibition of Colletotrichum nymphaeae after 3 (A), 6 (B), 9 (C) and 12 (D) days of incubation as affected by 0 (control), 25, 50, 100 and
250 ppm silver nanoparticles. Results are the means of three replications. Means sharing the same letter are not significantly different at the 5% probability level
using the Least Significant Different (LSD) test.

The assay was replicated three times (Karimi et al., 2016).
2.5. Essential oil extraction
Plant material was air-dried and 100 g of the dry plant material was
subjected to hydro-distillation in a Clevenger apparatus for 2 h using
500 ml of water, followed by drying over anhydrous sodium sulfate for
3 days. The layer of EO was then shaped at a temperature of 35 °C, using
a Vigreux column, subsequently removed and finally stored at 4 °C for
later analysis (Weisany et al., 2015).
2.6. Gas chromatography: composition of essential oils of thyme and dill
before and after encapsulation measured by mass spectroscopy
We used an Agilent 7890B gas chromatography (GC) system. It was
equipped with a split/splitless injector and a 5977A mass selective

4
W. Weisany, et al.
Fig. 6. Mycelium growth inhibition of Colletotrichum nymphaeae after 3 (A), 6 (B), 9 (C) and 12 (D) days of incubation as affected by dill (D) and thyme (T) essential
oils encapsulated in silver nanoparticles at 0 (control), 52, 145, 410 and 625 ppm. Results are the means of three replications. Means sharing the same letter are not
significantly different at the 5% probability level using the Least Significant Different (LSD) test.
detector system. Per sample, 1 μL was injected. The tray temperature
was set at 7 °C and the dwell time after injection was 4 s. We operated
the mass spectrometer (MS) in the EI mode, at 70 eV. The carrier gas
was helium (purity: 99.999%) at a flow rate of 1 mL min−1. The column
was kept at 40 °C for 4 min, subsequently heated to 250 °C and kept at
this temperature for 4 min. To carry out separation we used a
30 m × 0.25 mm DB 35 MS column. The quadrupole temperatures were
set at 150 °C, the ion source was set at 230 °C and the GC–MS interface
was set at 280 °C, respectively. The injector temperature was set at
280 °C. We identified the active compounds by comparing their mass
spectra and retention times with mass spectrum data stored in the Wiley
and NIST spectral library with and the mass spectra of standard com-
pounds.
2.7. Synthesizing silver nanoparticles
Silver nanoparticles (NPs) was prepared applying the method re-
ported by Tavallali and Pouresmaeil (2012). Briefly, we dissolved
0.265 mmol (45 mg) silver nitrate in 100 mL deionized water; subse-
quently, 10 mL of trisodium citrate aqueous solution (1%) was added
gradually to the mixture of reaction and stirred at room temperature for
30 minutes. Afterward, 0.2 mL of 0.005 M ascorbic acid was added to
the solution and mixed vigorously for a period of 1 hour until a yellow-
green silver nanoparticle colloid was formed. By diluting the original
stock solution, we created the following final concentrations of silver
NPs: 25, 50, 100 and 250 ppm. All solutions were kept at 4°C prior to
analysis. The synthesized silver nanoparticles was characterized by
scanning electron microscopy (SEM) (Fig. 1) and FT-IR (Fig. 2) tech-
niques. SEM images showed that Ag nanoparticles have a size of about
6-90 nm.
Percentage mycelial inhibition (%) = [(Dc – Dt)/Dc] × 100;
in which Dc is the diameter of the mycelium in the control and Dt is the
diameter of the mycelium of the treated plates.
2.8. Encapsulating essential oils in silver nanoparticles
After the silver nanoparticles had been synthesized, different
5
Industrial Crops & Products 141 (2019) 111808
quantities of the various stock solutions of the EOs (i.e. 0, 4, 12, 36 and
50 μL) were separately mixed with different quantities of silver particles
(0, 1.25, 2.5, 5 and 12.5 mL, respectively) realizing the following final
concentrations of EOs encapsulated in silver nanoparticles: 0, 52, 145,
410, and 625 ppm. The mixes of different concentrations were lightly
stirred using a glass rod allowing an even distribution of the essential
oils encapsulated in the silver nanoparticles in the mixture. Finally, the
mixtures were stored at 4 °C for later analysis.
2.9. Statistical analysis
We performed an analysis of variance with the statistical package
SAS version 9.1 (SAS Institute Inc., 2004). Through orthogonal com-
parisons, the treatment means were compared. Mean separation was
based on the Generalized Linear Model (GLM) method and on Least
Significant Different (LSD) at a probability level of 5%. Because the data
were normally distributed, transformation was not necessary.
3. Results and discussion
3.1. Essential oil release
Fig. 3 illustrates the release of both the thyme and dill EOs when
encapsulated or not encapsulated. The release was slower for en-
capsulated than for non-encapsulated EOs (Fig. 3). For thyme EOs, the
maximum release time for encapsulated EO was 550 min and for non-
encapsulated EO it was 500 min for 100% release (Fig. 3a). On the other
hand, for dill EOs the maximum release was observed after 600 min for
encapsulated and non-encapsulated EOs (Fig. 3b). After 450 and
500 min, approximately 95% of the non-encapsulated thyme and dill
EOs were released, however only 77% and 81% of the encapsulated
thyme and dill EOs were released, respectively (Fig. 3a). The slower
release might be attributed to the partial absorption of the EOs by na-
noparticles system; therefore, release is impeded by the slow oil diffu-
sion from the core across the membrane coating.
W. Weisany, et al.
Fig. 7. Conidia germination inhibition of Colletotrichum nymphaeae as affected
by 0 (control), 80, 240, 720 and 1000 ppm thyme (T) and dill (D) essential oil
(A), 0 (control), 25, 50, 100 and 250 silver nanoparticles (B) and dill (D) and
thyme (T) encapsulated in silver nanoparticles at 0 (control), 52, 145, 410 and
625 ppm (C). Results are the means of three replications. Means sharing the
same letter are not significantly different at the 5% probability level using the
Least Significant Different (LSD) test.
3.1.1. Bioassays of fungicidal activity of essential oils, silver nanoparticles
and essential oils encapsulated in silver nanoparticles
The essential oils extracted from the leaves of thyme plants and
from the seeds of dill plants proved to inhibit the growth of C. nym-
phaeae mycelium (Fig. 4). The highest EO concentration tested
(1000 mg/L) showed the largest effect. However, there was no sig-
nificant difference between 720–1000 ppm. The mycelium growth of C.
nymphaeae mycelium was also significantly reduced by silver nano-
particles. The percentage of inhibition of mycelium growth was in-
creased by 100, 94, 80 and 70% after 3, 6, 9 and 12 days of incubation
with EO encapsulated with silver NPs (625 ppm), respectively (Fig. 5).
Volatile compounds in plants, particularly EO are considered non-
phytotoxic and potentially effective against plant pathogenic fungi
(Pimentel et al., 2018). Causes for this reduction in growth could be
multiple, including changes in membrane permeability, resulting from
leakage of electrolytes and proteins (Cui et al., 2015), irregularities in
mitochondrial structure, plasma membrane outage, and decreases in
sugars, changes in cell wall structure, ATP and DNA. In many cases,
6
Industrial Crops & Products 141 (2019) 111808
blocking the generation of energy (ATP) and the enzymes involved in
that process causes cell demolition (Huang et al., 2014; Cui et al., 2015;
Li and Yu, 2015; Lakehal et al., 2016). In addition, Cristani et al. (2007)
suggested that the capability of EOs to disturb the lipid bilayer mem-
branes was linked with their antimicrobial activity (Cristani et al.,
2007). Since EOs are very hydrophobic, they interact with the fatty
acids in the membranes of the cells of the microbes (Blaszyk and Holley,
1998; Helander et al., 1998). Finally, EOs may inhibit the activities of
enzymes in the mitochondria, such as ATPase, malate dehydrogenase
and succinate dehydrogenase (Zeng et al., 2015).
In the present study, the efficiency of EO encapsulation on the re-
duction of C. nymphaeae mycelium growth was studied at concentra-
tions ranging from 52 to 625 ppm. The EOs of thyme and dill en-
capsulated in silver NPs were able to significantly inhibit mycelium
growth of C. nymphaeae, up to 100% at 3, 6, 9 and 12 days after in-
cubation (Fig. 6).
3.2. Assay of germination of conidia
The germination of conidia of C. nymphaeae was significantly re-
duced at 10 h after applying the EOs of thyme and dill, of the silver NPs
and of the EOs of thyme and dill encapsulated in silver NPs. Conidia
germination decreased by 82% in the case of the EOs from thyme and
71% in the case of the EOs from dill, compared with the control (Fig. 7).
Encapsulating the EOs of thyme and dill further increased the inhibition
of conidia germination by 8.5 and 18.3%, respectively. This is in line
with the inhibition of Fusarium oxysporum reported by Musarrat et al.
(2010). In our study, silver NPs strongly reduced the number of conidia
that germinated when compared with the control. The antifungal ac-
tivities of silver NPs are due to Ag+ ions. These ions can create punc-
tures in the membranes, through which leakage of cytoplasmic contents
out of the cell can occur; such leakage will destroy the H+ gradient
across the membrane and will ultimately result in the death of the cell
(Knetsch and Koole, 2011; Lara et al., 2010). NPs can prevent enzy-
matic deterioration of EOs, can alter them into a powder, can help to
create optimal timing and location of application and can make EOs
much more efficient. Bilia et al. (2014) called this the opportunity to
create an ideal pharmacokinetic profile. Combining the application of
antifungal EOs with the application of antifungal NPs creates synergy in
effectiveness by combining different spectra of mechanisms of action
(Rai et al., 2017).
3.2.1. Essential oils, silver nanoparticles and encapsulated essential oils
affected the development and morphology of the mycelium
The essential oils of thyme and dill, the silver nanoparticles and the
EOs of thyme and dill when encapsulated in silver nanoparticles da-
maged the C. nymphaeae mycelium when compared with the water
treatment (Fig. 8). The essential oils from thyme and dill, the silver
nanoparticles and the essential oils from thyme and dill when en-
capsulated in silver nanoparticles prevented the formation of the ap-
pressorium (Fig. 8B–F).
Applying EOs of thyme and dill caused some changes in the mor-
phology: we observed swelling and thickening of hyphae and some
hyphae showed twisted growth (Fig. 8B–C). At higher concentrations of
the EOs, we observed vesicle-like structures besides the mycelia of the
fungus. At high concentrations of silver NPs (625 ppm), we observed
shortened hyphae, vesicle-like structures and leakage of cytoplasmic
content (Fig. 8D). Treatments with EOs from thyme and oil en-
capsulated in silver nanoparticles resulted in multi-branched, coiled,
and thickened mycelia (Fig. 8E–F).
The untreated C. nymphaeae showed regular growth of the hyphae
(Fig. 9A), while EOs encapsulated with silver NPs, at 410 ppm, ap-
peared to create a coating around the fungal material and caused a
strong reduction of the growth of the hyphae (Fig. 9B). Moreover, the
hyphae showed all kinds of deformations (Fig. 9B). In most cases, hy-
phae died and became lesioned.
W. Weisany, et al.
Fig. 8. Morphological responses of Colletotrichum nymphaeae to the effects of control (A), dill (B), thyme (C), nano-silver (D), dill essential oil encapsulated in silver
nanoparticles (E) and thyme essential oil encapsulated in silver nanoparticles (F).
Silver NPs bind to the RNA and DNA of microbes and block DNA
duplication and cell division (Lara et al., 2010; Blecher et al., 2011;
Knetsch and Koole, 2011). In addition silver ions can act as anti-
microbials by producing reactive oxygen species (ROS), which are
harmful to both bacterial cells and eukaryotic host cells (Lara et al.,
2010; Knetsch and Koole, 2011). Moreover, Lara et al. (2010) reported
that silver ions prevent the synthesis of cell walls in gram-positive
bacteria.
Encapsulation of EOs from thyme and dill in silver nanoparticles
inhibited the growth of the mycelium of C. nymphaeae (Fig. 6). The
precise mechanism by which nano-encapsulated EOs cause damage to
and inhibit growth of C. nymphaeae mycelium is not well known. One
possibility is that they bind to the surface of the cell, subsequently in-
fluencing the permeability of the cell membrane (Rai et al., 2017).
Accumulation of EOs disrupts the structural integrity of the phospho-
lipid bilayer, and this disruption may interfere with cell metabolism,
ultimately causing the death of the cell. Moreover, Rai et al. (2017)
stated that various types of essential oils might activate channels in the
the cell membrane when mixed with other antifungal agents like NPs.
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Industrial Crops & Products 141 (2019) 111808
3.3. Essential oil composition
The main component of the EOs of thyme was thymol whereas the
main component of dill EOs was carvone. This was true both before and
after nano- encapsulation. Other components showed variation with
treatments (Tables 1 and 2). Analyzing the EOs with GC–MS indicated
that α-pinene (0.24%), p-cymene (3.07%), γ-terpinene (0.36%), iso-
borneol (0.24%), thymol (91.15%), carvacrol (1.79%) and (E)-Car-
yophyllene (1.08%) were the major components in T. daenensis EOs,
whereas α-phellandrene (1.85%), limonene (3.13%), p-cymene
(1.37%), dill ether (0.61%), cyclohexanon (2.71%), carvone (87.91%),
and apiol (1.52%) were dominant in A. graveolens EOs. After en-
capsulation with silver NPs, linalool (0.24%), camphene, carvacrol and
(E)-caryophyllene contents of thyme EOs improved respectively by
0.20, 0.49, 3.7 and 1.46% (Table 1). Moreover, encapsulating dill EOs
in silver nanoparticles caused an increase in the contents of dill ether,
carvone and cis-dihydrocarvone by 1.07, 4.14, and 88.99%, respec-
tively (Table 2). Nano-encapsulation of EOs decreased α-pinene and
thymol in T. daenensis EO, and α-phellandrene, limonene, p-cymene and
cyclohexanon in A. graveolens EO (Tables 1 and 2; Fig. 10–13 in
W. Weisany, et al. Industrial Crops & Products 141 (2019) 111808

Fig. 9. Scanning electron microscopy (SEM) of regular morphology of Colletotrichum nymphaeae hyphae (A) and effected by EOs encapsulated in silver nanoparticles
(B). The hyphae fungal morphology is deeply altered.

Table 1
The chemical composition essential oils (EO) in thyme leaves and essential oils of thyme encapsulated in silver nanoparticles.
Thyme EO Thyme EO encapsulated in silver nanoparticles

Compounds Area (%) Retention time (min) Compounds Area (%) Retention time (min) Confirmed by

α-Phellandrene 0.13 6.39 α-Pinene 0.13 7.86 STD, MS

α-Pinene 0.24 6.59 Camphene 0.19 8.64 STD, MS
Camphene 0.14 7.24 α-Terpipene 0.18 10.98 STD, MS
2-β-Pinene 0.06 8.10 1,8-Cineole 28.47 12.11 STD, MS
Myrcene 0.12 8.45 ƴ-Terpinene 0.18 12.48 STD, MS
α-Thujene 0.04 8.97 Linalool L 0.20 13.97 STD, MS
α-terpipene 0.18 9.28 2-Nonyne 5.44 16.59 STD, MS
Limonene 0.06 9.59 L-Camphor 0.50 17.28 STD, MS
β-Phellandrene 0.03 9.81 Camphene 0.49 17.79 STD, MS
p-Cymene 3.07 10.14 Thymyl Methyl Ether 0.24 18.16 MS, RI
ƴ-Terpinene 0.36 10.64 Thymol 58.05 20.34 STD, MS
Bicyclo[3.1.0]hexan-2-o 0.01 11.29 Carvacrol 3.70 20.66 STD, MS
Linalool 0.13 12.09 Trans-Caryophyllene 1.46 21.45 STD, MS
Camphene 0.18 14.44 β-Bisabolene 0.12 23.15 MS, RI
Isoborneol 0.24 14.56 CIS-α-Bisabolene 0.18 24.00 STD, MS
Terpinen-4-ol 0.10 14.69 Patchulane 0.10 25.93 STD, MS, RI
Isoterpinolene 0.09 15.33 Bicyclo[4.4.0] 0.07 26.97 STD, MS
Thymyl methyl ether 0.02 16.00 Tetramethylhexadecane 0.18 36.03 STD, MS
Cis-dihydrocarvone 0.02 16.08 STD, MS
Carvacrol methyl ether 0.19 16.16 RI, MS
Isobornyl acetate 0.04 17.43 STD, MS
Thymol 91.15 18.17 STD, MS
Carvacrol 1.79 18.31 RI, MS
Trans-Caryophyllene 1.08 19.31 STD, MS
Ledene 0.03 19.70 STD, MS
4,7,10-Cycloundecatriene 0.03 20.21 RI, MS
Alloaromadendrene 0.02 21.03 STD, MS
β-Bisabolene 0.07 21.09 STD, MS
CIS-α-bisabolene 0.14 21.97 RI, MS
3-Tridecen-1-yne 0.07 23.93 STD, MS

STD, MS = confirmed by injection of Standard and by Mass Spectra library; RI, MS = confirmed by n-alkanes Retention Index by Mass Spectra library;
MS = tentative identification = confirmation only by Mass Spectra library.

Supplementary files). effectively inhibited Colletotrichum nymphaeae; therefore, it can be used

4. Conclusions and future trends
EOs harvested from leaves of thyme and from seeds of dill very
as antifungal agents against C. nymphaeae. Encapsulation in silver na-
noparticles strongly improves the chemical stability of EOs when ex-
posed to light, air, high temperatures and moisture. Furthermore, nano-
phytopathological researches on the physiology of the pathogen and its

8
W. Weisany, et al. Industrial Crops & Products 141 (2019) 111808

Table 2
The chemical composition of essential oils (EO) of dill leaves and essential oils of dill encapsulated in silver nanoparticles.
Dill EO Dill EO encapsulated in silver nanoparticles

Compounds Area (%) Retention time (min) Compounds Area (%) Retention time (min) Confirmed by

α-Pinene 0.24 6.58 α-Phellandrene 0.10 8.99 STD, MS

α-Phellandrene 1.85 8.98 Limonene 0.67 9.60 STD, MS
Limonene 3.13 9.62 ƴ-Terpinene 0.18 9.82 STD, MS
β- Phellandrene 0.39 9.81 p-Cymene 0.83 10.12 STD, MS
p-Cymene 1.37 10.11 Dill ether 1.07 15.01 STD, MS
Dill ether 0.61 15.01 A-phellandrene epoxide 0.15 15.68 STD, MS
cis-Dihydrocarvone 0.28 15.78 cis-Dihydrocarvone 4.14 15.81 STD, MS
Cyclohexanon 2.71 16.11 Cyclohexanon 0.80 16.08 STD, MS
L-carvone 87.91 17.52 L-carvone 88.99 17.46 STD, MS
2-(4-hydroxy-2-butenyl)-2-nitroc 0.42 22.35 2-(4-hydroxy-2-butenyl)-2-nitroc 0.36 22.18 MS, RI
Myristicine 0.15 24.32 Myristicine 0.13 24.02 STD, MS
Camphene 0.08 25.11 Camphene 0.09 24.93 STD, MS
2,5-dimethyl-3-vinyl-hexa-1 0.39 25.36 2,5-dimethyl-3-vinyl-hexa-1 0.37 25.02 STD, MS
Apiol 1.52 25.87 Apiol 1.89 25.86 MS, RI
l-Phellandrene 0.29 30.12 l-Phellandrene 0.24 29.56 STD, MS

STD, MS = confirmed by injection of Standard and by Mass Spectra library; RI, MS = confirmed by n-alkanes Retention Index by Mass Spectra library;
MS = tentative identification = confirmation only by Mass Spectra library.

host, the infection process, the disease distinction and interactions can
aid in extending novel approaches to management of plant diseases,
including the use of nanoparticles that are less destructive to the en-
vironment than synthetic chemical pesticides. EOs from thyme and dill
took longer to be released when they were encapsulated in silver na-
noparticles than when they were not encapsulated. The use of nano-
encapsulated EOs with NPs sustained and controlled the release of the
active compounds and thus increased the bioavailability and usefulness
against C. nymphaeae. Therefore, nano-encapsulating essential oils may
enhance their application as antifungal agents.
Acknowledgements
The financial support by the University of Kurdistan, National Elites
Foundation, Iran, and by the Agriculture and Natural Resources
Research Center of the Kurdistan province, Iran, is gratefully ac-
knowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111808.
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