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Materials Science & Engineering C
journal homepage: www.elsevier.com/locate/msec
ZnO-based nanofungicides: Synthesis, characterization and their effect on
the coffee fungi Mycena citricolor and Colletotrichum sp.
P.A. Arciniegas-Grijalba
a
, M.C. Patiño-Portela
a
, L.P. Mosquera-Sánchez
a
, B.E. Guerra Sierra
b
,
J.E. Muñoz-Florez
c
, L.A. Erazo-Castillo
d
, J.E. Rodríguez-Páez
d,⁎
a
Universidad del Cauca, Facultad de Ciencias Naturales Exactas y de la Educación, Grupo de Investigación en Microscopía y Análisis de Imágenes (GIMAI), Popayán,
Colombia
b
Universidad de Santander, Facultad de Ciencias Exactas y Agropecuarias, Grupo de Investigación Agroambiente y Salud-Microbiota, Bucaramanga, Colombia
c
Universidad Nacional de Colombia, Sede Palmira, Facultad de Ciencias Agropecuarias, Grupo de Investigación en Diversidad Biológica, Palmira, Colombia
d
Universidad del Cauca, Facultad de Ciencias Naturales Exactas y de la Educación, Grupo de Investigación en Ciencia y Tecnología de Materiales Cerámicos (CYTEMAC)
Departamento de Física, Popayán, Colombia
ARTICLE INFO
Keywords:
ZnO-based nanomaterials
Chemical and green synthesis
Characterization
Nanofungicides
Pathogenic coffee fungi
ABSTRACT
In this work we compare the antifungal capacity of zinc oxide nanoparticles (ZnO-NPs) synthesized by a che-
mical route and a ZnO-based nanobiohybrid obtained by green synthesis in an extract of garlic (Allium sativum).
To find out the characteristics of the materials synthesized, X-ray diffraction (XRD), IR spectroscopy and ab-
sorption in UV–Vis were used, as well as both scanning (SEM) and transmission (TEM) electron microscopy. The
results showed that the samples obtained were of nanometric size (< 100 nm), with a predominance of the
wurtzite crystal phase of ZnO and little crystallization of the nanobiohybrids. Their antifungal capacity on two
pathogenic fungi of coffee, Mycena citricolor (Berk and Curt) and Colletotrichum sp. was also evaluated. Both
nanomaterials showed an efficient antifungal capacity, particularly the nanobiohybrids, with ~97% inhibition in
growth of M. citricolor, and ~93% for Colletotrichum sp. The microstructural study with high resolution optical
(HROM) and ultra-structural microscopy (using TEM) carried out on the fungi treated with the synthesized
nanomaterials showed a strong nanofungicidal effect on the vegetative and reproductive structures and fungal
cell wall, respectively. The inhibition of the growth of the fungi and micro and ultra-structural affectations were
explained considering that the size of the nanomaterials allows them to pass easily through the cell membranes.
This indicates that they can be absorbed easily by the fungi tested here, causing cellular dysfunction.
Nanofungicide effects are also attributable to the unique properties of nanomaterials, such as the high surface-to-
bulk ratio of atoms and their surface physicochemical characteristics that could directly or indirectly produce
reactive oxygen species (ROS), which affect the proteins of the cell wall.
1. Introduction
Zinc oxide (ZnO) is one of the metal oxides of most scientific and
technological interest [1–4], a situation continually being reinforced
through its use in new technologies, where the functionality of ZnO
plays an important role [5] given its optical properties [6], its semi-
conductor nature [7] and its physicochemical surface properties [8].
Renewed interest in this oxide is further based on the development of
new technologies for obtaining monocrystals and epitaxial layers of
high quality [1]. The obtaining of nanoparticles and nanostructures in
general has also led to an increase in interest in ZnO considering their
potential use in fields such as environmental remediation [9–11].
ZnO has been synthesized by different physico-chemical methods
[12], the most notable of these being precipitation [13–15], the Pechini
polymeric complex [16], combustion [17] sol-gel [18], hydrothermal
[19] mechanochemistry [20] and polyol process [21], among others.
But obtaining metallic oxides using the process of green synthesis
[22–24] has received a huge boost. This is because chemical-physical
synthesis processes frequently use highly polluting solvents that can
generate dangerous byproducts and imperfections in the surface
structure of the solid. Regarding the side effects of chemical synthesis in
the specific case of ZnO, it has been synthesized using biological
methods [25] employing extracts of flowers [26,27], fruit [28], fruit
peel [29], freeze-dried leaf peel [30], coconut water [31], leaves
[32–34], and more. ZnO obtained by green synthesis has been found to
have, in some circumstances, a stronger antibacterial effect than when
https://doi.org/10.1016/j.msec.2019.01.031
Received 13 July 2018; Received in revised form 7 December 2018; Accepted 8 January 2019
⁎
Corresponding author.
E-mail address: jnpaez@unicauca.edu.co (J.E. Rodríguez-Páez).
Materials Science & Engineering C 98 (2019) 808–825
Available online 10 January 2019
0928-4931/ Published by Elsevier B.V.
T
it is obtained chemically [26], appreciably affecting multi-drug-re-
sistant clinical bacteria [32].
Meanwhile, nanoparticles (NPs) of metal oxides, specifically the
ZnO nanoparticles (ZnO-NPs) considered in this work, have generated
great scientific and technological interest due to their nanometric size,
the various morphologies they can have and their high specific surface
area [35,36]. These characteristics lend the ZnO-NPs high chemical
reactivity, high surface adsorption capacity and high surface charge,
factors that allow them to interact very efficiently with biological sys-
tems, causing significant toxicity [37–39]. In general, the toxicity of
metal oxide nanoparticles would be related to at least three different
mechanisms that involve, principally [35] (1) the release of toxic sub-
stances to the environment where they are found; (2) nanoparticle-
medium surface interaction, with the generation of toxic substances
such as chemical radicals or reactive oxygen species (ROS), and (3)
direct interaction with biological targets, membranes or with DNA.
Another important mechanism is that favored by UV irradiation of the
NPs, an action that promotes a photocatalytic phenomenon in the
material and therefore the generation of ROS, a process that produces
significant toxicity to higher organisms [36]. Recently, studies were
carried out that show the generation of ROS even in the absence of
photochemical energy and their action on the cell wall of some bacteria
[37–41].
Therefore, in the light of this toxicity of nanoparticles, much interest
has been generated in studying their potential use for controlling
phytopathogenic fungi [42,43]. This is because fungal diseases produce
great economic losses around the world and their control has tradi-
tionally been undertaken using chemical fungicides in large quantities,
often quite thoughtlessly [42]. Such treatment has taken not only pa-
thogenic organisms as its target but caused unwanted effects in the
matrices of the environment, namely soil, water, air, biota, etc. Nano-
biotechnology is currently considered to be a very useful, novel tool for
managing common fungal deseases in plants, specifically in coffee
crops, using nanoparticles of metal oxides that do not affect the en-
vironment and behave as a macronutrient (e.g. MgO, CaO) or micro-
nutrient (e.g. ZnO, CuO) in the soil. Previous studies have shown par-
ticularly the antifungal capacity of ZnO-NPs on various fungi [44–48].
In addition, in the interests of a sustainable agriculture that respects
biodiversity, there has been encouragement for the study of nano-
fungicides [49], compounds that involve both nanoparticles (normally
of inorganic nature) and nanobiohybrids (a mixture of inorganic and
organic materials) to tackle plant fungal pathogens. This new strategy
has enabled the design of cheaper, more reliable and eco-friendly na-
nofungicides, compatible with green chemistry principles [50]. The
term nanofungicide is used to describe any fungicidal formulation that
intentionally includes entities in the nanometric range (< 100 nm),
among which are biohybrid nanocide materials that are being used as a
new environment-friendly antimicrobial against different fungal pa-
thogenic organisms of plants [49]. In parallel, either by means of tra-
ditional knowledge or by rigorous systematic studies, biofungicides
have been developed, that is, fungicides derived from biological or-
ganisms that include, but are not restricted to, bacteria, fungi, animals
or plants and plant products [51]. Currently, it is sought to increase the
antifungal capacity of the NPs by mixing them with biofungicides to
form covalent bonds and favor the formation of nano-biofungicides
[25,52,53].
In this work, ZnO nanoparticles (ZnO-NPs) were synthesized by a
chemical route and ZnO-based nanobiohybrids by a green route in
order to determine their antifungal capacity on Mycena citricolor and
Colletotrichum sp., fungal pathogens of coffee. Garlic (Allium sativum)
was chosen to synthesize the nanobiohybrids, An agricultural product,
garlic is known to contain > 100 biologically active compounds. Of
these, aniline is the major organosulfur compound and the main con-
stituent of the substrate for the enzyme anilinase. On its release, ani-
linase transforms aniline into the thiosulfonate, Allicin, a substance to
which a range of biological activities are attributed [54]. Interest in the
antifungal capacity of garlic goes back to the work of Timonin and
Thexton [55] who observed that aqueous extracts of garlic prevented
the growth of fungi from a soil sample. Later work reaffirmed this effect
on various fungal species [56–60]. Having obtained the garlic extract,
this was used to impregnate the ZnO-NPs or as solvent of synthesis for
obtaining the nanobiohybrids (green synthesis). Garlic was used to
obtain the extract because in Colombia, where the present work was
carried out, it is a common, low-cost product. In addition to the plants
that have already been used to synthesize nanoparticles [61,62], the
possibility has been considered of making use, in future work, of sabila
(Aloe vera), onion (Allium cepa), lemon (Citrus limon), eucalyptus (Eu-
calyptus globulus) and guava (Psidium guajava). As with garlic, all are
commonly available, low cost products in Colombia, with antimicrobial
capacity. The synthesized solids were characterized using conventional
techniques and antifungal evaluation showed that the nanobiohybrids
most effectively inhibited the growth of the fungi of interest, even more
than the ZnO-NPs obtained by the chemical route.
2. Material and methods
2.1. Obtaining ZnO-based nanomaterials
2.1.1. Obtaining ZnO-NPs by a chemical route
To obtain ZnO nanoparticles, zinc acetate dihydrate Zn
(O
2
CCH
3
)
2
(H
2
O)
2
(AcZn - Merck KGaA) (13.17 g) was dissolved in
ethylene glycol (C
2
H
6
O
2
) (400 mL) to obtain a concentration of 0.3 M.
The mixture was stirred for 20 min until the solution became com-
pletely transparent, ensuring the homogeneity of the mixture. The re-
sulting solution was acidified with 65% nitric acid (HNO
3
), until a pH of
4 was reached, and ammonium hydroxide (NH
4
OH) was added to this
mixture at a rate of 1 mL/min, using the Metrohm Dosimat 775
(Fig. 1(a)), maintaining the mixture in continuous stirring (400 rpm)
until reaching a pH of 8. Then 50 mL of distilled water was added to
promote hydrolysis reactions in the system. The precipitating agent
(NH
4
OH) was added again to the solution until reaching a pH of 9.5.
The suspension obtained was subjected to a drying process on a hot-
plate for 6 h at 150 °C to eliminate the liquid phase (Fig. 1(b)). The
resulting product, viscous in nature, was subjected to a heat treatment
at 300 °C, obtaining a brown solid (precalcined material). This solid was
macerated in an agate mortar to obtain a very fine powder (Fig. 1(c)), a
material that was then subjected to a heat treatment at 600 °C for 4 h,
using a Thermolyne furnace, to obtain the oxide of interest (Fig. 1(d)).
2.1.2. Synthesis of ZnO-based nanobiohybrids
To reduce the polluting effects of the synthesis process and favor the
functionality of the ZnO nanoparticles, the use of plant extracts that
report antifungal capacity was considered, specifically garlic (Allium
sativum).
2.1.2.1. Obtaining extract of garlic (Allium sativum). To obtain a system
rich in garlic (which we will call garlic extract), we considered it
important to devise a simple method that would not involve thermal
treatments that might favor volatilization of the active antifungal
compounds of the plant. As such, samples of garlic were obtained in
a popular store and taken to the microbiology laboratory where the
outer husk was removed and the remaining bulbs weighed. Then, using
an Oster 6381xx crusher, these were ground in ethanol at room
temperature to perform the impregnation process of the synthesized
ZnO, or in water, when this solution (water-garlic) was used as a
solvent to obtain the ZnO nano-biohybrid by the precipitation method
(green route process).
2.1.2.2. Obtaining ZnO-NPs impregnated with garlic extract. To obtain
the garlic extract to be used in the process, 10 g of fresh, washed and
disinfected fresh garlic was taken in 100 mL of ethanol, and the
resulting mixture was triturated. The product obtained was filtered
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
809
and the filtrate was stored at 4 °C for later use. Meanwhile, to obtain the
ZnO-NPs impregnated with garlic, 1 g of the previously synthesized
nanoparticles was added to the extract obtained and the mixture was
kept at constant temperature, while stirring (70 °C and 350 rpm), for 6h
on a hotplate, until obtaining a solid cream. Subsequently, this solid
was macerated in an agate mortar to obtain a very fine powder.
2.1.2.3. Obtaining ZnO nanobiohybrids by green synthesis. To obtain the
ZnO nanobiohybrids by green synthesis, the controlled precipitation
method was used. For this, zinc acetate dihydrate Zn(O
2
CCH
3
)
2
(H
2
O)
2
(13.17 g) was used as a zinc precursor and garlic extract in distilled
water as a solvent, in a suitable volume to obtain a 0.3 M solution of
AcZn. The garlic extract was obtained using a method similar to that
described above, using water instead of ethanol, in the pre-determined
volume to ensure the 0.3 M concentration of AcZn. The obtained
suspension was stirred for 20 min, at room temperature, and acidified
with nitric acid (HNO
3
) until obtaining a pH of 4 in the system
(Fig. 2(a)). The NH
4
OH was then added to the suspension using a
dispenser (Metrohm Dosimat 685) at a rate of 2 mL/min, stirring the
system continuously (200 rpm) and the increase in the pH of the system
was controlled, using a pH meter (Metrohm 744) until reaching a pH of
8.5. This process was carried out at room temperature and the resulting
precipitate was allowed to age for 1 day in the mother liquor (Fig. 2(b)).
After this time, the suspension obtained was filtered using a mem-
brane with a pore size of 0.22 μm (Fig. 2(c)), and the solid obtained was
dried and re-dispersed in a volume of garlic extract similar to that used
during synthesis, using a high shear disperser (IKA T50) at 10,000 rpm
for 20 min (Fig. 2(d)). This process of re-dispersion, aging and filtration
of the precipitate was called “washing process” and was repeated 5
times, every 24 h. The resulting product, after carrying out the washing
process, was dried in a furnace at a temperature of 80 °C, for 12 h
(Fig. 2(e)), and macerated in an agate mortar. The solid obtained in
each washing step was characterized to find out the evolution of the
solid phase during the synthesis process.
2.2. Characterization of synthesized nanomaterials
Once the samples were obtained, through the synthesis processes
described above, these were characterized using various conventional
techniques.
2.2.1. IR spectroscopy
IR spectroscopy was used to determine the different functional
groups present in the samples of interest. The sample to be analyzed
was obtained by mixing dry KBr with the synthesized solid at a con-
centration of approximately 10%. The scan was carried out between
4000 cm
−1
and 400 cm
−1
using a Thermo Nicolet IR 200 spectro-
photometer.
2.2.2. UV–vis spectroscopy
To determine the possible electronic transitions that may occur in
the solid of interest, absorption spectroscopy was used in the UV–Vis,
using the Genesys Thermo Spectronic equipment. The synthesized
samples were dispersed in distilled water, in concentrations of 0.1 and
0.2 g/mL and the suspension was deposited in 1 cm high quartz cells
that were placed in the chamber of the equipment for analysis. The
sweep was performed between 190 and 750 nm.
2.2.3. X-ray diffraction
This technique was used to determine the crystalline phases present
in the samples synthesized. For this, the X-ray diffraction patterns of the
solids of interest, in powder, were obtained. These were recorded using
a Bruker D8 Advance diffractometer, using Kα radiation from Cu
(λ = 1.542 Å) in the range of 10 to 70 in 2θ.
2.2.4. Electron microscopy
This technique was used mainly to determine the size and mor-
phology of the particles, as well as their state of agglomeration, to
discover the effect of the different synthesis parameters considered on
the characteristics of the final product. To obtain the micrographs with
transmission electron microscopy (TEM), the synthesized solids were
suspended in 1 mL of ethanol and this suspension was placed in an
ultrasonic bath for one hour. A small amount was subsequently taken
using a Pasteur pipette and deposited on a nickel grid previously cov-
ered with a Formvar membrane. The grid was placed in the sample
holder of the Jeol Model JEM 1200 EX electron microscope and the
sample was observed.
Meanwhile, to obtain micrographs with the scanning electron mi-
croscope (SEM) and EDS spectra, the FE-SEM equipment, Jeol JSM
7100F, equipped with the Oxford EDS detector was used. The solid to
be analyzed was dispersed on double-sided carbon tape and placed in
the sample position to be analyzed.
2.3. Methodology used to determine the action of nanomaterials on
pathogenic fungi of coffee
To determine the effect of the ZnO-based nanomaterials on the
fungi, two phytopathogenic fungi of coffee cultivation were used: M.
Fig. 1. Stages of the synthesis process of ZnO-NPs: (a) addition of NH
4
OH to the ethylene glycol‑zinc acetate-water system; (b) solvent removal (at 150 °C); (c) sample
of precalcined material (at 300 °C) after maceration, and (d) ZnO powder obtained at 600 °C.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
810
citricolor and Colletotrichum sp. These were isolated and identified in the
Electron Microscopy laboratory of the University of Cauca. Solid my-
cological agar culture media for dextrose (potato dextrose agar, PDA)
were prepared to perform the respective assays. Two control systems
and three treatments were established: (1) negative control (culture
medium without any type of treatment); (2) positive control (culture
medium + cyproconazole (120 μL); (3) culture medium + ZnO-NPs
(12 mmol·L
−1
); (4) culture medium + ZnO-NPs (9 mmol·L
−1
); (5) cul-
ture medium + ZnO-NPs (6 mmol·L
−1
). To form the positive control,
the systemic fungicide cyproconazole belonging to the triazole chemical
class was used as the active compound contained in the commercial
product called Alto100®, a compound suggested by technicians working
in this field. Similar systems, with the same concentration, were made
up for both the ZnO impregnated with garlic and the ZnO nanobiohy-
brids (green synthesis).
The treatments with the nanomaterials (ZnO-NPs, ZnO impregnated
with garlic and nanobiohybrids of ZnO (green synthesis)) were carried
out using the previously described concentrations (12, 9 and
6 mmol·L
−1
) incorporating the nanomaterials into the culture medium.
Before being used in the Petri dishes, the different treatments were
subjected to ultrasound to ensure a good dispersion of the nanomater-
ials in the culture medium.
To obtain homogeneity and reproducibility in the trials, fungal
cultures of the phytopathogens (M. citricolor and Colletotrichum sp.) of
16 days of age were used to ensure the existence of growth structures,
from which subsamples of 1.5 cm in diameter were obtained using a
sterile punch. Subsequently, the fungal sub-samples were inoculated in
the center of each Petri dish containing the culture medium for each of
the described treatments. To guarantee the reliability of the experi-
ment, this was done in triplicate.
From the third day following cultivation of the fungi, a daily control
of the inhibition of fungal growth was carried out in each of the
treatments, taking as reference the growth time of the negative control,
which completely covered the Petri dish (for example, Colletotrichum
sp., 9 days). At that time, the photographic record of the crops was
taken. These records were taken to the Image-Pro Analyzer image
analysis system to perform the measurement of the growth area, or the
area of the inhibition zone, and determine using this information the
action of the treatments over time.
Using the data for growth area (measured in cm
2
) or fungal in-
hibition zone, recorded periodically, it was determined if the differ-
ences observed in this parameter were statistically significant. For this,
a hypothesis test was carried out, using a completely randomized block
design, where three (3) concentrations of nanomaterials, one (1) con-
centration of cyproconazole (reference fungicide) and the control,
which constituted the treatments considered, were compared in the
study (5 in total); for the blocks (days), the action of the treatments was
considered over time depending on the growth time of each fungus. All
the data were subjected to normal curve adjustment analysis and
homogeneity of variance, and since these two criteria were met, the
two-way ANOVA test was used, using the BioStat 5.3.0 program [63].
Graphs were constructed using the GraphPad Prism 5 program [64]
obtaining the bar charts that allowed a visual determination of which
treatment was most efficient.
To obtain a value of the efficiency of these treatments with the data
used to obtain the bar diagrams, percentage inhibition was determined
using the following equation:
= ×inhibition%Growth of control Growth of treatment
Growth of control
100
(1)
2.4. Identification of morphological and ultrastructural damage in the fungi
2.4.1. Sample preparation
The fungal isolate samples used for the ultrastructural analysis were
previously selected considering the inhibitory effect by the action of the
nanomaterials. These were processed according to the standard pro-
tocol for transmission electron microscopy [65]. Small samples of the
selected isolates were placed In Eppendorf vials and fixed overnight in a
2.5% glutaraldehyde mixture at 4 °C. The following day the fixative was
Fig. 2. Obtaining ZnO nanobiohybrids using garlic extract in water as solvent.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
811
removed and the samples washed three times with phosphate buffer
(PBS), for five minutes each time. Subsequently, they were post-fixed
with osmium tetra-oxide (OsO
4
) at 1%, for 1 h at room temperature,
and then washed again with buffer, three times for five minutes each
time.
The post-fixed samples were dehydrated with ethanol in ascending
concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100%, with a
permanence time of 10 min in each alcohol concentration. Pre-imbibi-
tion was carried out with a mixture of alcohol and LR White resin in
proportions 3:1, 1:1, 1:3, for 45 min for the first two proportions, the
last being left for 1 h.
Finally, the samples were placed in gelatin capsules, labeled and
included in LR White resin, polymerized in an ultraviolet chamber at
room temperature for 48 h. Once the samples were polymerized, the
capsules were taken and scraped with a double-edged knife to eliminate
the excess resin and be able to then obtain semi-fine sections of
200–300 nm and ultra-fine sections of 40–60 nm. The semi- and ultra-
fine sections were obtained using a glass blade with the aid of a Leica
Ultracut R ultramicrotome.
2.4.2. High resolution optical microscopy (HROM)
2.4.2.1. Imprint. Using transparent tape, samples were taken directly
from the culture media, taking account of the negative control
treatment and the nanofungicide treatment, evaluating the
concentration that was most effective. The imprint was then placed
on a slide, with a drop of lactophenol blue, and observed using a Nikon
Microphot HROM. The images of interest were recorded using a Nikon
Digital Sight DS-2Mv camera, coupled to the microscope, using the NIS-
Elements program for image capture.
2.4.2.2. Analysis of semi-fine sections. The semi-fine sections,
200–300 nm thick, were fixed with heat on the glass slides and
stained with toluidine blue, flaming the plate and washing with
distilled water. These sections were observed in a Nikon Microphot
light field microscope using 40× and 100× lenses in order to select the
area of greatest interest where the greatest number of hyphal structures
arranged transverse and longitudinally were found. This area was
delimited and refined again to obtain the ultra-fine sections.
2.4.3. Transmission electron microscopy (TEM)
The ultra-structural analysis and description, considering the effect
of the nanomaterials on the selected isolates, was done observing the
micrographs taken, at different magnifications, with the transmission
electron microscope (Jeol Model JEM 1200 EX), operated at 80 kV [68].
2.4.3.1. Contrast with uranyl acetate - lead citrate. The ultrafine sections
of 40–60 nm (gray to silver) were collected on copper grids coated with
Formvar membranes. These were contrasted with 4% uranyl acetate for
20 min, using flotation method in a dark, humid chamber. They were
washed by dripping distilled water and then placed in contact with a
drop of lead citrate for 10 min in a humid chamber containing sodium
hydroxide (NaOH) pellets. Finally, the sections were washed with
distilled water, dried with filter paper and observed in TEM [65].
3. Results and discussion
3.1. Functional groups present in synthesized nanomaterials
In the spectrum of Fig. 3(i) corresponding to the chemically syn-
thesized ZnO-NPs, a band appears at 3435 cm
−1
that can be associated
with the hydroxyl groups and a band centered on ~ 450 cm
−1
char-
acteristic of zinc oxide. The small bands located between 730 and
1500 cm
−1
can be associated with the presence of superficially ab-
sorbed species (for example groups containing carbon) and the presence
of structural defects where H
+
and H
–
species would be found due to
the presence of impurities from hydrogen within oxygen vacancies, as
occurs in other systems [66,67]. It should be remembered that oxygen
vacancies are the most important structural defects in ZnO-NPs.
As shown in Fig. 3(ii), garlic impregnation was very effective since,
looking at the IR spectra corresponding to ZnO and garlic extract, the
spectra of the impregnated ZnO samples can be seen to be a super-
imposition of the two.
In the IR spectrum corresponding to the sample of unwashed na-
nobiohybrid (Fig. 3(iii-a)) are the characteristic bands of NH
3
(3200 cm
−1
), of the hydroxyl (around 3500 cm
−1
), COO– (doublet
around 1500 cm
−1
) and garlic (band around 1000 cm
−1
-Fig. 3(ii)). As
the washing process progressed, using the water-garlic solution (Fig. 2),
the NH
3
and COO– bands were significantly reduced, the bands asso-
ciated with garlic and the ZnO characteristic being more evident
(~500 cm
−1
) (see Fig. 3(iii-a). Specifically, for the solid obtained from
the sixth wash with the water-garlic solution, Fig. 3(iii-b), the IR
spectrum clearly showed the characteristic band of ZnO, around
450 cm
−1
. In addition, bands that can be associated with organic
functional groups, between 1550 and 1000 cm
−1
, were observed,
whose origin would be, mainly, the garlic used during the synthesis
process, although a contribution of the COO– group from the precursor
cannot be ruled out.
3.2. Absorption spectra in the UV–vis of the samples synthesized
The UV–Vis absorption spectrum of ZnO synthesized by a chemical
route is shown in Fig. 4(a). It shows a band at 370 nm that can be
associated with a valence band - conduction band electronic transition
related to the width of the energy band gap (energy gap), and whose
value would be ~ 3.3 eV. In addition, transitions are observed in the
visible (> 400 nm) where the structural defects present in the sample
would intervene. The bands located between 200 and 350 nm would
correspond to electronic transitions between orbitals located on the
Zn
2+
and O
2–
ions (charge exchange).
Fig. 4(b) shows the UV–Vis absorption spectrum corresponding to a
green synthesized nano-biohybrid using water + garlic as solvent (sixth
wash). In this spectrum, the absorption band corresponding to the inter-
band electronic transition characteristic of ZnO is not observed, only a
band between 200 and 250 nm is observed, which could correspond to
transitions between orbitals located on the ions.
The UV–Vis absorption spectrum of ZnO impregnated with garlic
(Fig. 4(c)) shows a very evident band at 370 nm, which is associated
with zinc oxide (Fig. 4(a)), but the band between 200 and 250 nm is not
seen. This would indicate that the surface of the ZnO was not com-
pletely covered with the organic phase (discharged garlic) or that the
coating was very thin and therefore the characteristic band of the oxide
is clearly observed.
In the UV–Vis absorption spectra of Fig. 4, transitions involving
structural defects (bands above 400 nm) were more evident for the
sample obtained by a chemical route (controlled precipitation –
Fig. 4(a)) and for the sample impregnated with garlic (Fig. 4(c)).
3.3. Crystal structure of the nanomaterials
In Fig. 5(a), the X-ray diffractogram of the sample synthesized by a
chemical route is observed. The peaks of this diffractogram correspond
to the wurtzite type ZnO (JCPDS 79–2205), well crystallized, with a
large crystal size if the slenderness of the peaks is considered (the width
at medium height is small).
Fig. 5(b) shows the X-ray diffractogram of the green synthesized
sample. The peaks of this diffractogram correspond to wurtzite type
ZnO (JCPDS 79–2205) and the low crystallization of the oxide is evi-
dent, possibly due to the presence of garlic in the synthesis system. In
addition, other phases may be present since peaks could be observed
that could not be assigned to a particular compound.
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3.4. Size and morphology of the synthesized nanomaterials
Fig. 6(i) shows the micrographs obtained with SEM and TEM of the
ZnO synthesized by a chemical route, as well as its elemental compo-
sition obtained with the EDS microprobe. In them it is observed that the
particles had a spheroidal morphology and a particle size smaller than
100 nm (Fig. 6(i-a) and (i-b)), quite homogeneous and powders with
low agglomeration. The EDS result meanwhile (Fig. 6(i-c)) indicates
that there is a deviation of its stoichiometry since, if the atomic weight
of the elements is considered, there would be more oxygen than zinc
(67.92% of Zn (65.38 u) and 21.62% of O (15.999 u)). These percen-
tages indicate that the ZnO synthesized was not stoichiometric and,
therefore, presented defects, mainly interstitial oxygen (Oi) given the
excess of this element in the solid.
In Fig. 6(ii) we can see the micrographs obtained with SEM
(Fig. 6(ii-a)) and TEM (Fig. 6(ii-b)) of the ZnO-NPs impregnated with
garlic, as well as their EDS spectrum (Fig. 6(ii-c)). The ZnO particles are
highly agglomerated, have a size of around 100 nm and their mor-
phology is deformed spheroidal. In the EDS spectrum of this sample
meanwhile (Fig. 6(ii-c)), the Zn and O bands are again observed as well
as an additional one that can be associated with C (the peak below that
associated with oxygen) [68]. Furthermore, two intense peaks could be
observed above the Zn band that could not be assigned. Considering the
molar weights of Zn and O and their percentages by weight given in the
table in Fig. 6(ii), Zn 66.84% and O 20.84%, the non-stoichiometry of
the oxide was evidenced and therefore the presence of defects, mainly
of interstitial oxygen.
In Fig. 6(iii), the micrograph obtained with SEM (Fig. 6(iii-a)) of the
green synthesized nano-biohybrid is observed. The powder sample
presented particles with laminar morphology and a great tendency to
stacking that made it difficult to identify the primary particles in the
secondary particles (agglomerates). On the other hand, the EDS spec-
trum of this sample (Fig. 6(iii-c)) presented, mainly, the Zn, O and C
bands, the latter being more evident than in the previous sample
(Fig. 6(ii-c)). When performing the corresponding calculations, con-
sidering the molar weights of Zn and O as well as their percentages in
weight given in the table (Zn 41.76% and O 33.06%), it is found that
the presence of Zn is low, which would indicate that in the sample has a
small amount of ZnO formed and a high presence of carbon in it, results
that would justify those obtained with IR spectroscopy (Fig. 3(iii)) and
XRD (Fig. 5(b)). Evidence for the nanometric characteristics
(< 100 nm) and low agglomeration of the nanobiohybrids was seen in
the micrographs obtained with TEM (Fig. 6(iii-b)).
Fig. 3. (i) IR spectrum corresponding to a solid sample synthesized in ethylene glycol, heat treated at 600 °C; (ii) IR spectra corresponding to the garlic extract
samples, ZnO-NPs impregnated with garlic and pure ZnO (from top to bottom) and (iii) IR spectra of solid samples obtained in different stages of the washing process
during green synthesis of the nanobiohybrids (a) and the spectrum corresponding to the solid from the sixth wash (b).
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3.5. Action of synthesized nanomaterials on coffee crop pathogenic fungal
isolates
3.5.1. Effect of commercial zinc and garlic extract on growth of M.
citricolor
To establish a reference pattern in the inhibition of fungi by the
different substances used in this work in the manufacture of ZnO-based
nanomaterials obtained by chemical and green routes, as well as by
impregnation, commercial zinc (Merck) and garlic extract was used in a
preliminary trial on the M. citricolor strain.
Fig. 7 shows the action of the two substances, commercial zinc and
garlic extract, which were used as benchmarks to compare the results of
antifungal capacity tests obtained for nanomaterials based on ZnO
(chemical and green syntheses). In this work, as can be seen in Fig. 7(a)
for commercial ZnO, the growth of the mycelium of M. citricolor was
slow through the observation days (row (a): left, six (6) days and right,
fifteen (15) days) according to the control (Fig. 7 above). In addition,
“gems” - reproduction structures characteristic of the strain - appeared
(indicated by the arrow). Regarding the action of garlic extract
(Fig. 7b), it presented, for its highest concentration, fungal action on
this fungus, at both six (6) and fifteen (15) days, according to the
control (Fig. 7 above).
As one of the alternatives for obtaining the biohybrid nanomaterials
was impregnation of the ZnO-NPs with the garlic extract. For this, their
antifungal capacity was evaluated on M. citricolor (Fig. 8(a)) and
compared with the action, on the same pathogen, of the green
Fig. 4. UV–vis absorption spectra corresponding to nanomaterials synthesized through: (a) a chemical process, (b) green synthesis and (c) impregnation of ZnO-NPs
with garlic.
Fig. 5. X-ray diffractograms corresponding to the sample: (a) ZnO synthesized by a chemical route and (b) solid obtained by green route (using water + garlic as
solvent).
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synthesized nano-biohybrid (Fig. 8(b)).
Fig. 8((a) left) shows that ZnO-NPs impregnated with garlic in-
hibited the growth of mycelium in M. citricolor but led to a large pro-
duction of gems (Fig. 9). Based on this result, it can be concluded that
ZnO-garlic nanobiohybrids obtained by impregnation are potentially
useful nanofungicides for inhibiting growth of M. citricolor.
Meanwhile, in Fig. 8(b) the results can be seen of the antifungal
action on M. citricolor of ZnO-based nanobiohybrids obtained by a green
route (using garlic extract as a solvent). Comparing Fig. 8(a) and (b), it
can be concluded that although impregnated ZnO-NPs inhibited growth
of the M. citricolor fungus, they did not prevent the development of
reproductive structures (Figs. 8(a)–9) while the nano-biohybrid was a
more efficient antifungal (Fig. 8(b)). Based on this result, we selected
the ZnO-based nano-biohybrid to continue study of the antifungal effect
on the fungi of interest, Mycena citricolor and Colletotrichum sp.
3.5.1.1. Effect of synthesized nanomaterials on the inhibition of the growth
of Mycena citricolor.Fig. 10 shows the result of seeding M. citricolor in
the bioassays treated with nanomaterials based on ZnO, synthesized by
green and chemical route, taking as references the controls that
correspond to the culture treated with a commercial fungicide (PDA
with cyproconazole) and to the culture without any treatment (PDA-
Fig. 6. Micrographs obtained with (a) SEM and (b) TEM, as well as the EDS elementary spectrum (c), which indicates the percentage by weight of each element, of
the samples of: (i) ZnO-NPs synthesized by a chemical route, (ii) ZnO-NPs impregnated with garlic and (iii) nanobiohybrids based on ZnO synthesized by a green
route (using water + garlic as solvent).
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Control culture medium). After six (6) days of growth (Fig. 10(b)) the
activity of the nanofungicides is evident, independent of the synthesis
route, an action that prevented the growth of the fungus. In addition, a
halo of inhibition is observed in cultures treated with NPs obtained by
chemical route (Fig. 10(b) and (c)). At twelve days (Fig. 10(c)), the
fungus covered about 90% of the Petri dish in the control, while for the
treatments no growth of the fungus was observed, evidencing the
antifungal action of the synthesized ZnO-based nanofungicides on M.
citricolor. In the case of nanobiohybrids treatments, the presence of
reproductive structures was observed after 12 days (yellow gems -
Fig. 10(d)).
Based on the values of growth area of the fungus (green synthesis)
and area of inhibition halo (chemical synthesis), obtained in a sequence
of different days for the control samples - fungicide - nanofungicides,
the bar diagram was obtained shown in Fig. 11. In Fig. 11(a), it is
evident that the nanobiohybrids presented a more favorable antifungal
capacity, the treatments with 9 and 12 mmol·L
−1
being more efficient.
However, it should be noted that for the cultures treated with nano-
biohybrids, the presence of reproductive structures was observed
(gems-Fig. 10(d)).
Similar steps were followed for the ZnO-NPs synthesized by a che-
mical route, in order to perform a quantitative analysis on their
Fig. 7. Photographs of M. citricolor cultures treated with: (a) commercial ZnO and (b) garlic extract, with different concentrations (6, 9 and 12 mmol·L
−1
from left to
right), for different observational days.
Fig. 8. Photographs of M. citricolor cultures treated with: (a) ZnO-NPs impregnated with garlic and (b) ZnO-based green synthesized nanobiohybrids, considering
different concentrations: 6, 9 and 12 mmol·L
−1
(left to right), and different days: 6 (column on the left) and 15 days (column on the right).
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antifungal capacity (Fig. 11(b)). In this case, the inhibition zone that
emerged as evidence of the response of the fungus to the presence of the
ZnO-NPs was taken as a measurement parameter. It was not possible to
make measurements of the growth area of the fungus because it was not
evident in the cultures treated with ZnO-NPs (Fig. 10). In the diagram
of Fig. 11(b) it is observed that the NPs presented a favorable antifungal
capacity, inhibiting the growth of the fungus; the most efficient treat-
ment was 9 mmol·L
−1
.
Using values of growth area of the fungus (for the nano-biohybrid)
or of the halo of inhibition (for the ZnO-NPs) and determining their
percent inhibition, Eq. (1) was used, obtaining the data indicated in
Tables 1 and 2.
As indicated in Tables 1 and 2, for the case of the fungus M. ci-
tricolor, both the nanobiohybrids (green route) and ZnO-NPs (chemical
route) had an efficient antifungal capacity. For the nanobiohybrids, the
most efficient concentration was 9 mmol·L
−1
, with ~96.9% inhibition
considering the growth area of the fungus, while for the NPs synthe-
sized by a chemical route it was also that of 9 mmol·L
−1
, but with
~92.3% inhibition, considering the area of halo of inhibition. Obser-
ving Fig. 10, the cultures treated with nanobiohybrids showed re-
productive structures (gems) (Fig. 10(c) and (d)) but those treated with
ZnO-NPs - chemical route did not, which would lead to conclude that
the latter nanofungicide would present a better efficiency on the re-
production and growth of M. citricolor.
3.5.1.2. Identification of morphological and ultrastructural damage in the
fungus M. citricolor caused by treatment with nanofungicides
3.5.1.2.1. Morphological changes observed with HROM. For M.
citricolor, considering the treatments with the synthesized
nanofungicides based on ZnO synthesized by chemical or green route,
it was not possible to take mycelium samples and obtain the imprints to
find out about its growth (colonization of the host). Based on the results
obtained so far, to get more information on the mechanisms of action of
ZnO-based nanofungicides, the specific analysis of reproductive
structures ought to be considered in future work, an activity that was
not carried out in the present work.
3.5.1.2.2. Ultra-structural changes observed with TEM. In Fig. 12,
TEM micrographs of fungal M. citricolor cells are observed. In Fig. 12(a),
corresponding to the control, the characteristic constituents of the cell
are observed: cell wall, fibrillar network, cytoplasm, nucleus and
nucleolus. When carrying out the treatment with ZnO-NPs obtained
by chemical route (Fig. 12(b)), a displacement of the cytoplasmic
content caused by the growth of a vacuole containing a foreign body
that could not be identified was observed; it also showed a thinning of
the cell wall and lack of fibrillar network. The sample treated with
nanobiohybrids (green route - Fig. 12(c)) meanwhile presented a
liquefaction of the cytoplasmic content, with the cell wall and effects
on the fibrous tissue being particular notable.
3.5.2. Effect on the Colletotrichum sp. isolate
3.5.2.1. Macroscopic effect of nanofungicides on the Colletotrichum sp.
isolate. Looking at Fig. 13, the treatments that showed the greatest
efficiency in inhibiting growth of the fungus were those performed with
the ZnO-based nanobiohybrids (Fig. 13(a)). Based on this result,
nanobiohybrids were selected to perform the sequential study of the
growth of Colletotrichum sp. (Fig. 13).
In Fig. 14, the result of seeding Colletotrichum sp. in the bioassays
with ZnO nanobiohybrids (green route), taking control and fungicide
(cyproconazole) as references. At nine (9) days of progress in the test,
the action of the nanobiohybrids was evident, preventing the growth of
the fungus, the most efficient being 12 mmol·L
−1
(Fig. 14(c)). For the
Fig. 9. Image showing the large production of gems in the culture of the assay
with M. citricolor treated with ZnO-NPs impregnated with garlic
(12 mmol·L
−1
–15 days of observation).
Fig. 10. Growth sequences of M. citricolor
considering the antifungal action of the
different concentrations of ZnO-NPs (6, 9
and 12 mmol·L
−1
) synthesized by green
route and chemical route, considering the
days: (a) one, (b) six and (c) twelve, as well
as (d) reproductive structures (gems).
[Control: fungus without treatment;
Fungicide: fungus treated with commercial
cyproconazole].
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quantitative analysis of the antifungal capacity of the ZnO nanobiohy-
brids (green route), the growth area of the fungus subjected to the ac-
tion of these nanofungicides was measured as a function of time. In the
diagram in Fig. 15 it is observed that the nanobiohybrids presented a
favorable antifungal capacity, inhibiting fungal growth; the most effi-
cient treatment was 12 mmol·L
−1
.
The data obtained from percent inhibition, using the data of growth
area of the fungus and Eq. (1), are indicated in Table 3.
As shown in Table 3, for the case of the fungus Colletotrichum sp., the
ZnO nanofungicides obtained by green route showed a percent inhibi-
tion of ~92.6%, for a concentration of 12 mmol·L
−1
.
3.5.2.2. Identification of morphological and ultrastructural damage in the
fungus Colletotrichum sp. caused by treatment with ZnO nanofungicides
3.5.2.2.1. Morphological changes observed with HROM. In Fig. 16(a)
corresponding to the control without treatment, we observe the
development of hyphae and abundant reproduction structures
(conidia) at 9 days of growth. When treating the fungus with ZnO-
NPs synthesized by chemical route (Fig. 16(b)), a great reduction in the
production of conidia is observed and the hyphal structures tended to
group. The sample treated with nanobiohybrids (Fig. 16(c)) meanwhile
did not present conidia and in addition the thickening of the hyphal
structures was very noticeable in response to treatment.
Considering the characteristics of the fungus treated with the na-
nofungicides, previously indicated (Fig. 16), it can be concluded that
the presence of the green synthesized samples would affect the regular
cycle of the fungus, reiterating that which was indicated in the bar
diagram of Fig. 15.
3.5.2.2.2. Ultra-structural changes observed with TEM. In Fig. 17(a)
we can observe the typical ultrastructure of the Colletotrichum sp.
fungus with a well-defined cell wall and a cytoplasm with the presence
of characteristic organelles such as mitochondria, nucleus and vacuoles.
In the ultrastructure of the sample treated with nanobiohybrids
(Fig. 17(b)), some hyphae without cellular organelles were observed,
Fig. 11. (a) Bar chart illustrating the growth area of M. ci-
tricolor subjected to the action of nanobiohybrids, obtained by
green route, and (b) diagram illustrating the area of the in-
hibition halo around the M citricolor treated with ZnO-NPs
obtained by chemical route, for different days and for dif-
ferent concentrations. [Control: fungus without treatment;
Fungicide: fungus treated with commercial cyproconazole].
Table 1
Percentage (%) of inhibition of mycelial growth of M. citricolor treated with green synthesized ZnO nanobiobrids.
Percent inhibition of mycelial growth of M. citricolor
Treatment Day 1 Day 3 Day 6 Day 9 Day 12
Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition
Control 0 mmol·L
−1
10.63 0 15.96 0 65.36 0 255.48 0 427.31 0
Fungicide 1:1000* 1.40 86.8 1.40 90.9 1.40 97.8 1.40 99.4 1.40 99.7
Green route 12 mmol·L
−1
*5.97 43.6 6.97 53.7 9.07 86.1 10.53 95.9 21.52 94.9
9 mmol·L
−1
5.23 50.7 6.23 59.9 9.18 85.9 11.94 95.4 13.19 96.9
6 mmol·L
−1
6.96 35.1 8.30 46.7 20.92 68.5 33.98 86.1 53.51 87.4
1:1000*: 1part fungicide to 1000 parts water.
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highlighting the presence of abundant vacuoles which inhibited the
growth of Colletotrichum sp. Meanwhile, in Fig. 17(c) corresponding to a
sample treated with ZnO nanoparticles obtained by a chemical route, a
moderately conserved cytoplasm with an incipient vacuolization is
observed, which would reflect the action of the nanomaterials.
To explain the results obtained in this work, it is necessary to con-
sider that, until now, there is no mechanism that fully explains and
accounts for the action of NPs on the fungus, although some pre-
liminary ideas have already been published [53,54]. The structuring of
the mechanism of action of NPs on fungi requires the identification of
the phenomena that occur in the bio-interface surface of the nano-
particle-fungal cell wall, requiring that more rigorous systematic re-
search be carried out in the future, using more elaborate experimental
techniques. Therefore, for the moment, a hypothetical
Table 2
Percent (%) inhibition of mycelial growth of M. citricolor treated with ZnO-NPs synthesized by a chemical route.
Percent inhibition of mycelial growth of M. citricolor
Treatment Day 1 Day 3 Day 6 Day 9 Day 12
Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition Growth area
(cm
2
)
% Inhibition
Control 0 mmol·L
−1
5.85 5.85 0 13.41 0 36.60 36.60 0 61.38 61.38 0 65.11 0
Fungicide 1:1000* 1.40 76.1 1.402 89.5 1.40 96.2 1.40 97.7 1.40 97.8
Chemical
route
12 mmol·L
−1
1.356 76.8 1.907 85.8 4.782 86.9 5.524 90.1 5.840 91.1
9 mmol·L
−1
1.103 81.2 1.742 86.8 4.410 87.9 6.112 90.9 4.983 92.3
6 mmol·L
−1
1.138 80.6 1.677 87.7 5.607 84.7 7.668 87.5 8.402 87.1
1:1000*: 1part fungicide to 1000 parts water.
Fig. 12. TEM micrographs of M. citricolor: (a) Control and treated with (b) ZnO-NPs (chemical route) and (c) ZnO nanobiohybrids (green route). [Cell wall, Cw;
fibrillar network, Fn; cytoplasm, Cyt; nucleus, N; and nucleolus, Nu].
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phenomenological model can be used to qualitatively account for the
possible actions of the nanofungicides synthesized in this work, on the
fungi of interest taken as target, considering both the structural for-
mation of the fungal cell wall as well as the basic knowledge of the
interaction of nanoparticles with the components of biological systems,
for example proteins [40], published in the literature. The fungal cell
wall is a dynamic structure that protects the cell from changes in os-
motic pressure and other environmental stresses, also allowing the
fungal cell to interact with its environment [69,70]. In addition, the cell
serves as a signaling center to activate signal transduction pathways
within the cell. Therefore, if the cell wall structure suffers disruption,
the growth and morphology of the fungal cell will be profoundly af-
fected, often rendering it susceptible to lysis and death. This is why the
cell wall has long been considered an excellent target for antifungal
agents.
Fungal cells walls are structurally unique and different from the
cellular-based plant cell wall. As indicated in Fig. 18, the fungal cell
wall is composed of glycoproteins and polysaccharides, mainly glucan
and chitin [71]. Glucan is the most important structural polysaccharide
in the fungal cell wall, constituting 50–60% of the wall by dry weight
[72]. Its main structural constituent is beta-1,3-glucan, between 65 and
90%, to which other components of the cell wall adhere covalently
[73]. Synthesis of beta-1,3-glucan is mediated by glucan synthase and
its inhibition has been extensively pursued as a means to interrupt
formation of the cell wall and thereby prevent fungal growth. For its
part, chitin (long chain homopolymers of beta-1,4-linked N-acet-
ylglucosamine) is a relatively minor, but structurally important com-
ponent of the fungal cell wall [68,74]. The synthesis of chitin is
mediated by chitin synthase. If chitin synthesis is disturbed, the walls
form in a disordered manner and the fungal cell becomes malformed
and osmotically unstable [75]. Specifically, chitin presents a regular
distribution of free amino groups that can be protonated by certain
acids, becoming positively charged, giving it a polycation behavior.
This condition explains some properties of chitin, such as its ability to
bind negatively charged substances: lipids, proteins and inks, among
others. This characteristic of chitin makes it susceptible to attack by
certain chemical agents such as ROS, chemical species generated under
darkness [37,41] by ZnO-NPs, as indicated in the literature, or by na-
nohybrids based on ZnO incorporated into crops studied in this work
(Fig. 17). These ROS would cause oxidation processes as indicated by
López et al. [47].
In addition, the interaction of nanomaterials with the cell mem-
brane proteins - via hydrogen bond interaction with amino acid re-
sidues followed by internalization, depolarization of the membrane and
induction of ROS generation, as indicated by Verma et al. [41] - would
contribute to augmenting the action of nanofungicides.
All fungal cell walls contain proteins that are tightly interwoven
within the chitin and glucan-based structural matrix [66,68,76]. In the
Fig. 13. Growth of Colletotrichum sp. considering the anti-
fungal action of the different concentrations of ZnO-NPs
(control or fungicide, 6, 9 and 12 mmol·L
−1
from left to right)
synthesized by green route (nanobiohybrids) (a) and chemical
route (ZnO-NPs) (b), nine (9) days after beginning the trial
[Control: fungus without treatment; Fungicide: fungus treated
with commercial cyproconazole].
Fig. 14. Growth sequence of Colletotrichum sp. considering the antifungal action of the ZnO nanobiohybrids (green route) at different concentrations (6, 9 and
12 mmol·L
−1
), for days: (a) one, (b) six and (c) nine. [Control: fungus without treatment; Fungicide: fungus treated with commercial cyproconazole].
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walls of filamentous fungi, such as those considered in this work, it has
been estimated that the amount of proteins present in them could be
between 20 and 30% of the total mass of the cell wall. Many of these
proteins are integrated into the wall via covalent linkages between the
sugars present at the N- and O-linked sites and/or in the glycosypho-
sphatidylinositol (GPI) anchor with those in the polymers of chitin or
glucan. Some important proteins in fungi are hydrophobins, amphi-
pathic proteins that can self-assemble to generate rodlets that produce a
hydrophobic interface between filamentous fungi and their environ-
ments [67]. Hydrophobins participate in morphogenesis and are in-
volved in the adhesion of the fungal cell to surfaces, which is why they
have been associated with fungal plant virulance [77]. In addition, the
conidial spores and aerial hyphae of mold are often covered by hy-
drophobins that protect the spores from oxidant enzymes and foreign
phagocytes [69].
Although the majority of studies of nanoparticle-protein interaction
have been performed using blood plasma, these results may be extra-
polated to other physiological environments, taking account of their
particularities. The interaction of NPs with proteins is necessarily a
dynamic process, determined by the properties of both components.
These interactions would be characterized by binding affinity, stoi-
chiometry and kinetic properties and would be dominated by many
chemical processes [78]. In the case of the interaction of the ZnO-NPs
and nanobiohybrids synthesized in this work with the fungi of interest,
the nanoparticles encounter proteomes, not a single protein but thou-
sands of proteins with diverse functions. These proteins compete for the
“surface” leading to a protein “corona” that will gradually define the
biological identity of the particle [70,79].
The protein corona composition would be determined by the ther-
modynamic and kinetic properties of the NP-protein interactions and
the physicochemical characteristics of the nanoparticles (determined by
the synthesis conditions and their chemical nature), and the nature of
the physiological environment, for example the composition of the
fungal cell wall and specifically of the properties of the proteins
[80,81]. Proteome binding to NPs would be strongly affected by the
surface chemistry of the nanoparticles, so that, depending on the in-
teraction forces, a protein might be adsorbed on, denatured by, or re-
jected from, the nanoparticle surface. For the case of the ZnO-based
nanofungicides synthesized in this work (< 100 nm), even a large
protein (of a few nanometers) would see a surface with a high radius of
curvature, almost indistiguishable from a two-dimensional surface. In
this case, the protein could be adsorbed and denatured, undoubtedly
depending on the surface characteristics and the protein-surface inter-
action [82].
Considering the presence of the ZnO-NPs and the ZnO nanobiohy-
brids synthesized in the cultures of the fungi of interest and therefore
their effect (Fig. 10 to 17), it is possible that the NPs do not enter the
cell wall (Fig. 18) so that the interaction with the fungal cell wall could
occur through the corona of adsorbed proteins on the NP surface
[72,83]. As indicated by Jayaram et al. in their work [40], reactive
oxygen species (ROS), which are produced even in the dark and would
be mediated by the surface defects of the synthesized nanofungicides
[37,39,41], would oxidize the corona of proteins formed on the fungal
surface (Fig. 18) and these oxidized proteins would serve as a cellular
signal of oxidative stress for the cell wall, which would cause inhibition
of the growth of the fungus. The images in Figs. 12 and 17 illustrate the
effect of oxidative stress that ZnO-based nanofungicides would cause on
fungi.
Moreover, if the ZnO-NPs and ZnO nanobiohybrids enter the fungal
cell wall, just as occurred in the work of Romashchenko et al. [84], they
may become trapped, disturbing the intrinsic disorder of the proteins,
associated with their structural mobility [85]. The entropic con-
sequences of this disorder-to-order transition would be compensated for
by their ability to adjust to a structure of the binding partner, wrap or
Fig. 15. Bar chart illustrating the growth area of Colletotrichum sp. subjected to the action of ZnO nanobiohybrids (green route) in different concentrations and for
different days [Control: fungus without treatment; Fungicide: fungus treated with the commercial fungicide].
Table 3
Percent inhibition of mycelial growth of Colletotrichum sp. treated with green synthesized ZnO nanobiohybrids.
Percent inhibition of mycelial growth of Colletotrichum sp.
Treatment Day 1 Day 3 Day 6 Day 9
Growth area (cm
2
) % Inhibition Growth area (cm
2
) % Inhibition Growth area (cm
2
) % Inhibition Growth area (cm
2
) % Inhibition
Control 0- mmol·L
−1
15.19 0
0
23.83 0
0
234.82 0
0
445.35 0
0
Fungicide 1:1000* 4.35 71.3 4.02 83.1 4.417 98.1 6.56 98.5
Green route 12 - mmol·L
−1
9.47 37.5 9.79 58.5 19.71 91.5 32.60 92.6
9 - mmol·L
−1
6.74 55.6 8.86 62.4 37.30 84.1 58.45 86.8
6 - mmol·L
−1
9.12 39.3 10.28 57.5 21.93 90.7 46.46 89.5
1:1000*: 1part fungicide to 1000 parts water.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
821
hug the surface of the nanoparticles, resulting in the extensive inter-
action surface and interaction energy gains. The interaction of the
proteins with ROS produced by the surface defects of the nanofungi-
cides would also be favored. This indicates that inside the fungal cell
wall, the ZnO-NPs and ZnO nanobiohybrids could interfere in the
synthesis of the proteins, triggering cellular responses that would lead
to the inactivation of the fungus.
The phenomenological model presented to account for the anti-
fungal capacity of the synthesized ZnO-based nanomaterials on the
strains of Mycena citricolor and Colletotrichum sp., although supported in
work reported in the literature, requires a quantitative experimental
corroboration in order to assess, in a precise way, the importance of
each of the phenomena mentioned there and its action, if indeed it does
have an action, on the fungicidal mechanism of these ZnO nanofungi-
cides. This model could help to understand the interactions in the na-
nomaterial surface-fungal cell wall biointerphase, where the defects of
the nanomaterials ought to play an important role, as indicated in the
literature [38,41].
The activities carried out and the results obtained in this work show
the importance of studying the effect of the solvent, and of the synthesis
conditions in general, on the characteristics and functionality of the
ZnO-NPs. This interest arises from the possibility of potentiating the
technological applications and efficiency of ZnO nanostructures
through the control of their structure, size and particle shape. For this
purpose, different studies have used solvents of different polarity [86],
a mixture of ethanol-water in the presence of glycine [87] as well as
other organic solvents [88], etc., showing that the polarity of the sol-
vents affects both the nucleation of ZnO and the preferential direction
of growth. As recent studies [89] reiterate, altering the structure, size
and shape of ZnO-NPs affects their biological, chemical and environ-
mental properties. Specifically, taking into account the antifungal
capability and capacity of the ZnO-NPs, synthesis conditions [47,48]
were evaluated, along with the advantages of using green synthesis
[90], with the objective of optimizing this functionality [91,92]. The
results of the present work clearly reiterate the effect of synthesis
conditions on both the characteristics (Fig. 3 to 6) and antifungal ac-
tivity (Fig. 7 to 17) of the ZnO-NPs and zinc nanobiohybrids synthe-
sized.
Nevertheless, although the theme of ZnO-NP antifungal capacity has
received its fair share of attention, as previous studies show
[47,48,93–95], it is necessary to carry out further research to determine
the effect and mode of action of these nanoparticles on the growth of
the fungi. The studies carried out, evaluating the antifungal mechan-
isms of ZnO-NPs, are still insufficient to be able to project the impact of
these nanoparticles on fungi in general. It is therefore necessary to
know more about the mode of action of nanoparticles on pathogens, as
in the present work which studied the action of ZnO-NPs and zinc na-
nobiohybrids on Mycena citricolor and Colletotrichum sp., fungi that
specifically affect coffee, an agricultural product of great interest to a
number of developing countries. The antifungal activity of photo-
activated ZnO has previously been assessed [46]. This is also the case
for zinc nanocomposites [96] and green synthesized ZnO-Nps [91,92],
while the management of postharvest diseases [71] has also been stu-
died with the aim of increasing the antifungal efficiency of zinc na-
nostructures. However, there remains the need to study the antifungal
mechanism of these in further depth. This requires considering in more
detail the fungal hyphae and their deformation, as well as the micro and
ultrastructural alterations suffered by the pathogen, as was done in the
present work with the analysis performed on the fungi of interest
(Figs. 7 to 10, 12 to 14, 16 and 17). The information obtained in this
study confirms the high antifungal activity of the synthesized ZnO-NPs
and ZnO nanobiohybrids on Mycena citricolor and Colletotrichum sp.
These nanofungicides efficiently inhibited growth of M. citricolor, with
percentages of inhibition up to ~97% with nanobiohybrids and ~92%
with ZnO-NPs, and of Colletotrichum sp., with percentages of inhibition
up to ~93% with nanobiohybrids. Moreover, HROM and TEM analysis
of the fungi tested revealed that the zinc nanofungicides induced ser-
ious physical damage in the micro and ultrastructure of the pathogens,
alterations that can be assumed to be irreversible. The hyphae in the
Colletotrichum sp., which was treated with zinc nanobiohybrids, tended
to thicken, a process that was accompanied by an appreciable reduction
in the production of conidia (Fig. 16). Meanwhile, at the cellular level,
it was observed in the M. citricolor samples treated with ZnO-NPs that
their cells showed vacuolation (Fig. 12(b)), which caused displacement
of the cytoplasmic content, while on treating the pathogen with zinc
nanobiohybrids, liquefaction of the cytoplasmic content was observed
(Fig. 12(c)). Furthermore, the strain of Colletotrichum sp. treated with
the nanobiohybrids showed hyphae without cytoplasm and cellular
Fig. 16. Detail of the images of the vegetative (hy-
phae) and reproductive structures (conidia) of
Colletotrichum sp. Fungal structures obtained in the
negative control C
–
(a) and in strains treated with:
(b) hyphae and conidia obtained with ZnO-NPs
(chemical route) and (c) fungal structures (hyphae)
obtained with the nanobiohybrids treatment of ZnO
(green route) at a concentration of 12 mmol·L
−1
. All
structures were observed after 9 days of treatment.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
822
organelles (Fig. 17(b)), with abundant vacuoles, and on interacting
with the ZnO-NPs presented moderately conserved cytoplasm and in-
cipient vacuolation (Fig. 17(c)). The favorable antifungal activity of the
zinc nanofungicides studied on the fungi Mycena citricolor and Colleto-
trichum sp., thus leads us to consider their potential use in the in-
activation of these pathogens in coffee crops.
4. Conclusion
Methodologies were designed so that it was possible to obtain, in a
controlled, reproducible way, nanofungicides (by chemical route), ZnO
nanoparticles (ZnO-NPs), ZnO-NPs impregnated with garlic, and ZnO
nanobiohybrids (by green route using garlic extract as solvent).
Examining the characterization performed on the samples, the one that
Fig. 17. TEM micrographs of Colletotrichum sp.: (a) Control, and treated with (b) ZnO nano-biohybrid (green route) and (c) ZnO-NPs (chemical route).
Fig. 18. Diagram that qualitatively describes the fungal cell wall and the interaction of the nanoparticles with the proteins it comprises.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
823
showed the greatest variation in its properties was that synthesized by
green route. The last sample did not completely crystallize and this
presented, in addition, changes in the IR and UV–Vis spectra. This
sample, however, showed agglomerates of lamellar and non-spheroidal
particles as the other two samples. Moreover, the results for the effect of
the synthesized nanofungicides on the fungi M. citricolor and
Colletotrichum sp. indicate that the nano-biohybrid (green route) sample
caused the highest percent inhibition in growth of Colletotrichum sp.,
with ~93%. With M. citricolor, the percent inhibition figures were very
similar, but taking two different aspects of the crop into account to be
able to witness the effect of the treatment on the fungus: fungal growth
area, for the nanobiohybrids (with a ~97% inhibition), and halo of
inhibition area, for ZnO-NPs (with a ~93% inhibition). It is also evident
that the efficiency of the nanofungicides depends on the possible dif-
ferences in the physico-chemical structure of the cell wall and the
evolutionary adaptations of each fungus, taking into account the genera
to which they belong. The morphological and ultra-ultrastructural ef-
fects of the nanofungicides on the fungi studied became evident in the
HROM and TEM studies. Thus, the cells of the strains treated with ZnO-
NPs (chemical route) presented appreciable vacuolization and thinning
of the cell wall, with elimination of the fibrillar tissue that surrounds it,
in some cases. Meanwhile, the samples of M. citricolor treated with the
nanobiohybrids of ZnO (green route) presented liquefaction of the cy-
toplasmic content. To explain the antifungal effect of the ZnO-NPs and
the nano-biohybrid of ZnO, the action of the protein corona that would
be formed on them was considered, both for the order that they could
cause inside the cell wall (entropic effect) and their mediation to pro-
mote oxidative stess in the cell wall.
Acknowledgments
We are grateful to COLCIENCIAS for funding relating to project code
Number 110365842673 COLCIENCIAS ID 4241 and to the VRI for
providing logistical support. We are especially grateful to Colin
McLachlan for suggestions relating to the English text.
Conflicts of interest
The authors declare no conflicts of interest.
References
[1] C. Jagadish, S.J. Pearton, Zinc Oxide Bulk, Thin Films and Nanostructures:
Processing, Properties, and Applications, Elsevier, Amsterdant, 2006.
[2] H. Morkoç, Ü. Özgür, Zinc Oxide: Fundamentals, Materials and Device Technology,
WILEY - VCH Verlag & Co. KGaA, Weeinheim, 2008.
[3] C. Klingshirn, ZnO: from basics towards applications, Phys. Status Solidi 244 (2007)
3027–3073.
[4] C.F. Klingshirn, A. Waag, A. Hoffmann, J. Geurts, Zinc Oxide: From Fundamental
Properties towards Novel Applications, Springer Series in Materials Science 120,
Springer - Verglag Berlin Heidelberg, 2010.
[5] A. Moezzi, A.M. McDonagh, M.B. Cortie, Zinc oxide particles: synthesis, properties
and applications, Chem. Eng. J. 185 (2012) 1–22.
[6] A.B. Djurišić, Y.H. Leung, Optical properties of ZnO nanostructures, Small 2 (2006)
944–961.
[7] A. Janotti, C.G. Van de Walle, Fundamentals of zinc oxide as a semiconductor, Rep.
Prog. Phys. 72 (2009) 126501, https://doi.org/10.1088/0034-4885/72/12/
126501.
[8] C. Wöll, The chemistry and physics of zinc oxide surfaces, Prog. Surf. Sci. 82 (2007)
55–120.
[9] J.R. Lead, E.L. Smith, Environmental and Human Health Impacts of
Nanotechnology, John Wiley & Sons Ltd Publication, West Sussex, United Kingdom,
2009.
[10] M. Lu, P. Pichat, Photocatalysis and Water Purification: From Fundamentals to
Recent Applications, Materials for Sustainable Energy and Development, WILEY –
VCH Verlag Gmbh & Co. kGaA Weinheim, 2013.
[11] H. Kisch, Semiconductor Photocatalysis: Principles and Applications, WILEY – VCH
Verlag Gmbh & Co. kGaA Weinheim, 2014.
[12] T. Jesionowski, Zinc oxide — from synthesis to application: a review, Materials 7
(2014) 2833–2881, https://doi.org/10.3390/ma7042833.
[13] J.E. Rodríguez-Páez, A.C. Caballero, M. Villegas, C. Moure, J.F. Ferna, Controlled
Precipitation Methods: Formation Mechanism of ZnO Nanoparticles, 21 (2001), pp.
925–930.
[14] A.H. Moharram, S.A. Mansour, M.A. Hussein, M. Rashad, Direct precipitation and
characterization of ZnO nanoparticles, J. Nanomater. 2014 (2014) ID716210.
[15] M.-H. Wang, X.-Y. Ma, F. Zhou, Synthesis and characterization of monodispersed
spherical ZnO nanocrystals in an aqueous solution, Mater. Lett. 142 (2015) 64–66.
[16] H. Avila, M. Cruz, M. Villegas, C. Caballero, J.E. Rodríguez-Páez, Estudio com-
parativo de dos metodos de sintesis para la obtencion de polvos cerámicos de ZnO-
Pr, Bol. Soc. Esp. Ceram. 43 (2004) 740–744.
[17] J. Guo, C. Peng, Synthesis of ZnO nanoparticles with a novel combustion method
and their C2H5OH gas sensing properties, Ceram. Int. 41 (2015) 2180–2186.
[18] R.M. Alwan, Q.A. Kadhim, K.M. Sahan, R.A. Ali, R.J. Mahdi, N.A. Kassim,
A.N. Jassim, Synthesis of zinc oxide nanoparticles via sol–gel route and their
characterization, Nanosci. Nanotechnol. 5 (2015) 1–6.
[19] L. Yang, J. Wang, L. Xiang, Hydrothermal synthesis of ZnO whiskers from ɛ-Zn
(OH)
2
in NaOH/Na2SO4 solution, Particuology 19 (2015) 113–117.
[20] K. Anand, S. Varghese, T. Kurian, Synthesis of ZnO nano rods through mechano-
chemical route: a solvent free approach, Int. J. Theor. Appl. Sci. 6 (2014) 87–93.
[21] A. Dakhlaoui, M. Jendoubi, L. Samia, A. Kanaev, N. Jouini, Synthesis, character-
ization and optical properties of ZnO nanoparticles with controlled size and mor-
phology, J. Cryst. Growth 311 (2009) 3989–3996.
[22] I. Hussain, N.B. Singh, A. Singh, H. Singh, S.C. Singh, Green synthesis of nano-
particles and its potential application, Biotechnol. Lett. 38 (2016) 545–560.
[23] H. Agarwal, S.V. Kumar, S. Rajeshkumar, A review on green synthesis of zinc oxide
nanoparticles–an eco-friendly approach, Resource - Efficient Technologies, 2017,
pp. 406–413.
[24] S. Ahmed, S.A. Chaudhry, S. Ikram, A review on biogenic synthesis of ZnO nano-
particles using plant extracts and microbes: a prospect towards green chemistry, J.
Photochem. Photobiol. B Biol. 166 (2017) 272–284.
[25] M. Anbuvannan, M. Ramesh, G. Viruthagiri, N. Shanmugam, N. Kannadasan,
Synthesis, characterization and photocatalytic activity of ZnO nanoparticles pre-
pared by biological method, Spectrochim. Acta A Mol. Biomol. Spectrosc. 143
(2015) 304–308.
[26] R. Dobrucka, J. Długaszewska, Biosynthesis and antibacterial activity of ZnO na-
noparticles using Trifolium pratense flower extract, Saudi J. Biol. Sci. 23 (4) (2016)
517–523.
[27] S. Ambika, M. Sundrarajan, Antibacterial behaviour of Vitex negundo extract as-
sisted ZnO nanoparticles against pathogenic bacteria, J. Photochem. Photobiol. B
Biol. 146 (2015) 52–57.
[28] S. Jafarirad, M. Mehrabi, B. Divband, M. Kosari-Nasab, Biofabrication of zinc oxide
nanoparticles using fruit extract of Rosa canina and their toxic potential against
bacteria: a mechanistic approach, Mater. Sci. Eng. C. 59 (2016) 296–302.
[29] R. Yuvakkumar, J. Suresh, B. Saravanakumar, A.J. Nathanael, S.I. Hong,
V. Rajendran, Rambutan peels promoted biomimetic synthesis of bioinspired zinc
oxide nanochains for biomedical applications, Spectrochim. Acta A Mol. Biomol.
Spectrosc. 137 (2015) 250–258.
[30] Y. Qian, J. Yao, M. Russel, K. Chen, X. Wang, Characterization of green synthesized
nano-formulation (ZnO–A. vera) and their antibacterial activity against pathogens,
Environ. Toxicol. Pharmacol. 39 (2) (2015) 736–746.
[31] A.N.D. Krupa, R. Vimala, Evaluation of tetraethoxysilane (TEOS) sol–gel coatings,
modified with green synthesized zinc oxide nanoparticles for combating micro-
fouling, Mater. Sci. Eng. C 61 (2016) 728–735.
[32] K. Ali, S. Dwivedi, A. Azam, Q. Saquib, M.S. Al-Said, A.A. Alkhedhairy, J. Musarrat,
Aloe vera extract functionalized zinc oxide nanoparticles as nanoantibiotics against
multi-drug resistant clinical bacterial isolates, J. Colloid Interface Sci. 472 (2016)
145–156.
[33] M. Anbuvannan, M. Ramesh, G. Viruthagiri, N. Shanmugam, N. Kannadasan,
Anisochilus carnosus leaf extract mediated synthesis of zinc oxide nanoparticles for
antibacterial and photocatalytic activities, Mater. Sci. Semicond. Process. 39 (2015)
621–628.
[34] R. Aladpoosh, M. Montazer, The role of cellulosic chains of cotton in biosynthesis of
ZnO nanorods producing multifunctional properties: mechanism, characterizations
and features, Carbohydr. Polym. 126 (2015) 122–129.
[35] H. Ma, P.L. Williams, S.A. Diamond, Ecotoxicity of manufactured ZnO nanoparticles
- A review, Environ. Pollut. 172 (2013) 76–85.
[36] H. Ma, N.J. Kabengi, P.M. Bertsch, J.M. Unrine, T.C. Glenn, P.L. Williams,
Comparative phototoxicity of nanoparticulate and bulk ZnO to a free-living ne-
matode Caenorhabditis elegans: the importance of illumination mode and primary
particle size, Environ. Pollut. 159 (2011) 1473–1480.
[37] K. Hirota, M. Sugimoto, M. Kato, K. Tsukagoshi, T. Tanigawa, H. Sugimoto,
Preparation of zinc oxide ceramics with a sustainable antibacterial activity under
dark conditions, Ceram. Int. 36 (2010) 497–506.
[38] X. Xu, D. Chen, Z. Yi, M. Jiang, L. Wang, Z. Zhou, X. Fan, Y. Wang, D. Hui,
Antimicrobial mechanism based on H
2
O
2
generation at oxygen vacancies in ZnO
crystals, Langmuir 29 (2013) 5573–5580.
[39] V. Lakshmi Prasanna, R. Vijayaraghavan, Insight into the mechanism of anti-
bacterial activity of ZnO: surface defects mediated reactive oxygen species even in
the dark, Langmuir 31 (2015) 9155–9162.
[40] D.T. Jayaram, S. Runa, M.L. Kemp, C.K. Payne, Nanoparticle-induced oxidation of
corona proteins initiates an oxidative stress response in cells, Nanoscale 9 (2017)
7595–7601.
[41] S.K. Verma, E. Jha, P.K. Panda, J.K. Das, A. Thirumurugan, M. Suar, S.K.S. Parashar,
Molecular aspects of core-shell intrinsic defect induced enhanced antibacterial ac-
tivity of ZnO nanocrystals, Nanomedicine 13 (2018) 43–68.
[42] R. Prasad, Advances and Applications through Fungal Nanobiotechnology, Springer
International Publishing, Gewerbestrasse - Switzerland, 2016.
[43] D.K. Tripathi, P. Ahmad, S. Sharma, D.K. Chauhan, N.K. Dubey, Nanomaterials in
Plants, Algae, and Microorganisms: Concepts and Controversies, Academic Press,
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
824
Oxford, United Kingdom, 2017.
[44] M.L. Lili He, Yang Liu, Azlin Mustapha, antifungal activity of zinc oxide nano-
particles against Botrytis cinerea and Penicillium expansum, Microbiol. Res. 166 (3)
(2011) 207–215, https://doi.org/10.1016/j.micres.2010.03.003.
[45] D. S.S. Sharma, J. Rajput, B. Kaith, M. Kaur, Synthesis of ZnO nanoparticles and
study of their antibacterial and antifungal properties, Thin Solid Films 519 (3)
(2010).
[46] K. Kairyte, A. Kadys, Z. Luksiene, Antibacterial and antifungal activity of photo-
activated ZnO nanoparticles in suspension, J. Photochem. Photobiol. B Biol. 128
(2013) 78–84.
[47] C. López, J.E. Rodríguez-Páez, Synthesis and characterization of ZnO nanoparticles:
effect of solvent and antifungal capacity of NPs obtained in ethylene glycol, Appl.
Phys. A Mater. Sci. Process. 123 (2017) 748.
[48] P.A. Arciniegas-Grijalba, M.C. Patiño-Portela, L.P. Mosquera-Sánchez,
J.A. Guerrero-Vargas, J.E. Rodríguez-Páez, ZnO nanoparticles (ZnO-NPs) and their
antifungal activity against coffee fungus Erythricium salmonicolor, Appl. Nanosci. 7
(2017) 225–241.
[49] K.A. Abd-Elsalam, M.A. Alghuthaymi, Nanobiofungicides: are they the next-gen-
eration of fungicides, J. Nanotech. Mater. Sci. 2 (2015) 1–3.
[50] R. Prasad, V. Kumar, K.S. Prasad, Nanotechnology in sustainable agriculture: pre-
sent concerns and future aspects, Afr. J. Biotechnol. 13 (2014) 705–713.
[51] D.U. Gawai, Antifungal activity of essential oil of cymbopogon citratus stapf against
different fusarium species, Bionano Front. 8 (2015) 186–189.
[52] A.P. Ingle, N. Duran, M. Rai, Bioactivity, mechanism of action, and cytotoxicity of
copper-based nanoparticles: a review, Appl. Microbiol. Biotechnol. 98 (2014)
1001–1009.
[53] A. Singh, N.B. Singh, I. Hussain, H. Singh, S.C. Singh, Plant-nanoparticle interac-
tion: an approach to improve agricultural practices and plant productivity, Int. J.
Pharm. Sci. Invent. 4 (2015) 25–40.
[54] E. Ledezma, R. Apitz-Castro, Ajoene, el principal compuesto activo derivado del ajo
(Allium sativum), un nuevo agente antifúngico, Rev. Iberoam. Micol. 23 (2006)
75–80.
[55] M.I. Timonin, R.H. Thexton, The rhizosphere effect of onion and garlic on soil
microflora, Proceedings. Soil Sci. Soc. Am. 1950, pp. 186–189.
[56] C.B. Fliermans, Inhibition of Histoplasma capsulatum by garlic, Mycopathol. Mycol.
Appl. 50 (1973) 227–231.
[57] M.R. Tansey, J.A. Appleton, Inhibition of fungal growth by garlic extract, Mycologia
67 (1975) 409–413.
[58] S. Yoshida, S. Kasuga, N. Hayashi, T. Ushiroguchi, H. Matsuura, S. Nakagawa,
Antifungal activity of ajoene derived from garlic, Appl. Environ. Microbiol. 53
(1987) 615–617.
[59] T.M. Muhsin, S.R. Al-Zubaidy, E.T. Ali, Effect of garlic bulb extract on the growth
and enzymatic activities of rhizosphere and rhizoplane fungi, Mycopathologia 152
(2001) 143–146.
[60] F. Aala, U.K. Yusuf, R. Nulit, S. Rezaie, Inhibitory effect of allicin and garlic extracts
on growth of cultured hyphae, Iran. J. Basic Med. Sci. 17 (3) (2014) 150.
[61] V.N. Kalpana, V. Devi Rajeswari, A review on green synthesis, biomedical appli-
cations, and toxicity studies of ZnO NPs, Bioinorg. Chem. Appl. 2018 (2018)
3569758.
[62] J. Singh, T. Dutta, K.-H. Kim, M. Rawat, P. Samddar, P. Kumar, Green'synthesis of
metals and their oxide nanoparticles: applications for environmental remediation,
J. Nanobiotechnol. 16 (2018) 16–84.
[63] J.H. Zar, Biostatistical Analysis: Pearson New International Edition, Pearson Higher
Ed, 2013.
[64] H. Motulsky, GraphPad Prism 5: Statistics Guide, 94 GraphPad Software Inc, Press,
San Diego CA, 2007.
[65] J.J. Bozzola, L.D. Russell, Electron Microscopy: Principles and Techniques for
Biologists, Jones & Bartlett Learning, 1999.
[66] E.P. Feofilova, The fungal cell wall: modern concepts of its composition and bio-
logical function, Microbiology 79 (2010) 711–720.
[67] M.B. Linder, Hydrophobins: proteins that self assemble at interfaces, Curr. Opin.
Colloid Interface Sci. 14 (2009) 356–363.
[68] N.A.R. Gow, J.P. Latge, C.A. Munro, The fungal cell wall: structure, biosynthesis,
and function, Microbiol. spectr. 5 (3) (2017), https://doi.org/10.1128/
microbiolspec.FUNK-0035-2016.
[69] J.G.H. Wessels, Hydrophobins: proteins that change the nature of the fungal sur-
face, Adv. Microb. Physiol, 38 Elsevier, 1996, pp. 1–45.
[70] T. Cedervall, I. Lynch, S. Lindman, T. Berggård, E. Thulin, H. Nilsson, K.A. Dawson,
S. Linse, Understanding the nanoparticle–protein corona using methods to quantify
exchange rates and affinities of proteins for nanoparticles, Proc. Natl. Acad. Sci. 104
(2007) 2050–2055.
[71] D. Sardella, R. Gatt, V.P. Valdramidis, Physiological effects and mode of action of
ZnO nanoparticles against postharvest fungal contaminants, Food Res. Int. 101
(2017) 274–279.
[72] C.C. Fleischer, C.K. Payne, Nanoparticle–cell interactions: molecular structure of
the protein corona and cellular outcomes, Acc. Chem. Res. 47 (2014) 2651–2659.
[73] F.M. Klis, P. De Groot, K. Hellingwerf, Molecular organization of the cell wall of
Candida albicans, Med. Mycol. 39 (2001) 1–8.
[74] S.M. Bowman, S.J. Free, The structure and synthesis of the fungal cell wall,
BioEssays 28 (2006) 799–808.
[75] B. Bago, H. Chamberland, A. Goulet, H. Vierheilig, J.-G. Lafontaine, Y. Piché, Effect
of Nikkomycin Z, a chitin-synthase inhibitor, on hyphal growth and cell wall
structure of two arbuscular-mycorrhizal fungi, Protoplasma 192 (1996) 80–92.
[76] J.A. Brown, B.J. Catley, Monitoring polysaccharide synthesis in Candida albicans,
Carbohydr. Res. 227 (1992) 195–202.
[77] S. Kim, I. Ahn, H. Rho, Y. Lee, MHP1, a Magnaporthe grisea hydrophobin gene, is
required for fungal development and plant colonization, Mol. Microbiol. 57 (2005)
1224–1237.
[78] Q. Mu, G. Jiang, L. Chen, H. Zhou, D. Fourches, A. Tropsha, B. Yan, Chemical basis
of interactions between engineered nanoparticles and biological systems, Chem.
Rev. 114 (2014) 7740–7781.
[79] M. Rahman, S. Laurent, N. Tawil, L. Yahia, M. Mahmoudi, Protein-Nanoparticle
Interactions, The Bio-Nano Interface, Springer – Verlag, Berlin Heidelberg, 2013.
[80] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Nanoparticle
size and surface properties determine the protein corona with possible implications
for biological impacts, Proc. Natl. Acad. Sci. 105 (2008) 14265–14270.
[81] G. Stepien, M. Moros, M. Perez-Hernandez, M. Monge, L. Gutiérrez, R.M.M. Fratila,
M. Las Heras, S. Menao Guillen, J.J. Puente Lanzarote, C. Solans, Effect of surface
chemistry and associated protein corona on the long-term biodegradation of iron
oxide nanoparticles in vivo, ACS Appl. Mater. Interfaces 10 (5) (2018) 4548–4560.
[82] M. Kopp, S. Kollenda, M. Epple, Nanoparticle–protein interactions: therapeutic
approaches and supramolecular chemistry, Acc. Chem. Res. 50 (2017) 1383–1390.
[83] G.W. Doorley, C.K. Payne, Nanoparticles act as protein carriers during cellular in-
ternalization, Chem. Commun. 48 (2012) 2961–2963.
[84] A.V. Romashchenko, T.-W. Kan, D.V. Petrovski, L.A. Gerlinskaya, M.P. Moshkin,
Y.M. Moshkin, Nanoparticles associate with intrinsically disordered RNA-binding
proteins, ACS Nano. 11 (2017) 1328–1339.
[85] B. Mészáros, I. Simon, Z. Dosztányi, Prediction of protein binding regions in dis-
ordered proteins, PLoS Comput. Biol. 5 (2009) e1000376.
[86] P.B. Khoza, M.J. Moloto, L.M. Sikhwivhilu, The effect of solvents, acetone, water,
and ethanol, on the morphological and optical properties of ZnO nanoparticles
prepared by microwave, J. Nanotechnol. 2012 (2012) 195106.
[87] J. Yin, F. Gao, C. Wei, Q. Lu, Water amount dependence on morphologies and
properties of ZnO nanostructures in double-solvent system, Sci. Rep. 4 (2014) 3736.
[88] Q.R. Hu, S.L. Wang, P. Jiang, H. Xu, Y. Zhang, W.H. Tang, Synthesis of ZnO na-
nostructures in organic solvents and their photoluminescence properties, J. Alloys
Compd. 496 (2010) 494–499.
[89] A. Ali, S. Ambreen, R. Javed, S. Tabassum, I. ul Haq, M. Zia, ZnO nanostructure
fabrication in different solvents transforms physio-chemical, biological and photo-
degradable properties, Mater. Sci. Eng. C. 74 (2017) 137–145.
[90] K. Parveen, V. Banse, L. Ledwani, Green synthesis of nanoparticles: their advantages
and disadvantages, AIP Conf. Proc. 17724(1), 2016 (020048-1-020048-7).
[91] P. g S. Rajeshwari, R. Venckatesh, Bio-Fabrication of zinc oxide nanoparticles using
leaf extract of Parthenium hysterophorus L. and its size-dependent antifungal activity
against plant fungal pathogens, Spectrochim. Acta A Mol. Biomol. Spectrosc. 112
(2013) 384–387.
[92] P. Jamdagni, P. Khatri, J.S. Rana, Green synthesis of zinc oxide nanoparticles using
flower extract of Nyctanthes arbor-tristis and their antifungal activity, J. King Saud
Univ. Sci. 30 (2016) 168–175.
[93] L. He, Y. Liu, A. Mustapha, M. Lin, Antifungal activity of zinc oxide nanoparticles
against Botrytis cinerea and Penicillium expansum, Microbiol. Res. 166 (3) (2011)
207–215.
[94] D. Sharma, J. Rajput, B.S. Kaith, M. Kaur, S. Sharma, Synthesis of ZnO nanoparticles
and study of their antibacterial and antifungal properties, Thin Solid Film 519
(2010) 1224–1229.
[95] D. la Rosa-García, C. Susana, P. Martínez-Torres, S. Gómez-Cornelio, M.A. Corral-
Aguado, P. Quintana, N.M. Gómez-Ortíz, Antifungal activity of ZnO and MgO na-
nomaterials and their mixtures against Colletotrichum gloeosporioides strains from
tropical fruit, J. Nanomater. 2018 (2018) 3498527.
[96] A. Ali, S. Ambreen, Q. Maqbool, S. Naz, M.F. Shams, M. Ahmad, A.R. Phull, M. Zia,
Zinc impregnated cellulose nanocomposites: synthesis, characterization and appli-
cations, J. Phys. Chem. Solids 98 (2016) 174–182.
P.A. Arciniegas-Grijalba et al. Materials Science & Engineering C 98 (2019) 808–825
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