agronomy

Article
Preparation and In Vitro Characterization of Chitosan
Nanoparticles and Their Broad-Spectrum Antifungal
Action Compared to Antibacterial Activities against
Phytopathogens of Tomato
Jae-Wook OH 1 , Se Chul Chun 2 and Murugesan Chandrasekaran 3, *
1 Department of Animal Biotechnology, Konkuk University, Seoul 05029, Korea; ohjw@konkuk.ac.kr
2 Department of Bioresource and Food Science, Konkuk University, Seoul 05029, Korea; scchun@konkuk.ac.kr
3 Department of Food Science and Biotechnology, Sejong University, 209 Neungdong-ro, Gwangjin-gu,
Seoul 05006, Korea
* Correspondence: chandrubdubio@gmail.com; Tel.: +82-2-3408-4026; Fax: +82-2-3408-4319




Received: 28 November 2018; Accepted: 3 January 2019; Published: 8 January 2019


Abstract: The present study was to prepare chitosan nanoparticles (CNPs) from chitosan (CS) to
evaluate their in vitro antimicrobial activities against phytopathogens of tomato. We prepared and
characterized CNPs for their particle size, polydispersity index, and structures. The antifungal
properties of CS and CNPs against phytopathogenic fungi namely Colletotrichum gelosporidies,
Phytophthora capsici, Sclerotinia sclerotiorum, Fusarium oxysporum, Gibberella fujikuori were investigated.
CNPs showed the maximum growth inhibitory effects on mycelial growth of F. oxysporum followed
by P. capsici. We also studied antibacterial activities against phytopathogenic bacteria, such as three
strains of Erwinia carotovora subsp. carotovora and one strain of Xanthomonas campestris pv. vesicatoria.
Our results showed that both CS and CNPs markedly inhibited the growth of the both Xanthomonas
and Erwinia strains. From our study, it is evident that both CS and CNPs have tremendous potential
against phytopathogens of tomato for further field screening towards crop protection.

Keywords: chitosan; chitosan nanoparticles; phytopathogens; antibacterial activity;

antifungal activity

1. Introduction
Nanotechnology has been revolutionizing almost all realms in life sciences, especially agriculture.
Precision in agriculture was emphasized by many researchers employing nanoparticles against
diagnosis and fertilizers application [1]. Chitosan (CS), a naturally occurring linear biopolymer
composed of D-glucosamine and N-acetyl glucosamine residues, is derived from the complete or partial
deacetylation of chitin [2,3]. In the plant system, chitosan has been reported to induce multifaceted
disease resistance [4,5]. CS is a competent elicitor in agriculture mediating plant immunity by
microbe-associated molecular patterns [6]. Current research stresses the utility of chitosan nanoparticles
(CNPs) in agriculture as a comprehensive strategy towards sustainability in productivity and high
yield in plant protection prospects throughout the world in agriculture [7–10]. NPs prepared from
natural sources possess advantages, such as availability of replenishable resources, biocompatibility,
biodegradability, and ecological safety. In this regard, chitosan-based nanoparticles are preferably
used for various applications owing to their biodegradability, high permeability toward biological
membranes, non-toxicity to human, cost-effectiveness, and broad antimicrobial activities [11–15].
Seed treatment with CNPs and as a biofertilizer application efficiently abates fungal infection and
aids plant growth [16]. CS polymeric nanoparticles (NPs) are biodegradable and are utilized for

Agronomy 2019, 9, 21; doi:10.3390/agronomy9010021 www.mdpi.com/journal/agronomy

Agronomy 2019, 9, 21 2 of 12

the controlled release of NPK fertilizers [17]. CS polymethacrylic acid colloidal suspension helps
in sustained release because of a high affinity towards calcium phosphate rather than potassium
chloride and urea due to high anionic charges [18]. CNPs also regulate upgraded micronutrient
supplementation 1-naphthylacetic acid under various pH and temperature enhancing uptake of
plant growth hormones [19]. Cu-CS complemented NPs show diverse antifungal efficacy combating
Alternaria alternata, Macrophomina phaseolina, and Rhizoctonia solani [14]. Acetamprid laden alginate-CS
nanocapsules are employed for agrochemical supply with efficient delivery and liposome-based
application [20]. Further, CNPs significantly enhanced plant immunity [21], demonstrated efficient
activity against phytopathogens [22], and produced high yields [23]. CNPs act as carriers for
encapsulating herbicides for escalated efficiency and release kinetics with reduced toxicity levels
for the combinatorial herbicides, Imazapic and Imazapyr [24]. Thus, CNPs have been regarded
as an effective modality compared to other combined applications for improved efficiency and
agricultural efficacy pertaining to the arrest of phytopathogens. Nevertheless, CS conventionally
has been largely used to increase plant productivity, for crop protection and plant defense, and to
enhancement of shelf life of fruits [25]. The potential of chitosan to protect plants from fungal diseases
and bacterial diseases has been reported [2–4,25–27]. The chelating property of chitosan towards
various organic and inorganic compounds makes it a suitable biopolymer for improvement in stability,
solubility, and biocidal activity. However, the insolubility of bulk chitosan in aqueous media limits
its wide spectrum application as an antifungal agent [10,14]. Compared to bulk CS, CNPs imbues
versatility in biological activities due to altered physicochemical characteristics, such as size, surface
area, cationic nature, active functional groups, and higher encapsulation efficiency, etc., alone and/or
through the blending of other components [10,14]. With this important prospect, we undertook the
present study to assess the combinatorial research in alienating the effects of CNPs over CS to combat
phytopathogens in tomato. Up to date, most research concerning CNPs is concentrated on fungal
pathogens. The present report analyzes its effect against various fungal pathogens encountered in
tomato as well as bacterial pathogens causing leaf spot and soft rot disease in tomato. Therefore, in the
present investigation, CNPs were prepared which were further examined against phytopathogenic
bacteria such as Erwinia carotovora subsp. carotovora and Xanthomonas campestris pv. vesicatoria and
fungi viz. Colletotrichum gelosporidies, Phytophthora capsici, Sclerotinia sclerotiorum, Fusarium oxysporum,
and Gibberella fujikuori.

2. Materials and Methods

2.1. Preparation of Chitosan Nanoparticles

CNPs were prepared based on the ionic gelation of chitosan (Mw ≈ 190–370 kDa, deacetylation
degree ≥75%) with sodium tripolyphosphate (TPP) anions. CS was dissolved at 0.1% level (w/v) in 1%
(v/v) acetic acid followed by overnight stirring on a magnetic stirrer at 200 rpm and filtered through
a PVDF syringe filter (pore size 0.22 µm). TPP was dissolved at 0.25% level (w/v) in sterile distilled
water and filtered through a PVDF membrane syringe filter (pore size 0.22 µm). The cross-linking of
chitosan with TPP at equal volume was performed drop by drop under a magnetic stirrer at 700 rpm.
The resulting formulation was centrifuged for 10 min at 10,000 rpm, and the pellet was resuspended in
sterile distilled water followed by ultra-sonication at 28% pulse ratio for 100 s at 4 ◦ C. Centrifugation
followed by ultrasonication was repeated three times, and the precipitated nanoformulation was
freeze-dried and stored in a desiccator for further analysis.

2.2. Characterization of Nanoparticles

2.2.1. UV-Visible Spectra and Dynamic Light Scattering (DLS) Measurements

UV-visible spectra were recorded using a Shimadzu UV-visible1800 spectrophotometer for the
confirmation of nanoparticle formation. Dynamic light scattering (DLS) was used for the measurement
Agronomy 2019, 9, 21 3 of 12

of average particle size, and polydispersity index (PDI) on a high-performance particle Zetasizer
HPPS-5001 (Malvern, UK). Each sample was analyzed in triplicate at 25 ◦ C at a scattering angle of
90 ◦ C. Pure water was used as a reference for dispersing medium. The results are given as the average
particle size obtained from the analysis of three different batches, each of them measured three times.

2.2.2. Fourier Transform Infrared (FTIR) Analysis

To confirm the synthesis of nanoparticles, Fourier transform infrared (FTIR) analysis was done.
For FTIR, each sample was prepared in potassium bromide (KBr) as a pellet under 1:99 ratio of sample
to KBr, and was recorded by ABB FTLA 2000-100 (ABB Co., Quebec, Canada) at a resolution limit of
16 cm−1 .

2.2.3. Scanning Electron Microscopy (SEM) Observation

The scanning electron microscope (SEM) was used to study the surface morphology of NPs.
The samples were dried by critical point drying (CPD, Emitech) and mounted on aluminium stubs
and then coated with gold using a Sputter coater model E-1010 (Emitech). The samples were
examined using a scanning electron microscope model S 2700 (Hitachi Ltd, Tokyo, Japan) with
15 kV accelerating voltage.

2.3. Bacterial Strains

2.3.1. Bacterial Strains and Growth Conditions

To obtain the antibacterial activity of CS and CNPs, one strain of X. campestris pv. vesicatoria
(KACC1154) and three strains of E. carotovora subsp. carotovora (KACC 113114, 113154, and 133061) that
cause leaf spot disease and soft rot disease of Solanum lycopersicum, respectively. The bacterial strains
were cultured on nutrient agar medium at 30 ◦ C. After 48 h of incubation, each bacterial suspension
was prepared in Luria–Bertani broth (LB).

2.3.2. Antibacterial Activities

The inhibition activity of both CS and CNPs against Xanthomonas and Erwinia phytopathogenic
bacteria were evaluated by measuring optical density (OD) 600 nm. Xanthomonas and Erwinia strains
were cultivated in nutrient agar and incubated at 28 ◦ C. A representative colony was picked off and
placed in plus-LB (peptone 10 g, yeast extract powder 5 g, NaCl 5 g, distilled water up to 1000 mL,
glucose 1 g, pH 7.2) and incubated overnight on the rotary shaker at 180 rpm, 30 ◦ C. Different
concentrations (0.5, 1.0, and 5.0 mg/mL, (w/v)) of CS and CNPs were added to the cultured bacterial
suspension and then the mixture was incubated at 30 ◦ C on a rotary shaker at 180 rpm for 48 h while
the same volume of sterile distilled water was added to the controls. The absorbance of samples was
measured at 600 nm.

2.4. Fungal Strains

2.4.1. Fungal Strains and Growth Conditions

The antifungal properties of CS and CNPs against phytopathogenic fungi namely C. gelosporidies,
P. capsici, S. sclerotium, F. oxysporum, and G. fujikori were investigated at various concentrations of CS
and CNPs ranging from 0.1 to 5.0 mg/mL. Potato dextrose agar (PDA) medium was prepared and
poured in Petri dishes with above-mentioned percentages of various CS and CNPs, separately.

2.4.2. Antifungal Activities

Antifungal assay of CS was conducted for both the radial growth determination of fungi. For
radial growth determination, the sterile CS and CNPs solution were added to PDA at a concentration
of 0.1, 0.5, 1.0, 3.0, and 5.0 mg/mL, (w/v). Mycelial agar plugs from the actively growing peripheral
Agronomy 2019, 9, 21 4 of 12

end of uniform size (diameter, 5.0 mm) were taken from the 7-day old culture of the test pathogens
and inoculated in the center of plates supplemented with different concentrations of CS and CNPs.
All the Petri dishes were incubated at 28 ◦ C for 10 days, and the observation of radial mycelial
Agronomy 2019, 9, x FOR PEER REVIEW
growth
4 of 13
was recorded when controlling Petri dish was covered with full growth. Wherein, S. sclerotiorum
platesend of uniform
were incubated size for
(diameter,
5 days,5.0 mm) were
owing taken
to their from
fast the 7-day
growth. Allold
theculture of the consisted
treatments test pathogens
of three
and inoculated in the center of plates supplemented with different concentrations
replications. The inoculated plates were compared with control (without CS and CNPs) to calculate of CS and CNPs.
All the Petriinhibition
the percentage dishes were incubated
rate at 28of
of mycelia °Cthe
for 10 days, andThe
pathogen. the observation
percentages ofof
radial mycelial
growth growthwere
inhibition
was recorded when controlling Petri dish was covered with full growth. Wherein, S. sclerotiorum
calculated relative to control using the following formula:
plates were incubated for 5 days, owing to their fast growth. All the treatments consisted of three
replications. The inoculated plates were compared with control (without CS and CNPs) to calculate
% Inhibition rate = (Mc − Mt) Mc × 100
the percentage inhibition rate of mycelia of the pathogen. The percentages of growth inhibition were
calculated relative to control using the following formula:
where Mc is the mycelial growth in control, Mt is the mycelial growth in treatment.
% Inhibition rate = (Mc − Mt) Mc × 100
where Mc is the mycelial growth in control, Mt is the mycelial growth in treatment.
2.5. Statistical Analysis
2.5. of
All Statistical Analysis
the experiments were conducted in triplicate and results were tabulated as the mean ±
standard All
deviation
of the experiments were
(SD). Data analyzed by
were conducted analysis and
in triplicate of variance (ANOVA).
results were Student’s
tabulated t-test
as the mean ± and
the probability values of p < 0.05 were considered to be significant.
standard deviation (SD). Data were analyzed by analysis of variance (ANOVA). Student’s t-test and
the probability values of p < 0.05 were considered to be significant.
3. Results and Discussion
3. Results and Discussion
The optical properties of biopolymer materials were analysis by UV-Visible spectroscopy and are
shown in The optical
Figure 1. Aproperties
UV-Visible of spectrum
biopolymer ofmaterials wereaanalysis
CS obtained by UV-Visible
broad absorption bandspectroscopy and
intensity compared
are shown in Figure 1. A UV-Visible spectrum of CS obtained a broad absorption
with CNPs (sharp intensity), and the absorption peak wavelength was at 320 nm in the UV region. band intensity
compared with CNPs (sharp intensity), and the absorption peak wavelength was at 320 nm in the UV
But in the case of CNPs, a higher intensity level than CS biopolymer was observed, which is due to
region. But in the case of CNPs, a higher intensity level than CS biopolymer was observed, which is
the formation of NPs. CS capped AuNPs were proved to uptake analytes, such as amorphous carbon
due to the formation of NPs. CS capped AuNPs were proved to uptake analytes, such as amorphous
nanotubes,
carboncopper oxide,
nanotubes, and Zinc
copper oxide,sulfate, which
and Zinc indirectly
sulfate, which shows theshows
indirectly abilitythe
of CNPs
ability to
ofneutralize
CNPs to the
adverse
neutralize the adverse effects of chemical fertilizers [28]. Hence, CNPs, apart from providing plant and
effects of chemical fertilizers [28]. Hence, CNPs, apart from providing plant protection
crop productivity,
protection andhavecrop aproductivity,
neutralizinghaveeffect on the uptake
a neutralizing of harmful
effect chemical
on the uptake remnants
of harmful in the soil.
chemical
As organic farming
remnants in the is largely
soil. stressed
As organic theseisdays,
farming green
largely nanotechnology
stressed will nanotechnology
these days, green have positive outcomes
will
have
without positive outcomes
deleterious outcomes without deleterious
to Mother outcomes to Mother Earth.
Earth.

Figure 1. UV-visible
Figure spectrum
1. UV-visible spectrumofofsynthesized chitosanand
synthesized chitosan andchitosan
chitosan nanoparticle.
nanoparticle.

The particles size

The particles distribution
size distribution of theCNPs
of the CNPswere
were measured
measured by and
by DLS DLSisand is shown
shown in Figurein2. Figure
The 2.
CNPs showed
The CNPs showed characteristic
characteristic chemical
chemical changes thatthat
changes occurred during
occurred the formation
during of CNPsoffrom
the formation CNPs thefrom
CSbiopolymer
the CS biopolymer source
source at
at different
differentexperimental
experimental conditions.
conditions.TheThe
sizesize
of CNPs was was
of CNPs in the
in diameter
the diameter
range of ~100 to 1000 nm, with an early correlation coefficient decay curve and polydispersity
range of ~100 to 1000 nm, with an early correlation coefficient decay curve and polydispersity index.index.
Escalated antifungal activity against Fusarium Head Blight (FHB) of wheat has been attributed to low
Escalated antifungal activity against Fusarium Head Blight (FHB) of wheat has been attributed to low
molecular weight CNPs, and they could possibly replace the use of chemical fertilizers in the
molecular weight CNPs, and they could possibly replace the use of chemical fertilizers in the abatement
Agronomy 2019, 9, 21 5 of 12
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9, xx FOR
FOR PEER
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abatement
of FHB [29].of
abatement FHB
ofThe [29].
FHBDLS The
The DLS
[29].methodDLSin method in
in the
the emancipation
the emancipation
method of antifungal
emancipation of
of antifungal characteristics
characteristics
antifungal in
in broad-
in broad-spectrum
characteristics broad-
spectrum
activity
spectrum in activity in
in S.
S. lycopersicum
S. lycopersicum
activity also showsalso
lycopersicum shows
a similar
also aa similar
similar effect.
showseffect. effect.

Figure
Figure2.2.
Figure (a)
2.(a) Dynamic
(a)Dynamic
Dynamic light
light
light scattering
scattering
scattering (DLS)
(DLS) of theof
(DLS) the
the synthesized
ofsynthesized
synthesized
chitosanchitosan
chitosan nanoparticles.
nanoparticles.
nanoparticles. (b)
(b) The
The
(b) The effective
effective zeta-potential
effective zeta-potential
zeta-potential in aqueous
insolution
in aqueous solution
aqueouswere
solution were measured
were measured
measured by
by particleby particle characterizer.
particle characterizer.
characterizer.

These
These results
Theseresults were
resultswere
were demonstrated
demonstrated
demonstrated by
by Kheiri
by Kheiri et al. et
Kheiri [29]al.
et [29]
al.under under
[29] field
under field
trails for trails
field for
effective
trails effective
forcrop crop
protection
effective crop
protection
strategies strategies
protectionwith the use
strategies with the
the use
of CNPs.
with of
of CNPs.
useEarlier, CNPs
CNPs. Earlier,
withCNPs
Earlier, a size with
CNPs aa size
of less
with thanof
size less
of400 than
than 400
400 suppressed
suppressed
less wilt diseasewilt
suppressed in
wilt
disease
tomatoes in tomatoes
caused by caused
F. by
oxysporum F. oxysporum
f. sp. f.
Lycopersicisp. Lycopersici
with a with
corresponding a corresponding
yield increase
disease in tomatoes caused by F. oxysporum f. sp. Lycopersici with a corresponding yield increase [30]. yield increase
[30]. The [30].
present
The
The present
study shows study
present shows
shows the
the significant
study significant
scale
the scale
of outcome
significant scaleevenof
of outcome
at a range
outcome even at
at aa Hence,
of 100.
even range
range of 100.
ofthe Hence,
initial
100. DLSthe
Hence, initial
analysis
the initial
DLS
DLS analysis
intrigues us and
analysis intrigues
can be the
intrigues us and
us basis
and forcan be
be the
canfuture basis
basis for
combinatorial
the for future combinatorial
analysis
future for various analysis
combinatorial analysis forfor various
phytopathogens not
various
phytopathogens
phytopathogens not only in tomato but also against a variety of crop plants to determine the
only in tomato butnot
alsoonly in
against tomato
a varietybut
of also
crop against
plants to a variety
determine of thecrop plants
reliability to
for determine
CNPs. CS and
the
reliability
CNPs
reliability for
for CNPs.
functional groups
CNPs. CS
CS and CNPs
CNPs functional
are studied
and using FTIR
functional groups are
are studied
spectroscopy.
groups Theusing
studied FTIRFTIR
using spectra
FTIR spectroscopy. The
of CS and CNPs
spectroscopy. The FTIR
are
FTIR
spectra
shown
spectrainof CS
CS and
ofFigureand3.CNPs
CNPs areare shown
shown in in Figure
Figure 3. 3.

Figure
Figure 3. Fourier transform infrared (FTIR) spectrum of chitosan and chitosan nanoparticle.
Figure 3.
3. Fourier
Fourier transform
transform infrared
infrared (FTIR)
(FTIR) spectrum
spectrum of
of chitosan
chitosan and
and chitosan
chitosan nanoparticle.
nanoparticle.
A peak at 3500 to 3300 cm−−11 was observed for the main functional group of chitosan and is due
A peak at
at 3500 to
to 3300
3300 cm −1 was
was observed for
for the main functional group of
of chitosan and −
is1 due
to theAO-H
peakgroup 3500
of stretchingcm observed
vibrations. the of
The presence main functional
absorption group
peaks at 1630 chitosan
and 1531and
cm is−1due
are
to the O-H group of stretching vibrations. The presence of absorption peaks at 1630
to the O-H group of stretching vibrations. The presence of absorption peaks at 1630 and 1531 cm areand 1531 cm −1 are
due to the N-H bending vibration of protonated amino (−NH2 ) group and C-H bending vibration of
due
due to
to the
the N-H
N-H bending
bending vibration
vibration of
of protonated
protonated amino
amino (−NH
(−NH 2) group and C-H bending vibration of
) group and C-H bending vibration of
the alkyl group. The absorption peaks at 1059 and 886 cm−−11 are recognized due to the anti-symmetric
2
the alkyl group. The absorption peaks at 1059 and 886 cm are recognized due to
the alkyl group. The absorption peaks at 1059 and 886 cm are recognized due to the anti-symmetric
−1 the anti-symmetric
stretching vibration of C-O-C bridges and assigned to glucopyranose ring in chitosan matrix. A similar
stretching
stretching vibration
vibration ofof C-O-C
C-O-C bridges
bridges and
and assigned
assigned to to glucopyranose
glucopyranose ringring in
in chitosan
chitosan matrix.
matrix. A A
similar
similar study
study by
by Saharan
Saharan etet al.
al. [14]
[14] employing
employing Cu-CNPs
Cu-CNPs against
against pathogenic
pathogenic fungi
fungi ofof tomato
tomato showed
showed
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that spectral
study by Saharan peaksetforal.Cu-CNPs were sharp
[14] employing and a shift
Cu-CNPs waspathogenic
against observed atfungi 1631 and 1536 cm
of tomato −1. Whereas,
showed that
the present
spectral peaks results corroborate
for Cu-CNPs wereasharpminimaland variation
a shift was which suggests
observed at 1631 that
and Cu1536
bonding − 1
cm .influences
Whereas, the the
variation. It can be deduced that the nanoencapsulation of CNPs will
present results corroborate a minimal variation which suggests that Cu bonding influences the variation. have varied distribution
Itfrequencies
can be deduced that might
that theinfluence the optimalof
nanoencapsulation benefits
CNPs willfor agricultural practices. The
have varied distribution present study
frequencies that
involves
might CNPs the
influence withoutoptimalanybenefits
furtherfor encapsulation. The results
agricultural practices. Theaffirm
present thatstudy
CNPs without
involves CNPs any
encapsulation
without any further can be of significantThe
encapsulation. benefit.
resultsHence,
affirm wethatpropose
CNPs withoutthe use of encapsulation
any the beneficialcan nano-be
encapsulation
of significant benefit.particleHence,
whichwe would boostthe
propose green
use ofagriculture and provide
the beneficial multiple benefits
nano-encapsulation particleowing
which to
Nanotechnology in Agriculture. On the contrary, chitosan characteristic
would boost green agriculture and provide multiple benefits owing to Nanotechnology in Agriculture. peaks are shifted to 3359,
2874,
On the1576, 1412, chitosan
contrary, 1021, 877,characteristic
and 700 cm−1peaks , whenare compared
shifted to with corresponding
3359, 2874, 1576, 1412, CNPs.1021,This 877,
indicates
and
thatcm
700 − 1
an interaction
, when compared between chitosan and treatment
with corresponding nanoparticles
CNPs. which that
This indicates is an an
indication
interactionof a between
chemical
reaction.and
chitosan However,
treatment there need to be further
nanoparticles whichtrials
is anfor effectiveoffield
indication applications.
a chemical In this
reaction. regard, there
However, it can
be concluded
need to be further that the encapsulation
trials for effectiveof twoapplications.
field herbicides namely,
In thisImazapic
regard, itand canImazapyr
be concluded with that
CNPs theis
an effective strategy
encapsulation of two for sustainable
herbicides agriculture
namely, Imazapicwith andreduced
Imazapyr harm
withtoCNPs
humans is anand the environment.
effective strategy for
The mode of
sustainable action of this
agriculture withformulation
reduced harm wastoquestioned
humans and forthe
a molecular
environment. dissection
The mode study for affirming
of action of this
the positivity
formulation was ofquestioned
its role [24]. forAlthough
a molecular there are several
dissection studyreports pertaining
for affirming to nanotechnology
the positivity of its role [24].in
progressive agriculture, there are still unanswered questions in the field
Although there are several reports pertaining to nanotechnology in progressive agriculture, there are of CNPs, such as the choice
of material
still unanswered for nanoencapsulation
questions in the field which aggregates
of CNPs, such aswiththeCNPs
choicewith consideration
of material towards a clear
for nanoencapsulation
and efficient
which aggregates resultwith forCNPs
boosting
with crop production
consideration and productivity
towards together
a clear and efficient withfor
result crop protection
boosting crop
which has minimal detrimental effects on soil. We have reviewed numerous
production and productivity together with crop protection which has minimal detrimental effects on reviews and researches
and We
soil. believe
havethat the field
reviewed of Nanotechnology
numerous reviews andin Agriculture
researches andisbelieve
still underexploited.
that the field of Nanotechnology
The CNPs peak shifted
in Agriculture is still underexploited. to 1576 cm −1 is due to the wagging of NH2 bond and the strong peak at

1412Thecm−1CNPs is due to C-H

peak bending
shifted to 1576 cm−1 isof
vibration duethetoalkyl group. The
the wagging of NHionic interaction
2 bond and thewith thepeak
strong treated at
molecules
1412 cm is− 1 indicates
due to C-H the conversion of chitosan
bending vibration polymer
of the in the nano
alkyl group. forminteraction
The ionic that forms awith cross-link with
the treated
the treatedindicates
molecules molecules. the The surface of
conversion morphological
chitosan polymer structure
in thewasnano further examined
form that forms by SEM analysis.
a cross-link with
Thetreated
the SEM images molecules.of CSThe andsurface
CNPs morphological
are displayed atstructure
the higher wasand lowerexamined
further magnification
by SEM scale of 200
analysis.
andSEM
The 100 nm images as shown
of CS and in Figure
CNPs are 4. The morphological
displayed structure
at the higher of CSmagnification
and lower shows the large scaleparticle
of 200 andsize
and agglomerated state. The SEM images of CNPs clearly show
100 nm as shown in Figure 4. The morphological structure of CS shows the large particle size and a spherical-like structure and
particles in the
agglomerated agglomerated
state. The SEM images state. Further,
of CNPs the uniformity
clearly of the size of structure
show a spherical-like the nanoparticles
and particlescan in be
observed.
the agglomerated state. Further, the uniformity of the size of the nanoparticles can be observed.

Figure4.4.Scanning
Figure Scanningelectron microscope
electron (SEM)
microscope images
(SEM) of chitosan
images (A,C)(A,C)
of chitosan and chitosan nanoparticles
and chitosan (B,D).
nanoparticles
(B,D).
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The above results clearly indicate that the CNPs show a highly porous surface due to
agglomeration
The above attributes.
results The porous
clearly nature
indicate andthe
that agglomeration
CNPs show capabilities of the CNPs
a highly porous surfacerender
due them
to
agglomeration
useful attributes.
as a critical The porous
chitosan-based nature and agglomeration
bio-nanopesticide capabilitieshas
[15]. Agglomeration of the CNPs
been render them
considered as the
useful phenomenon
primary as a critical chitosan-based
for synthesis bio-nanopesticide
for novel CNPs for [15]. Agglomeration
biomedical has been
applications andconsidered
nanomedicineas the[31].
Theprimary phenomenon
same concept holds for
for synthesis for novel
agricultural. Hence,CNPs for biomedical
the porous nature canapplications and nanomedicine
harbor quenching molecules to
[31]. The same concept holds for
effectively adsorb harmful chemicals in soil. agricultural. Hence, the porous nature can harbor quenching
molecules to effectively
This study adsorb
was carried outharmful chemicals
to evaluate in soil.
different concentrations of CS and CNPs against plant
pathogenic fungi and bacteria under laboratory conditions. The ofmain
This study was carried out to evaluate different concentrations CS and CNPsof
concept against plant is
this study
pathogenic fungi and bacteria under laboratory conditions. The main concept of this study is
dependent on the comparison between the effectiveness of CS and nano-CS concentrations to inhibit
dependent on the comparison between the effectiveness of CS and nano-CS concentrations to inhibit
phytopathogens. Antifungal effects of CS and CNPs were evaluated against plant pathogenic fungi.
phytopathogens. Antifungal effects of CS and CNPs were evaluated against plant pathogenic fungi.
The NPs inhibited the radial growth of pathogens at different concentration levels, and all the tested
The NPs inhibited the radial growth of pathogens at different concentration levels, and all the tested
cultures showed a clear significant effect compared with the control treatment (Figures 5 and 6).
cultures showed a clear significant effect compared with the control treatment (Figures 5 and 6). In
In the present study, we also found some differences in the antifungal activity in both plate assay and
the present study, we also found some differences in the antifungal activity in both plate assay and
percentage
percentageinhibition
inhibitionofofCS
CSand
andCNPsCNPs particularly Colletotrichumgelosporidies
againstColletotrichum
particularly against gelosporidies
andand Gibberella
Gibberella
fujikuori.
fujikuori. These differences in the activity of the fungal species might be due to the presence of some of
These differences in the activity of the fungal species might be due to the presence
some resistance
resistance mechanism
mechanism against
against thesethese compounds.
compounds. The differences
The differences we found
we found in theinpercentage
the percentage
of
of inhibition
inhibitionprove
provethat
thatnot
notall
allbiological
biologicalsystems
systemsexhibit
exhibit similar behavior under the influence of thethe
similar behavior under the influence of
same external agent.
same external agent.

Figure 5. Antifungal activity of chitosan nanoparticles (NPs)-Plate assay. (A) Colletotrichum

Figure 5. Antifungal activity of chitosan nanoparticles (NPs)-Plate assay. (A) Colletotrichum
gelosporidies-Chitosan, (B)Colletotrichum
gelosporidies-Chitosan,(B) gelosporidies-ChitosanNPs,
Colletotrichum gelosporidies-Chitosan NPs,(C)(C) Phytophthora
Phytophthora capsici-Chitosan,
capsici-Chitosan,
(D) Phytophthora capsici-Chitosan NPs, (E) Sclerotium sclerotiorum -Chitosan, (F) Sclerotium
(D) Phytophthora capsici-Chitosan NPs, (E) Sclerotium sclerotiorum -Chitosan, (F) Sclerotium sclerotiorum-
sclerotiorum-Chitosan NPs, (G)
Chitosan NPs, (G) Fusarium Fusarium oxysporum-Chitosan,
oxysporum-Chitosan, (H) Fusarium oxysporum-chitosan
(H) Fusarium oxysporum-chitosan NPs,
NPs, (I) Gibberella
Gibberella fujikori-Chitosan,
(I)fujikori-Chitosan, (J) Gibberella fujikuori-Chitosan NPs. All the Petri dishes were incubated
(J) Gibberella fujikuori-Chitosan NPs. All the Petri dishes were incubated at 28 °C for
at 10 ◦ C for 10 days whereas Sclerotinia sclerotiorum plates were incubated for 5 days, owing to their
28days whereas Sclerotinia sclerotiorum plates were incubated for 5 days, owing to their fast growth.
fast growth.
Agronomy 2019, 9, 21 8 of 12
Agronomy 2019, 9, x FOR PEER REVIEW 8 of 13

Managementofoffungal
Management fungaldiseases
diseasesininfood
foodcrops
cropsisiseconomically
economicallyimportant.
important. Recently,
Recently,aagreater
greater
emphasishas
emphasis hasbeen
beengiven
giventotothe
thedevelopment
developmentofofsafe
safemanagement
managementmethods
methodsthat
thatpose
poseless
lessdanger
dangertoto
humans and animals, with a focus on overcoming the deficiencies of synthetic fungicides. CNPs
humans and animals, with a focus on overcoming the deficiencies of synthetic fungicides. CNPs show show
broad-spectrumactivities,
broad-spectrum activities,such
suchasasplant
plantgrowth
growthpromotion,
promotion,biocide,
biocide,and
andplant
plantprotection
protection[32].
[32].

Figure 6. Antifungal activity of chitosan nanoparticles-Inhibition studies (i) Colletotrichum

Figure 6. Antifungal activity of chitosan nanoparticles-Inhibition studies (i) Colletotrichum
gelosporidies-A. Chitosan B. Chitosan NPs. (ii) Phytophthora capsici-A. Chitosan B. Chitosan NPs.
gelosporidies-A. Chitosan B. Chitosan NPs. (ii) Phytophthora capsici-A. Chitosan B. Chitosan NPs. (iii)
(iii) Sclerotinia sclerotiorum-A. Chitosan B. Chitosan NPs. (iv) Fusarium oxysporum-A. Chitosan B.
Sclerotinia sclerotiorum-A. Chitosan B. Chitosan NPs. (iv) Fusarium oxysporum-A. Chitosan B. Chitosan
Chitosan NPs. (v) Gibberella fujikuori-A. Chitosan B. Chitosan NPs. The percentages of growth inhibition
NPs. (v) Gibberella fujikuori-A. Chitosan B. Chitosan NPs. The percentages of growth inhibition were
were calculated relative to control.
calculated relative to control.
In this study, CS and CNPs markedly inhibited the growth of the one Xanthomonas strain and
In this study, CS and CNPs markedly inhibited the growth of the one Xanthomonas strain and
three Erwinia strains as seen by measuring the OD value at 600 nm. The reduction in the OD of cell
three Erwinia strains as seen by measuring the OD value at 600 nm. The reduction in the OD of cell
suspension depends on the type of CS and the species of bacteria. CS and CNPs and bacterial species
suspension depends on the type of CS and the species of bacteria. CS and CNPs and bacterial species
significantly affected the surviving cell numbers (Figures 7 and 8). This result revealed that the growth
significantly affected the surviving cell numbers (Figures 7 and 8). This result revealed that the
of X. campestris and E. carotovora was inhibited by CS and CNPs regardless of the kinds of CS and the
growth of X. campestris and E. carotovora was inhibited by CS and CNPs regardless of the kinds of CS
species of bacteria, which implies that the two kinds of CS were good bactericide for the control of
and the species of bacteria, which implies that the two kinds of CS were good bactericide for the
control of bacterial disease of tomato. Results from this study indicated that the addition of chitosan
Agronomy 2019, 9, 21 9 of 12
Agronomy 2019, 9, x FOR PEER REVIEW 9 of 13

at 0.5 to 5.0
bacterial mg/mL
disease to the two
of tomato. bacterial
Results strains
from this caused
study a reduction
indicated that theinaddition
the OD600nm afterat480.5
of chitosan h to
of
incubation.
5.0 mg/mL The reduction
to the percentage
two bacterial ofcaused
strains CS in the OD600nmin
a reduction ranged from 4.4%after
the OD600nm to 86.97%
48 h ofasincubation.
compared
to the
The control while
reduction the reduction
percentage of CS inpercentage
the OD600nmin most of the
ranged strains
from 4.4%were more than
to 86.97% 50.00% (Figure
as compared to the
7).
control while the reduction percentage in most of the strains were more than 50.00% (Figure 7).

Figure 7.7.Effect
Effectofof
chitosan
chitosanon on
the the
growth of Xanthomonas
growth campestris
of Xanthomonas pv. vesicatoria
campestris and Erwinia
pv. vesicatoria andcartovora
Erwinia
subsp. carotovora
cartovora pathogenic
subsp. carotovora to Solanum
pathogenic lycopersicum.
to Solanum (A) Erwinia
lycopersicum. (A) cartovora subsp. carotovora
Erwinia cartovora 113114.
subsp. carotovora
(B) Erwinia
113114. (B) cartovora
Erwinia subsp. carotovora
cartovora
Agronomy 2019, 9, x FOR PEER REVIEW subsp. 113154. (C)
carotovora Erwinia
113154. cartovora
(C) subsp.
Erwinia carotovora
cartovora subsp.YKB133061.
carotovora
10 of 13
(D) Xanthomonas
YKB133061. campestris pv.campestris
(D) Xanthomonas vesicatoriapv.
11154.
vesicatoria 11154.

This result was consistent with the previous result, which found CS had strong antibacterial
activity against nine strains of X. arboricola pv. poinsettiicola and X. axonopodis pv. poinsettiicola from
different geographic sources based on the colony count method [33]. Similarly, the addition of CNPs
to four bacterial strains caused a reduction in the OD600nm after 48 h of incubation. The reduction
percentage in the OD600nm ranged from 10.61% to 84.75% as compared to the control while the
reduction percentage in strain 13114 was more than 40.00% (Figure 8).

Figure 8. Effect of chitosan nanoparticles on the growth of Xanthomonas campestris pv. vesicatoria
Figure 8. Effect of chitosan nanoparticles on the growth of Xanthomonas campestris pv. vesicatoria and
and Erwinia carotovora subsp. carotovora pathogenic to Solanum lycopersicum. (A) Erwinia carotovora
Erwinia carotovora subsp. carotovora pathogenic to Solanum lycopersicum. (A) Erwinia carotovora subsp.
subsp. carotovora 113114. (B) Erwinia carotovora subsp. carotovora 113154. (C) Erwinia carotovora subsp.
carotovora 113114. (B) Erwinia carotovora subsp. carotovora 113154. (C) Erwinia carotovora subsp.
carotovora YKB133061. (D) Xanthomonas campestris pv. vesicatoria 11154.
carotovora YKB133061. (D) Xanthomonas campestris pv. vesicatoria 11154.

However, in this study, the three Erwinia strains, in general, showed a difference in the
sensitivity to CS and CNPs. In particular, the reduction percentage of CS in the OD600nm of strain
13114, 113154, and 133061 was 45.42%, 53.72%, and 51.33% respectively, while the reduction
percentage of CNPs in the OD600nm of strain 13114, 113154 and 133061 was 43.31%, 55.37%, and
48.26% respectively, as compared to the corresponding control. In contrast, strain X. campestris 1154
Agronomy 2019, 9, 21 10 of 12

This result was consistent with the previous result, which found CS had strong antibacterial
activity against nine strains of X. arboricola pv. poinsettiicola and X. axonopodis pv. poinsettiicola from
different geographic sources based on the colony count method [33]. Similarly, the addition of CNPs
to four bacterial strains caused a reduction in the OD600nm after 48 h of incubation. The reduction
percentage in the OD600nm ranged from 10.61% to 84.75% as compared to the control while the
reduction percentage in strain 13114 was more than 40.00% (Figure 8).
However, in this study, the three Erwinia strains, in general, showed a difference in the sensitivity
to CS and CNPs. In particular, the reduction percentage of CS in the OD600nm of strain 13114, 113154,
and 133061 was 45.42%, 53.72%, and 51.33% respectively, while the reduction percentage of CNPs
in the OD600nm of strain 13114, 113154 and 133061 was 43.31%, 55.37%, and 48.26% respectively,
as compared to the corresponding control. In contrast, strain X. campestris 1154 showed susceptibility
to both CS and CNPs, which caused the reduction in OD600nm by 54.53% and 53.03%, respectively,
as compared to the control. The difference in the sensitivity of bacteria to CS may be attributed to
the complexity of interaction between the two kinds of chitosan and these Erwinia and Xanthomonas
strains. The above results show prominent antibacterial activities. Cationic properties of CS rather than
CNPs induce positively charged quaternary groups to hydroxyl or amino groups that are primarily
responsible for antibacterial activities [34]. This aspect clearly depicts that antibacterial activity is an
inherent property of CS rather than CNPs. Hence the evidence of broad-spectrum antifungal activity
of CNPs and chitosans and their effective antibacterial activity might open up a new perspective in the
analysis of CNPs vs. CS when addressing antifungal and antibacterial potentials.

4. Conclusions
In the present work, it was demonstrated that chitosan has significant antifungal activity against
the fungi tested, viz. C. gelosporidies, P. capsici, S. sclerotiorum, F. oxysporum, and G. fujikori, and this
was much higher than when chitosan nanoparticles were used independently. Thus, CNPs can be
effectively used against plant phytopathogenic fungi ensuring a plethora of positive outcomes ranging
from antifungal, antibacterial activities, plant growth promotion, biocidal activities, and reduction in
harmful effects to humans and environment due to chemical fertilizers. This research also addresses
the fact that CNPs are responsible for broad-spectrum activity against phytopathogens of tomato.
This opens up new research in the search for an effective combination of CNPs with other elemental
forms to ensure broad-spectrum antimicrobial activity affirmatively.

Author Contributions: M.C., J.-W.O. and S.C.C. planned and designed the research; M.C. performed the
experiments; M.C. and J.-W.O. wrote the manuscript together with assistance from S.C.C.
Funding: This research received no external funding.
Conflicts of Interest: Authors declare there is no conflict of interest.

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