Journal of Integrative Agriculture 2015, 14(4): 691697

Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Inhibitory effect of chitosan on growth of the fungal phytopathogen,

Sclerotinia sclerotiorum, and sclerotinia rot of carrot
WANG Qing, ZUO Jin-hua, WANG Qian, NA Yang, GAO Li-pu
Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Fruits and
Vegetable Storage and Processing/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China),
Ministry of Agriculture/Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 100097, P.R.China

Abstract
The antifungal activity of chitosan on a common fungal phytopathogen, Sclerotinia sclerotiorum, and the control effect on
sclerotinia rot of carrot were investigated. Mycelial growth and fungal biomass were strongly inhibited by chitosan. Using
propidium iodide stain combined with fluorescent microscopy, the plasma membrane of chitosan-treated S. sclerotiorum
mycelia was observed to be markedly damaged. Concomitantly, protein leakage and lipid peroxidation was also found
to be significantly higher in chitosan-treated mycelia compared to the control. Chitosan provided an effective control of
sclerotinia rot of carrot, with induction of activity of defense-related enzymes including polyphenoloxidase and peroxidase.
These data suggest that the effects of chitosan on sclerotinia rot of carrot may be associated with the direct damage to the
plasma membrane and lipid peroxidation of S. sclerotiorum, and the elicitation of defense response in carrot.
Keywords: antifungal activity, carrot, chitosan, plasma membrane, Sclerotinia sclerotiorum

1. Introduction
Sclerotinia sclerotiorum is a necrotrophic fungal pathogen
that can infect over 400 species of plants including a wide
range of vegetables, thus causing great losses in yield (Boland and Hall 1994). For example, sclerotinia rot, caused
by S. sclerotiorum, is an economically important disease
of carrot and can manifest as both a preharvest epidemic
occurring in the field as well as a postharvest epidemic
occurring during storage (Kora etal. 2003). Sclerotia, that

Received 31 March, 2014 Accepted 26 May, 2014

WANG Qing, E-mail: wangqing@nercv.org;
Correspondence GAO Li-pu, Tel/Fax: +86-10-51503051,
E-mail: gaolipu@nercv.org
2015, CAAS. All rights reserved. Published by Elsevier Ltd.
doi: 10.1016/S2095-3119(14)60800-5

are very resistant to abiotic stresses, function as a survival structure in this species and thus pose a significant
challenge for management measures (Zeng etal. 2012).
Currently, the use of synthetic chemical fungicides is the
main method of control for sclerotinia disease. However,
public concern over the potential impact of fungicides on
the environment and human health has created an interest
in exploring new alternatives for disease management
(Bautista-Baos etal. 2006).
As a natural polysaccharide, chitosan (poly -(14)
N-acetyl-D-glucosamine) represents a promising alternative
treatment for postharvest disease management due to its
antifungal activity and elicitation of defense response in
the plant host (Terry and Joyce 2004; Bautista-Baos etal.
2006). Previous reports have indicated that chitosan can
inhibit the growth of several postharvest fungal pathogens,
including Alternaria alternata (Reddy etal. 1997), Botrytis
cinerea (Chien and Chou 2006), Penicillium expansum (Liu
etal. 2007), Penicillium digitatum (Pacheco etal. 2008),

WANG Qing et al. Journal of Integrative Agriculture 2015, 14(4): 691697

2. Results
2.1. Effect of chitosan on mycelial growth and biomass accumulation of S. sclerotiorum
The antifungal activity of chitosan against S. sclerotiorum
is shown in Fig.1. Mycelial growth was markedly inhibited
by 1% chitosan at all time points assayed (Fig.1-A). After
5 d of incubation, colonies of S. sclerotiorum grew up to
7.87 cm in diameter on potato dextrose agar (PDA) while
the diameter of chitosan-treated colonies was only 0.92
cm, and the growth inhibition rate reached 88.31%. Mycelial biomass was also strongly inhibited by 1% chitosan
(Fig.1-B). The mycelial biomass of untreated cultures increased dramatically during the first 2 d of incubation in PDB.
In contrast, mycelial biomass remained at a low level and
never increased in potato dextrose broth (PDB) containing
1% chitosan. And the growth inhibition rate increased to
97.40% after 2 d of incubation.

2.2. Effect of chitosan on plasma membrane integrity

of S. sclerotiorum
As indicated in Fig.2, no evidence of plasma membrane

damage was observed in hyphae of S. sclerotiorum cultured

in PDB. In contrast, the integrity of the plasma membrane
declined over the period of incubation time when S. sclerotiorum was cultured in PDB containing chitosan. This was
evidenced by increasing intensity of staining of the hyphae
by propidium iodide as determined with fluorescence microscopy.

2.3. Effect of chitosan on protein leakage and lipid

peroxidation
Leakage loss of protein from mycelia into the culture medium
was substantial when S. sclerotiorum was cultured in PDB
containing 1% chitosan (pH 5.0). The protein concentration
in the medium rapidly increased during the first 24 h and then
continued to increase over the measurement period (Fig.3).
In contrast, the level of protein in PDB medium without chitosan remained low during the entire measurement period.
The pattern of lipid peroxidation when S. sclerotiorum was
cultured in PDB with and without 1% chitosan (pH 5.0) was

A 9.0

Colony diameter (cm)

Rhizopus stolonifer (Hernndez-Lauzardo etal. 2008),

Alternaria kikuchiana and Physalospora piricola (Pacheco
etal. 2008). The antimicrobial activity of chitosan has
been commonly considered to be closely associated with
its molecular weight, degree of deacetylation, pH, and the
sensitivity of the target microorganism (Xu etal. 2007).
Regarding the antimicrobial mechanism of chitosan, it has
been proposed that positively charged chitosan reacts with
negatively charged molecules on the cell surface of the
target organism altering cell permeability which results in
material being leaked from the cell and/or material being
inhibited from entering the cell (Kushwaha etal. 2010).
Cheah etal. (1997) reported that fungal mycelium of
S. sclerotiorum exposed to chitosan appeared to be deformed and dead through microscope studies. Molloy etal.
(2004) measured the susceptibility of carrots, which were
treated postharvest with a 0.2% (w/v) chitosan hydrolysate,
to the storage pathogen S. sclerotiorum and found that it reduced the frequency and size of rot compared to untreated
controls. However, limited information is available on the
inhibitory effect, or the possible mode of action of chitosan
against S. sclerotiorum. The objectives of this study were
to investigate the antifungal activity of chitosan against
S. sclerotiorum in vitro and in carrot, and its possible mode
of action by evaluating plasma membrane integrity, protein
leakage and lipid peroxidation in S. sclerotiorum and the
elicitation of defense enzymes, including polyphenoloxidase (PPO) and peroxidase (POD) in carrot by chitosan.

Control
Chitosan
6.0

3.0

0.0

B 3.0

Mycelial biomass (g)

692

1
2
3
4
Time after treatment (d)

Control
Chitosan

2.0

1.0

0.0

1
2
3
4
Time after treatment (d)

Fig. 1 Effect of chitosan on mycelial growth (A) and biomass

accumulation (B) of S. sclerotiorum. Data presented are the
means of pooled data from three experiments consisting of three
biological replicates. Error bars indicate standard deviations of
the means (n=9). The same as below.

693

Solube protein (mg g1 fresh weight)

WANG Qing et al. Journal of Integrative Agriculture 2015, 14(4): 691697

1.2

Control
Chitosan

1.0
0.8
0.6
0.4
0.2
0.0

1
2
3
Time after treatment (d)

Fig. 3 Effect of chitosan on protein leakage of S. sclerotiorum

over a 4-d period of incubation.

Fig. 2 Effect of chitosan on plasma membrane integrity of

S. sclerotiorum hyphae over a period of 60 min as determined
by staining with propidium iodide (PI).

similar to the results obtained from protein leakage (Fig.4).

Malondialdehyde (MDA) content remained at a relatively
low level in control mycelia compared to mycelia exposed
to the chitosan treatment.

2.4. Effect of chitosan on S. sclerotiorum rot and

defense-related enzyme activity
After 2 d of inoculation, S. sclerotiorum rot began to appear.
The disease incidence of carrot control reached 100% at
4 d, while that of chitosan-treated carrot kept under 20%
(Fig.5-A). Lesion diameter showed the similar change
pattern (Fig.5-B). Chitosan-treated carrot had significantly
(P<0.05) lower lesion diameter compared to non-treated
control at each day. In order to investigate whether chitosan had inductive effects on carrot host, defense-related
enzymes of PPO and POD were detected. Generally, POD
activity increased gradually from 0 to 4 d of storage, and
chitosan treatment significantly induced this enzyme activity (Fig.6-A). PPO activity in control carrot dramatically
increased from 1 to 4 d, while that in chitosan treatment
had significantly higher activity at each day except 4th d
(Fig.6-B).

3. Discussion
In the present study, chitosan was shown to be effective in
inhibiting the growth and biomass production of S. sclerotiorum (Fig.1). These results were similar to previous findings
on the antifungal activity of chitosan against several other

MDA (g g1 fresh weight)

4.0
Control
Chitosan

3.5
3.0
2.5
2.0
1.5
1.0

Time after treatment (d)

Fig. 4 Effect of chitosan on lipid perioxidation of S. sclerotiorum

over a 4-d period of incubation.

fungal pathogens, such as Aspergillus parasiticus (Cota-Arriola etal. 2011), Geotricum candidum (El-Mougy etal.
2012), A. alternata (Reddy etal. 2000), Fusarium solani
and Sclerotium rolfsii (Eweis etal. 2006). Several complex
mechanisms were proposed in these previous studies to
explain the antifungal activity of chitosan. The report by Liu
etal. (2007) discussed the effect of chitosan on both fungal
cell walls and cell membranes, and suggested that injury
to the plasma membrane may contribute to the fungicidal
effect of chitosan. In the present study, plasma membrane
integrity was assessed in order to better understand the
antifungal action of chitosan against S. sclerotiorum. The
results of PI staining indicated that the plasma membrane
integrity increasingly declined with the increase in time of
exposure to chitosan (Fig.2). This result was consistent
with a previous study on the effect of chitosan on plasma
membrane integrity of B. cinerea and P. expansum (Liu
etal. 2007). The plasma membrane damage may be due
to the interaction of the positive amino groups of chitosan
with the negative charged residues of macromolecules on
the fungal cell membrane, which changed the permeability

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WANG Qing et al. Journal of Integrative Agriculture 2015, 14(4): 691697

80
60

Lesion diameter (mm)

40
20
0

b
a
1

60

50
40
30

20

b
b

10
0

a
1

3
2
1

2
3
Time after treatment (d)

1
2
3
Time after treatment (d)

1
2
3
Time after treatment (d)

0.3
0.24
0.18
0.12
0.06
0

a
4

Fig. 5 Effect of chitosan on disease incidence (A) and lesion

diameter (B) of S. sclerotiorum rot of carrot.

of the plasma membrane (Laflamme etal. 1999).

It is well known that the integrity of the plasma membrane
is essential for maintaining fungal viability, and that membrane damage could lead to the leakage of intracellular components (Wei etal. 2008). Protein is one of the basic and
functional constituents (Lopez-Diez and Bone 2000). In this
regard, exposure of S. sclerotiorum cultures to 1% chitosan
(pH 5.0) in PDB resulted in a significant leakage of proteins
from mycelia into the surrounding medium comapred to
S. sclerotiorum cultured in PDB alone (Fig.3). Moreover,
exposure to chitosan also resulted in a substantial level of
lipid peroxidation in S. sclerotiorum mycelia compared to
mycelia growing in PDB alone (Fig.4). To the best of our
knowledge, this is the first report that chitosan can decrease
the integrity of the plasma membrane of S. sclerotiorum,
increase lipid peroxidation, and cause a substantial loss of
proteins thus inhibiting growth and biomass accumulation.
Our study also indicates that chitosan effective controlled
S. sclerotiorum rot in carrot (Fig.5), along with induction of
defense-related enzymes PPO and POD (Fig.6). Ojaghian
etal. (2013) reported that the activities of PPO and POD
increase in the inoculated carrots after application of different

Chitosan

4
B

Control

0
2
3
Time after treatment (d)

PPO activity (U g1 FW min1)

Disease incidence (%)

100

Chitosan

POD activity (U g1 FW min1)

Control

Fig. 6 Effect of chitosan on enzyme activity of POD (A) and

PPO (B) of carrot.

chitosans. Similar inductive effect of chisan was obtained

on other horticultural products, such as tomato (Liu etal.
2007), cherry (Chailoo and Asghari 2011) and pear (Meng
etal. 2010).

4. Conclusion
The present study shows that chitosan directly inhibited the
growth of S. sclerotiorum, and potentially induced defense
reaction in carrot. This suggests that chitosan is a promising
natural fungicide to manage postharvest diseases of vegetable. However, further studies with whole plants growing in
soil will be needed to determine the effectiveness of chitosan
application in controlling S. sclerotiorum and determining the
optimum timing and method of its application.

5. Materials and methods

5.1. Chitosan
Chitosan (Qingdao Honghai Bio-Tech Co., Ltd., Shandong,
China), with approximately a 90% level of deacetylation and
an average molecular weight of 350 kDa, was prepared at

WANG Qing et al. Journal of Integrative Agriculture 2015, 14(4): 691697

a starting concentration of 2.5% (w/v) in 1% HCl by stirring

at room temperature.

5.2. Carrot
Carrots were harvested at commercial maturity, and
uniform sized carrots without wounds or rot were selected
for use. The carrots were disinfected with 2% (v/v) sodium
hypochlorite for 2 min, rinsed with tap water and dried in
air prior to treatment.

5.3. Assay of mycelial growth of S. sclerotiorum

S. sclerotiorum was isolated from infected carrot and
maintained on potato dextrose agar (PDA) (Beijing
Borunlaite Sciense & Technology Co., Ltd., China). The
effect of chitosan on mycelial growth was assayed according
to Yao and Tian (2005). Mycelial disks (5 mm in diameter)
from 10-d-old cultures, grown at 25C, were placed in the
centre of Petri dishes (90 mm in diameter) containing 20 mL
of PDA supplemented with 1% chitosan. The concentration
of chitosan was selected based on preliminary experiments
conducted at pH 5.0. PDA without chitosan served as a
control. Mycelial growth was determined by measuring
colony diameter at daily intervals for 5 d.

5.4. Assay of mycelial biomass of S. sclerotiorum

Three mycelial disks (5 mm in diameter) from 10-d-old
cultures were inoculated into conical flasks (250 mL)
containing 100 mL of potato dextrose broth (PDB) (Oxoid)
with or without 1% chitosan (pH 5.0) at 180 r min1 at 25C.
Fungal biomass was collected, washed thoroughly three
times with sterile double-distilled water, and then placed
on filter paper for 2 h. The fresh weight was assayed each
day for 5 d.

5.5. Assay of plasma membrane integrity

Mycelia from 2-d-old cultures of S. sclerotiorum grown in
PDB were collected, washed and dehydrated on filter paper.
The obtained mycelia were then transferred into conical
flasks (250 mL) containing 50 mL of sterile water (control)
or 1% chitosan (pH 5.0), and incubated at 180 r min1 at
25C for 20, 40 and 60 min. The mycelia were collected on
filter paper, and rinsed with 50 mmol L1 sodium phosphate
buffer (pH 7.0) twice to remove residual medium. Fresh
mycelia were stained with 10 g mL1 propidium iodide
(PI) for 5 min at 30C (Liu etal. 2007). Mycelia were
then collected and washed twice with the buffer to remove
residual dye. The mycelia were observed with a Zeiss
Axioskop 40 microscope (Carl Zeiss, Germany) equipped

695

with an individual fluorescein rhodamine filter set (Zeiss No.

15: excitation BP 546/12 nm, emission LP 590 nm). Three
fields of view from each cover slip were chosen randomly,
and the experiment was repeated twice.

5.6. Determination of protein leakage and lipid peroxidation

Mycelia from 2-d-old cultures of S. sclerotiorum growing on
PDA were collected, washed and dehydrated on filter paper
as above. 3 g of mycelia were then transferred into conical
flasks (250 mL) containing 50 mL of sterile double-distilled
water (control) or 1% chitosan (pH 5.0), and incubated at
180 r min1 at 25C .
The mycelia were removed by filtration through 0.2-m
pore size membrane after 1, 2, 3 and 4 h of incubation,
respectively. The filtrate was collected for determining
protein leakage (Liu etal. 2010), and the mycelia were
used in the malondialdehyde (MDA) assay for lipid
peroxidation. Soluble protein content in the filtrate was
determined according to Bradford (1976) with bovine serum
albumin (Sigma-Aldrich, Germany) as a standard. Protein
leakage was expressed as mg g1 fresh weight of mycelia.
For assaying lipid peroxidation, a method based on the
reaction of thiobarbituric acid with MDA derived from lipid
peroxidation was employed. Detection of thiobarbituric acid
species was carried out by a colorimetric assay (Ritter etal.
2008). The lipid peroxidation was expressed as g g1 fresh
weight of mycelia.

5.7. Effect of chitosan on S. sclerotiorum rot of carrot

Carrots were wounded (5 mm deep and 3 mm wide) with a
sterile nail at the equator. Then 50 L of 1% chitosan and
sterile distilled water as the control were placed into each
wound. Carrots were air-dried for 2 h, a mycelial disk was
added to each wound. Treated carrots were put in 200 mm
130 mm50 mm plastic boxes with sterile water to maintain
a high relative humidity (about 95%) and stored at 25C.
Disease incidence and lesion diameter of carrot caused by
S. sclerotiorum was determined each day after inoculation.
Each treatment contained three replicates with 10 carrots
per replicate and the experiment was repeated three times.

5.8. Determination of enzyme activity

For enzyme assays, carrots were wounded, and 50 L of
1% chitosan was added to each wound as described above,
carrots wounded with water addition served as controls.
Flesh samples surrounding the wounds of 10 carrots were
kept at 0, 1, 2, 3 and 4 d, respectively, at 25C. Each
treatment contained three replicates and the experiment

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WANG Qing et al. Journal of Integrative Agriculture 2015, 14(4): 691697

was repeated twice.

PPO and POD were extracted by the method of Chen
etal. (2000), with some modifications. Tissue samples (5 g)
of each treatment were homogenized with 10 mL of
100 mmol L1 dium phosphate buffer (pH 6.4) containing 0.2 g
of polyvinyl polypyrrolidone (PVPP) and ground at 4C.
The homogenate as centrifuged at 15 000g for 30 min at
4C and the supernatant was used for the enzyme assay.
PPO activity was determined by adding 0.1 mL of enzyme
preparation to 3.0 mL of catechol substrate (500 mmol L1,
in 100 mmol L1 dium phosphate buffer, pH 6.4) and the
increase in absorbance 398 nm was measured immediately.
POD activity was determined using guaiacol as substrate
(Ippolito etal. 2000). The reaction mixture consisted of
0.1 mL crude extract, 2 mL of guaiacol (8 mmol L1, in 100
mmol L1 sodium phosphate buffer, pH 6.4), incubated for
30 min at 30C. The increase in absorbance at 460 nm
was measured after 1 mL H2O2 (24 mmol L1) was added.
The activities of PPO and POD were expressed as U mg1
protein, where one unit was expressed as the increase
rate of absorbency per mass of protein per min. Protein
content was determined according to Bradford (1976) with
bovine serum albumin (Sigma-Aldrich, Shanghai, China)
as standard.

5.9. Data analysis

All statistical analyses were performed with SPSS ver.
13.0 (SPSS Inc., Chicago, IL, USA). Data of chitosan
treatment and non-treatment control were compared in
Students t-test. Differences at P<0.05 were considered to
be significant. Each treatment consisted of three replicates
and the experiment was repeated three times. There
were no significant interactions between treatments and
experiments, and data presented in this paper were pooled
across three independent repeated experiments.

Acknowledgements
This research was supported by grants from the National
Natural Science Foundation of China (31101364), the
Ministry of Agriculture of China (CARS-25-E-01 and
201203095) and the Beijing Academy of Agriculture and
Forestry Sciences, China (CXJJ201304).

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