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Chitinase and β-1,3-glucanase enzymatic activities in response to infection by

Alternaria alternata evaluated in two stages of development in different
tomato fruit varieties

Article in Scientia Horticulturae · March 2007

DOI: 10.1016/j.scienta.2006.12.005

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 Scientia Horticulturae 112 (2007) 42–50
www.elsevier.com/locate/scihorti

Chitinase and b-1,3-glucanase enzymatic activities in response to

infection by Alternaria alternata evaluated in two stages of
development in different tomato fruit varieties
I.E. Cota a, R. Troncoso-Rojas a,*, R. Sotelo-Mundo b, A. Sánchez-Estrada a,
M.E. Tiznado-Hernández a
a
Dirección de Tecnologı́a de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo,
A.C. Apartado Postal No. 1735, Hermosillo, Sonora 83000, Mexico
b
Dirección de Tecnologı́a de Alimentos de Origen Animal, Centro de Investigación en Alimentación y Desarrollo,
A.C. Apartado Postal No. 1735, Hermosillo, Sonora 83000, Mexico
Received 27 January 2006; received in revised form 20 August 2006; accepted 4 December 2006

Abstract
Chitinase (ChA) and b-1,3-glucanase (GA) activity had been related with plant defense mechanism against pathogen attack in vegetative
tissues. Scarce information is available about the behavior of these enzymes in response to different stages of development and fungi infection in
fruits. The changes in ChA and GA activities in response to Alternaria alternata infection were evaluated in mature green (MG) and red ripe (RR)
developmental stages of Sunpride, Geronimo and Charleston varieties of tomato fruit. Tomato fruits were inoculated with a conidial suspension of
A. alternata and stored for 10 days at 25 8C and 90–92 H.R.%. The degree of fruit infection was measured by a hedonic scale every 2 days. ChA
activity was determined fluorometrically by quantifying the release of 4-methylumbelliferyll (4-MU) from 4-methylumbelliferyll b-D-N,N0 N00 -
triacetylchitotrioside, and GA activity was measured quantifying the release of glucose from b-1,3-glucan (laminarin) by HPLC. Tomato fruit in
RR stage was more susceptible to fungi infection than MG stages. Geronimo was the most resistant variety, whereas Sunpride was the most
susceptible for both stages of development (MG and RR). Higher levels of ChA and GA activities were observed for mature green stage in
Charleston variety at the end of the storage period. An induction in ChA and GA in response to infection by A. alternata was observed in all
varieties. Particularly high levels of ChA were found for inoculated Geronimo in RR stage and inoculated Charleston in MG stage which correlated
with low levels of fungi infection. Higher levels of GA induction in response to fungi infection were recorded for Sunpride variety in RR stage,
whereas no substantial induction was observed for Geronimo and Charleston varieties at the same stage of development. This GA induction
correlated negatively with the resistance showed by the different varieties to fungi infection. We concluded that chitinase and b-1,3-glucanase
induction are part of the tomato fruit defense mechanism against A. alternata infection with a different behavior depending upon stage of
development and variety.
# 2007 Elsevier B.V. All rights reserved.

Keywords: Chitinase; b-1,3-Glucanase; Alternaria alternata; Ripening; Varieties; Tomato fruit

1. Introduction activation of host defense mechanism. This includes an

Fungal diseases have been one of the most important causes
of crop losses ever since humans started to cultivate plants
(Harvey, 1978). Plants are endowed with several defense
mechanisms that protect them from fungal infection. Physical
contact of the fungal pathogens on plant cell surface results in
* Corresponding author. Tel.: +52 662 280 0422; fax: +52 662 280 0422.
E-mail address: rtroncoso@cascabel.ciad.mx (R. Troncoso-Rojas).
0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2006.12.005
induction of genes encoding enzymes such as phenylalanine
ammonia-lyase, 4 coumarate-CoA ligase, cinnamic acid-4-
hydroxylase, cinnamyl alcohol dehydrogenase, chalcone
synthase, chalcone isomerase, among others, which are
involved in synthesis of low-molecular-weight substances like
phytoalexins, phenols, lignins, tannins, and melanins with
antifungal activity (Calvo et al., 2002). Another group of
proteins worth to mention are the pathogenesis-related (PR)
proteins. Included in this group of proteins, there are chitinases
(EC 3.2.1.14) and b-1,3-glucanases (EC 3.2.1.39) which
 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
belongs to the PR-2 and PR-3 families respectively (van Loon
and van Strien, 1999). Chitinases and b-1,3-glucanases
catalyze the hydrolysis of chitin and b-1,3-glucan, respectively,
both polymers major components of the fungi cell walls
(Webster, 1986). Typically they are expressed constitutively at
low levels in plant cell and accumulate in response to fungal
(Leubner-Metzger and Meins, 2000; Veronese et al., 2003),
bacterial, viral attack, or other inducers of acquired resistance
(Krishnaveni et al., 1999; Salles et al., 2002).
Although chitinase and b-1,3-glucanase had been described
in several plants including lemon (Citrus limon) (Fanta et al.,
2003), tomato plants (Danhash et al., 1993; Domingo et al.,
1994; Harikrishna et al., 1996; Lawrence et al., 2000; Wu et al.,
2001), and tomato seeds (Wu and Bradford, 2003), the
information related to the behavior of these enzymes in fruits as
a response to fungi infection and different stages of
development is scarce. To the best of our knowledge, only
two studies about the chitinase and others antifungal proteins
during fruit ripening and in response to fungi attack had been
reported. The first study was done in grape fruit (Salzman et al.,
1998). The authors found an increase in the abundance of
several defense proteins (osmotin, lipid-transfer protein and
both basic and acidic chitinase) during ripening of grape
berries. Also, the basic chitinase and grape osmotin showed
inhibitory activities against G. bidwelli and B. cinerea (based
on in vitro growth assays). The second study was done with
kiwifruit, were the effect of fruit harvest maturity, phenolics
and hydrolytic enzymes on Botrytis fruit rot using resistant and
susceptible kiwifruit cultivars was evaluated (Wurms, 2004).
The authors found that the phenotype of resistance to Botrytis
cinerea is associated with chitinases and phenolic compounds.
There is evidence that defense reactions can change as a
function of the host age and developmental stage. Neale et al.
(1990) found that chitinase, b-1,3-glucanase and osmotin were
induced by viral infection in explants during flower formation,
but not in explants forming vegetative shoots. Terry and Joyce
(2004) mentioned that during development of plant organs and
in postharvest, natural disease resistance generally declines
leading to inevitable infection, disease and ultimately death.
Tomato fruit is commonly infected by Alternaria alternata
(Fr.Fr) Keissl sp. lycopersici, after harvest and extended
storage. The fungus is a pathotype capable of developing
primary infection of leaves, stems and fruit of susceptible
tomato cultivars (Grogan et al., 1975). Based on this, the
pathosystem tomato fruit—Alternaria alternata can be a good
model to study the fruit response to fungi infection. The
knowledge of natural fruit defense mechanisms in different
tomato fruit varieties will allow elaboration of strategies to
improve or to acquire efficient protection mechanism against
phytopathogens. Furthermore, this knowledge will allow
providing target enzymes to genetically engineer new disease
resistant varieties of tomato.
Based on the above mentioned, the objective of this study was
to evaluate the change in enzymatic activities of chitinase and b-
1,3-glucanase at different stages of development and in response
to fungi infection in three different varieties of tomato fruit, using
as a model the pathosystem tomato fruit—A. alternata.
43
2. Materials and methods
2.1. Plant material
Three varieties of tomato fruits (Lycopersicon esculentum
Mill var. ‘Sunpride’, ‘Geronimo’ and ‘Charleston’) were
selected based on different levels of resistance to different fungi
infection. Sunpride (Seminis Vegetable Seeds, Co.) is a mid-
season variety resistant to Alternaria stem canker (Alternaria
alternata f.sp. lycopersici), Fusarium wilt (Fusarium oxy-
sporum f.sp. lycopersici races 1–3), Verticillium dahliae Kleb
race 1, and tomato mosaic virus; whereas Geronimo (De Ruiter
Seeds, Hybrid seeds, Co.) is a variety with a good shelf-life and
high resistant to tomato mosaic virus, Fusarium oxysporum
f.sp. lycopersici race 1, Fusarium oxysporum f.sp. radicis-
lycopersici, Verticillium alboatrum Reinke & Berth and
Verticillium dahliae. Charleston is a large shelf-life variety
resistant to Fusarium oxysporum f.sp. lycopersici races 1 and 2,
Cladosporium fulvum races 1–5; Verticillium and tomato
mosaic virus (Ahern International Seeds, Inc.).
2.2. Alternaria alternata culture and conidial suspension
An isolate of Alternaria alternata (Fr.Fr) Keissl f.sp.
lycopersici was collected from tomatoes (Lycopersicon
esculentum Mill) in the Fall 2002 harvest at commercial
orchards in Sinaloa, México. The isolate was cultured on potato
dextrose agar (PDA; Difco Laboratories, Detroit, MI), the
colony and sporulation characteristics were studied according
to Simmons (1995) and Pryor and Michailides (2002).
Cultures were grown at 26 8C for about 10 days. Conidial
suspension of A. alternata was prepared according to French
and Hebert (1982). Briefly, conidia from the surface of several
plates of 10 days-old cultures were harvested by suspending
them in distilled water. Conidial concentration was determined
with a Neubauer chamber.
2.3. Inoculation process
Freshly harvested tomatoes of Sunpride, Geronimo and
Charleston varieties at the commercial mature green and red
ripe stage were obtained from local growers. Classification of
tomato ripening stage was done according the tomato color
chart reported by McGlasson et al. (1985). Fruits were sorted
out to get homogenous size, color and it was selected those free
from bruising and rotting. Tomatoes were divided into twelve
groups (which come from two stages of development and three
different varieties which were either inoculated or non-
inoculated) of 80 fruits each. Six groups were dipped into a
200 mg L1 sodium hypochlorite solution for 30 s, washed
with sterile distilled water and air dried at room temperature.
Inoculation was done by fruit immersion in a conidial
suspension A. alternata (3.5  106 conidia mL1) for three
min. Six control groups were included: mature green non-
inoculated and red ripe non-inoculated per variety. Fruits from
all the treatments were stored for 10 days at 25 8C with 90–92%
relative humidity, to induce A. alternata disease development.
44 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
The severity of infection was evaluated twice on ten fruits
previously marked from each treatment, every 2 days. The
extent of infection was assessed visually based on the area of
lesions. A rating of () represents no lesion, (+) slight, (++)
moderate, and (+++) high lesion. Tomato samples (without
locular material and seeds) were taken from the fungi
inoculated tissue of 10 fruits every 2 days, and stored at
20 8C until the enzymatic analysis. All experiment were
repeated at least twice and proved to be reproducible.
2.4. Preparation of crude enzyme extracts and protein
determination
Crude tissue extracts were obtained according to Sela-
Buurlage et al. (1993) with some modifications, described next.
Infected tomato and control tissues (100 g) were homogenized
at 4 8C in a Waring blender with 100 mM sodium acetate at pH
5.2 and 1% (v/v) b-mercaptoethanol in a ratio 2:1 (tomato
tissue: buffer). The homogenate was filtered through organza
fabric and centrifuged at 37,565  g for 15 min. The super-
natant was concentrated by ultrafiltration with YM10
membranes (Amicon, Millipore). Protein was determined
according to Bradford (1976) method using the Bio-rad protein
assay reagent with bovine serum albumin (BSA) as a standard.
2.5. Chitinase activity assay
Total chitinase activity was determined fluorometrically
according to Tung-Shin et al. (2002) with some modifications.
The reaction mixture contained 50 ml of concentrated tomato
crude extract, 1 ml of 4-methylumbelliferyll b-D-N,N0 N00 -
triacetylchitotrioside 1.3 mM (Sigma Chemical Co., St. Louis),
and 50 mM sodium phosphate buffer (pH 6) was added to get
1.5 ml of final reaction volume. Incubation was carried out for
5 min. Release of free 4-methylumbelliferone (4-MU) was
measured by fluorescence spectroscopy using a QM-2003
spectrofluorometer (PTI-Photon Technology Intl.), using an
excitation wavelength of 325 nm and emission wavelength of
446 nm. The linearity of the enzymatic reaction during the five
minutes was evaluated using chitinase from Streptomyces
griseus (Sigma). Also, a control was included by measuring the
fluorescence of the reaction mixture without the enzyme
extract. The r2 for 4-MU standard curve was 0.9984. Enzymatic
activity was expressed as mM of 4-MU min1 mg1 of protein.
2.6. Assay of b-1,3-glucanase activity
Total b-1,3-glucanase activity was assayed with laminarin
as substrate according to Siefert et al. (1994) and Hinton and
Pressey (1980) with some modifications. The reaction mixture
contained 100 ml of the concentrated crude extract (diluted 1:5
with sodium acetate buffer, 0.1 M, pH 5.5) mixed with 100 mg
laminarin in 100 ml of sodium-phosphate buffer (50 mM, pH
6.0). Incubation was carried out for 30 min with shaking at
37 8C. Enzyme reactions were stopped by cooling the samples
immediately on ice followed by protein denaturation at 95 8C
for 3 min. After this time, the mixture was centrifuged at
6000 rpm in a microfuge (Eppendorf North America, Westbury,
NY). Glucanase activity was measured by the release of
reducing sugars from the b-1,3-glucan laminarin using an
HPLC equipped with a refractive index detector, according to
López-Hernandez et al. (1994). A Supelcosil LC-NH2 column
(Sigma Chemical Co., St. Louis; 250 m length, 4.6 mm of
internal diameter and 5 mm of particle size) with a LC-NH2
guard column were used and the mobile phase was acetonitrile/
water (80:20, v/v). One peak, identified as glucose, was
observed in the chromatogram of the enzymatic reaction
products in all samples. Quantification was carried out with a
standard curve done by injecting into the HPLC equipment
solutions of glucose with different concentrations. The r2 for
glucose standard curve was 0.9508. b-1,3-Glucanase activity is
expressed as mM glucose min1 mg1 of protein.
2.7. Statistical analysis
The effect of fruit varieties, Alternaria alternata infection
and stage of development on enzymatic activities was studied
by analysis of variance with a level of significance of 5% based
in a split-plot design in which the main plots was the tomato
fruit variety, the subplots were the stage of development at
which the fruit was infected and the sub–subplot the fungi
infection. A fully randomized block design for the storage time
effect was included to eliminate the two-way and three-way
interactions effect due to the factors tomato fruit variety, stage
of development and infection by A. alternata. The differences
among specific means were found using Duncan’s multiple
range test ( p < 0.05). All statistical analysis was carried out
using SAS software package Version 8.2 (SAS Institute, 2000).
3. Results
3.1. Severity of infection caused by A. alternata
The effect of variety and stage of development on fungi
infection in tomato fruit inoculated with A. alternata during
storage for 10 days at 25 8C, is shown in Table 1. No infection was
observed in the non-inoculated tomato fruits during the 10 days of
storage which further supports the results of tomato fruit response
to A. alternata infection found in this study. The degree of tomato
fruit infection development by A. alternata was different
depending upon the maturation stage and variety. Inoculated
tomato fruits of Sunpride variety in mature green stage, showed
slight symptoms of the disease after 8 days of storage, whereas
Geronimo and Charleston varieties did not show any signs of
disease development when inoculated at mature green stage. In
red ripe stage, Sunpride and Charleston varieties showed the first
symptoms of the Alternaria disease after 4 and 6 days of storage,
respectively, whereas Geronimo showed a slight fungi growth
after 8 days of storage. After 6 days of storage, Sunpride showed
moderate lesions whereas Charleston was still showing slight
lesions. This result suggest that Sunpride variety was more
susceptible to disease caused by A. alternata as compared with
the other varieties evaluated; while Geronimo variety was more
resistant to disease caused by the pathogen.
 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50 45

Table 1
Effect of variety and ripening on disease incidence in control and Alternaria alternata inoculated tomato fruits stored for 10 days at 25 8C
Days at 25 8C Sunpride Geronimo Charleston
Control Inoculated Control Inoculated Control Inoculated
a
MG RR MG RR MG RR MG RR MG RR MG RR
0            
2            
4    +        
6    ++        +
8   + ++    +    ++
10   + +++    +    +++
Disease incidence was recorded visually as (+). A rating of () represents non-lesion, (+) represents slight lesion, (++) moderate lesion, and (+++) high lesion.
a
MG: mature green stage; RR: red ripe stage.

Tomato fruits from all varieties evaluated were ripening
during the storage time. Maturation rate was visually evaluated
in the fruits by color changes (from green to red), following the
color chart reported by McGlasson et al. (1985). In the case of
mature green tomatoes, the first change noticed was from green
to pink and later to red color during the storage time. Color
changes in mature green stage ocurred first in Sunpride tomato,
showing a light red color after 6 days of storage. By other side,
Charleston and Geronimo tomatoes showed the same change
after 8 and 10 days of storage, respectively (data not reported).
These differences in maturation rate could affect the suscept-
ibility to Alternaria rot observed among varieties.
3.2. Chitinase activity
Changes in chitinase activity in the different varieties during
ripening of tomato fruit inoculated with A. alternata and stored
for 10 days at 25 8C, are shown in Fig. 1. Differences in levels
of chitinase activity were found depending upon varieties,
maturity stage at the time of inoculation and fungi infection.
The behavior of this enzyme during fruit ripening can be
analyzed in the control fruits.
The constitutive level of chitinase activity in non-inoculated
tomato Sunpride in mature green stage was similar throughout
the storage time, with an activity of 37.08–30.62 mM 4-
MU min1 mg1 protein at 0 and after 10 days of storage,
respectively (Fig. 1a). No statistical differences ( p < 0.05)
were found in chitinase activity as a response to infection by A.
alternata with respect to control fruit with the exception of day
8 of storage. Chitinase activity in control fruit of tomato
Sunpride in red ripe stage (Fig. 1b) was similar throughout the
storage time with an activity of 61.44–54.6 mM 4-
MU min1 mg1 protein at 0 and after 10 days of storage,
respectively. Fungi infection induced higher levels of this

Fig. 1. Effect of ripening and fungi infection on chitinase activity (mM 4-MU min1 mg1 protein) in different varieties of tomato fruit. Control (*) and Alternaria
alternata inoculated tomato fruits (*) were stored for 10 days at 25 8C. Each point represents the mean of 10 fruits and vertical lines the standard deviation.
46 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
enzyme for most of the time points sampled although statistical
difference ( p < 0.05) were found only after 2 and 6 days of
storage.
The constitutive level of chitinase activity in non-inoculated
tomato Geronimo in mature green stage changed from 78.45 to
41.91 mM 4-MU min1 mg1 protein at 0 and after 10 days of
storage, respectively (Fig. 1c). Fungi infection induced higher
levels ( p < 0.05) of chitinase enzyme, after 2 and 8 days of
storage. In the case of control fruits of Geronimo variety in red
ripe stage (Fig. 1d), chitinase activity showed a reduction from
38.82 to 16.43 mM 4-MU min1 mg1 protein at 0 and after 10
days of storage, respectively. The level of chitinase activity in
inoculated fruit was significatively higher ( p < 0.05) as
compared to the non-inoculated fruit after 2 days and it
remained higher until the 8th day of storage time. After 10 days
of storage, chitinase activity was similar in inoculated and non-
inoculated fruits of Geronimo variety in red ripe stage.
Charleston variety showed the highest values in chitinase
activity and its behavior was different with respect to Sunpride
and Geronimo varieties. Chitinase activity in control fruit was
59.09 mM 4-MU min1 mg1 proteins at the beginning of the
storage in mature green stage. A slight decrease was observed at
day 2, and it remained constant until day 6th of storage
(Fig. 1e). Thereafter, chitinase activity showed a large increase
from 8th to 10th day of storage, until reaching 218.74 mM 4-
MU min1 mg1 proteins by the end of storage time. During
the first 4 days of storage, a similar behavior was observed for
fruit inoculated at mature green stage of Charleston variety as
compared with the control fruits. However, after 6 days of
storage time, the level of chitinase was significatively higher
Fig. 2. Effect of ripening and fungi infection on b-1,3-glucanase activity (mM glucose min1 mg1 protein) in different varieties of tomato fruit. Control (*) and
Alternaria alternata inoculated tomato fruits (*) were stored for 10 days at 25 8C. Each point represents the mean of 10 fruits and vertical lines the standard
deviation.
( p < 0.05) and remained higher after 8 days, showing at this
time, a large increase ( p < 0.05) with an activity of 259.73 mM
4-MU min1 mg1 protein. This increase was 5-and 2.5-fold
higher than Sunpride and Geronimo varieties, respectively. The
level of chitinase activity was similar between inoculated and
control fruits by the end of the storage time. The changes in
chitinase activity for the Charleston variety at red ripe stage,
inoculated and control, throughout the storage are shown in
Fig. 1f. In control fruit, the level of chitinase activity was
constant, with the exception of day 8 in which the chitinase
activity was higher with an activity of 90.79 mM 4-
MU min1 mg1 protein. An increase in chitinase activity
was observed in inoculated fruit as a response to infection by A.
alternata, being significatively higher ( p < 0.05) after 4 and 6
days of storage time with an activity of 116.22 and 122.46 mM
4-MU min1 mg1 protein, respectively. No differences were
found ( p > 0.05) for the remaining days of storage time.
3.3. b-1,3-Glucanase activity
Changes in b-1,3-glucanase activity in the different varieties
analyzed for control and inoculated fruits with A. alternata at
two stages of maturity and stored for 10 days at 25 8C are shown
in Fig. 2. Differences in levels of this enzymatic activity were
found depending upon varieties, stage of development and
pathogen infection.
Non-inoculated Sunpride variety in mature green stage
showed an increase in b-1,3-glucanase activity (116.94 mM
glucose min1 mg1 protein) after 2 days of storage at 25 8C
(Fig. 2a). Thereafter, the enzymatic activity was constant until
 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
day 8 of storage. However, a small increase in the b-1,3-
glucanase activity (147.76 mM glucose min1 mg1 protein)
was observed at the end of the storage. Inoculated Sunpride
variety in mature green stage showed an increase in b-1,3-
glucanase activity, after 4 days of storage, in which the level of
activity (223.33 mM glucose min1 mg1 protein) was higher
( p < 0.05) with respect to control fruit. Although slightly higher
values of b-1,3-glucanase activity were recorded after 2, 6 and 8
days of storage, they were not statistically significant ( p > 0.05).
Non-inoculated Sunpride variety in red ripe stage showed a lower
level of b-1,3-glucanase activity with respect to mature green
stage (Fig. 2b). The level of b-1,3-glucanase activity in control
fruit showed a slight increase throughout the storage time, with
an activity from 63.49 to 96.34 mM glucose min1 mg1 protein
after 0 and 10 days of storage, respectively. For inoculated fruits
at red ripe stage, the level of b-1,3-glucanase activity showed an
increase with respect to control fruit, after 4, 6 and 8 days
of storage with activities of 156.30, 155.41 and
238.08 mM glucose min1 mg1 protein, respectively. At the
end of the storage, the b-1,3-glucanase activity felt down to
98.44 mM glucose min1 mg1 protein ( p > 0.05).
Non-inoculated tomato Geronimo variety in mature green
stage (Fig. 2c) showed a slight increase in b-1,3-glucanase
activity after 2 days of storage at 25 8C. Thereafter, the
enzymatic activity was constant throughout the storage time.
The b-1,3-glucanase enzymatic activity levels were similar as
compared with non-inoculated Sunpride variety. Inoculated
Geronimo variety in mature green stage showed higher values
of b-1,3-glucanase activity as compared with controls,
although were found after 2, 4 and 6 days of storage. In
non-inoculated red ripe stage of Geronimo variety, it was
recorded a reduction in b-1,3-glucanase activity throughout the
storage time, from 75.09 to 21.97 mM glucose min1 mg1
protein, after 0 and 10 days of storage, respectively (Fig. 2d).
Also, the level of enzymatic activity was lower with respect to
the mature green stage. Infection of tomato fruit variety
Geronimo with A. alternata induced higher values of b-1,3-
glucanase activity, when compared to the controls throughout
the storage time. However, statistical differences ( p < 0.05)
were found after 2, 8 and 10 days of storage time.
Non-inoculated tomato fruit of Charleston variety in mature
green stage (Fig. 2e) showed at the beginning of the storage
a level of b-1,3-glucanase activity of 158.48 mM glucose
min1 mg1 protein. This enzymatic activity decreased after 2
days of storage and thereafter an increase was observed getting a
maximum level of 294.93 and 283.85 mM glucose min1 mg1
protein after 6 and 8 days of storage, respectively. Non-
inoculated fruits of Charleston variety in mature green stage
showed the highest level of b-1,3-glucanase activity as compared
to Sunpride and Geronimo varieties. Inoculation of Charleston
variety induced a large increase in the level of b-1,3-glucanase
activity (Fig. 2e). Furthermore, at day 6 of storage, the level of b-
1,3-glucanase activity in inoculated fruits showed a sudden
and large increase with an activity of 508.40 mM glucose
min1 mg1 protein and were similar at 8 days of storage
(497.97 mM glucose min1 mg1 protein). The b-1,3-gluca-
nase activity fell down to 278.85 mM glucose min1 mg1
47
protein, at the end of storage. The differences between control
and inoculated fruits were statistically significant ( p < 0.05) for
most of the days of storage with the exception of day 4th. Non-
inoculated fruit of Charleston variety in red ripe stage showed an
activity of 46.84 mM glucose min1 mg1 protein, at the
beginning of the storage (Fig. 2f). This activity increased to
89.02 and 87.52 mM glucose min1 mg1 protein after 2 and 4
days of storage, respectively. Thereafter, the activity decreased to
40.25 mM glucose min1 mg1 protein by the end of storage
time. In general, the red ripe stage of Charleston variety showed
lower levels of b-1,3-glucanase activity than mature green stage
(Fig. 2e). A slight increase in the activity of this enzyme was
observed in inoculated fruit for most of the days of storage, with
the exception of day 10. Furthermore, particularly after 4 days of
storage, it was recorded an activity of 152.82 mM
glucose min1 mg1 protein. Statistical differences were found
for samples taken after 4, 6 and 8 days of storage.
4. Discussion
Tomato fruit is susceptible to infection by A. alternata
(Grogan et al., 1975; Troncoso-Rojas et al., 2005; Ruelas et al.,
2006) which supports the use of tomato-Alternaria alternata
pathosystem as a model to study the responses of fruit to fungi
infection. In this study, the susceptibility of tomato fruit to
Alternaria infection was different depending upon the variety
and the stage of development at the time of inoculation. The
classical black mold lesions were not observed in mature green
stage in inoculated tomato of Geronimo and Charleston
varieties, whereas they were observed in Sunpride variety at
mature green stage, as well as in all varieties tested in red ripe
stage. These results agree with those reported by Prusky (1996)
who concluded that ripe fruits are more readily infected than
mature green fruits. According to the mentioned author, the
resistance of unripe fruits to fungi infection is due to the
presence of inducible and preformed antifungal compounds
that decline in ripe fruits, as well as the lack of activation of
fungal pathogenicity factors during fruit ripening (Prusky,
1996). Sunpride variety in mature green stage showed the black
mold lesions after 8 days of the storage time. This behavior
could be due to the fact that this variety has a faster ripening rate
than the other varieties tested which probably led to the lack of
other antifungal compounds (Cowan, 1999; Calvo et al., 2002),
normally present in a less mature tomato fruit.
Differences in levels of chitinase and b-1,3-glucanase
activity were found in this experiment associated with the stage
of development in which the fruits started to ripen when stored
for 10 days at 25 8C. Our results clearly show that both enzymes
chitinase and b-1,3-glucanase activities were higher in mature
green stage of non-inoculated Charleston fruit at the end of the
storage time, as compared with the others varieties. Also, in this
variety both enzymatic activities were higher in mature green
stage of non-inoculated fruit at the end of the storage time, as
compared with red ripe stage. Besides, in Geronimo variety the
constitutive levels of chitinase activity were higher in mature
green stage of non-inoculated fruit as compared with red ripe
stage, showing a reduction in activity as fruit advance in
48 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
ripening. These results do not agree with the study reported
(Hinton and Pressey, 1980), in which it was found an increase in
b-1,3-glucanase activity during ripening of tomato fruit variety
‘Walter’. The differences observed could be ascribed to the
tomato fruit varieties used in the two studies under comparison
or to the differences in the techniques used to measure the b-
1,3-glucanase activity.
The induction of chitinase and b-1,3-glucanase activity by
different fungi and abiotic stress had been reported in vegetative
tissues of plants (Ramammoorthy et al., 2002; Rivera et al.,
2002; Fanta et al., 2003; Baldé et al., 2006). In tomato leaves,
chitinases and b-1,3-glucanases can be induced by methyl
jasmonic acid, salicilyc acid, b-aminobutyric acid, fusicoccin
(Wu and Bradford, 2003), as well as by wounding (Wu and
Bradford, 2003), and by Alternaria solani infection (Lawrence
et al., 1996, 2000). Lawrence et al. (1996) reported that four
isozymes of chitinase (26, 27, 30 and 32 kDa) were induced in
leaves of resistant tomato genotypes and susceptible cultivars to
Alternaria solani infection. The same authors concluded that
only basic isozymes of chitinase and b-1,3-glucanase were
inhibitory to A. solani. Besides, Lawrence et al. (2000), found
that higher levels of both total chitinase and b-1,3-glucanase
activity in resistant tomato genotype was detected when
compared to the susceptible tomato genotype, and these levels
increased in both genotypes following A. solani inoculation
(Lawrence et al., 2000). There are few experiments with the
objective to elucidate whether fruit respond in the same way. In
our study, we observed an increase in both enzymatic activities
in tomato fruit as a response to infection caused by Alternaria
alternata. These data agree with the results reported by
Salzman et al. (1998), who found an accumulation of basic
chitinase and thaumatin activity in grape fruits cv Concord, as a
response to infection by Botrytis cinerea and Guignardia
bidwellii. Also, our results agree with those reported by El
Ghaouth et al. (2003), who evaluated the ability of the
biocontrol agent, the yeast Candida saitoana, to induce
systemic resistance in apple fruit against Botrytis cinerea.
They found that C. saitoana increased chitinase and b-1,3-
glucanase activities with a higher accumulation in fresh
(recently harvested apples) than in stored apples. In fresh
apples, the onset of systemic resistance to B. cinerea correlated
with the increase in chitinase and b-1,3-glucanase activity in
systemically protected tissue. The authors concluded that C.
saitoana is capable of inducing systemic resistance in apple
fruit and indirectly suggest that these hydrolases are involved in
the observed systemic protection.
The susceptibility to A. alternata was different among
tomato fruit varieties. Results clearly show that Sunpride
variety, which is classified as Alternaria stem canker resistant
(Seminis Vegetable Seeds, Co.), showed an increase in b-1,3-
glucanase activity as a response to infection by the pathogen in
red ripe fruit. Furthermore, this increase in enzymatic activity
occurred at the same time period when the first disease
symptoms were observed. However, in mature green fruits, the
peak of b-1,3-glucanase activity was observed earlier than the
development of the infection symptoms. In the case of
chitinase, for Sunpride fruits inoculated at mature green stage,
the first symptoms of fungi infection were observed after 8 days
of storage correlating with a statistical significance increase of
chitinase enzyme activity. Furthermore, for those inoculated at
the red ripe stage, slight induction in chitinase activity was
recorded from day 6th of storage until the end. At the same
time, disease incidence changed from slight lesions to high
lesions. Sunpride variety showed the lowest induction in
chitinase activity in response to the fungi attack, as compared
with the other two varieties evaluated, which can explain the
highest advance in the development of the fungi infection.
Geronimo and Charleston, classified as large shelf-life
varieties, resistant to others fungi and non-resistant to
Alternaria spp. (De Ruiter Seeds and Ahem International
Seeds Inc., respectively), were less affected by A. alternata as
compared with Sunpride variety. Indeed, Geromino showed the
first symptoms of fungi infection by the end of the storage in the
red ripe stage; whereas no damage was observed in mature
green stage. Statistically significant higher values of b-1,3-
glucanase activity, for most of the storage time, were found for
this variety in both mature green and red ripe stages of
development in inoculated fruits as compared to non-inoculated
fruits. Also, higher levels of chitinase activity were recorded
after 2 and 8 days of the storage for mature green fruits and for
most of the storage time in the case of red ripe stage. However,
in the case of red ripe fruits of Geronimo, a trend of decreasing
in activity was observed by the end of the storage time with no
differences between control and fungi infected fruits, correlat-
ing with the development of lesions due to the fungi infection.
In the case of Charleston variety, it showed the highest levels of
chitinase and b-1,3-glucanase activity in mature green stage as
a response to infection by A. alternata. On the other side, in red
ripe stage, Charleston variety began to show fungi lesions when
the chitinase activity started to decrease at the same time that
statistically higher levels of b-1,3-glucanase were recorded.
Sunpride variety inoculated at red ripe stage showed the
highest susceptibility for the fungi infection albeit that this
variety also showed the highest level of b-1,3-glucanase
activity induced by the fungi attack. In contrast, Geronimo was
the most resistant variety when inoculated at the red ripe stage
with a much lower induction in the b-1,3-glucanase activity in
response to the fungi attack. On the hand, chitinase activity
showed a larger induction for Geronimo variety as compared to
Sunpride variety when inoculated at the red ripe stage of
development. This data seems to indicate that chitinase enzyme
plays a more critical role in the tomato fruit defense from fungi
attack than b-1,3-glucanase enzyme.
An experiment testing the antifungal activity in vitro of
several chitinase and b-1,3-glucanase isoforms reported that
there are some isoforms of both enzymes with no antifungal
activity (Sela-Buurlage et al., 1993). Although chitinase and b-
1,3-glucanase isoforms can not be determined by the methods
used in this assay, a possible explanation of the data found in
this study could be that A. alternata infection induce an isoform
of b-1,3-glucanase with no antifungal activity and an isoform of
chitinase with a strong antifungal activity in tomato fruit,
although more experimental evidences are needed to support
this statement.
 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50
Based on the above mentioned, we concluded that chitinase
and b-1,3-glucanase activities decreased during tomato fruit
ripening, particularly in Charleston variety that was one of the
resistant varieties evaluated. Also, chitinase and b-1,3-
glucanase induction are part of tomato fruit defense mechanism
against A. alternata infection with a different behavior
depending upon stage of development and variety.
Acknowledgment
The authors wish to extend their thanks to M.S.I. Alfonso
Aguilar for his technical assistant in the graphics creation
included in this manuscript.
References
Baldé, J.A., Francisco, R., Queiroz, A., Regalado, A.P., Ricardo, C.P., Veloso,
M.M., 2006. Immunolocalization of a class III chitinase in two muskmelon
cultivars reacting differently to Fusarium oxysporum f.sp. melonis. J. Plant
Physiol. 163 (1), 19–25.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of proteins utilizing the principle of protein dye
binding. Anal. Biochem. 72, 248–254.
Calvo, A.M., Wilson, R.A., Woo, J.B., Keller, N.P., 2002. Relationship between
secondary metabolism and fungal development. Microbiol. Mol. Biol. R 66
(3), 447–459.
Cowan, M.M., 1999. Plant Products as antimicrobial agents. Clin. Microbiol.
Rev. 12 (4), 564–582.
Danhash, N., Wagemakes, C.A.M., van Kan, J.A.L., de Wit, P.J.G.M., 1993.
Molecular characterization of four chitinase cDNAs obtained from Cla-
dosporium fulvum-infected tomato. Plant Mol. Biol. 22, 1017–1029.
Domingo, C., Conejero, V., Vera, P., 1994. Genes encoding acidic and basic
class III b-1,3-glucanases are expressed in tomato plants upon viroid
infection. Plant Mol. Biol. 24, 725–732.
El Ghaouth, A., Wilson, C.L., Wisniewski, M., 2003. Control of postharvest
decay of apple fruit with Candida saitoana and induction of defense
responses. Phytopathology 93, 344–348.
Fanta, N., Ortega, X., Pérez, L.M., 2003. The development of Alternaria
alternata is prevented by chitinases and b-1,3-glucanases from Citrus
limon seedlings. Biol. Res. 36, 411–420.
French, E.R., Hebert, T.T., 1982. Métodos de Investigación Fitopatológica.
Instituto Interamericano de Cooperación para la Agricultura, San José,
Costa Rica, pp. 168–185.
Grogan, R.G., Kimble, A., Misaghi, I., 1975. A stem canker disease of tomato
caused by Alternaria alternata f. sp. lycopersici. Phytopathology 65, 880–
886.
Harikrishna, K., Jampates-Beale, R., Milligan, S.B., Gasser, C., 1996. An
endochitinase gene expressed at high levels in the stylar transmitting tissue
of tomatoes. Plant Mol. Biol. 30, 899–911.
Harvey, J.M., 1978. Reduction of losses in fresh market fruits and vegetables.
Annu. Rev. Phytopathol. 16, 321–341.
Hinton, D.M., Pressey, R., 1980. Glucanases in fruits and vegetables. J. Am.
Soc. Horticult. Sci. 105 (4), 499–502.
Krishnaveni, S., Muthukrishnan, S., Liang, G.H., Wilde, G., Manickam, A.,
1999. Induction of chitinase and b-1,3-glucanases in resistant and suscep-
tible cultivars of sorghum in response to insect attack, fungal infection and
wounding. Plant Sci. 144 (1), 9–16.
Lawrence, C.B., Joosten, M.H.A.J., Tuzun, S., 1996. Differential induction of
pathogenesis-related proteins in tomato by Alternaria solani and the
association of a basic chitinase isozyme with resistance. Physiol. Mol.
Plant Pathol. 48, 361–377.
Lawrence, C.B., Singh, N.P., Qiu, J., Gardner, R.G., Tuzun, S., 2000. Con-
stitutive hydrolytic enzymes are associated with polygenic resistance of
tomato to Alternaria solani and may function as an elicitor release mechan-
ism. Physiol. Mol. Plant P 57, 211–220.
49
Leubner-Metzger, G., Meins Jr., F., 2000. Sense transformation reveals a novel
role for class I b-1,3-glucanases in tobacco seed germination. Plant J. 23,
215–221.
López-Hernandez, J., Gonzalez-Castro, M.J., Vazquez-Blanco, M.E., Vazquez-
Odoriz, M.L., Simal-Lozano, J., 1994. HPLC determination of sugars and
starch in green beans. J. Food Sci. 59 (5), 1048–1049.
McGlasson, W.B., Beatti, B.B., Kavanagh, E.E., 1985. Tomato Ripening Guide,
Agfact, H8.4.5, NSW Department of Agricultures (cited by Wills, R.,
McGlasson B., Graham, D., Joyce D., 1998. Postharvest: An Introduction
to the Physiology & Handling of Fruit, Vegetables & Ornamental, 4th ed.
CAB international, p. 118).
Neale, A.D., Wahleithner, J.A., Lund, M., Bonnett, H.T., Kelly, A., Meeks-
Wagner, R., Peacock, W.J., Dennis, E.S., 1990. Chitinase, b-1,3-glucanase,
osmotin, and extensin are expressed in tobacco explants during flower
formation. Plant Cell 2, 673–684.
Prusky, D., 1996. Pathogen quiescence in postharvest diseases. Annu. Rev.
Phytopathol. 34, 413–434.
Pryor, B.M., Michailides, T.J., 2002. Morphological, pathogenic, and molecular
characterization of Alternaria isolates associated with Alternaria late blight
of pistachio. Phytopathology 92, 406–416.
Ramammoorthy, V., Raguchander, T., Samiyappan, R., 2002. Induction of
defense-related proteins in tomato roots treated with Pseudomonas fluor-
escens Pf1 and Fusarium oxysporum f. sp. Lycopersici. Plant Soil 239, 55–
68.
Rivera, M.E., Codina, J.C., Olea, F., De Vicente, A., Pérez-Garcia, A., 2002.
Differential expression of b-1,3 glucanase in susceptible and resistant
melon cultivars in response to infection by Sphaerotheca fusca. Physiol.
Mol. Plant P 61, 257–265.
Ruelas, C., Tiznado-Hernández, M.E., Sánchez-Estrada, A., Robles-Burgueño,
M.R., Troncoso-Rojas, R., 2006. Changes in phenolic acid content during
Alternaria alternata infection in tomato fruit. J. Phytopathol. 154, 236–
244.
Salles, I.I., Blount, J.W., Dixon, R.A., Schubert, K., 2002. Phytoalexin induc-
tion and b-1,3-glucanase activities in Colletotrichum trifolii infected leaves
of alfalfa (Medicago sativa L.). Physiol. Mol. Plant P 61, 89–101.
Salzman, R.A., Tikhonova, I., Bordelon, B.P., Hasegawa, P.M., Bressan, R.A.,
1998. Coordinate accumulation of antifungal proteins and hexoses consti-
tutes a developmentally controlled defense response during fruit ripening in
grape. Plant Physiol. 117, 465–472.
SAS, 2000. Institute SAS user’s guide. Version 8.2., SAS Institute Inc., Cary,
NC, USA.
Sela-Buurlage, M.B., Ponstein, A.S., Bres-Vloemans, S.A., Melchers, L.S.,
Elzen, P.J.M., Cornelissen, B.J.C., 1993. Only specific tobacco (Nicotiana
tabacum) chitinases and b-1,3-glucanases exhibit antifungal activity. Plant
Physiol. 101, 857–863.
Siefert, F., Langebartels, C., Boller, T., Grossmann, K., 1994. Are ethylene and
1-aminocyclopropane-1-carboxylic acid involved in the induction of chit-
inase and b-1,3-glucanase activity in sunflower cell-suspension cultures?
Planta 192, 431–440.
Simmons, E.G., 1995. Alternaria taxonomy: current status, viewpoint, chal-
lenge. In: Chelkowski, J., Visconti, A. (Eds.), Alternaria Biology, Plant
Diseases and Metabolites. Elsevier Science Publishers, Amsterdam, pp. 1–
35.
Terry, L.A., Joyce, D.C., 2004. Elicitors of induced disease resistance in
postharvest horticultural crops: a brief review. Postharvest Biol. Technol.
32, 1–13.
Troncoso-Rojas, R., Sánchez-Estrada, A., Ruelas, C., Garcı́a, H.S., Tiznado-
Hernández, M.E., 2005. Effect of benzyl isothiocyanate on tomato fruit
infection development by Alternaria alternata. J. Sci. Food Agric. 85,
1427–1434.
Tung-Shin, H., Ya-Min, C., Hsien-Yi, S., Chen-Tien, C., 2002. Purification and
characterization of hydrolase with chitinase and chitosanase activity from
commercial stem bromelain. J. Agric. Food Chem. 50, 4666–4673.
van Loon, L.C., van Strien, E.A., 1999. The families of pathogenesis-related
proteins, their activities and comparative analysis of PR-1 type proteins.
Physiol. Mol. Plant P 55, 85–97.
Veronese, P., Ruiz, M.T., Coca, M.A., Hernandez-Lopez, A., Lee, H., Ibeas, J.I.,
Damsz, B., Pardo, J.M., Hsegawa, P.M., Bressan, R.A., Narasimhan, M.I.,
50 I.E. Cota et al. / Scientia Horticulturae 112 (2007) 42–50

2003. In defense against pathogens. Both plants sentinels and foot soldiers
need to know the enemy. Plant Physiol. 131, 1580–1590.
Webster, J., 1986. Introduction to Fungi. Cambridge University Press, USA, pp.
57–64.
Wu, Ch., Bradford, K.J., 2003. Class I chitinase and b-1,3-glucanase are
differentially regulated by wounding, methyl jasmonate, ethylene, and
gibberellin in tomato seeds and leaves. Plant Physiol. 133, 263–273.
Wu, Ch., Leubner-Metzger, G., Meins, F., Bradford, K.J., 2001. Class I b-1,3-
glucanase and chitinase are expressed in the micropylar endosperm of
tomato seeds prior to radicle emergence. Plant Physiol. 126, 1299–
1313.
Wurms, K.V., 2004. The incidence of Botrytis cinerea and expression of
putative host defenses in green-and golden-fleshed kiwifruit of differing
harvest maturity. NZ Plant Prot. 57, 125–129.

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