4. Chitosan : Definition
Chitosan, deacetylated chitin, is currently obtained from the outer shell of
crustaceans such as crabs, krills and shrimps.
Chitin and chitosan are polysaccharides, chemically similar to cellulose differing
only by the presence or absence of nitrogen. Chitosan is a low acetyl form of chitin
mainly composed of glucosamine, 2-amino-2-deoxy-ββ-d-glucose.
The positive charge of chitosan confers to this polymer numerous and unique
physiological and biological properties with great potential in a wide range of
industries.
Chemical structure of
chitosan.
7. Related history
Dates back to the
1980s
1989 : Against
freezing stress
The Mir space station September 1997 :
chitosan induces increased biomass and
pathogen resistance due to elevated levels of
beta 1-3 glucanase enzymes within plant cells.
2008 : Elicitor
2009 : Foliar spray
8. Mechanism of chitosan action
Direct effects of chitosan oligomers on defense gene transmission are
proposed to occur by :
Alterations of DNA helical structure, single strand cleavage and removal of
histones H2A and H2B
Chitosan may compete with histones for sensitive DNA sites allowing
stalled DNA polymerase complexes to continue to transcribe through the
open reading frames of PR genes.
Lee A. Hadwiger, 2013
9. Summation of proposed chitosan roles in plant defense
Some chitosan points of origin and their proposed effects on the
regulation of plant defense genes (PR genes)
Lee A. Hadwiger, 2013
11. Multiple effects of chitosan on plant defense system
systems
Plant cellular
responses to
chitosan
• Activationof MAP-
kinases
• Oxidative burst
• callose apposition
• PR protein synthesis
• Hypersensitive
response
Chitosan and “the
renaissance of
elicitors”
• Biochemical defence
response in preharvest
studies
• Biochemical defence
response in
postharvest studies
Effect of chitosan
on pre and
postharvest disease
• Control of bacterial ,
fungal and viral
diseases
• Control of postharvest
diseases
Lee A. Hadwiger, 2013
12. Chitosan and “the renaissance of elicitors”
chitosan has currently been labeled as a
“PAMP”
A chitosan-binding protein with lectin
activity has been isolated from non-heading
Chinese cabbage leaves
Lee A. Hadwiger, 2013
13. Oligochitosan : A plant diseases vaccine
Fig. The effect of oligochitosan on TMV control
(A) control; 1 ppm, 10 ppm
25 ppm 50 ppm 100 ppm
14. (A) The cells loaded with H2DCF-DA.
(B) Bright field image of the cells loaded
with H2DCF-DA.
(C) The cells loaded with H2DCF-DA
before treatment with oligochitosan.
(D) Bright field image of the cells loaded
with H2DCF-DA before treatment with
oligochitosan.
(E) The cells loaded with H2DCF-DA and
elicited by oligochitosan in the presence
of the CAT.
(F) Bright field image of the cells loaded
with H2DCF-DA and elicited by
oligochitosan in the presence of the CAT.
(G) The cells loaded with H2DCF-DA and
elicited by oligochitosan in the presence
of the DPI.
(H) Bright field image of the cells loaded
with H2DCF-DA and elicited by
oligochitosan in the presence of the DPI.
Laser scanning confocal microscopy of
oligochitosan-induced production of H2O2
in epidermal cells of tobacco leaf.
15. (A) The cells loaded with DAF-2 DA.
(B) Bright field image of the cells loaded with
DAF-2 DA.
(C) The cells loaded with DAF-2 DA before
treatment with oligochitosan.
(D) Bright field image of the cells loaded with
DAF-2 DA before treatment with oligochitosan.
(E) The cells loaded with DAF-2DA and elicited by
oligochitosan in the presence of the CPTIO.
(F) Bright field image of the cells loaded with
DAF-2DA and elicited by oligochitosan in the
presence of the CPTIO.
(G) The cells loaded with DAF-2DA and elicited by
oligochitosan in the presence of the l-NAME.
(H) Bright field image of the cells loaded with
DAF-2DA and elicited by oligochitosan in the
presence of the l-NAME.
Laser scanning confocal microscopy
of oligochitosan-induced production of
NO in epidermal cells of tobacco leaf.
16. In this study high molecular weight chitosan had the lowest inhibitory effect on the
fungus tested.
Contrary to these results, the inhibitory effect on mycelial growth of Fusarium
oxysporum f. sp. vasinfectum, and Alternaria solani occurred when these fungi grew
on media with high molecular weight chitosan (2.0×105 g/mol) (Guo et al., 2006).
21. (a) Non-inoculated control; labelling is
evenly distributed over host
primary cell walls and middle
lamella matrices.
(b) Pathogen growth within the host
wall was followed by the disruption of
wall fibrillar structure. The swollen host
wall
appeared almost free from labelling .
(c,d) The host cell wall facing invading
hypha is almost free from labelling.
Very few scattered gold particles could
be detected over the altered walls.
Note the association of gold particles
with the
innermost unaltered wall layer (c,
arrow).
The host wall in contact with fungal
cell
(c) displayed a loosened fibrillar
structure
Fig. 1. Transmission electron micrographs of
untreated bell pepper tissue 72 h after
inoculation with Botrytis cinerea. Labelling is
with Aplysia gonad lectin (AGL)-gold complex
for the localization of molecules containing El Ghaouth et al ., 1997
22. (a) Fungal cells in the ruptured
epidermal cell layer displayed
severe cellular alterations.
(b) (b–d) The host wall in the
proximity of (b and d) or
appressed (c) against severely
altered fungal cells appeared
well preserved and showed no
apparent sign of disintegration.
(c) Labelling was light and
scattered over host walls.
Fig. 2. Transmission electron micrographs
of chitosan-treated bell pepper tissue 72 h
after inoculation with B. cinerea. Labelling
is
with the gold complexed-AGL. El Ghaouth et al ., 1997
23. (a) Non-inoculated control;
labelling was evenly
and abundantly distributed over
the host cell wall.
The host wall in contact (b) or
penetrated by fungal cell (c)
displayed a complete disruption of
cellulose labelling pattern
(arrows).
(d) Intramural growth of the
pathogen caused the
disintegration of the wall into
a network of labelled fibrils. Some
areas of the walls were almost
free from labelling (arrow).Fig. 3. Transmission electron micrographs
of untreated bell pepper tissue 72 h after
inoculation with Botrytis cinerea. Labelling
is
with the gold complexed exoglucanase for El Ghaouth et al ., 1997
24. (a) The host wall in the proximity of a
severely altered hyphal cell appeared well
preserved
and showed no apparent sign of
disintegration.
(b) A higher magnification of (a) A slight
reduction in labelling intensity is noticeable
over the outermost wall portion
facing fungal cells.
The host walls in close contact with a
highly altered (c) and normal (d) invading
fungal cell
displayed an intense cellulose labelling.
Fig. 4. Transmission electron micrographs
of chitosan-treated bell pepper tissue 72 h
after inoculation with B. cinerea. Labelling
is
with the gold-complexed exoglucanase. El Ghaouth et al ., 1997
25. Transmission electron micrographs of
B. cinerea cells in chitosan-treated and
non-treated bell pepper fruit tissue.
Fungal cells show various degree of
alterations that range from:
a) cell wall loosening
b) Vacuolation
c) cytoplasm retraction followed by
deposition of material in the paramural
spaces
d) In the control tissue, fungal cells are
delimited by a thin wall.
26. Effects of chitosan on control of postharvest diseases
and physiological responses of tomato fruit
27. Effects of chitosan on spore germination of B. cinerea
and P. expansum in vitro
Fig. 1. Effects of chitosan concentration on spore germination (A) and germ
tube elongation (B) of Botrytis cinerea and Penicillium expansum 12 h after
incubation at 25 °C. Bars represent standard deviations of the means.
Values followed by different letters are significantly different according to
Duncan's multiple range test at P < 0.05.
Liu et al., 2007
28. Effects of chitosan on mycelial growth of B. cinerea
and P. expansum in vitro
Fig. 2. Effects of chitosan concentration on mycelial growth of B. cinerea and P.
expansum 3 days after incubation at 25 °C. Bars represent standard deviations
of the means. Values followed by different letters are significantly different
according to Duncan's multiple range test at P < 0.05.
Liu et al., 2007
29. Effects of chitosan on plasma membrane integrity of
the spores
Fig. 3. Effects of chitosan on plasma membrane integrity of the spores of B.
cinerea (A) and P. expansum (B). Pathogen spores were cultured in PDB
containing 0.5% chitosan or in PDB without chitosan as the control at 25 °C.
Bars represent standard deviations of the means
Liu et al., 2007
30. Effects of chitosan on postharvest diseases of
tomato fruit
Fig. 4. Effects of chitosan on postharvest diseases caused by B. cinerea and
P. expansium in tomato fruit stored 3 days at 25 °C (A) and 21 days at 2 °C
(B). Bars represent standard deviations of the means. Values followed by
different letters are significantly different according to Duncan's multiple range
test at P < 0.05.
Liu et al., 2007
31. Elicitation of the enzyme activities and phenolic
compounds by chitosan treatment
Fig. 5. Changes of PPO (A and B), POD activities (C and D), and phenolic
compounds (E and F) in tomato fruit. Fruit were treated with 1% chitosan, and stored
at 25 (A, C and E) and 2 °C (B, D and F), respectively. Fruit wounded and treated
with water, and non-wounded served as controls. Bars represent standard deviationsLiu et al., 2007
32. Effect of chitosan coating combined with postharvest calcium
treatment on strawberry (Fragaria ananassa) quality during
refrigerated storage
Parameters studied : -
1) Loss of fruit due to visible
fungal growth .
2) Respiration rate of fruits.
3) Weight loss of fruits.
Mun˜oz et al., 2008
33. Loss of fruit due to visible fungal growth
Fig. 1. Percentage of infected strawberries as a function of storage
time at
10 C for control and 1% CS coated samples. Vertical bars indicate
standard
deviation. Mun˜oz et al., 2008
34. Respiration rate
Fig. 2. Respiration rate of control and chitosan-coated strawberries as a
function of storage time at 10 C. Vertical bars indicate standard
deviation.
Mun˜oz et al., 2008
35. Weight loss of fruits
Fig. 3. Loss of weight of strawberries as a function of storage time at
10 C. Vertical bars indicate standard deviation.
Mun˜oz et al., 2008
36. Evaluation of the effectiveness of following natural
compounds and resistance inducers:
Treatments Gray mold Blue mold Rhizopus rot
Commercial
chitosan
79%, 90% 84%
Benzothiadiazole 73% 84% -
Calcium with
organic acids
70% 71% 79%
Romanazzi et al., 2013
40. Chitinase activities detected in
strawberry fruits after sodium
dodecyl sulphate – polyacrylamide
gel electrophoresis
Extracts of cut strawberries
treated
With :
1) Water : stored for 12 and 48 h
2) Chitosan : stored for 12 and 48
h
Subjected to SDS – PAGE
containing glycol chitin as a
substrate for chitinase activity.
Chitinase activity was
visualized by staining with
Calcoflour white M2R
El Ghaouth et al ., 1997
41. Chitinase activity detected in strawberry fruits
after separation in two dimensional gel
electrophoresis
El Ghaouth et al ., 1997
43. a) Control PDA
plates, b–f chitosan-
supplemented PDA
plates (v/v):
b) 0.5%
c )1%
d )1.75%
e) 2.5%
f) 5%.
Fig. Effect of chitosan on the radial growth
of Botrytis cinerea.
Fungal growth decreased as chitosan
concentration increased Barka et al., 2004
44. Fig. 4a–f Microscopic structural changes in B.
cinerea mycelium
in response to chitosan.
a-b : Control mycelium
c–f : mycelium
sampled from cultures
grown on PDA
supplemented
with 1.75% (v/v)
chitosan.
Both small and large
vesicles appeared in the
mycelium
as result of chitosan.
In other cases, the
cytoplasm is devoid of
any
organelle.
Barka et al., 2004
45. a. Uninoculated control b. Plant challenged with B. cinereac. Plant leaves were sprayed
with chitosan before
inoculation with B. cinerea
d. Plant growing on chitosan amended
medium challenged with the fungus
e. plant growing on
chitosan-amended medium.
Fig. 5a–e
Phytopathogenicity
assay of B. cinerea on
grapevine plants.
Barka et al., 2004
46.
47. Fig. Effect of pre-harvest chitosan spray treatments on the decay of
strawberry fruit stored at 3 and 13°C.
Bhaskara et al ., 2000
Control
2 g/l
4 g/l
6 g l
48. Fig. Zero order kinetics for decay of strawberry fruit
sprayed with chitosan before harvest and stored at 3°C
Bhaskara et al ., 2000
49.
50. Effect of chitosan and oligochitosan with different
concentrations on decay incidence caused by A.
kikuchiana and P. piricola in pear stored at 25⁰C.
Meng et al. , 2010
51. Effects of chitosan or oligochitosan on lesion growth of pear fruit
caused by A. kikuchiana (A and B) and P. piricola (C and D) at 96
and 120 h after inoculation
Meng et al ., 2010
52. Table : IC50 of chitosan or oligochitosan on disease
incidence caused by A. kikuchiana and P. piricola in pear
fruit.
IC50 : half maximal inhibitory
concentration Meng et al. , 2010
53. Fig. Effects of chitosan or oligochitosan on activities of
POD (A), PPO (B), CHI (C) and b-1,3-glucanase (D) of
pear fruit.
Meng et al. , 2010
55. Changes in rishitin content of tomato fruit
Chitosa + A. alternata
A. alternata
Chitosan
Bhaskara et al., 200
56. Recent innovative uses of chitosan in crop/product protection
An ideal alternative to chemically synthesized pesticides.
It both reduced the growth of decay and induced resistance in the host tissue.
Help protect the safety of edible products.
Chitosan protection by exclusion occurs with soybean seed treatments.
Protection from insects such as agarotis, ypsilon, soybean pod borer, and
soybean aphids.
The chitosan treatment developed an antifeedant rate of greater than 80% against
all these insects.
The treatment was accompanied by increases in seed germination, plant growth
and soybean yield.
This chitosan application fulfilled the major objective of replacing high toxicity
pesticides
Lee A. Hadwiger, 2013
57. Environmental friendly
Use in wound healing
Weight control nutrient
Carrier for pharmaceuticals.
Approved as an additive to pesticides as a “sticker” (adjuvant) by
the National Organic Program (NOP).
The United States Environmental Protection Agency (EPA) has
reported chitosan indicating that chitosan use : no adverse effects
within the environment.
Lee A. Hadwiger, 2013
58. Whether chitosan to be
preferred or not…..
Elicitor
Barrier
Sticker
Increase
yields
cosmetology (lotions, hair additives, facial and body creams)
food (coating, preservative, antioxidant, antimicrobial)
biotechnology (chelator, emulsifier, flocculent)
pharmacology and medicine (fibers, fabrics, drugs, membranes, artificial organs) and
agriculture (soil modifier, films, fungicide, elicitor)
1997 NASA : chitosan induces increased biomass and pathogen resistance due to elevated levels of beta 1-3 glucanase enzymes within plant cells.
Chitosan is one of the most abundant carbohydrate biopolymers in the world. Oligochitosan prepared from chitosan is a potent plant immunity regulator. The efficacy of oligochitosan on plant disease control is presented in this review. This paper summarizes recent progress made on oligochitosan activated plant innate immunity, including: signal perception; signal transduction; oligochitosan response genes and proteins; oligochitosan induced defense-related secondary metabolites accumulation. Based on published papers and our former results, we deduce that the mode of oligochitosan act on plant is similar with general vaccines act on human and animals. So we conclude that oligochitosan is a plant disease vaccine.
NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems.
Bars0.5 and 0.25 mm. Abbreviations: AM, amorphous material; Cy, cytoplasm; F, fungal cell; FN, fibrillar network; FS,
fungal shell; FW, fungal wall; HCW, host cell wall; IS, intercellular space; M, mitochondria; ML, middle lamella; PM, plasma
membrane; S, septum; Va, vacuole; Ve, vesicle
(b–d) Inoculated control.
(b–d) Inoculated control. Note the pronounced alteration and the
swelling of the cell wall.
Note the intense and regular cellulose labelling over host walls facing the fungal cell.
The antifungal activity of chitosan at different concentrations on spore germination and germ tube elongation of B. cinerea and P. expansum is shown in Fig. 1. Spore germination of P. expansum was significantly inhibited by chitosan at all concentrations, while that of B. cinerea was significantly inhibited only when the concentration of chitosan was higher than 0.01% (P < 0.05). Chitosan completely inhibited spore germination of P. expansum at 0.5% and B. cinerea at 1%. Germ tube elongation of both pathogens was significantly inhibited when the chitosan concentration was higher than 0.01% (P < 0.05).
As shown in Fig. 2, chitosan at different concentrations markedly inhibited mycelial growth of B. cinerea and P. expansum, with the greater inhibitory effects at the higher concentration (P < 0.05). The mycelial growth of B. cinerea was more sensitive to chitosan as compared to that of P. expansum. The mycelial growth of B. cinerea was completely inhibited by chitosan at 0.5%, while the colony diameter of P. expansum was 13.1 mm at the same concentration.
The results in Fig. 4 indicate that chitosan at 0.5 and 1% could significantly decrease gray mould and blue mould caused by B. cinerea and P. expansum in tomato fruit stored at 25 and 2 °C, respectively (P < 0.05). Control effects of chitosan on both diseases were enhanced when the concentration of chitosan increased from 0.5 to 1%. In addition, gray mould was better controlled than blue mould in tomato fruit treated with chitosan at both temperatures.
The research was carried out on the strawberry cultivar ‘Camarosa’, where fruits were produced according to the Organic Farming. Fruits were selected for the absence of defects, uniformity in size, and with ripening degree of 2/3 red on the surface
Results showed that there were no significant differences among practical grade chitosan dissolved in the acid solutions and the water-soluble commercial chitosan formulation, all treatments with chitosan resulted effective in the control of gray mold and Rhizopus rot in strawberries stored at 20±1°C for 3 days. In general, treatment with chitosan reduced the gray mold infection from 73 to 61% and the Rhizopus rot infection from 88 to 77% according to the solution. Good results were obtained with chitosan dissolved in acetic acid, chitosan dissolved in formic acid, and commercial chitosan formulation. However, the practical grade chitosan dissolved in acid solution and the commercial chitosan formulation differ profoundly in their preparation technique. The chitosan dissolved in acid solution must be prepared 2 days before application to monitor and adjust the solution pH, while the commercial chitosan formulation can be prepared 2 hours before application, just by dissolving the powder in water. For this reason, the water-soluble commercial chitosan formulation, being applied more easily in the field, satisfies better the feasibility request of growers.
Consequently, the possibility of stimulating
internal plant defenses has become an interesting option
for enhancing natural disease resistance. Several lines of evidence have shown that activation of
the natural plant defense systems could occur upon
exogenous applications of chitin and chitosan oligosaccharides
(Benhamou 1996). Similarly to what has been
observed for plants growing on chitogel-amended medium,
exogenous foliar applications of chitogel sensitize
grapevines to react efficiently to B. cinerea attack as
compared to water-treated plantlets
Alternaria
kikuchiana Tanaka and Physalospora piricola Nose are two kinds of
fungal pathogens of pear fruit in storage. Using these two fungi
as subject, this study aimed at the difference between chitosan
and oligochitosan (1) as fungicides by comparison their half maximal
inhibitory concentration (IC50) on spore germination, germ
tube elongation and mycelial growth; (2) as elicitor by comparison
the inductive effect on the activities of related enzymes of host; (3)
as preservatories on decay control of pear fruit.
Moreover, the disease control effects
of chitosan and oligochitosan were concentration-dependent and
weakened over inoculated time. The IC50 of chitosan on disease
incidence caused by A. kikuchiana was 1.32 g/L after inoculation
72 h, 7.94 g/L after inoculation 96 h and 10.7 g/L after inoculation
120 h, meanwhile the IC50 of oligochitosan was 1.75 g/L after inoculation
72 h and 10 g/L after inoculation 96 h (Table 3). The black
spot disease already appeared on all oligochitosan-treated pear
fruit after inoculation 120 h. Relatively, the IC50 of chitosan and
oligochitosan on disease incidence caused by P. piricola was respectively,
1.57 and 7.47 g/L after inoculation 96 h, while 2.05 and
8.79 g/L after inoculation 120 h (Table 3). Therefore, compare to
oligochitosan, chitosan was more effective on disease control,
especially to that caused by P. piricola.
Peroxidase and polyphenol oxidase were two kinds of redox enzymes.
The activities of PPO and POD in flesh around wound of
pear fruit were raised first and then decreased from 24 h (Fig. 4A
and B). However, compare to oligochitosan, chitosan inoculation
significantly enhanced the activities of POD and PPO in flesh
around wound of pear fruit, especially to POD (Fig. 4A and B).
Chitinase and b-1,3-glucanase are two kinds of pathogenesis-related
protein. The CHI and b-1,3-glucanase activities in flesh
around wound of control pear fruit increased with storage time,
while chitosan and oligochitosan inoculation obviously enhanced
the activities of both enzymes (Fig. 4C and D). Furthermore,
oligochitosan inoculation was more effective than chitosan, for
example, the activities of CHI and b-1,3-glucanase in oligochitosan-
treated fruit after 48 h were almost 1.5-fold to those in control
fruit.
The safety of chitosan to the environment, the agricultural
worker and the consumer should not be questioned
Because of the biological, physical and chemical properties discussed above, chitosan is not readily identified as a polymer with a single function
