Effect of pre-harvest chitosan sprays on post-harvest infection by Botrytis cinerea quality of strawberry fruit

Abstract

The effect of pre-harvest sprays of chitosan on post-harvest decay and quality of strawberries stored at 3 and 13°C was investigated. Strawberry plants were sprayed with 2, 4 and 6 g l−1, chitosan solutions as the fruit were turning red. A second spray was performed after 10 days. Fruit were picked 5 and 10 days after each spray. Harvested fruit from chitosan sprayed plants were challenged with Botrytis cinerea. Chitosan sprays significantly reduced post-harvest fungal rot and maintained the keeping quality of the fruit compared with control. The incidence of decay decreased with increased chitosan concentration and increased with storage period and temperature. The second spray of chitosan extended the protective effect against decay of fruit from subsequent picks. Fruit from chitosan sprayed plants were firmer and ripened at a slower rate as indicated by anthocyanin content and titratable acidity than berries from non-treated plants. Chitosan sprays were not phytotoxic at all the concentrations tested. Chitosan sprays at 6 g l−1 concentration performed twice, 10 days apart, protected the fruit from decay and kept the fruit quality at an acceptable level throughout the storage period of 4 weeks in fruit stored at 3°C. The protective effect of chitosan sprays was more pronounced for fruit from pick 1 than pick 2. Kinetic data on decay and ripening characteristics provided quantitative evidence that chitosan compensates for higher storage temperature and protects against deterioration of lower quality fruit from the second harvest.

Full text

Postharvest Biology and Technology 20 (2000) 39–51
Effect of pre-harvest chitosan sprays on post-harvest
infection by Botrytis cinerea and quality of strawberry fruit
M.V. Bhaskara Reddy, Khaled Belkacemi, Ronan Corcuff,
Franc¸ois Castaigne, Joseph Arul *
Department of Food Science and Nutrition and Horticultural Research Center,La6al Uni6ersity,Sainte-Foy,
Que., Canada G
1
K
7
P
4
Received 15 December 1999; accepted 23 April 2000
Abstract
The effect of pre-harvest sprays of chitosan on post-harvest decay and quality of strawberries stored at 3 and 13°C
was investigated. Strawberry plants were sprayed with 2, 4 and6gl
−1
, chitosan solutions as the fruit were turning
red. A second spray was performed after 10 days. Fruit were picked 5 and 10 days after each spray. Harvested fruit
from chitosan sprayed plants were challenged with Botrytis cinerea. Chitosan sprays significantly reduced post-harvest
fungal rot and maintained the keeping quality of the fruit compared with control. The incidence of decay decreased
with increased chitosan concentration and increased with storage period and temperature. The second spray of
chitosan extended the protective effect against decay of fruit from subsequent picks. Fruit from chitosan sprayed
plants were firmer and ripened at a slower rate as indicated by anthocyanin content and titratable acidity than berries
from non-treated plants. Chitosan sprays were not phytotoxic at all the concentrations tested. Chitosan sprays at 6
gl
−1
concentration performed twice, 10 days apart, protected the fruit from decay and kept the fruit quality at an
acceptable level throughout the storage period of 4 weeks in fruit stored at 3°C. The protective effect of chitosan
sprays was more pronounced for fruit from pick 1 than pick 2. Kinetic data on decay and ripening characteristics
provided quantitative evidence that chitosan compensates for higher storage temperature and protects against
deterioration of lower quality fruit from the second harvest. © 2000 Elsevier Science B.V. All rights reserved.
Keywords
:
Strawberry; Chitosan; Pre-harvest spray; Botrytis cinerea; Post-harvest quality; Decay and ripening kinetics
www.elsevier.com/locate/postharvbio
1. Introduction
Strawberries (Fragaria ananassa Duchesne) are
especially perishable fruit, being susceptible to
mechanical injury, desiccation, decay and physio-
logical disorders during storage. Botrytis cinerea
and Rhizopus sp. are the two major storage patho-
gens of strawberry (Barkai-Golan, 1981; Mass,
1981), and the infection of fruit by B.cinerea can
be traced to the infection of floral parts in the
field (Powelson, 1960) or by contact with rotting
berries. Control of Botrytis during storage can be
* Corresponding author. Tel.: +1-418-6562839; fax: +1-
418-6563353.
E-mail address
:
joseph.arul@aln.ulaval.ca (J. Arul).
0925-5214/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0925-5214(00)00108-3

M.V.Bhaskara Reddy et al.
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Posthar6est Biology and Technology
20 (2000) 39–51
40
achieved by physical and chemical methods.
Modified atmospheres with elevated CO
2
levels and
low temperatures are effective in reducing the
incidence of decay (Mass, 1981; Reyes and Smith,
1986; Brown, 1992). However, prolonged exposure
of berries to high CO
2
concentrations can cause off-
flavor development (Li and Kader, 1989; Ke et al.,
1994) and low temperature alone is not an effective
means of control (Brown, 1992). Although prophy-
lactic field sprays with systemic benzimidazoles are
effective in controlling post-harvest fungal infec-
tions (Dennis, 1975), there is an increased concern
among consumers about the potentially harmful
health effects of chemical residues (Klein and Lurie,
1991), and development of chemical tolerance in
post-harvest pathogens (Spotts and Cervantes,
1986). Thus alternative approaches are necessary to
maintain the marketable quality of strawberries.
Chitosan, a high molecular weight b-(1,4)-glu-
cosamine polymer, is an important structural com-
ponent of the cell wall of some plant-pathogenic
fungi, especially Zygomycetes (Bartnicki-Garcia,
1970). It is also produced from the chitin compo-
nents of arthropod exoskeletons; by deacetylation.
Chitosan has been shown to have anti-fungal
activity against a wide range of fungi (El Ghaouth et
al., 1992a,b). Chitosan coating of harvested straw-
berries protected them from infection by B.cinerea
and improved their quality (El Ghaouth et al.,
1991a). Chitosan coating also reduced the incidence
of decay of tomato, bell pepper and cucumber,
delayed ripening of apple and tomato fruits and
reduced the desiccation of pepper and cucumber (El
Ghaouth et al., 1991c; Davis and Elson, 1994).
Chitosan-induced glucanohydrolase activity in
strawberries was inhibitory against B.cinerea (El
Ghaouth et al., 1991b). The present study reports
the effects of prophylactic pre-harvest chitosan
sprays applied at intervals on the decay and keeping
quality of harvested strawberries stored at 3 and
13°C.
2. Materials and methods
2
.
1
.Culti6ar
Runner roots of the cultivar Seascape were
transplanted to 30 cm diameter plastic pots
(three roots per pot) filled with potting mixture
containing four parts by volume of pasteurized
organic soil, one part of peatmoss and one part
of perlite. The pots were randomized on green
house benches, all within one large chamber.
Plants were maintained at 25°C 12/12 h, light/
dark cycles and watered at regular intervals. At
the time of flowering plants were agitated
to simulate bee transfer of the pollen for opti-
mum fruit setting. A total of 63 plants per treat-
ment in three replications of 21 each were
maintained in a randomized complete block de-
sign (RCBD).
2
.
2
.Chitosan sprays
Shrimp-shell chitosan was purchased from
Nova-Chem Ltd. (Dartmouth, Nova Scotia,
Canada) and ground into a fine powder. The
purified chitosan was prepared by dissolving chi-
tosan in 0.25 N HCl, and the undissolved parti-
cles were removed by centrifugation (15 min,
10 000×gat 24°C). The solution was neutral-
ized with 2.5 N NaOH to a pH of 8.0 to pre-
cipitate the chitosan. The precipitated chitosan
was recovered by filtration, washed extensively
with deionized water to remove salts and was
subsequently lyophilized. Chitosan stock solution
(10gl
−1
) was prepared by dissolving chitosan
in 0.05 N HCl and pH was adjusted to 5.6.
Different chitosan concentrations of 2, 4, and 6 g
l
−1
were prepared in water. Plants were sprayed
with chitosan solution from a hand sprayer when
the fruit were just turning red. The spray was
continued until the deposition of chitosan
droplets was uniform on the fruit surface. A set
of plants was also sprayed with sterile water as
control. Sprays were repeated after an interval of
10 days.
2
.
3
.Har6esting and storage
Fruit were harvested 5 and 10 days after each
spray (designated as pick 1 and pick 2, respec-
tively). Berries of uniform size, free of physical
damage and fungal infection were selected. For
quality evaluation, 30 fruit were randomly dis-

M.V.Bhaskara Reddy et al.
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Posthar6est Biology and Technology
20 (2000) 39–51
41
tributed into replicates of 10 fruit for each treat-
ment, temperature, spray, pick and storage inter-
val.
2
.
4
.Inoculation of chitosan sprayed fruits
B.cinerea was isolated from infected strawber-
ries and maintained on potato dextrose agar
(PDA). Conidia of B.cinerea were recovered by
flooding 2-week-old cultures with sterile water
containing 0.1% (v/v) Tween 80 and filtering the
mycelial suspension through three layers of ster-
ile cheese cloth. The concentration of the
conidial suspension was adjusted to 2×10
5
conidia per ml. Berries to be inoculated were
transferred to plastic containers (in three repli-
cates of ten each) with three layers of moistened
blotters at the bottom. Fruit were inoculated in-
dividually with 20 ml of conidial suspension. The
boxes were closed with perforated lids and subse-
quently transferred from ambient temperat-
ure to 3 or 13°C for storage. Strawberries were
evaluated weekly for disease symptoms, and
spoiled fruit were discarded to avoid secondary
infection.
2
.
5
.Assessment of quality
The effect of pre-harvest chitosan sprays on
post-harvest quality of strawberries was assessed
each week. A sample of seven to nine berries was
randomly removed from each replicate and ana-
lyzed for firmness, titratable acidity and an-
thocyanin content. For firmness, berries were
sliced into halves and each half was punch tested
on a texture analyzer using a 4-mm flat plunger
(Texture Technologies Corp., Scarsdale, NY).
Acidity was determined using a 10 g aliquot of
puree in 40 ml of deionized water and titrating
with 0.1 N NaOH to an end point of pH 8.1.
Titratable acidity was expressed as g of citric
acid per l. Anthocyanins were extracted with aci-
dified ethanol froma2galiquot of homogenate
according to the method of Fuleki and Francis
(1968). Anthocyanin content was expressed as
mg anthocyanin per g fresh weight of strawberry
homogenate.
2
.
6
.Experimental design and analysis
The experiment was a completely randomized
4×2×2×4 factorial design. The factors were
chitosan concentration, number of sprays, num-
ber of picks and storage interval. The experiment
was conducted twice, and within each repetition
the treatment order was random. Analysis was
carried out with triplicate data from each repeti-
tion. Data were pooled across number of sprays
and picks (2 sprays×2 picks) and S.E.M. were
determined.
2
.
7
.Kinetics of fruit quality deterioration/change
Shelf life based on a specific criterion of qual-
ity acceptability at a specified environmental con-
dition can be estimated from kinetic models
(Labuza, 1982):
−dq
dt=k[q]
n
(1)
where [q] is any quality characteristic, tthe time,
kthe rate constant, and nis the order of the
quality deterioration/change. The application of
chitosan is expected to modify the kinetics of the
deterioration/change of strawberry quality during
storage. Therefore, the rate of quality loss can be
modeled as:
−dq
dt=(k
0
−k
i
c)[q]
n
(2)
where cis chitosan concentration, k
0
the rate
constant at 0 chitosan concentration, and k
i
is
the inhibition constant at different chitosan con-
centrations. The value of ncan range from 0
(zero order kinetics) up to 2 (second order) for
various reactions. Integration of Eq. (2) yields
Eqs. (3)– (5) for zero, first and second order ki-
netics, respectively.
[q]=[q
0
]−(k
0
−k
i
c)t,n=0 (3)
ln[q]=ln[q
0
]−(k
0
−k
i
c)t,n=1 (4)
1
[q]=1
[q
0
]+(k
0
−k
i
c)t,n=2 (5)
with

M.V.Bhaskara Reddy et al.
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20 (2000) 39–51
42
k
c
=k
0
−k
i
c(6)
where k
c
is the rate constant at different chitosan
concentrations.
At zero chitosan concentration, (Eq. (2))
becomes,
−dq
dt=k
0
[q]
n
(7)
On integration, Eq. (7) yields Eqs. (8)– (10) for
zero, first and second order kinetics, respectively:
[q]=[q
0
]−k
0
t,n=0 (8)
ln[q]=ln[q
0
]−k
0
t,n=1 (9)
1
[q]=1
[q
0
]+k
0
t,n=2 (10)
Where [q
0
] is the value of the quality attribute
at time 0. From the experimental data k
0
,k
c
and
k
i
ccan be determined.
The sensitivity of a food material or a reaction
to temperature changes can be expressed by Q
10
,
the degree by which the process is accelerated by
a rise of 10°C. Expressing a modified Q
10
* factor
for various chitosan concentrations with respect
to 0 chitosan concentration,
Q
10
*=k
c
at (T+10°C)
k
0
at (T°C) (11)
Mean inhibition constant k
(
i
is given by,
k
(
i
=
%k
i1
c
1
+k
i2
c
2
+k
i3
c
3
+…
%c
1
+c
2
+c
3
+…
(12)
2
.
8
.Determination of kinetic parameters
The quality characteristics considered include
decay, texture, anthocyanin and titratable acidity.
An optimization routine uses the values of the
independent variable (the tvalues) to predict the
value of a dependent variable (the [q] value). The
routine uses the Marquardt– Levenberg algorithm
(Marquardt, 1963) to find the parameters of the
independent variable (t) that give the best fit
between the model and the data.
The model used obeys the following differential
equation:
g
d[q]
dt=k[q]
n
(13)
with the initial condition at, t=0; [q]=[q
0
]; with
g=1, if [q] increases with time and g=−1, if [q]
decreases with time.
The agreement between experimental and pre-
dicted values was judged acceptable when the
mean deviation was less than the mean experi-
mental error.
3. Results
3
.
1
.Efficacy of pre-har6est sprays of chitosan in
controlling post-har6est decay and quality of
fruits
The general trend of development of decay in
strawberry fruit from chitosan sprayed plants and
stored at 3 and 13°C is shown in Fig. 1. The decay
of fruit decreased with increasing chitosan con-
centration, and increased with storage interval
and temperature. The rate of decay development
was higher in fruit stored at 13°C compared with
3°C at all chitosan concentrations tested.
The effect of chitosan spray on firmness of fruit
during storage is shown in Fig. 2. The mainte-
nance of texture was dependent on chitosan spray
concentration, storage time and temperature. Chi-
tosan concentration and storage interval played
an important role in texture maintenance at both
3 and 13°C storage temperatures. The fruit tex-
ture was firmer with increasing chitosan concen-
tration, and it decreased with storage temperature
and time. The mean of fruit texture from week 0
to 4 across number of sprays and picks decreased
from 3.4 to 1.1 N at 3°C compared with 3.5– l.3 N
at 13°C. The fruit texture was firmer with increas-
ing chitosan concentrations.
The effect of chitosan sprays on the evolution
of anthocyanin in strawberry fruit during storage
at 3 and 13°C is shown in Fig. 3. The anthocyanin
development was dependant on chitosan spray
concentration, storage temperature and time. The
rate of pigment development was lower with in-
crease in chitosan concentration and was higher
with increases in storage temperature and dura-
tion. In general, the rate of increase in an-

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20 (2000) 39–51
43
thocyanin content was lower during the initial
storage period of up to 2 weeks for all treatments.
Anthocyanin content of fruit from control plants
was higher than from chitosan treated plants.
The citric acid content of fruit from chitosan
sprayed and control plants is shown in Fig. 4. The
acidity of fruit was dependant on chitosan con-
centration, storage temperature and time. The
citric acid content of fruit decreased with increase
in storage temperature and time, and the rate of
decrease was lower with increase in chitosan
concentration.
3
.
2
.Kinetics of fruit quality deterioration/change
The data of fruit quality parameters (decay,
anthocyanin content, tissue texture and titratable
acidity) of fruit pooled across two sprays (sprays
1 and 2) and two picks (picks 1 and 2) and two
repetitions were linearly transformed and plotted
against storage time to describe the changes in
quality by kinetic models. Kinetic models repre-
senting the goodness of fit of the data of fruit
stored at 3°C are shown in Figs. 5 and 6. The
data for 13°C storage showed similar fit except
Fig. 1. Effect of pre-harvest chitosan spray treatments on the decay of strawberry fruit stored at 3 (S.E.M.91.58) and 13°C
(S.E.M.92.28). Control (),2gl
−1
(2), 64 g l
−1
()and6gl
−1
(). Data were pooled across the number of sprays and picks
and repetitions.
Fig. 2. Effect of pre-harvest chitosan spray treatments on the texture of strawberry fruit stored at 3 (S.E.M.90.21) and 13°C
(S.E.M.90.18). Control (),2gl
−1
(2),4gl
−1
()and6gl
−1
(). Data were pooled across the number of sprays and picks
and repetitions.

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20 (2000) 39–51
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Fig. 3. Effect of pre-harvest chitosan spray treatments on anthocyanin content of strawberry fruit stored at 3 (S.E.M.90.57×10
−2
)
and 13°C (S.E.M.90.84×10
−2
). Control (),2gl
−1
(2),4gl
−1
()and6gl
−1
(). Data were pooled across the number
of sprays and picks and repetitions.
Fig. 4. Effect of pre-harvest chitosan spray treatments on titratable acidity of strawberry fruit stored at 3 (S.E.M.90.038) and 13°C
(S.E.M.90.18). Control (),2gl
−1
(2),4gl
−1
()and6gl
−1
(). Data were pooled across the number of sprays and picks
and repetitions.
that the changes in quality parameters were more
pronounced. Linear regression coefficients (R
2
)
for the kinetic plots of quality parameters are
given in Tables 1 and 2. Zero order reaction best
described the progress of decay and anthocyanin
development. First order reaction was appropriate
for titratable acidity changes, whereas a second
order reaction best described texture loss during
storage.
Microbial growth typically follows first order
reaction kinetics with respect to microbial popula-
tion. In this work, decay was not evaluated by
either severity of infection or microbial popula-
tion, but by number of infected berries or rate of
disease incidence. The infection process involves
secretion of enzymes by the pathogen, which de-
polymerize the insoluble pectic polymers of the
plant cell wall, leading to tissue maceration
(Bateman, 1968). Tissue maceration is the key
step for successful infection since it results in
adequate supply of nutrients required for patho-
gen growth. Thus pathogenesis is ultimately an
enzymatic process which determines the rate of
infection. The general feature of enzyme catalyzed
reactions is that, as the substrate concentration
increases, the reaction order diminishes from first

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20 (2000) 39–51
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Table 1
Rate constants k
0
a
and k
c
b
, and modified Q
10
* factor for decay of strawberry fruit picked from plants sprayed with different concentrations of chitosan and stored at
3 and 13°C for 4 weeks
Regression coefficient
c
Quality factor Spray 1Chitosan (g l
−1
) Spray 2
R
2
Pick 1 Pick 2 Pick 1 Pick 2
13°C Q
10
* 3°C 13°C Q
10
* 3°C 13°C Q
10
* 3°C 13°C3°C 13°C Q
10
* 3°C
6.86 4.2 1.33Decay (per day) 6.710 5.0 1.67 7.00 4.2 0.978 0.9061.58 6.00 3.8 1.65
6.50 4.1 0.88 4.00 3.0 1.09 4.511.45 2.73.2 0.963 0.886Zero order 2 1.00 5.14
3.86 2.4 0.64 3.11 2.3 1.09 3.30 2.0 0.984 0.9374 0.82 3.43 2.2 1.41
3.75 2.4 0.55 2.18 1.6 1.82 2.57 1.6 0.984 0.8841.306 0.78 3.29 2.1
a
k
0
is the rate constant at zero chitosan concentration.
b
k
c
is the rate constant at different chitosan concentrations.
c
R
2
was determined from pooled data of spray 1 and 2 and pick 1 and 2 for a given storage temperature.

M.V.Bhaskara Reddy et al.
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Table 2
Rate constants k
0
a
and k
c
b
, and modified Q
10
* factor for quality factors of strawberry fruit picked from plants sprayed with different concentrations of chitosan and
stored at 3 and 13°C for 4 weeks
Regression coefficient
c
Quality factor Spray 1Chitosan (g l
−1
) Spray 2
R
2
Pick 1 Pick 2 Pick 1 Pick 2
13°C Q
10
* 3°C 13°C Q
10
* 3°C 13°C Q
10
* 3°C 13°C3°C 13°C Q
10
* 3°C
77Anthocyanin (g kg
−1
per day) 1.40 23 55 2.8 37 83 2.2 0.981 0.98647 62 1.3 57
54 0.9 18 43 1.9 22 5744 1.50.9 0.920 0.982Zero order 2 39 42
36 0.6 12 32 1.4 16 41 1.14 0.972 0.94827 30 0.6 35
27 0.5 11 25 1.1 16 2828 0.8 0.978 0.9430.524226
35 1.6 16 28Texture (N
−1
per day) 1.80 32 41 1.3 0.983 0.93414 23 1.6 22
28 1.3 8 18 1.1 16 2618 0.8Second order 0.975 0.9611.21762
20 0.9 7 13 0.8 8 20 0.64 0.990 0.9896 12 0.9 10
15 0.7 6 10 0.6 8 1510 0.5 0.987 0.9630.71046
29 1.2 13 25 1.9 15 30 2.0 0.997 0.955Citric acid (kg m
−3
per day) 0 14 23 2.1 24
22 0.9 7 20 1.5 12 2318 1.51.3 0.996 0.876First order 2 10 18
17 0.7 5 15 1.2 8 17 1.1 0.977 0.8584 8 10 0.7 14
15 0.6 4 12 0.9 7 15 1.0 0.969 0.810136 7 10 0.7
a
k
0
is the rate constant at zero chitosan concentration.
b
k
c
is the rate constant at different chitosan concentrations.
c
R
2
was determined from pooled data of spray 1 and 2 and pick 1 and 2 for a given storage temperature.

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order to zero order. Since the substrates for mac-
erating enzymes i.e. insoluble pectic polymers are
not limiting, the reaction order should be 0, in
accordance with our observation. Likewise an-
thocyanin synthesis is also an enzymatic process.
The starter molecules for anthocyanin synthesis
such as cinnamic, p-coumaric and caffeic acids are
not limiting with tissue ripening (Harborne, 1964;
Wardale, 1973).
Organic acids make a major contribution to the
typical acidity of many unripe and ripe fruits.
Citric and malic acids are the two major acids
component of strawberry fruit (Haard, 1976). The
loss of acidity which accompanies ripening ap-
pears to result, in part, from the use of the acids
as respiratory substrates, via Krebs cycle. In addi-
Fig. 6. Zero order kinetics for anthocyanin development of
strawberry fruit sprayed with chitosan before harvest and
stored at 3°C (A). First order kinetics for loss of titratable
acidity of strawberry fruit sprayed with chitosan before har-
vest and stored at 3°C (B). Control (),2gl
−1
(2),4gl
−1
() and 6 g l
−1
(). Data were pooled across the number of
sprays and picks and repetitions. Lines represent the fit of the
data to the kinetic model.
Fig. 5. Zero order kinetics for decay of strawberry fruit
sprayed with chitosan before harvest and stored at 3°C (A).
Second order kinetics for texture loss of strawberry fruit
sprayed with chitosan before harvest and stored at 3°C (B).
Control (),2gl
−1
(2),4gl
−1
()and6gl
−1
(). Data
were pooled across the number of sprays and picks and
repetitions. Lines represent the fit of the data to the kinetic
model.
tion, malate may also be decarboxylated by malic
enzyme as shown in apples and pears (Hulme and
Rhodes, 1971). Thus the rate of acidity loss is
related to the concentration of organic acids at
any time, in agreement with the observed first
order kinetics.
Loss of texture is dependent on both cell wall
degradation and loss of turgidity of the tissue.
Cell wall degradation during ripening is an enzy-
matic process and follows increase in the activity
of endogenous cell wall degrading enzymes such
as polygalacturonase and cellulase. All cell wall
polymers are susceptible to enzyme action if
brought into contact with relevant enzymes. Ex-
posure of cell wall associated pectin and cellulose
to endogenous enzyme action is facilitated by free

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48
radicals generated by aerobic respiration, which
can participate in the derangement of the intact
supramolecular structures of pectin (Kon and
Schwimmer, 1977). Although the supply of sub-
strate for these enzymes is not a limitation, their
exposure to enzymes is limiting, and thus this
process can be described by first order. The other
component to tissue softening is loss of turgor
pressure which falls with loss of water or desicca-
tion due to transpiration and respiration (Bourne,
1983). Since water loss from produce is typically
of first order, it follows that the rate of loss of
turgidity should also be first order. Thus loss of
texture depends on two independent processes
described by first order behavior, and therefore, it
is expected to follow second order kinetics overall.
The rate constants of decay and ripening pro-
cess indicators of stored fruit picked at two inter-
vals following two chitosan sprays at
concentrations between 0 and6gl
−1
are listed in
Tables 1 and 2. Pre-harvest chitosan treatments
reduced the rate of both decay and ripening.
Significant reduction in the rates was observed
with4gl
−1
chitosan concentration, beyond
which the rates leveled off. At4gl
−1
chitosan
concentration, the rates were reduced typically
from 40 to 60% at both 3 and 13°C storage
temperatures. The decay rate constants were one
order of magnitude higher than pigment develop-
ment, and two orders of magnitude higher than
texture loss and acidity. This suggests that straw-
berry fruit are quite susceptible to infection, more
so at 13°C, and this is a major limiting factor for
their preservation. Generally, the rates of decay
and ripening were higher for pick 2 fruit than pick
1, and a double application of chitosan reduced
these rates more effectively than a single
application.
The rates of decay development and ripening
were significantly lower at 3°C compared with
13°C storage temperature. The Q
10
* values for
various quality characteristics at different chi-
tosan concentrations with respect to zero chitosan
concentration were calculated from the reaction
rate constants at 3 and 13°C (Eq. (11)). The decay
was more sensitive to temperature than the ripen-
ing parameters in general. The Q
10
* values for
pigment development, texture loss or titratable
acidity were typically B2.0 but \2.0 for decay
(Tables 1 and 2). Chitosan treatment reduced the
sensitivity of the fruit to higher storage tempera-
ture. For example, the Q
10
* values for decay de-
creased to values between 2.0 and 2.5 at4gl
−1
concentration, while the Q
10
* values for ripening
indicators decreased to :1.0.
The protective effect of chitosan can also be
evaluated from the mean inhibition constants of
chitosan for various fruit quality parameters. The
mean inhibition constants (Eq. (12)) expressing
the capacity of chitosan in controlling decay, an-
thocyanin accumulation, texture loss and titrat-
able acidity of stored fruits are presented in Table
3. The protective effect of chitosan against decay
and in delaying ripening was more pronounced at
13°C than at 3°C. Likewise, the follow-up spray
was found more effective in protecting berries
Table 3
Mean inhibition constant (k
i
) of chitosan in the decay and ripening characteristics (anthocyanin accumulation, texture loss and
titratable acidity) of strawberry fruits picked from plants sprayed with different concentrations of chitosan and stored at 3 and 13°C
for 4 weeks
Spray 1Quality factor Spray 2
Pick 1 Pick 2 Pick 1 Pick 2
13°C3°C 3°C13°C 13°C3°C13°C3°C
17.8 51.2 6.6 53.0 16.0 90.4 16.7 88.4Decay (m
3
kg
−1
per day)×10
−2
4.4 7.4 5.4 9.6Anthocyanin (g m
3
kg
−1
per day)×10
−2
2.4 8.0 4.6 10.2
2.1 5.32.6 2.2 3.5 2.2 3.7Texture (m
3
N
−1
kg
−1
per day)×10
−3
5.3
1.5 2.92.5 2.2Citric acid (per day)×10
−3
2.71.4 1.8 2.4

M.V.Bhaskara Reddy et al.
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20 (2000) 39–51
49
from decay and in slowing down ripening. Chi-
tosan offered stronger protection to fruits from
pick 2, which are more perishable than pick 1 fruit.
It is clear that chitosan compensates for a higher
storage temperature and it offers protection
by slowing decay and ripening. However, the
protective effect is highly significant against
infection.
4. General discussion
Pre-harvest fungicide sprays are undertaken to
control the initial infection in the field and to
obtain fruits free of infection (Aharoni and Barkai-
Golan, 1987). Present study shows that prophylac-
tic sprays of chitosan are effective in controlling
the infection of B.cinerea in strawberries (Fig. 1).
A previous study (El Ghaouth et al., 1991a) re-
ported that dipping of strawberry fruit in chitosan
solution protected the fruits from decay as effec-
tively as dipping them in the fungicide, Rovrol
®
.
Chitosan inhibits the growth of several fungi,
induces chitinase activity, and elicits phytoalexins
and defense barriers in the host tissues (Hirano and
Nagao, 1989; El Ghaouth et al., 1992a,b, 1994,
1997). The control of decay in strawberries ob-
served in this study could be attributed to either its
fungistatic property, ability to induce defense en-
zymes and phytoalexins in plants, or a combina-
tion of these factors. Although the severity of
decay was decreased significantly by chitosan
sprays in pick 1, an increase in decay was observed
with increase in storage time and subsequent picks.
Decay was greater at 13°C than at 3°C. The
lowered efficacy of chitosan sprays against fungal
spoilage in pick 2 could be due to the higher
susceptibility of second flush fruit to diseases.
Browne et al. (1984) have shown that freshly
harvested strawberries from the first harvest in
each season have greater shelf-life compared with
subsequent harvests since the first harvest is less
susceptible to molds. The present study indicates
that loss of chitosan spray effectiveness in subse-
quent harvests can be compensated by a follow-up
spray at a maximum interval of 10 days.
The results from this study showed pre-harvest
chitosan sprays also had a beneficial effect on
flesh firmness, titratable acidity and in slowing
the synthesis of anthocyanins in strawberries
stored at both 3 and 13°C. This could be due to
the formation of a chitosan film on fruit which
can act as a barrier for O
2
uptake thereby slow-
ing the metabolic activity, and consequently the
ripening process. El Ghaouth et al. (1991b) have
also observed retention of firmness, higher titrat-
able acidity and reduced rate of anthocyanin pro-
duction in chitosan coated strawberries. Coating
of fruits with semipermeable films can retard
ripening by modifying the internal CO
2
,O
2
and
ethylene levels (Lowings and Cutts, 1982). Chi-
tosan coating is likely to modify the internal
atmosphere without causing anaerobic respira-
tion, since chitosan films are selectively more per-
meable to O
2
than to CO
2
(Bai et al., 1988). In
addition, chitosan coating can reduce desiccation
by providing a moisture barrier (El Ghaouth et
al., 1991c). The suppressive effect on decay by
chitosan can in part, be attributed to delaying the
senescence process, since resistance to fungal in-
fection can be greater as a result of slower senes-
cence (Eckert, 1975). Our results showed that
strawberry from chitosan sprayed plants main-
tained keeping quality for 4 weeks at 3°C.
Kinetic data on decay and ripening characteris-
tics provide quantitative evidence that chitosan
compensates for higher storage temperature and
provides additional protection against deteriora-
tion of lower quality fruit from pick 2. It is
difficult to coat strawberry fruit individually, but
pre-harvest spraying is highly feasible. Many
studies, including this one have shown the high
potential of chitosan for preserving fresh fruits
and vegetables, but translating this into practice
requires optimizing chitosan concentrations for
individual crops and improving chitosan coating
formulations for integration with CA or MA dur-
ing transport and storage of perishable
commodities.
Acknowledgements
We acknowledge financial supports of Natural
Sciences and Engineering Research Council
(NSERC) of Canada-Strategic Grants Program

M.V.Bhaskara Reddy et al.
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Posthar6est Biology and Technology
20 (2000) 39–51
50
and of Conseil des Recherches en Peˆche et en
Agroalimentaire du Que´bec (CORPAQ).
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