Agron. Sustain. Dev.

DOI 10.1007/s13593-014-0252-3

REVIEW ARTICLE

Chitosan antimicrobial and eliciting properties for pest control

in agriculture: a review
Ke Xing & Xiao Zhu & Xue Peng & Sheng Qin

Accepted: 9 September 2014

# INRA and Springer-Verlag France 2014

Abstract In agriculture, current control of pathogens Contents

relies mainly on chemical fertilizers and pesticides. 1. Introduction
However, alternative solutions are needed due to con- 2. Antimicrobial activities of chitosan
cerns for public health, environmental protection, and 2.1 Against fungi
development of resistant pests. Chitosan is a nontoxic, 2.2 Against bacteria
biodegradable biopolymer showing antimicrobial and 2.3 Against viruses
plant-immunity eliciting properties. Here, we review 3. Antimicrobial mechanisms of chitosan
chitosan antimicrobial activities, modes of action, and 3.1 Electrostatic interactions
the elicitation of plant defense responses. The major 3.2 Membrane damage mechanism
points are the following: (1) Chitosan exhibits various 3.3 Chitosan-DNA/RNA interactions
inhibitory efficiency against bacteria, fungi, and viruses; 3.4 Metal chelation capacity of chitosan
(2) the five main modes of action of chitosan are 3.5 Deposition onto the microbial surface
electrostatic interactions, plasma membrane damage 4. Elicitation of plant defense responses by chitosan
mechanism, chitosan-DNA/RNA interactions, metal che- 4.1 Pathogenesis-related proteins
lation capacity of chitosan, and deposition onto the 4.2 Defense-related enzymes
microbial surface; (3) the elicitation of plant defense 4.3 Defense-related secondary metabolites accumulation
responses by chitosan may be related to various 4.3.1 Phytoalexins
pathogenesis-related proteins, defense-related enzymes, 4.3.2 Lignin
and secondary metabolites accumulation, as well as the 4.3.3 Suberization
complex signal transduction network. The facing problems 4.3.4 Phenolic compounds
and strategies for antimicrobial mechanism research and agri- 4.3.5 Callose
cultural application of chitosan are also discussed. 4.4 Signal Transduction
4.4.1 Extracellular signal perception of chitosan
4.4.2 Intracellular signal perception of chitosan
Keywords Chitosan . Plant diseases . Antimicrobial . 4.4.3 Signal transduction of chitosan with other signal
Defense responses . Signal transduction . Agriculture molecules
5. Conclusion and future perspectives

K. Xing : X. Zhu : X. Peng (*)

School of Life Science, Jiangsu Normal University,
Xuzhou 221116, Jiangsu, People’s Republic of China
1 Introduction
e-mail: pengxue@jsnu.edu.cn
In agriculture, pathogens cause many important plant diseases
S. Qin (*) and are responsible for losses in crop yield and quality in all
The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu
Province, Jiangsu Normal University, Xuzhou 221116, Jiangsu,
parts of the world. Besides that, many pathogenic fungi can
People’s Republic of China also produce kinds of harmful toxins and metabolites in the
e-mail: shengqin@jsnu.edu.cn infection process, which is a great threat to the safety of
 K. Xing et al.

agricultural products. The end result of pathogens infection is 2009), antitumor activity (Toshkova et al. 2010), and
a reduction in plant growth, lower yield, inferior product immune-enhancing effect (Li et al. 2013c; Zaharoff et al.
quality, and huge economic loss. Therefore, plant diseases 2007). These properties make chitosan a promising candidate
need to be controlled to maintain the quality and safety of for medicine (Tan et al. 2013), food (Dutta et al. 2009; Qiu
agricultural products. During the past 100 years, crop protec- et al. 2014), cosmetic (Ray 2011), water treatment (Bhatnagar
tion has relied heavily on chemical fertilizers and pesticides. and Sillanpää 2009), and biomedical engineering industries
However, the chemical pesticide is a double-blade sword. (Silva et al. 2012; Upadhyaya et al. 2013), as well as for many
Excessive use of pesticides and fertilizers helps farmers raise agricultural uses (Cota-Arriola et al. 2013; El-Hadrami et al.
productivity significantly, but it also harms biological diversi- 2010). In fact, a number of commercial applications of chito-
ty, natural and agricultural systems, and public health and san benefit from its antimicrobial activity. As a versatile
leads to the development of resistant strains (Sun et al. material, chitosan exhibits proved antimicrobial activities
2012). As relatively recent, terms, genetic engineering, and against fungi, bacteria, and viruses and acts as an elicitor of
genetic modification are ad hoc approaches that could im- plant defense mechanisms. With the wide-spectrum antimi-
prove plant traits, such as disease resistance and production of crobial activities, chitosan has been utilized to reduce or
useful goods. However, in the face of public concerns about prevent the spread of pathogens (Li et al. 2013a; Mansilla
the safety of the genetically modified crops, alternative et al. 2013; Fig. 2) or to enhance plant innate immunity
methods should be provided to solve the real problems in defenses (El-Ghaouth et al. 1994; Amborabé et al. 2008;
agricultural production. Therefore, it is important to develop Fondevilla and Rubiales 2012). The interplay of antimicrobial
environmentally friendly pesticides and techniques that can be and eliciting properties makes chitosan a potential antimicro-
used to reduce pesticide use while ensuring the healthy devel- bial agent to control plant disease caused by pathogens. Fur-
opment of plants and sustainable agriculture. Natural products thermore, chitosan is an abundant and biodegradable biopoly-
are an excellent alternative to synthetic pesticides as a means mer derived from chitin, which is the second large renewable
to reduce negative impacts to human health and the resource after cellulose in the world. In addition, toxicity tests
environment. that reported the lethal dose for 50 % of test animals (LD50) of
Chitosan, β-(1,4)-2-amino-2-deoxy-D-glucose, is a natural chitosan in laboratory mice exceed 16 g/day/Kg body weight,
versatile biopolymer derived by partially deacetylation of which is very close to that of salt or sugar (Dodane and
chitin (Fig. 1), mainly as the structural component of the Vilivalam 1998; Singla and Chawla 2001). Therefore, the
exoskeletons of crustaceans and insects, as well as in some development of chitosan pesticide has potential social and
fungal cell walls (Sanford 2003). In 1859, French C. Rouget economic benefits.
reported finding chitosan after boiling chitin in potassium Based on the current state of research and progress in
hydroxide. From then on, chitosan has attracted considerable corresponding areas, this review is organized into sections
interest in various fields due to its unique biological activities, discussing the antimicrobial properties of chitosan against
such as biocompatibility (Hsu et al. 2011; Mi et al. 2002), plant pathogens (including fungi, bacteria, and viruses), the
biodegradability (Kim et al. 2011), nontoxicity (Shi et al. modes of action as antimicrobial compounds, and the ability
2006), antimicrobial activity (Li et al. 2008; Rabea et al. to elicit natural plant defense responses.

Fig. 1 The structure of chitin and

chitosan. Chitin and chitosan are
nitrogenous polysaccharides. The
structure of the chitin molecule is
similar to that of cellulose, but it is
composed of the units of 2-
acetylamino-2-deoxy-D-
glucopyranose bound by a
glycosidic bond. In contrast to
chitin, chitosan amino groups are
not mostly acetylated
Chitosan antimicrobial and eliciting properties for pest control

kikuchiana Tanaka and Physalospora piricola Nose at 5.0 g/

L in vitro (Meng et al. 2010). In pear fruit, treatments with
chitosan reduced the disease incidence and inhibited the lesion
expansion caused by these two fungal pathogens (Meng et al.
2010). In commercial winegrapes, chitosan effectively
inhibited growth of Botrytis cinerea in liquid culture and
suppressed gray mold on detached grapevine leaves and
bunch rot (Reglinski et al. 2010). Chitosan exhibited strong
antifungal activity against Rhizoctonia solani, the rice sheath
blight pathogen. Two types of acid-soluble chitosan (with
different degrees of deacetylation) caused a 60–91 % inhibi-
tion in mycelial growth, 31–84 % inhibition of disease inci-
dence, and 66–91 % inhibition in lesion length (Liu et al.
2012).
Chemical modifications as an approach are efficient in
enhancing the biological activity against some economic plant
Fig. 2 Chitosan counteracted Pto DC3000 bacterial colonization in pathogenic fungi and bacteria and widening their applications
tomato seedlings. Disease phenotype of seedlings pretreated with (Guo et al. 2006). Chitosan hydrochlorides, even at the lowest
10 μg/mL chitosan (+Chitosan) or 0.001 % (v/v) acetic acid (−Chitosan)
and then immersed in Pto DC3000 cell suspension (+Pto DC3000) or
test concentration of 0.0025 %, inhibited growth of the
sterile distilled water containing 0.025 % (v/v) SILWET L-77 plus 10 mM Candida species significantly (Seyfarth et al. 2008). In the
MgCl2 (−Pto DC3000). Photographs were taken at 7 days postinocula- bioassay of Fusarium oxysporum and Pythium debaryanum,
tion. Tomato seedlings were healthy (−Chitosan and −Pto DC3000); it N-(benzyl) chitosan derivatives exhibited high inhibition per-
could induce the characteristic symptom of bacterial speck of tomato and
make the seedling wilting and tawny (−Chitosan and + Pto DC3000);
centage of spore germination at 1,000 mg/L (Rabea et al.
tomato seedlings remain healthy which showed that chitosan was harm- 2009).
less to plants (+Chitosan and −Pto DC3000); it significantly decreased As evaluated by leakage of proteinaceous and other UV-
bacterial damages in cotyledons which revealed that chitosan contributed absorbing material, there was no significant increase in leak-
to counteract bacterial growth in tomato seedlings (+Chitosan and + Pto
DC3000)
age and any apparent symptoms of phytotoxicity when plants
were grown in the presence of chitosan, even at a higher
chitosan concentration (Kong et al. 2010), which showed that
2 Antimicrobial activities of chitosan chitosan was harmless to plants.

In 1979, the first study reported that chitosan showed a broad 2.2 Against bacteria
range of activities and a high inactivation rate against both
Gram-positive and Gram-negative bacteria (Allan and Chitosan and its derivatives inhibited the growth of a wide
Hadwiger 1979). Since then, many studies on the antimicro- variety of bacterial plant pathogens (Liu et al. 2001;
bial properties of chitosan and its derivatives have been re- Wiśniewska-Wrona et al. 2007; Rabea and Steurbaut 2010;
ported (No et al. 2002; Xing et al. 2008; Lee and Je 2013). In Badawy et al. 2014; Table 1). Based on the available evi-
modern agriculture, lots of plant pathogens have been found to dences, bacteria appear to be generally less sensitive to the
be sensitive to chitosan (Manjunatha et al. 2008; Rabea et al. antimicrobial action of chitosan than fungi (Kong et al. 2010).
2009; Li et al. 2013a, b, c). Although chitosan has been proved In various microbial species, the antibacterial efficiency of
to be effective against bacteria, fungi, and viruses, it exhibits chitosan against Gram-positive and Gram-negative bacteria
different inhibitory efficiencies against different microbial is different, however, somewhat controversial. Several re-
species. searchers have demonstrated that chitosan exhibited higher
inhibition effects on Gram-positive bacteria than on Gram-
2.1 Against fungi negative bacteria (No et al. 2002; Tayel et al. 2010; Lee and Je
2013). Concerning the bacteria surface structure, Gram-
As a broad-spectrum fungicide, chitosan has been shown to be positive bacteria tend to have a loose cell wall, while Gram-
fungicidal against several fungal plant pathogens (Liu et al. negative bacteria have an outer membrane structure in the cell
2001; Wiśniewska-Wrona et al. 2007; Rabea and Steurbaut wall. As a polymeric macromolecule, chitosan is unable to
2010; Table 1). Chitosan can effectively inhibit the develop- pass through the outer membrane of Gram-negative bacteria,
ment of phytopathogenic fungi at different life-cycle stages. since this membrane functions as an efficient outer permeabil-
For instance, chitosan completely inhibited spore germination, ity barrier against macromolecules (Helander et al. 2001).
germ tube elongation, and mycelial growth of Alternaria While in other studies, Gram-negative bacteria were more
 K. Xing et al.

Table 1 The minimum growth

inhibitory concentrations (MIC) Microorganisms Chitosan samples MIC (ppm)
of native chitosan or its deriva-
tives against fungal and bacterial Fungi
plant pathogens Botrytis cinerea Chitosan 10
Drechstera sorokiana Chitosan 10
Fusarium oxysporum Chitosan 100
Micronectriella nivalis Chitosan 10
Piricularia oryzae Chitosan 5,000
Rhizoctonia solani Chitosan 1,000
Trichophyton equinum Chitosan 2,500
Bacteria
Agrobacterium tumefaciens N-(o,o-dichlorobenzyl) chitosan 500
Agrobacterium tumefaciens Quaternary N-(benzyl) chitosan 500
Agrobacterium tumefaciens N-(benzyl) chitosan 800
Clavibacter michganensis subsp. michganensis Chitosan 1,000
Erwinia carotovora Chitosan 200
Erwinia carotovora N-(o,o-dichlorobenzyl) chitosan 480
Erwinia carotovora Quaternary N-(benzyl) chitosan 600
Erwinia carotovora N-(benzyl) chitosan 700
Erwinia carotovora N-(α-methylcinnamyl) chitosan 1,025
Erwinia carotovora subsp. carotovora Chitosan 5,000
Xanthomonas campestris Chitosan 500

susceptible to chitosan (Park et al. 2004; Du et al. 2009). They seedlings with 10 μg/mL chitosan before Pseudomonas
suggested that hydrophilicity in Gram-negative bacteria is syringae pv. tomato DC3000 (Pto DC3000) inoculation sig-
significantly higher than that in Gram-positive bacteria, mak- nificantly decreased bacterial damages in cotyledons com-
ing them more sensitive to chitosan (Chung et al. 2004). pared with control (Mansilla et al. 2013). Not only does
Moreover, the Gram-negative cell envelope contains an addi- chitosan inhibit planktonic cell growth but also it affects the
tional outer membrane composed by phospholipids and lipo- already established biofilms. Unexpectedly, log reductions
polysaccharides, which face the external environment. The were in some cases higher for biofilm than for planktonic
highly charged nature of lipopolysaccharides confers an over- cells, deserving further more detailed work (Orgaz et al.
all negative charge to the Gram-negative cell wall. Therefore, 2011).
Gram-negative bacteria with high electronegative charge will
interact more effectively with the polycationic chitosan com- 2.3 Against viruses
pared with Gram-positive bacteria. Besides microorganism
species, diverse consequences may be due to various initial Compared with the studies of antibacterial and antifungal
reaction material and conditions, such as pH, molecular activity of chitosan, relatively few research studies of its
weight, and degree of deacetylation of chitosan, etc. (Kong antiviral activity have been reported (Su et al. 2009). The
et al. 2010; Younes et al. 2014). antiviral activity of chitosan in animals, microorganisms,
The in vitro antibacterial effect of chitosan and its ability in and plants has been reviewed (Chirkov 2002; Wang et al.
protection of watermelon seedlings from Acidovorax citrulli 2012). Chitosan inhibited viral infections in animal cells and
were evaluated. The disease index of watermelon seedlings prevented the multiplication of bacteriophages in infected
planted in soil and the death rate of seedlings planted in perlite cultures of microorganisms (Chirkov et al. 2001; Chirkov
were significantly reduced by chitosan at 0.40 mg/mL com- 2002). In plants, chitosan induced resistance toward viral
pared with the pathogen control (Li et al. 2013b). Chitosan diseases and inhibited the systemic spreading of viruses and
solution at 0.10 mg/mL markedly inhibited the growth of viroids so that most or all plants treated with chitosan did not
Xanthomonas pathogenic bacteria from different geographical develop systemic viral infection (Chirkov 2002; Rabea et al.
origins. The surviving cell numbers in the chitosan solution 2003). Low-molecular chitosan inhibited the formation of
decreased more than 3.86 log10 CFU/mL compared with the local necroses induced by tobacco mosaic virus for 50–90 %
control after 6 h of incubation regardless of the bacterial strain (Davydova et al. 2011). Actually, the direct inhibitory effect of
(Li et al. 2008). As shown in Fig. 2, pretreatment of tomato chitosan on viruses was mainly manifested in the inactivation
Chitosan antimicrobial and eliciting properties for pest control

of viruses. Chitosan was effective in inhibiting coliphage chitosan derivatives with increasing positive charge render
infection and the replication of 1–97 A phage in Bacillus the prevailing electrostatic explanations questionable, since
thuringiensis culture. When added to a phage suspension, chitosan-thioglycolic acid (slightly positive zeta potential)
chitosan decreased its titer. Electron microscopic observations had superior effects compared with trimethyl chitosan (highly
showed that chitosan caused structural changes in phage par- positive zeta potential) with all microbes tested (Geisberger
ticles and damaged their integrity (Chirkov 2002). Electron et al. 2013). However, these observations did not repudiate
microscope photographs of tobacco mosaic virus suspension electrostatic interactions of chitosan-pathogens totally and
showed that the number of virus particles was notably de- revealed that the antimicrobial action of water-soluble
creased and most of them twisted together and bound into a chitosans was dependent on the degree of deacetylation and
bundle (Hu et al. 2009). the substituted group (Je and Kim 2006). It shed light on the
modes of action of chitosan that is probably more complex,
involving a series of molecules that may ultimately lead to a
killing process.
3 Antimicrobial mechanism of chitosan The Gram-positive bacterial cell wall is made up of thick
peptidoglycan layer that is rich in teichoic acids, which are
Ever since the wide-spectrum antimicrobial activity of chito- negatively charged because of the presence of phosphate
san was discovered, great interests in this polymer and its groups in the structure. While in Gram-negative bacteria,
derivatives have increased in recent years due to their unique lipopolysaccharides impart a strongly negative charge to the
properties. Undoubtedly, more and more research studies bacterial surface. Also, there are similar negatively charged
proved their potential use in agriculture, medical industry, compounds (e.g., proteins and glycoproteins) in the fungal cell
food industry, and so on. As we all know, research of antimi- membrane and viral envelope. Thus, the positively charged
crobial mechanisms is an absolutely necessary stage of the chitosan molecules potentially interact with negatively
microbicide development. However, the exact mechanisms of charged pathogen surfaces, which is termed as electrostatic
the antimicrobial activities of chitosan and its derivatives are interactions, can destroy the cell structure, cause extensive cell
still unknown, which limit their further application to some surface alterations, and increase membrane permeability
extent. In the past decades, various mechanisms of action have (Rabea et al. 2003; Chung et al. 2004; Liu et al. 2004), leading
been proposed to explain the antimicrobial activity of chitosan to the leakage of intracellular substances and ultimately
(Table 2). On the basis of present research studies, the antimi- resulting in impairment of pathogen vital activities (Helander
crobial mechanisms of chitosan and its derivatives can be et al. 2001; Zakrzewska et al. 2005; Je and Kim 2006).
summarized as follows. To verify the possible involvement of teichoic acids of
Staphylococcus aureus in chitosan’s antimicrobial activity
3.1 Electrostatic interactions and to analyze their role in chitosan susceptibility, Raafat
et al. (2008) tested S. aureus strain SA113 together with four
Polycationic polymer chitosan has so many reactive amino mutants lacking one or more genes involved in teichoic acids
groups in its structure that can be protonated, and thus the biosynthesis. The minimum growth inhibitory concentration
polymer will bear positive charge, while chitin as an N- (MIC) of chitosan for wild-type S. aureus SA113 was 84.8 μg/
acetylglucosamine polymer does not show any antimicrobial mL. The S. aureus SA113ΔtagO deletion mutant, a complete-
activity. Differences in the structures might account for their ly lacked wall teichoic acids, was the most resistant of the
varying inhibition effects, which also suggest that the presence strains to chitosan, with an MIC at 545.5 μg/mL (more than 5-
of amino groups is the base of the antimicrobial activity of fold higher than the wild type). The S. aureus SA113ΔdltA
chitosan. Because of the stable crystalline structure, chitosan mutant, which lacked the D-alanine modification in teichoic
is normally insoluble in water, but soluble in dilute aqueous acids, as a result of which the cells carried an increased
acidic solutions below its pKa (∼6.3), in which amine (−NH2) negative surface charge, was almost 100 times more suscep-
groups in glucosamine units are converted into the soluble tible to the action of chitosan, with an MIC as low as 0.9 μg/
protonated form (−NH3+) (Madihally and Matthew 1999; mL. These data clearly indicated that teichoic acids played a
Pillai et al. 2009; Silva et al. 2012). It was observed that major role in the chitosan-bacteria interaction since the lack of
dimethylaminoethyl-chitosan 90 prepared from 90 % teichoic acids in Staphylococcus resulted in a less negatively
deacetylated chitosan had more activity than charged cell wall and increased resistance to chitosan, and
dimethylaminoethyl-chitosan 50 prepared from 50 % further substantiated the hypothesis that the polycationic na-
deacetylated chitosan (Je and Kim 2006), which meant that ture of chitosan is a major factor contributing to its antimicro-
the amino group (NH3+) as the active functional group was bial activity.
found to be essential to the antibacterial activity of chitosan To clarify the possible role of phospholipids, the main
(Chung and Chen 2008). However, a selection of three composition of teichoic acids, involved in the antimicrobial
Table 2 Antimicrobial action of chitosan and 1,329 its derivatives

Chitosan sample Microorganism Mechanism Reference

ε-Polylysine-chitosan Escherichia coli, Staphylococcus aureus Disrupted bacterial cell membranes with release of cellular cytoplasm Liang et al. 2014
Gallic acid-g-chitosan Foodborne pathogens Increased the release of intracellular components; disrupted cell membranes Lee and Je 2013
Chitosan Saccharomyce. cerevisiae deletion mutants Disrupted protein synthesis and membrane integrity Galván et al. 2013
Chitosan Pseudomonas syringae Electrostatic interactions; caused morphological changes and damage in Mansilla et al. 2013
bacterial surfaces
Chitosan-thioglycolic acid Streptococcus sobrinus; Candida albicans; Affected cell wall integrity and intracellular ultrastructure Geisberger et al. 2013
Neisseria subflava
Chitosan-arginine Pseudomonas fluorescens; E. coli Increased membrane permeability resulted from chitosan-membrane interaction Tang et al. 2010
Chitosan Beauveria bassiana; Pochonia chlamydosporia; Membrane fluidity determines sensitivity of filamentous fungi to chitosan Palma-Guerrero et al. 2010
F. oxysporum f. sp. radicis-lycopersici;
Neurospora crassa wild-type strain;
N. crassa fatty acid desaturase mutant
Chitosan Rhizopus stolonifer Induced K+efflux, inhibited H+-ATPase activity García-Rincóna et al. 2010
Chitosan N. crassa Permeabilized the plasma membrane and killed cells in an energy- Palma-Guerrero et al. 2009
dependent manner
Chitosan nanoparticles E. coli; S. aureus Damaged cell membrane structures and putative bind to extracellular or Xing et al. 2009b
intracellular targets
Chitosan E. coli; S. aureus Electrostatic interactions, destroyed cell structures, induced the leakage of Chung and Chen 2008
enzymes and nucleotides
Chitosan microspheres E. coli Influenced the structure and permeability of membrane; caused cellular leakage Kong et al. 2008
Chitosan S. simulans; S. aureus Electrostatic interactions; TA might represent a “target” for chitosan’s action Raafat et al. 2008
Chitosan Aspergillus fumigatus; B. cinerea Aspergillus. Had an affinity for plasma membrane lipids Park et al. 2008
parasiticus; F. oxysporum; F. solani;
Penicillium verrusosum var. verrucosum
Chitosan E. coli; S. aureus Killed bacteria through cell membrane damage Liu et al. 2004
Chitosan E. coli; P. aeruginosa; Salmonella typhimurium Disrupted the outer membrane of Gram-negative bacteria Helander et al. 2001
K. Xing et al.
Chitosan antimicrobial and eliciting properties for pest control

action of chitosan, lecithin and Na3PO4 were used to simulate microscopy clearly demonstrated oleoyl-chitosan nanoparti-
the effect of phospholipids and phosphate groups in the cyto- cles with intact spherical structure adhered to the surface of
plasmic membrane. Results showed that no matter whether E. coli and S. aureus and efficiently permeabilized bacterial
treated with lecithin or phosphate groups, chitosan could cell membranes (Xing et al. 2009a; Fig. 3). The morphological
inhibit the growth of Escherichia coli effectively. It meant changes were observed more obviously as the contact time
that lecithin or phosphate groups did not influence the inter- increased continuously (Xing et al. 2009a).
action between chitosan and E. coli. While in the case of Besides morphological changes, detection and quantification
S. aureus, the addition of lecithin or phosphate groups appar- of amino acids residues in membrane proteins reflected the
ently influenced the inhibition rate (Xing et al. 2009b). There-
fore, it is presumable that phospholipids might be a target
molecule in the chitosan-pathogen interaction that occurred at
the cell surface of S. aureus. The different effects of lecithin
and phosphate groups on the antibacterial activity against
E. coli and S. aureus have proved once again that the mech-
anisms of the antimicrobial activity of chitosan were different
for Gram-positive and Gram-negative bacteria.

3.2 Membrane damage mechanism

A major function of the cell wall and cell membrane is to

protect the interior substances so that they would not leak to
the cell exterior. The electrostatic interaction, between the
positively charged amino groups of chitosan and the negative-
ly charged residues of macromolecules exposed at the micro-
bial surfaces, changed the permeability of cell membranes and
thereby caused the death of bacteria (Helander et al. 2001).
Chitosan was found to react with both the cell wall and cell a
membrane, but not simultaneously, indicating that the inacti-
vation of pathogens by chitosan occurs via a two-step sequen-
tial mechanism, i.e., an initial separation of the cell wall from
its cell membrane, followed by destruction of the cell mem-
brane (Chung and Chen 2008).
Light and electron microscope investigations revealed that
growth inhibition of F. oxysporum as a response to chitosan
was accompanied by marked cellular changes, which included
hyphal swelling, increased vacuolation, retraction, and alter-
ation of the plasma membrane, cytoplasm aggregation, and
abnormal cell wall deposition (Benhamou 1992). In electron
micrographs, the outer membrane of chitosan-treated E. coli
was disrupted and covered by an additional tooth-like layer. In
micrographs of chitosan-treated S. aureus, the membrane of
dividing cells was disrupted in the constricting region with the
loss of cell contents (Liu et al. 2004). Similar results were
reported by lots of research studies with different chitosan
b
derivatives and tested strains (Lee and Je 2013).
However, whether such remarkable modification is result-
Fig. 3 Transmission electron microscope of E. coli (a) and S. aureus (b)
ed from the direct effect of chitosan is unknown. This is cells treated with 300 mg/L oleoyl-chitosan nanoparticles for up to
because chitosan solution cannot be directly observed in elec- 30 min. a Untreated cell displayed a smooth and compact surface. b
tron micrographs, which makes it difficult to investigate the Some nanoparticles with intact spherical structure (arrows pointed to)
mode of action of chitosan on microbes. Our previous work adhering to the surface of cell after nanoparticles treated for 5 min. c Deep
roughening and collapse of the cell surface was found after 15 min. d
gave a direct evidence for such interaction, which employed Apparent holes and loss of cell contents (arrows pointed to) were ob-
oleoyl-chitosan nanoparticles, combined with E. coli and served in lysed bacteria, surrounded by dark floccules instead of spherical
S. aureus to explore the antibacterial interaction. Electron nanoparticles after 30 min
 K. Xing et al.

integrity of cell membranes indirectly. When antibacterial agents 3.3 Chitosan-DNA/RNA interactions
interacted with cell membranes, the conformation of membrane
proteins would be changed, and then Tyr residues located inside Chitosan with lower molecular weight is assumed to be able to
the membrane would be exposed to the surface (Ye et al. 2007). pass through the bacterial cell wall (Sudarshan et al. 1992;
After treatment with oleoyl-chitosan nanoparticles, the fluores- Goy et al. 2009), destroy intracellular components from col-
cence intensity of Tyr residues increased in E. coli and S. aureus, loidal state to flocculation and degeneration, disrupt the nor-
which indicated that chitosan influenced the structure of cell mal physiological metabolic activity of bacteria, or directly
membranes by interacting with proteins on the cell membrane of interfere with genetic materials (Come et al. 2003; Issam et al.
the bacteria (Xing et al. 2009b). Accordingly, it is speculated that 2005), and then inhibit the reproduction of bacteria, resulting
membrane proteins would be one of the target molecules on cell in the death of microorganisms ultimately. It is presumable
surfaces for chitosan’s action. that chitosan could bind with DNA and inhibit synthesis of
The efflux of potassium ions was identified as an early messenger RNA (mRNA) through penetration toward the
response of the cell to the presence of some cationic com- nuclei of the microorganisms and interfere with the synthesis
pounds. A rapid efflux of potassium depended on the chitosan of mRNA and proteins (Sudarshan et al. 1992; Rabea et al.
concentration was observed. In addition, there was an impor- 2003). Fluorescence micrographs evidenced that the fluores-
tant inhibitory effect of chitosan on H+-ATPase activity in the cein isothiocyanate labeled chitosan oligomers were observed
plasma membrane of Rhizopus stolonifer. The decrease in the at the inside of the cell. Permeated chitosan oligomers (mo-
H+-ATPase’s activity could provoke the accumulation of pro- lecular weight=8,000 and 5,000) were suggested to block the
tons inside the cell, which would result in the inhibition of the transcription from DNA to inhibit the growth of bacteria (Liu
chemiosotic driven transport that allows the H+/K+ exchange et al. 2001) and then disrupt the related protein synthesis.
(García-Rincóna et al. 2010). Our previous studies indicated that chitosan nanoparticles
As we mentioned above, the plasma membrane protected efficiently permeabilized bacterial cell membranes and ad-
the cell from harmful substances present in the external envi- hered to the bacterial surface (Xing et al. 2009a) and then
ronment from entering into the interior. Why does the plasma penetrate into the bacteria with the contact time increased
membrane form a barrier to chitosan in some species but not in (Xing et al. 2009b). As we discussed above, the phosphate
others? By imaging fluorescently labeled chitosan, a recent group might be an extracellular target contributing to its
work shed new light on this question. It was observed that interaction with the positively charged chitosan, ultimately
chitosan bound to the conidial surfaces of all species tested but resulting in impairment of vital bacterial activity. There are
only consistently permeabilizes the plasma membranes of also phosphate groups in the main chain of nucleic acid
chitosan-sensitive fungi. This suggested that the plasma mem- (DNA/RNA). It is possible that the amino groups of chitosan
brane formed a barrier to chitosan in chitosan-resistant fungi that possess positive charges would attract the negatively
but not in chitosan-sensitive fungi. Fatty acid analysis re- charged phosphate groups of DNA/RNA. In vitro chitosan-
vealed that the plasma membranes of chitosan-sensitive fungi DNA/RNA interaction obviously inhibited electrophoretic
were shown to have more polyunsaturated fatty acids than mobility of bacterial genomic DNA or total RNA on agarose
chitosan-resistant fungi, suggesting that their permeabilization gel. The brightness of bands weakened gradually as the con-
by chitosan may be dependent on membrane fluidity. More- centration of chitosan nanoparticles increased, showing the
over, a fatty acid desaturase mutant of Neurospora crassa with aggravation of chitosan-DNA/RNA interactions. The possible
reduced plasma membrane fluidity exhibited increased resis- reason might be that negative charges of DNA/RNA had been
tance to chitosan. These findings suggested a new strategy for counteracted by chitosan so that they could not move in
antifungal therapy by increasing plasma membrane fluidity to electric field accordingly. The gel-retardation experiment
make fungi more sensitive to fungicides such as chitosan pointed out that DNA and RNA might be the intracellular
(Palma-Guerrero et al. 2010). targets of chitosan (Xing et al. 2009b).
Fluorescently labeled chitosan was found to be taken up In a recent work, about 4,600 nonessential gene deletion
and accumulated in bacteria and fungi by many researchers. mutants of S. cerevisiae were employed to investigate the
Little is known, however, about its mode of endocytical antifungal mechanism of low molecular weight chitosan. It
internalization by fungal cells. A study focused on the inter- was found that 31 % of the 107 mutants most sensitive to
nalization of chitosan by living cells made a number of novel chitosan had deletions of genes related primarily to functions
findings (Palma-Guerrero et al. 2009). Sodium azide and low involving protein synthesis. As the chitosan concentration
temperature (4 °C), two standard treatments to inhibit ATP ranged from 0.35 to 1.25 mg/mL, the β-galactosidase activity
production (Atkinson et al. 2002), prevented the endocytic was reduced from 32 to 13 % of no-chitosan controls, which
marker FM4-64 uptake by chitosan-treated conidia indicating could be the result from interference with transcription effi-
that chitosan-induced permeabilization of the plasma mem- ciency and other processes in addition to translation (Galván
brane was ATP-dependent but did not involve endocytosis. et al. 2013).
Chitosan antimicrobial and eliciting properties for pest control

3.4 Metal chelation capacity of chitosan polymer film to damage the physiological metabolism process
of the bacteria.
In the cell wall of Gram-positive bacteria, peptidoglycan
accounts for about 50–80 % of the cellular dry weight, as well
as a large number of special ingredients like teichoic acids.
Phosphate groups of teichoic acids are able to attract divalent 4 Elicitation of plant defense responses by chitosan
metal cations (Lambert 2002), especially Mg2+ and Ca2+, to
maintain enzymatic functions and the stability of cytoplasmic Nowadays, chitosan is considered to be a promising antimi-
membranes (Elsenhans et al. 1983). For Gram-negative bac- crobial agent owing to its antibacterial, antifungal, and antivi-
teria, lipopolysaccharides not only increase the negative ral activities. This has led to the exploitation of its properties in
charge of the cell membrane but also have a strong affinity various aspects of agriculture. Since the 1980s, the study of
for cations such as Mg2+ and Ca2+. The combination of metal chitosan has been changed from a general sewage treatment
ions and chelating agents, such as ethylenediaminetetraacetic agent to plant growth regulator, fruits and vegetables
acid (EDTA), released lipopolysaccharides and led to the antistaling agent, soil conditioner, and seed coating agent,
collapse of the outer membrane (Vaara 1992). As a kind of especially in the disease control in agricultural production.
complexing reagent, chitosan is able to chelate some essential Lots of studies showed that chitosan is not only an antimicro-
nutrients, metal ions, and trace elements necessary for the bial agent but also an effective elicitor of plant systemic
growth of bacteria and fungi. When the pH is below 6.0, acquired resistance to pathogens (Table 3). Even applied on
protonated NH+ groups of chitosan compete with divalent plants together with the biological control agents, chitosan
metal ions for phosphate groups in teichoic acids or lipopoly- enhanced the efficacy in the control of pathogens (Vallance
saccharide molecules. In the presence of chitosan, the cell wet et al. 2011; Abro et al. 2013). It is possible for chitosan as a
weight of P. syringae pv. tomato DC3000 decreased 50 % new type of green pesticides to play an important role in
compared with control. However, the addition of MgCl2 res- agriculture owing to its nontoxic, biodegradable, and
cued the values of the chitosan-treated group (Mansilla et al. nonpollution characteristics.
2013). The addition of Mg2+ or Ca2+ increased the concentra-
tion of positive charges in the system and weakened the 4.1 Pathogenesis-related proteins
bacteriostatic action that mainly depends on electrostatic
forces. Therefore, the antibacterial activity of chitosan de- In many plant species, response to infection by plant patho-
creased obviously in a dose-dependent manner when Mg2+ gens or various abiotic stresses is accompanied by the synthe-
and Ca2+ were added to the culture medium. It suggested that sis of low molecular weight compounds, proteins, and pep-
disruption of the barrier properties of the outer membrane is tides with antimicrobial activities, which are termed as
the first step for chitosan to exhibit antimicrobial effects. pathogenesis-related proteins (Bol et al. 1990; Selitrennikoff
2001). These pathogenesis-related proteins were first detected
by Van Loon and Van Kammen (1970), when they observed
3.5 Deposition onto the microbial surface accumulation of various novel proteins in leaves of tobacco
after tobacco mosaic virus infection. Since then, chitosan has
High molecular weight chitosan can deposit onto the bacterial been described as an elicitor to induce plants produce a wide
surface and form a dense polymer film. Chitosan-treated cells range of pathogenesis-related proteins with antimicrobial ac-
exhibited altered outer membranes, the surface of which was tivity to protect themselves from pathogen infection. Some of
covered by numerous vesicular structures and an additional these pathogenesis-related proteins are hydrolytic enzymes
layer of material, causing the cell envelope to appear consid- that target cell walls, such as chitinase and β-1,3-glucanase,
erably thickened (Helander et al. 2001). The thickened cell the markers of plant defense responses. Since there are spe-
envelope prevents nutrients from entering the cell, as well as cific hydrolytic enzymes but no corresponding substrate in
the extracellular transport of metabolite excretion. Similar to plants, these enzymes may have been retained throughout
chitosan, chito-oligomers caused blockage of nutrient flow evolution for the purpose of confronting challenges by insects
and were responsible for the growth inhibition and lysis of and fungi (Hadwiger 2013).
E. coli, which were evidenced by scanning electron micros- Since insect exoskeletons and fungal cell walls contain
copy (Vishu et al. 2005). The deposition of cationic oligomers chitin and/or β-D-glucans as major structural components,
on to the cell surface is more prominent than membrane chitinase and β-1,3-glucanase are capable of catalyzing the
disruption as in the case of Gram-positive bacteria, owing to hydrolysis of chitin and β-D-glucans, decomposing cell walls
stronger association of O-chains to the outer membrane struc- of fungi, thus preventing the growth of fungi on the plant (El-
ture (Vishu et al. 2005). Therefore, another possibility for the Ghaouth et al. 1992; Abbasi et al. 2009). Furthermore,
antimicrobial activity of chitosan is based on the formation of chitinase and β-1,3-glucanase very often act synergistically
 K. Xing et al.

Table 3 Listing of some variable applications of chitosan as an elicitor of plant defense responses

Plant/crop Disease/condition Efficacy Reference

Jute Stem rot Enhanced the activity of defense-related enzymes Chatterjee et al. 2014
Rice Leaf streak, leaf blight Accumulated defense-related enzymes Li et al. 2013a
Watermelon Fruit blotch disease Direct killing effect Li et al. 2013b
Peach Brown rot Enhanced antioxidant and defense-related enzymes Ma et al. 2013
Pine Pitch canker Upregulated the expression level of defense-related enzymes Fitza et al. 2013
Camellia Anthracnose Accumulated H2O2, defense-related enzymes, and soluble protein Li and Zhu 2013
Broccoli Native microflora Antimicrobial coating served as carriers for bioactive compounds Alvarez et al. 2013
Sycamore – Enhanced the production of H2O2 and nitric oxide Malerba et al. 2012
Rice Sheath blight Induced activity of defense-related enzymes Liu et al. 2012
Safflower; sunflower Salt stress Induced the activity of antioxidant enzymes Jabeen and Ahmad 2013
Tomato – Accumulated phosphatidic acid and nitric oxide Raho et al. 2011
Hypericum perforatum – Produced xanthone-rich extracts with antifungal activity Tocci et al. 2011
Apricot Fruit rot Direct inhibition activity Lou et al. 2011
Radish Cadmium stress Promoted the uptake of nutrients, nitrogen, potassium and Farouk et al. 2011
phosphorous, decreased cadmium concentration
Barley Mildew Induced stomatal closure Koers et al. 2011
Pear Fungal pathogens in storage Significantly increased defense-related enzymes activity Meng et al. 2010
Grape Botrytis bunch rot Direct antifungal activity and induction of defense-related Reglinski et al. 2010
enzymes activities
Sweet cherry Short shelf life Maintained quality attributes and extended the postharvest Dang et al. 2010
life by inducing defense-related enzymes activities
Fresh-cut mangoes Short shelf life Combined effects of postharvest heat treatment and chitosan Djioua et al. 2010
coating on quality and antimicrobial proprieties of fresh-
cut mangoes
Maize Low-temperature stress Increased the chilling tolerance of maize seedlings and Guan et al. 2009
induced higher activities of antioxidative enzymes
Pearl millet Downy mildew Elevated nitric oxide accumulation and activated early Manjunatha et al. 2009
defense reactions
Pearl millet Downy mildew Increased the level of the defense-related enzymes Manjunatha et al. 2008
Tobacco Tobacco necrosis virus Elicited callose apposition and abscisic acid accumulation Iriti et al. 2006

in the chitin-glucan degradation of fungal cell walls. Not effects on the plant’s DNA conformation that influenced gene
unexpectedly, increased resistance could be achieved in plants transcription in turn (Hadwiger 1999). In oat leaves, chitosan
simultaneously expressing high levels of both enzymes (Du- strongly activated the expression of general defense response
mas-Gaudot et al. 1996). Many reports revealed that chitosan genes, such as pathogenesis-related 10 (Hoat et al. 2013). In
was able to induce resistance in the host by increasing rice seedlings, chitosan triggered a set of defense responses,
chitinase and β-1,3-glucanase activities in cucumbers, pears, including the transcriptional upregulation of defense-related
and peaches (El-Ghaouth et al. 1994; Meng et al. 2010; Ma genes (β-1,3-glucanase and chitinase) and accumulation of
et al. 2013). More interestingly, El-Ghaouth et al. (1992) pathogenesis-related protein 1. Furthermore, chitosans of
found that chitosan only induced chitinase activity in wound- low molecular weight were more effective at inducing the
ed strawberry fruit but not in intact fruit and suggested that the described defense responses than those of higher molecular
nonporous strawberry cuticle might have physically separated weight (Lin et al. 2005).
chitosan from the tissue and, therefore, prevented chitosan
from inducing chitinase (Romanazzi et al. 2009). 4.2 Defense-related enzymes
Chitosan-mediated induction resulted in the rapid activa-
tion of a subset of genes called pathogenesis-related genes, As an exogenous elicitor, chitosan can induce resistance in the
generally regarded as the genes that functionally develop host by increasing the activities of several defense-related
disease resistance. Chitosan appeared to employ multiple enzymes, such as phenylalanine ammonia-lyase, peroxidase,
modes to increase pathogenesis-related gene function, includ- polyphenol oxidase, catalase, and superoxide dismutase
ing activating cell surface or membrane receptors and internal activity.
Chitosan antimicrobial and eliciting properties for pest control

Phenylalanine ammonia-lyase is an enzyme that catalyzes et al. 2009). It suggested that seed priming with chitosan
the biotransformation of L-phenylalanine to ammonia and might accelerate their germination speed and improve their
trans-cinnamic acid (MacDonald and D’Cunha 2007). As tolerance to stress conditions. Similar increase of chitosan-
the key enzyme of phenyl propanoid pathway, phenylalanine induced catalase activity in peach suggested that chitosan
ammonia-lyase is induced in host tissues following pathogen exhibited antioxidant capability (Ma et al. 2013), as enhance-
infection of plant tissues and by abiotic elicitor treatments, ment of catalase is helpful to eliminate free radicals (Chen
such as chitosan (Khan et al. 2003). Phenylalanine ammonia- 2008). Thus, it was speculated that chitosan might delay
lyase activity in the skin of table grape berries sprayed with repining and senescence of plant by regulating antioxidant
1.0 % chitosan was 2-fold higher than that in the untreated enzyme.
control. Both preharvest and postharvest chitosan treatments
significantly reduced the incidence of gray mold and were 4.3 Defense-related secondary metabolites accumulation
effective to control decay of table grapes (Romanazzi et al.
2002). Similar induced activity of phenylalanine ammonia- Secondary metabolites are not directly involved in growth or
lyase was also reported to increase in response to elicitation reproduction, but they are often involved with plant defense.
with chitosan in rice and wheat (Li et al. 2013a). Elicitation is a tool extensively used for enhancing secondary
Peroxidase is widely distributed in higher plants and con- metabolite yields. Chitosan is an example of elicitors inducing
tributes to the oxidization of phenolic and enodiolic defense-related secondary metabolites accumulation in plant
cosubstrates to quinones and generates hydrogen peroxide tissue.
(Borsani et al. 2001). While the exact mechanisms have yet
to be elucidated, peroxidase is known to play a part in increas- 4.3.1 Phytoalexins
ing plants’ defenses against pathogens (Karthikeyan et al.
2005). Chitosan treatment significantly increased peroxidase Phytoalexins are antifungal and antioxidative compounds syn-
activity in flesh around wound of pear fruit (Meng et al. 2010). thesized by plants in response to a pathogen challenge or
Peroxidase activity in the peach treated with 5 g/L chitosan induced by treatment with elicitors such as chitosan. In a
reached the peak at 24 h, and it was almost 3-fold as that in narrow sense, phytoalexins tend to fall into several classes
control fruit. Moreover, peroxidase gene expression in including terpenoids, isoflavonoid, and alkaloids; however,
chitosan-treated fruit maintained relatively higher than that researchers often find it convenient to extend the definition
in control fruit (Ma et al. 2013). to include all phytochemicals that are part of the plant’s
Polyphenol oxidase, catalyzing the phenolic substances to defensive arsenal.
synthesize lignin, is ubiquitous among angiosperms and as-
sumed to be involved in plant defense by promoting the Hadwiger and Beckman (1980) demonstrated that chitosan
formation of lignin that contributes to the reinforcement of at concentration as low as 0.9 μg/mL elicited phytoalexin
the cell wall structure preventing the penetration of pathogen induction and inhibited germination of macroconidia. When
(Chen et al. 2000; Li and Steffens 2002; Li and Zhu 2013). chitosan was applied to pea pod tissue with or prior to Fusar-
Chitosan significantly increased polyphenol oxidase activity ium solani, the tissue was protected from infection. Similar to
in rice seedlings following inoculation of two rice pathogens the inhibitory effect, phytoalexin production was affected by
(Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola) molecular weight and degree of acetylation of chitosan. The
(Li et al. 2013a). When injected into date palm roots at three highest phytoalexin production was achieved in grapevine
concentrations (0.1, 0.5, and 1 mg/mL), chitosan elicited leaves within 48 h of incubation with chitosan at 200 μg/mL
peroxidase expression activity, particularly at the concentra- with a molecular weight of 1,500 and a degree of acetylation
tion of 1 mg/mL, and increased the level of phenolic com- of 20 % (Aziz et al. 2006). It was observed that pretreating
pounds (El-Hassni et al. 2004). Plant phenolics have been in cottonseeds with chitosan markedly increased cotton resis-
the center of a myriad of discoveries related to plant defenses tance to vascular wilt caused by F. oxysporum f. sp.
to different pathogens (Nicholson and Hammerschmidt 1992; vasinfectum. All chitosan derivates tested significantly stimu-
Treutter 2006). lated phytoalexin (gossypol) production in roots more than
Catalase, which is involved in the degradation of H2O2 into stems, which greatly increased with a maximum of 1.16 mg/
H2O and O2, is the major H2O2-scavenging enzyme in all 5 g in chitosan-treated fresh root tissue (Awadalla and
aerobic organisms. Accumulating evidence indicated that cat- Mahmoud 2005).
alase played an important role in plant defense, aging, and
senescence (Yang and Poovaiah 2002). The increase of cata- Therefore, chitosan can be extensively used for inducing
lase activity was detected both in the chilling-sensitive and phytoalexin accumulation in plant tissue and enhancing
chilling-tolerant maize seedlings after priming with chitosan secondary metabolite yields (Komaraiah et al. 2003;
at three concentrations (0.25, 0.50, and 0.75 %, w/v) (Guan Eilenberg et al. 2010). Ruta graveolens L. accumulated
 K. Xing et al.

various types of secondary metabolites, such as coumarins 4.3.4 Phenolic compounds

and alkaloids; both of them could be regarded as phyto-
alexin and defense tools for plants against pathogenic fungi. Phenylalanine ammonia-lyase is the key enzyme in the
Chitosan induced a severalfold increase in the concentra- phenylpropanoid pathway and is involved in the synthesis of
tions of coumarins and fluoroquinolone alkaloids. Such a phenolic compounds, which are associated with the expres-
dramatic increase suggested that chitosan might be partici- sion of disease resistance (Treutter 2006). Since chitosan
pating in the natural resistance mechanisms of Ruta produced elevated phenylalanine ammonia-lyase activity in
graveolens. The application of chitosan as elicitors may plant, the levels of total phenolic content may also increase
be considered a promising prospect in the biotechnological following chitosan treatments.
production of biologically active phytoalexins and other
secondary metabolites (Orlita et al. 2008). Increase in phenylalanine ammonia-lyase activity on chi-
tosan treatment and subsequent augmentation of total phenolic
contents has been previously reported in soybean leaves.
4.3.2 Lignin Application of chitosan led to elevated activity of phenylala-
nine ammonia-lyase in soybean leaf tissues but markedly
Lignin is closely associated with cellulose and hemicellu- declined at 48 h. It was observed the total phenolic content
lose in hardening and strengthening of plant cell wall was elevated at 60 h in chitosan-treated plants, showing a
(Rajan et al. 2005). Lignification renders the cell wall positive correlation between enzyme activity and total pheno-
more resistant to mechanical pressure during penetration lic content (Romanazzi et al. 2002). In Eurasian traditional
by fungal appressoria as well as more water resistant and medicine Greek oregano, 200 and 500 ppm chitosan oligo-
thus less accessible to cell wall degrading enzymes. Thus, saccharide treatments promoted plant height growth, whereas
it forms a barrier offering protection against microbial and 50 and 200 ppm chitosan oligosaccharide upregulated the
chemical degradation. In the plant-pathogen interaction, the content of polyphenols significantly (38 and 29 %, respective-
lignification of infected plant cell walls is a mechanism ly) (Yin et al. 2012). Chitosan also increased total phenolics in
for disease resistance and provides plants with effective date palm seedlings of two cultivars, Jihel (JHL, susceptible)
protection against pathogens. The synthesis of precursors and Bousthami noire (BSTN, resistant). The highest phenolic
of lignin and phenolic acids having antimicrobial activity levels were recorded at a chitosan concentration of 1 mg/mL
in wheat seeds was stimulated by chitosan treatment. 30 days after incubation, when they were about three times
Chitosan also inhibited fungal transmission to the primary higher than in the control roots (Nicholson and
roots of germinating seedlings. Results suggested that chi- Hammerschmidt 1992). As the major phenolic compound in
tosan controlled seed-borne Fusarium graminearum infec- sweet basil, rosmarinic acid has been reported to have various
tion and increased the resistance in seedlings by stimulat- bioactive properties such as antioxidant, antimicrobial, and
ing the accumulation of phenolics and lignin (Bhaskara anti-inflammatory activities. The total amount of phenolic
Reddy et al. 1999). Treatment of wounded wheat leaves compounds significantly increased after chitosan treatments,
with a partially acetylated chitosan hydrolysate elicited especially rosmarinic acid that increased 2.5 times by 0.1 %
lignification at wound margins and invoked significant chitosan treatment. Therefore, due to the significant induction
increases in phenylalanine ammonia-lyase, peroxidase ex- of phenolic compounds, the corresponding antioxidant activ-
pression, and catalase activities (Mitchell et al. 1994). ity increased at least 3.5-fold (Kim et al. 2005).

Chlorogenic acid, another phenolic compound, is an

4.3.3 Suberization important biosynthetic intermediate, for example in lignin
biosynthesis. Studies showed that chlorogenic acid
Suberization is another common mechanism of cell wall for displayed antibacterial and antifungal activity against cer-
disease resistance in plants. Suberization is a tissue-specific tain microorganisms (Sung and Lee 2010; Hemaiswarya
process, whereby cell walls become impregnated with a poly(- et al. 2011; Atanasova-Penichon et al. 2012). In ginseng
phenolic) matrix coincident with the deposition of a callus cultures, accumulation of phenolic compounds was
poly(aliphatic) matrix between the plasmalemma and carbo- increased 3-fold within 12 h after 1 % chitosan treatment.
hydrate cell wall (Bernards et al. 1999). As a biogenic elicitor, HPLC analysis revealed significantly higher levels of
chitosan locally and systemically stimulated wound healing in chlorogenic acid. Enhanced activity of phenylalanine
potato tuber tissues by increasing the number of wound peri- ammonia-lyase and peroxidase and enhanced levels of phe-
derm layers, accelerating the development of cork cambium nolic compounds, for example, chlorogenic acid, all point
(p he llog en ), a nd ind uc ing pr ote ina se i nhi bito rs to an enhanced defense response in ginseng rusty roots
(Ozeretskovskaia et al. 2009). (Rahman and Punja 2005).
Chitosan antimicrobial and eliciting properties for pest control

4.3.5 Callose (Miya et al. 2007). Petutschnig et al. (2010) suggested that
CERK1 was not only required for chitin but also for chitosan
Callose exists in the cell walls of a wide variety of higher perception. However, a recent work showed that defense
plants. It plays important roles during many processes in plant response genes were upregulated by chitosan, both in wild-
development and in response to numerous biotic and abiotic type and in the chitin-insensitive cerk1 mutant, indicating that
stresses, such as wounding and pathogens infection. Callose- chitosan is perceived through a CERK1-independent pathway
containing cell-wall appositions, called papillae, are effective (Povero et al. 2011).
barriers that are induced at the sites of attack during the
relatively early stages of pathogen invasion (Luna et al. A lectin specific for glucosamine oligomers has been puri-
2011). Chitosan was known to have eliciting activities leading fied by chitosan affinity chromatography from cultured cells
to callose formation in host plants in response to microbial of Rubus. Sodium dodecyl sulfate-polyacrylamide gel
infections (Iriti and Faoro 2008; El-Hadrami et al. 2010; electroploresis (SDS-PAGE) showed that the lectin appeared
Jabeen and Ahmad 2013). After treatments with 0.1 % chito- as a membrane-bound protein of molecular weight 67 kDa
san, tobacco plants significantly reduced tobacco necrosis with two apparent binding sites, i.e., the tetrasaccharide and
virus-induced necrotic lesions and enhanced inducible de- the hexasaccharide, but did not exhibit any affinity for the
fenses, which was associated with a network of callose de- cellotetraose, N-acetylchitotetraose, and maltotetraose. Con-
posits, micro-oxidative bursts, and micro-hypersensitive re- sidering its affinity for chitosan, the lectin may be a receptor
sponses (Bol et al. 1990). In fact, chitosan induced callose for chitosan-derived oligomers with elicitor activity, which
deposition at pathogen entry points during the initial hours of ultimately trigger plant defense reactions (Liénart et al. 1991).
pathogen inoculation (Iriti et al. 2006). The elicited callose
apposition in plant tissues exerted a determinant role in limit- Unfortunately, research papers about the binding protein or
ing microbial spread in the early phase of pathogen infection receptor of chitosan are few. To our knowledge, the lectin is
(Iriti and Faoro 2008). the only receptor discovered that is likely to bind to chitosan.
However, whether there are binding proteins for chitosan on
4.4 Signal transduction other plants remains to be further studied.

During the long-term coevolution, plants and pathogens have 4.4.2 Intracellular signal perception of chitosan
evolved an intricate relationship. Pathogens have developed
an array of offensive strategies to parasitize plants, and in turn, Besides the signal perception via cell surface or membrane
plants have evolved a complex multilayered defense system to receptors, many researchers (Hadwiger et al. 1989; Hadwiger
prevent infection (Nurnberger et al. 2004; Chisholm et al. 1999; Dumas-Gaudot et al. 1996) demonstrated that chitosan
2006). Based on the mechanisms mentioned above and other exhibited internal effects on the plant’s DNA conformation
literatures, chitosan can behave like a general elicitor, and regulated at the chromatin level directly since chitosan
exhibiting a wide variety of defense responses to pathogens entered most regions of the cell. The highly positively charged
infestation, including increases in chitinase and β-1,3- chitosan possessing a strong affinity for the negative charged
glucanase, defense-related enzymes, phytoalexins, and sec- phosphates of the DNA backbone, especially the minor
ondary metabolites by expressing related responsive genes groove of DNA (Liu et al. 2005), may compete with histone
and defense genes. There appears to be multiple modes by proteins containing lower densities of positive charges (Isaac
which chitosan can increase these gene expressions and func- et al. 2009). Chitosan treatments to the pea endocarp tissue
tions, including activating cell surface or membrane receptors resulted in subtle DNA fragmentation of the pea DNA within
and internal effects on the plant’s DNA conformation that 2.5 h, indicating that it can affect DNA in vivo (Hadwiger
influence gene transcription in turn (Hoat et al. 2013). It is et al. 1997). As a pathogenesis-related gene elicitor, chitosan
attractive to elucidate the role of chitosan in plant immunity may alter chromatin via competition with basic nuclear pro-
regulation. teins for DNA attachment sites, potentially displacing H2A/
H2B histones (Hadwiger 2008).
4.4.1 Extracellular signal perception of chitosan
4.4.3 Signal transduction of chitosan with other signal
The first step in the elicitor-induced transduction pathway is molecules
the recognition of the signaling molecule by a specific recep-
tor (Benhamou 1996). In the dicotyledonous model plant When the extracellular signaling molecule chitosan activates
Arabidopsis, chitin elicitor receptor kinase 1 (CERK1), a the specific receptor on the cell membrane or located intracel-
LysM receptor kinase, has been shown to play a critical role lular, one or more second messengers transmit the signal into
in fungal microbe-associated molecular pattern perception the cell and create a series of physiological responses. In the
 K. Xing et al.

above process termed as signal transduction, a single signal list of plant-pathogen interactions (Klüsener et al. 2002;
can be amplified and develop a complex signaling networks. Lamattina et al. 2003; Neill et al. 2003). Chitosan treatment
According to published literatures, reactive oxygen species showed downy mildew disease protection of 79.8 % over the
(ROS), Ca2+, nitric oxide (NO), ethylene (ET), jasmonic acid untreated control and elevated NO accumulation in pearl
(JA), salicylic acid (SA), and abscisic acid (ABA) all involved millet seedlings beginning from 2 h postinoculation. Howev-
in chitosan-mediated signal pathway. er, the degree of protection was reduced after NO scavenger c-
PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-
The oxidative burst, a rapid and transient production of huge 1-oxyl-3-oxide potassium salt] or NO synthase inhibitor L-
amounts of ROS, is one of the earliest responses to microbial NAME (N-nitro-L-arginine methyl ester hydrochloride) treat-
pathogen attack (Wojtaszek 1997) and has been shown to occur ment; this indicated the possible involvement of NO in
upon chitosan elicitation (Luna et al. 2011). The production of chitosan-induced resistance (Hadwiger 2013). In tomato cells,
ROS included hydrogen peroxide (H2O2), superoxide (O2−), chitosan induced a rapid NO production, as well as the for-
hydroxyl radicals (OH−), and so on. As the important signals mation of phosphatidic acid by activating both phospholipase
mediating defense gene activation, ROS are centrally involved D and phospholipase C/diacylglycerol kinase. Pretreatment
in the induction of plant disease resistance responses. H2O2 with NO scavenger c-PTIO inhibited the activation of either
served as a signal of oxidative stress and activation of signaling phospholipase-mediated signaling pathway. This indicated
cascades as a result of the early response of the plant to biotic that NO was required for phosphatidic acid generation via
stress (Mejía-Teniente et al. 2013). In sycamore cultured cells, both the phospholipase D and phospholipase C/diacylglycerol
0.01 % chitosan induced an accumulation of H2O2 reaching kinase pathway during plant defense response in chitosan-
about 50 nmol/g fresh weight after 24 h (Jabeen and Ahmad elicited cells (Tocci et al. 2011).
2013). In Arabidopsis cell suspension cultures, chitosan in-
duced the accumulation of H2O2 within 1 h. The addition of Phytohormones are not only instrumental in regulating
ascorbic acid (a H2O2 scavenger) blocked the formation of the developmental processes in plants but also play important
brown coloration (chemical interactions took place in the pres- roles for the plant’s responses to biotic and abiotic stresses
ence of H2O2) confirming that chitosan induced H2O2 accu- (Halim et al. 2006). For example, disease resistance in
mulation in the Arabidopsis cell cultures (Ndimba et al. 2003). Arabidopsis is regulated by multiple signal transduction
Similar results were obtained in chitosan-treated sweet peppers pathways in which SA, JA, and ET function as key signal-
and tomatoes (Orozco-Cardenas and Ryan 1999; Mejía- ing molecules in mediating or orchestrating biotic/abiotic
Teniente et al. 2013). stress responses. SA is involved in the systemic acquired
resistance in which a pathogenic attack on one part of the
Calcium metabolism is intimately related to ROS signaling. plant induces resistance in other parts, whereas JA and ET
Increase in cytosolic Ca2+ is also one of the fastest responses are central signaling molecules in the induced systemic
upon pathogen infection, and the use of specific inhibitors resistance. JA, the terminal product of the octadecanoid
showed that Ca2+ influx was required for ROS production pathway, has been proposed to be part of a signal trans-
after elicitation (Blume et al. 2000; Grant et al. 2000). It was duction pathway that regulates the induction of defense-
demonstrated that the polycationic nature of chitosan might response genes in plants against pathogen invasion. In rice,
lead to membrane disturbance through its interaction with chitosan caused a rapid increase in the endogenous JA level
negatively charged membrane phospholipids (Shibuya and within 3 min. Furthermore, the rise in JA level by chitosan
Minami 2001). According to published reports, treatments was again significantly higher upon wounding, and reached
that disrupt plasma membrane integrity are often accompanied a peak at 60 min versus 30 min in wounded leaves,
by alterations of cell Ca2+ signaling (Pizzo et al. 2002). In suggesting that this observed increase is a specific response
suspension-cultured cells of Glycine max, synthesis of callose to applied chitosan (Rakwal et al. 2002). An oilseed rape
started within 20 min of treatment with chitosan and parallels cDNA microarray containing 8,095 expressed sequence
over hours of the accumulation of 1,3-linked glucose in the tags was used to analyze the Brassica napus gene expres-
wall. However, chitosan-induced callose formation was not sion changes elicited by oligochitosan. Transcript levels for
possible without the presence of external Ca2+ and partly 136 genes were induced 2-fold or more in oligochitosan-
recovered upon restoration of 15 μM Ca2+ (Köhle et al. treated seedlings compared with control seedlings. Results
1985). In Arabidopsis, chitosan induced transient elevations of semiquantification RT-PCR showed that an important JA
in the concentration of free cytosolic Ca2+ and stomatal clo- synthase gene, a JA-mediated defense required for kinase
sure in guard cells (Klüsener et al. 2002). gene, an ET receptor gene, and two ET responsive element
binding protein genes were induced by oligochitosan, sug-
NO, another second messenger recently established in gesting that oligochitosan activated the plant self-defense
plants, is involved in the plant defense response of a growing through JA/ET signaling pathway (Yin et al. 2006).
Chitosan antimicrobial and eliciting properties for pest control

Recently, in a series of plant pathosystems, it has been antimicrobial mechanisms. Combined transcriptome and pro-
shown that the intensity and speed of callose deposition are teome analysis of key defense genes and proteins will enhance
regulated by ABA. ABA, also called abscisin II and dormin, is our understanding of the complicated chitosan-mediated sig-
now known to be the case only in a small number of plants. nal pathway and enable better biotechnological applications in
ABA-mediated signaling transduction also plays an important plant disease control. A wider comprehensive knowledge of
role in plant responses to environmental stress and plant the mechanism of action of chitosan in pathogens and plants
pathogens (Seo and Koshiba 2002). Chitosan treatment re- will increase the chance of its successful application to control
duced tobacco necrosis virus lesion area per leaf by 95.2 % in disease spread in plants. We also suggest comprehensive
respect to untreated controls. Furthermore, chitosan applica- cooperation among global chemists, microbiologists,
tion elicited both callose apposition and ABA accumulation in phytophysiologists, and agronomists to better exploit
leaf tissues, at 12 and 24 h after treatment, respectively. Be- chitosan’s antimicrobial properties, plant innate immunity
sides, treatment with the ABA inhibitor nordihydroguaiaretic elicited activity, and biotechnological potential for agricultural
acid, before chitosan application, reduced both callose depo- sustainable development.
sition and plant resistance to the virus, thus indicating the
involvement of ABA in chitosan-mediated processes. It was Acknowledgments This work was supported by grants from the Na-
indicated that the increase of ABA synthesis induced by tional Natural Science Foundation of China (31101502, 31370062), the
chitosan played an important role in enhancing callose depo- Program of Natural Science Foundation of the Jiangsu Higher Education
sition (Iriti and Faoro 2009). Institutions of China (11KJD210002), Qing Lan Project of Jiangsu
Province (2014), and the Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions (PAPD).
Based on the above analysis, chitosan activated the plant
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