Chitosan as a potential natural compound to control

CHITOSAN AS A POTENTIAL NATURAL

COMPOUND TO MANAGE POST HARVEST

DISEASES OF HORTICULTURAL CROPS

Submitted By :

Puja Pandey

41124

Statistics of post-harvest losses in India……

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.

Preparation of

chitosan??????????

Fig.1. Schematic presentation of Chitosan productionAbu Bakr Siddique , 2010

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

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

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)



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 defenceresponse in preharveststudies

• Biochemical defenceresponse in postharvest studies

Effect of chitosan on pre and postharvest disease

• Control of bacterial , fungal and viral diseases

• Control of postharvest diseases


Chitosan and “the renaissance of elicitors”

chitosan has currently been labeled as a “PAMP”

A chitosan-binding protein with lectinactivity has been isolated from non-heading

Chinese cabbage leaves


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

(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.

(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.

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).

Badawy and Rabea, 2009


Case

studies………….

El Ghaouth et al ., 1997

(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

polygalacturonic acid.


(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

(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

localization of cellulosic b-1,4-glucan. El Ghaouth et al ., 1997

(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

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.

Effects of chitosan on control of postharvest diseases

and physiological responses of tomato fruit

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

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

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

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

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 deviations

of the means.

Liu et al., 2007

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

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

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.


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.


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

Romanazzi et al., 2013


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


Chitinase activity detected in strawberry fruits

after separation in two dimensional gel

electrophoresis


Chitosan protects Vitis vinifera L. against Botrytis

cinerea

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

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

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

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

Fig. Zero order kinetics for decay of strawberry fruit

sprayed with chitosan before harvest and stored at 3°C

Bhaskara et al ., 2000

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

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

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

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

Blackmold rot development in chitosan-treated and

control tomato fruit. Bhaskara et al., 2000

Changes in rishitin content of tomato fruit

Chitosa + A. alternata

A. alternata

Chitosan

Bhaskara et al., 2000

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


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.


Whether chitosan to be

preferred or not…..

Elicitor

Barrier

Sticker

Increase yields

CONCLUSION

Science

Chitosan as a potential natural compound to control