International Journal of Biological Macromolecules 62 (2013) 677–683

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Synthesis of chitosan based nanoparticles and their in vitro evaluation

against phytopathogenic fungi
Vinod Saharan a,∗ , Akanksha Mehrotra a , Rajesh Khatik a ,
Pokhar Rawal b , S.S. Sharma b , Ajay Pal c
a
Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology,
Udaipur, Rajasthan, India
b
Department of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India
c
Department of Biochemistry, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar, Haryana, India

a r t i c l e i n f o a b s t r a c t

Article history: The main aim of present study was to prepare chitosan, chitosan-saponin and Cu-chitosan nanopar-
Received 8 July 2013 ticles to evaluate their in vitro antifungal activities. Various nanoparticles were prepared using ionic
Received in revised form gelation method by interaction of chitosan, sodium tripolyphosphate, saponin and Cu ions. Their parti-
24 September 2013
cle size, polydispersity index, zeta potential and structures were confirmed by DLS, FTIR, TEM and SEM.
Accepted 11 October 2013
The antifungal properties of nanoparticles against phytopathogenic fungi namely Alternaria alternata,
Available online 16 October 2013
Macrophomina phaseolina and Rhizoctonia solani were investigated at various concentrations ranging
from 0.001 to 0.1%. Among the various formulations of nanoparticles, Cu-chitosan nanoparticles were
Keywords:
Antifungal activity
found most effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alter-
Chitosan nata, M. phaseolina and R. solani, respectively in in vitro model. At the same concentration, Cu-chitosan
Plant fungi nanoparticles also showed maximum of 87.4% inhibition rate of spore germination of A. alternata. Chi-
Saponin tosan nanoparticles showed the maximum growth inhibitory effects (87.6%) on in vitro mycelial growth
Nanoparticle of M. phaseolina at 0.1% concentration. From our study it is evident that chitosan based nanoparticles par-
ticularly chitosan and Cu-chitosan nanoparticles have tremendous potential for further field screening
towards crop protection.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction questions of their application in crops [12–19,5]. These issues have

The advancement in nanotechnology has markedly extended
the application of nanomaterials in plants. From past 5 to 8 years,
nanomaterials have also been utilized in agriculture fields mostly
in crop protection [1,2]. Plant pathogenic fungi cause plant and
seed diseases to the most economically important crops and result
in quantitative and qualitative losses in agriculture [3]. Due to
excessive use of chemical fungicides, the environmental hazards to
human, flora and fauna have raised major concerns over the years.
Moreover, the uncontrolled use of chemical agents can cause devel-
opment of resistance in phytopathogenic fungi against fungicides.
More surface area, activation of novel reactive groups and unique
physico-chemical properties enable nanoparticles more effective
against microbes at very low dose [4,5]. Among the nanoparti-
cles, use of metal-based nanoparticles were initiated widely in
crop protection [6–13]. But the possible environmental toxicity due
to unpredicted nature of metal nanoparticles have raised serious
∗ Corresponding author. Tel.: +91 9461180586; fax: +91 294 2420447.
E-mail address: vinodsaharan@gmail.com (V. Saharan).
ignited considerable interest among scientists and researchers in
search for bio-based nanomaterials for crop protection [19,20].
Chitosan based nanoparticles are preferably used worldwide for
various applications owing to their biodegradability, high perme-
ability, non-toxicity to human and cost effectiveness [20]. It has
been shown that chitosan has broad antifungal activities [21–24].
However, the insolubility of bulk chitosan in aqueous media limits
its wide spectrum application as an antifungal agent. Moreover, it
has relatively low antifungal activity in bulk form [20]. Therefore,
various strategies have been employed to enhance its antifun-
gal activity [23,25,26]. The chelating property of chitosan towards
various organic and inorganic compounds makes it a suitable
biopolymer for improvement in stability, solubility and biocidal
activity [20]. Saponins, the complex glycosidic compounds, are
known for their fungistatic activities [27] and their self assem-
ble property in aqueous media have been successfully exploited in
chitosan-saponin nanoformulation against cancer cells [28]. Cop-
per (Cu) compounds are well known for their antifungal nature and
have been used with chitosan for antibacterial [29] and antifungal
activities [30]. Mainly, the biocidal studies of chitosan nanopar-
ticles have been carried out on bacteria and fungi and a meagre

0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2013.10.012
678 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
study has been reported on pathogenic plant fungi [29,31,32]. In
present investigation, chitosan based nanoparticles viz. chitosan,
chitosan-saponin and Cu-chitosan nanoparticles were prepared
by blending of saponins and Cu which were further examined
against phytopathogenic fungi viz. Alternaria alternata, Macrophom-
ina phaseolina and Rhizoctonia solani.
2. Materials and methods
2.1. Materials
Chitosan (80% N-deacetylation) and saponin were obtained from
HiMedia, Mumbai, India. Sodium tripolyphosphate was supplied by
SRL, Mumbai, India. PVDF (Poly vinylidene difluoride) syringe filters
(pore size 0.22 ␮m) were purchased from HiMedia, Mumbai, India.
Three fungal species, Alternaria alternate, Macrophomina phaseolina
and Rhiszoctonia solani were obtained from Department of Plant
Pathology, Rajasthan College of Agriculture, Maharan Pratap Uni-
versity of Agriculture, Udaipur.
2.2. Preparation of nanoparticles
2.2.1. Preparation of chitosan nanoparticles
Chitosan nanoparticles were prepared based on the ionic gela-
tion of chitosan with TPP anions [33]. For scale-up synthesis of
nanoparticles, the given method was modified. Chitosan was dis-
solved at 0.1% level (w/v) in 1% (v/v) acetic acid followed by
overnight stirring on magnetic stirrer (Remi Laboratory Instru-
ments, Mumbai, India) at 200 rpm and filtered through a PVDF
syringe filter (pore size 0.22 ␮m). TPP was dissolved at 1% level
(w/v) in ultrapure water and filtered through PVDF membrane
syringe filter (pore size 0.22 ␮m). The cross-linking of chitosan with
TPP at equal volume was performed by Pediatric set (Romsons,
Agra, India) under magnetic stirrer at 700 rpm. The resulting for-
mulation was subjected to centrifugation for 10 min at 10,000 rpm
and the pellet was re-suspended in ultrapure water followed by
ultra-sonication (with 3.0 mm probe, Sonicator, Lark Innovative,
Chennai, India) at 80% pulser ration for 100 s at 4 ◦ C. Centrifuga-
tion followed by ultra-sonication was repeated three times and the
precipitated nanoformulation was lyophilized and stored at 4 ◦ C for
further analysis.
2.2.2. Preparation of chitosan-saponin nanoparticles
Chitosan-saponin nanoparticles were prepared by ionic cross-
linking using TPP, as described earlier with certain modifications
[34]. Saponin was dissolved at 0.5% level (w/v) in ethanol and stirred
mix with chitosan solution [0.1% (w/v) in 1% (v/v) acetic acid] in
a volumetric ratio of 10:1. TPP [1% (w/v) in aqueous] was mixed
with chitosan-saponin solution using Pediatric set (Romsons, Agra,
India) under magnetic stirrer at 700 rpm. Further purification of
nanoparticles was done as described in chitosan nanoparticles
preparation.
2.2.3. Preparation of Cu-chitosan nanoparticles
Cu-chitosan nanoparticles were prepared as described earlier
by Jaiswal [35]. The procedure as used for preparation of chi-
tosan nanoparticles was used with extra addition of 0.01% CuSO4
(HiMedia, Mumbai, India) solution into the formulation before the
finishing of cross-linking reaction. The amount of entrapped Cu ions
into the chitosan nanoparticles was determined by double beam
Atomic Absorption Spectrophotometry (AAS4141 model, Electron-
ics Corporation of India Ltd, India). After centrifugation steps, as
discussed previously, the left-over supernatant was subjected to
AAS analysis to quantify free copper [36].
2.3. Characterization of nanoparticles
2.3.1. Dynamic light scattering (DLS) measurements
DLS was used for the measurement of average particle size,
polydispersity index (PDI) and zeta potential of nanoparticles on
high performance particle Zetasizer HPPS-5001 (Malvern, UK). Each
sample was analyzed in triplicate at 25 ◦ C at a scattering angle of
90◦ [37]. 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.
2.3.2. Fourier transform infrared (FTIR) analysis
To confirm the synthesis of various nanoparticles, FTIR analysis
was done. For FTIR each sample was prepared in potassium bromide
(KBr) as a pellet under 1:99 ratio of sample and KBr, and it was
recorded by ABB FTLA 2000-100 (Quebec, Canada) at a resolution
limit of 16 cm–1 [29].
2.3.3. Transmission electron microscopy (TEM) observation
In TEM, the various nanoparticles were diluted in ultrapure
water and mixed with 2% uranyl acetate solution; a drop of the
mixture was deposited onto a standard copper grid covered by a
holey carbon film, and dried at ambient temperature before obser-
vation. After evaporation of the liquid, the samples were fixed on
the carbon film of the grid, and visualized under transmission elec-
tron microscopy model H-7650 (Hitachi, Japan) with 40–120 kV
accelerating voltage [38].
2.3.4. Scanning electron microscopy (SEM) observation
Scanning electron microscope was used to study the surface
morphology of nanoparticles. 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 scanning electron
microscope model S 2700 (Hitachi, Japan) with 15 kV accelerating
voltage [28].
2.4. Antifungal activities
Poison food technique was used to measure the antifungal activ-
ity [39]. Different concentrations (0.001, 0.005, 0.01, 0.02, 0.06 and
0.1%, w/v) of various nanoparticles in aqueous solution were used
in antifungal activity test against three fungus species viz. A. alter-
nate, M. phaseolina and R. solani. Potato dextrose agar medium
(HiMedia, Mumbai, India) was prepared and poured in Petri dishes
(90 mm × 15 mm, HiMedia, Mumbai, India) with above mentioned
percentages of various nanoparticles, separately. Mycelial bit from
peripheral end of uniform size (diameter, 5.0 mm) was taken from
7 days old culture of test pathogens and placed in the centre of test
Petri dishes. All the Petri dishes were incubated at 28 ± 1 ◦ C for 7
days and the observation of radial mycelial growth was recorded
when control Petri dish cover full growth (90 mm). All the treat-
ments consisted of three replications and experiment was repeated
twice. The inoculated plates were compared with control (without
nanoparticles) to calculate the % inhibition rate of mycelia of the
pathogen by using the formula given by Vincent [40]
Mc − Mt
%Inhibition rate = × 100
Mc
where Mc is the mycelial growth in control, Mt is the mycelial
growth in treatment.
 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
Fig. 1. The size distribution by intensity in aqueous solution. (a) Chitosan NPs, (b) Chitosan-saponin NPs, and (c) Cu-chitosan NPs.
2.5. Spore germination method
The antifungal activities of nanoparticles (0.001, 0.005, 0.01,
0.02, 0.06 and 0.1%, w/v) on spore germination of A. alternata
were tested. Spore suspension (1.0 × 103 spores/ml) of A. alternata
was prepared aseptically from 7 days old pure culture; 50 ␮l of
spore suspension and 50 ␮l of nanoparticle at above mentioned
concentrations in aqueous were taken on glass slides (HiMedia,
Mumbai, India) in 10 replicates. All the treatments were maintained
at 28 ± 1 ◦ C for 8 h and the observations were made under micro-
scope to calculate the % inhibition rate by counting the number of
spore germinated compared to control.
Gc − Gt
%Inhibition rate = × 100
Gc
where Gc is the germination in control and Gt is the germination in
treatment.
2.6. Statistical analysis
Statistical analysis for the data was performed with JMP soft-
ware version 8 [41] using Turkey–Kramer HSD test for determining
significant differences among treatment at p = 0.05 level. Each
679
experiment was repeated twice and each treatment consists of
three replicates.
3. Results and discussion
3.1. Particle size, polydispersity index and zeta potential
The size distribution profile, shown in Fig. 1a–c, represents
mean diameter of chitosan, 192.2 ± 2.5 nm (ranges from 150.1 to
390.2 nm); chitosan-saponin, 373.9 ± 4.1 nm (ranges from 200.8
to 990.6 nm) and Cu-chitosan nanoparticles, 196.4 ± 2.2 nm (range
from 180.0 to 487.9 nm). Subsequently, chitosan, chitosan-saponin
and Cu-chitosan nanoparticles showed 0.6, 1.0 and 0.5 PDI
values, respectively. The PDI data indicate that chitosan and
Cu-chitosan nanoparticles showed narrow size distribution com-
pared to chitoan-saponin nanoparticles. Zeta potential of chitosan
(+45.33 mV) and Cu-chitosan (+88 mV) was higher than that of
chitosan-saponin nanoparticles (+31 mV). Zeta potential is an
important parameter for stability of nanoparticles in aqueous
media, therefore chitosan and Cu-chitosan nanoparticles with
higher zeta potential showed stability in aqueous solution in
present study [29,32].
680 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
Fig. 2. FTIR spectra: (a) chitosan, (b) chitosan NPs, (c) chitosan-saponin NPs, and (d) Cu-chitosan NPs.
3.2. FTIR study
FTIR analysis was performed to confirm the interaction of
chitosan, TPP, saponin and Cu. Bare chitosan was characterized
with some specific peaks located at 1643, 903 and 3456 cm–1
which relate to amide ( CONH2 ), anhydro glucosidic ring and pri-
mary amine ( NH2 ), respectively (Fig. 2a). In the case of chitosan
nanoparticles, the observed peaks 1643 and 3456 cm–1 get shifted
from higher wave number region to lower wave number region as
1636 and 3410 cm−1 (Fig. 2b). The reduction in stretching frequency
could be attributed to the TPP interaction with ammonium group
of chitosan and more hydrogen bonding in chitosan–TPP complex
[33]. The saponin interaction with chitosan was confirmed by the
presence of a new peak at 1560 cm–1 which corresponds to an
amide linkage between saponin and chitosan nanoparticles [28].
There was a sharp peak at 3430 cm–1 region which assures that
interaction of saponin with chitosan is more of hydrogen bonding
than ionic interactions (Fig. 2c). The peaks at 1636 cm–1 ( CONH2 )
and 1550 cm–1 ( NH2 ) in the spectrum of Cu-chitosan nanoparti-
cles were sharper and shifted to 1631 and 1536 cm–1 . Therefore, Cu
bonding with chitosan induces redistribution of vibration frequen-
cies (Fig. 2d).
3.3. Structure analysis by TEM and SEM
TEM image of single (Fig. 3a, in inset) and aggregate chitosan
nanoparticles (Fig. 3a) shows spherical shaped nano struc-
tures, further SEM micrograph confirmed the nano-organization
of chitosan formulation (Fig. 3b). Shape of chitosan-saponin
nanoparticles were smooth-rounded with variable sizes as exam-
ined using a TEM (Fig. 3c), where as in SEM micrograph,
chitosan-saponin nanoparticles were quite similar to chitosan
nanoparticles (Fig. 3d). TEM and SEM micrograph shows compact
polyhedron shaped of Cu-chitosan nanoparticles (Fig. 3e and f).
4. Antifungal activity
In vitro mycelial growth of A. alternata was comprehensively
controlled by 0.06 and 0.1% concentrations of all the nanoparticles.
A maximum 89.5% inhibition rate was recorded at 0.1% concen-
tration of Cu-chitosan nanoparticles followed by 82.2% at 0.1%
chitosan nanoparticles (Fig. 4). In the case of chitosan-saponin
nanoparticles, 80.9% of mycelial growth was inhibited at 0.1% con-
centration (Table 1).
Chitosan nanoparticles displayed strong inhibition of mycelial
growth of M. phaseolina. A maximum 87.6% inhibition rate was
recorded at 0.1% of chitosan nanoparticles. However, 0.01–0.1%
concentrations showed similar growth inhibition rate as per
the statistical analysis (Table 1). Chitosan-saponin nanoparticles
showed a dose dependent effect on mycelial growth. The high-
est inhibition rate (66.2%) was recorded at 0.1% concentration
while the other concentrations (0.001–0.06%) gave inhibition rate
from 30.8 to 45.2% (Table 1). Cu-chitosan nanoparticles at 0.001 to
0.02% concentrations inhibited growth from 35.3 to 54.0% whereas
0.06 and 0.1% formulation inhibited 62.5–63.0% of mycelia growth
(Table 1).
The radial growth of R. solani was reduced by all concentrations
of chitosan nanoparticles in a dose dependent manner. The highest
growth inhibition (34.4%) was observed at 0.1% chitosan nanopar-
ticles followed by 32.2% at 0.06% concentration. Chitosan-saponin
nanoparticles also showed a dose dependent effect. The highest
concentration (0.1%) of chitosan-saponin nanoparticles inhibited
 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
Fig. 3. TEM and SEM micrographs: (a) aggregated chitosan NPs; single NP in inset in TEM and, (b) SEM micrograph of chitosan NPs, (c) chitosan-saponin NPs in TEM, and (d)
SEM, (e) Cu-chitosan NPs in TEM, and (f) SEM.
fungal growth by 27.7%, and the same inhibition rate was recorded
at 0.06% concentration (Table 1). Among the various concentra-
tions of Cu-chitosan nanoparticles, highest inhibition rate (60.1%)
was recorded at a dose of 0.1%. The concentrations of Cu-chitosan
nanoparticles ranging from 0.01 to 0.06% showed fairly stable
growth inhibition from 38.1 to 43.1%.
A maximum 87.4% spore germination was inhibited by 0.1%
concentration of Cu-chitosan nanoparticles followed by chitosan
nanoparticles (87.1%) at 0.1%. Taken as a whole, all the examined
nanoparticles were found effective in controlling spore germina-
tion of A. alternata (Table 2). Bulk chitosan, bulk saponin and CuSO4
at 0.1% level were found less effective for inhibition of mycelial
growth and spore germination as compared to our synthesized
nanoparticles (Tables 1 and 2).
Stable, non-toxic and organic solvent free synthesis of chitosan
nanoparticles have been achieved using ionic gelation technique by
various independent group of researchers [42,43]. This technique
681
is based on the ionic interactions between the positively charged
primary amino groups of chitosan and the negatively charged TPP
and avoids the use of toxic chemical and emulsifying agents which
are often toxic to non-targeted organisms [43]. Until now, the syn-
thesis of chitosan nanoparticles have been well documented for
particle size and stability by modifying chitosan concentration, chi-
tosan to TPP weight ratio, rate of mixing and pH of the reaction
mixture [37]. In this study, the existing protocols for synthesis of
chitosan nanoparticles were modified to successfully achieve sta-
ble and speedy fabrication of nanoparticles. We have standardized
the synthesis process by precise control of cross linking reaction
using Pediatric set (100 ml volume) under room temperature that
produced 98 ± 1 mg of nanomaterials in a single reaction in 3 h
which can further be scaled up by using higher capacity of Pedi-
atric set. The chitosan nanoparticles were further modified into
chitosan-saponin and Cu-chitosan nanoparticles and their effect on
fungal growth was evaluated. None of the screened fungi showed
682 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683

Table 1
Effect of various chitosan NPs on in vitro mycelial growth of phytopathogenic fungi.

Treatments (%) % Inhibition ratea

A. alternata M. phaseolina R. solani

Chitosan NPs
0.001 11.5 ± 0.8 e 62.5 ± 3.7 c 13.1 ± 1.5 b
0.005 49.0 ± 1.0 d 76.8 ± 1.9 b 17.2 ± 2.2 b
0.01 52.7 ± 0.5 c 84.0 ± 2.8 a 17.4 ± 1.5 b
0.02 65.3 ± 0.7 b 87.3 ± 0.6 a 18.1 ± 0.7 b
0.06 80.1 ± 0.9 a 87.5 ± 0.3 a 32.2 ± 1.6 a
0.1 82.2 ± 0.5 a 87.6 ± 0.1 a 34.4 ± 1.1 a
Controlb 0.00 ± 0.0 f 0.00 ± 0.0 d 0.0 ± 0.0 c
Chitosan-saponin NPs
0.001 15.7 ± 1.0 d 30.8 ± 0.4 d 12.2 ± 0.7 c
0.005 59.8 ± 0.8 c 32.3 ± 0.5 d 16.0 ± 3.1 bc
0.01 62.8 ± 0.6 b 38.8 ± 0.9 c 16.4 ± 1.7 bc
0.02 70.2 ± 2.1 b 39.7 ± 0.6 c 22.8 ± 0.7 ab
0.06 78.3 ± 0.5 a 45.2 ± 0.7 b 27.7 ± 0.6 a
0.1 80.9 ± 0.7 a 66.2 ± 0.4 a 27.7 ± 0.5 a
Controlb 0.00 ± 0.0 e 0.00 ± 0.0 e 0.00 ± 0.0 d
Cu-chitosan NPs
0.001 9.9 ± 1.2 e 35.3 ± 0.4 d 22.3 ± 0.5 c Fig. 4. A representative photograph of antifungal bioassay. Effect of Cu-chitosan
0.005 40.3 ± 0.1 d 45.2 ± 2.6 c 28.5 ± 2.4 c nanoparticles on mycelial growth of A. alternata.
0.01 53.0 ± 0.9 c 45.8 ± 2.4 c 38.1 ± 0.3 b
0.02 69.3 ± 0.5 b 54.0 ± 2.5 b 38.7 ± 2.4 b
0.06 82.1 ± 1.5 a 62.5 ± 0.9 a 43.1 ± 1.6 b
0.1 89.5 ± 0.7 a 63.0 ± 0.3 a 60.1 ± 1.5 a
significant improvement in growth inhibition rate by chitosan-
Controlb 0.00 ± 0.0 f 0.00 ± 0.0 e 0.00 ± 0.0 d
Chitosan (0.1%)c 21.3 ± 0.3 18.0 ± 0.2 16.7 ± 0.7 saponin treatment indicating that chitosan-saponin nanoparticles
Saponin (0.1%)d 31.1 ± 0.5 25.6 ± 0.6 13.2 ± 0.3 do not deserve a good antifungal agent in present study. Moreover,
CuSo4 (0.1%)d 20.8 ± 0.0 16.7 ± 0.0 18.4 ± 0.0 higher PDI value and lower zeta potential make chitosan-saponin
a
Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by nanoparticles less stable and less effective against fungi used in
same letter in column of each treatment is not significant difference at p = 0.05 by present study. Albeit, saponin-chitosan nanoparticle showed excel-
Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germina- lent growth inhibition on cancer cell in earlier report [28]. Copper
tion of the control (0%).
b
compounds are known for their antifungal nature and are being
Control without any formulation.
c
Dissolved in 0.1% acetic acid. effectively used to control fungal diseases. However, due to uncon-
d
Dissolved in water, NPs-nanoparticles. trolled application, their concentrations have exceeded the limits
specified for heavy metals [44]. Therefore, in present study Cu-
Table 2
chitosan nanoparticles were prepared to improve the antifungal
Effect of various chitosan NPs on in vitro spore germination of A. alternata. activity and overcome the existing problem of heavy dose. The
protocol for synthesis of Cu-chitosan nanoparticles was standard-
Treatments (%) % Inhibition ratea
ized in the same way as chitosan nanoparticles and the developed
Chitosan NPs method delivered highly stable nanoparticles with low PDI and
0.001 14.6 ± 1.0 e
high zeta potential as compared to previous reports [29,32]. AAS
0.005 51.0 ± 0.8 d
0.01 57.3 ± 0.7 c result shown that 70% of copper embedded in chitosan formulation
0.02 68.5 ± 0.4 b in Cu-chitosan nanoparticles synthesis. Among all the treatments,
0.06 84.4 ± 0.6 a Cu-chitosan nanoparticles were found very effective at 0.1% con-
0.1 87.1 ± 0.1 a centration and showed 89.5, 63.0 and 60.1% growth inhibition of
Controlb 0.00 ± 0.0 f
A. alternata, M. phaseolina and R. solani, respectively in vitro. Fur-
Chitosan-saponin NPs
0.001 13.2 ± 1.2 c ther, the Cu-chitosan nanoparticles at 0.1% concentration showed
0.005 62.8 ± 0.6 b the strongest growth inhibitory effects (87.4%) on spore germi-
0.01 63.6 ± 2.6 b nation of A. alternata. The observed higher antifungal activities of
0.02 73.1 ± 5.1 ab
Cu-chitosan nanoparticles could be explained by the higher surface
0.06 78.3 ± 0.1 a
0.1 82.9 ± 0.8 a
charge density (higher zeta potential) which provides them greater
Controlb 0.00 ± 0.0 d binding affinity for negatively charged fungal membrane. Further,
Cu-chitosan NPs Cu(II) undergoes reduction to Cu(I) in fungi which produces toxic
0.001 10.6 ± 1.8 e H2 O2 which in turn destructs the cell viability [32]. In general,
0.005 43.2 ± 0.0 d
0.06–1% concentrations of nanoparticles were found effective to
0.01 52.0 ± 0.6 c
0.02 69.3 ± 0.2 b control fungal growth in present study. In a prior report, higher
0.06 83.3 ± 1.3 a doses ranging from 750 to 6000 mg/l of bulk chitosan were found
0.1 87.4 ± 0.2 a effective against plant fungi [26], and these concentrations were
Controlb 0.00 ± 0.0 f
higher compared to nanoparticles concentrations used in present
Chitosan (0.1%)c 21.1 ± 0.4
Saponin (0.1%)d 31.4 ± 0.1
study. The use of bulk chitosan has been restricted due to its poor
CuSO4 (0.1%)d 23.1 ± 1.3 solubility in water and poor antifungal activity [20]. Chemically
modified chitosans viz. triethylene diamine dithiocarbamate chi-
a
Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by
same letter in column of each treatment is not significant difference at p = 0.05 by tosan and o-hydroxyphenylaldehyde thiosemicarbazone chitosan
Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germina- have previously been evaluated and found to show maximum 60.4
tion of the control (0%). and 52.6% growth inhibition of F. oxysporum and R. solani at 500 and
b
Control without any formulation.
c
50 mg/l concentration [26,45]. Therefore, the results of our study
Dissolved in 0.1% acetic acid.
d
Dissolved in water, NPs-nanoparticles.
clearly indicate the pronounced effect of chitosan nanoparticles
 V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
over bulk and chitosan derivatives on growth inhibition of impor-
tant plant pathogenic fungi.
5. Conclusions
Chitosan, chitosan-saponin and Cu-chitosan nanoparticles were
prepared and their structures were well characterized by DLS, FTIR,
TEM and SEM. The lower PDI value and higher zeta-potential of chi-
tosan and Cu-chitosan nanoparticles proved their uniform size and
stability which may contribute to their higher antifungal activity
against A. alternata, M. phaseolina and R. solani in in vitro studies.
Cu-chitosan nanoparticles also showed maximum inhibition rate
of spore germination of A. alternata. Compared to chitosan and Cu-
chitosan nanoparticles, the chitosan-saponin nanoparticles were
found poor in antifungal activity. The higher PDI value and lower
zeta-potential decreased their uniform size and stability in aque-
ous solution. The efficacy of chitosan based nanoparticles mainly
chitosan and Cu-chitosan could further be tested on other plant
pathogenic fungi in in vitro and in vivo models. The present study
is a effort to explore potential of chitosan based nanoparticles and
the findings of present study could be utilized for development of
nano fungicide for crop protection in future.
Acknowledgements
The authors wish to thank Directorate of Research, MPUAT for
their support of this work. The kind co-operation extended by Dr.
Ramesh Raliya, Research Scientist, CAZRI, Jodhpur and Dr. N. Ilai-
yaraja, Scientist, Defence Food Research Laboratory, Mysore, India
for their technical help in FTIR and DLS studies, respectively is
highly acknowledged.
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