International Journal of Food Microbiology 221 (2016) 5460

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Efcacy of salicylic acid to reduce Penicillium expansum inoculum and

preserve apple fruits
Argus Cezar da Rocha Neto a,, Caroline Luiz a, Marcelo Maraschin b, Robson Marcelo Di Piero a,
a
b

Laboratory of Plant Pathology, Crop Science Department, Federal University of Santa Catarina, 88040-900 Florianpolis, Santa Catarina, Brazil
Laboratory of Morphogenesis and Plant Biochemistry, Federal University of Santa Catarina, 88040-900 Florianpolis, Santa Catarina, Brazil

a r t i c l e

i n f o

Article history:
Received 13 September 2015
Received in revised form 6 January 2016
Accepted 11 January 2016
Available online 13 January 2016
Keywords:
Apple
Penicillium expansum
Postharvest
Salicylic acid

a b s t r a c t
Apples are among the most commonly consumed fruits worldwide. Blue mold (Penicillium expansum) is one of
the major diseases in apples postharvest, leading to wide use of fungicides and the search for alternative products
to control the pathogen. In this context, this study aimed to evaluate the potential of salicylic acid (SA) as an alternative product to control blue mold and to preserve the physicochemical characteristics of apple fruit postharvest. The antimicrobial effect of SA was determined both in vitro and in situ, by directly exposing conidia to
solutions of different concentrations SA or by inoculating the fruit with P. expansum and treating them curatively,
eradicatively, or preventively with a 2.5 mM SA solution. The physiological effects of SA on fruit were determined
by quantifying the weight loss, total soluble solids content, and titratable acidity. In addition, the accumulation of
SA in the fruit was determined by HPLC. SA (2.5 mM) inhibited 100% of fungal germination in vitro and also controlled blue mold in situ when applied eradicatively. In addition, HPLC analysis demonstrated that SA did not persist in apple fruit. SA also maintained the physicochemical characteristics of fruit of different quality categories.
Thus, SA may be an alternative to the commercial fungicides currently used against P. expansum.
2016 Elsevier B.V. All rights reserved.

1. Introduction
The food industry is one of the most prominent in the world economy. In Brazil, this industry represents one of the most important segments of the market (Chitarra and Chitarra, 2005), including
production of more than 1,300,000 tons of apples in 2012 (FAOSTAT,
Food and Agriculture Organization of the United Nations Statistical
Database, 2012).
Apples (Malus domestica Borkh.) are susceptible to a wide range of
pathogenic microorganisms, especially those that can produce
pectinolytic enzymes able to degrade the apple tissues (Spadaro et al.,
2002; Vilanova et al., 2014a; Daniel et al., 2015). Moreover, mechanical
damage during the incorrect handling of the fruit postharvest may contribute to the development of different types of rot (Vilanova et al.,
2014b), even when the fruit is stored at low temperatures, which can
slow but cannot prevent the development of pathogenic fungi (Buronmoles et al., 2012).
Blue mold caused by Penicillium expansum is a very destructive disease of apples. P. expansum can produce high numbers of conidia that
can spread quickly, causing major losses of fresh and processed fruits
(Sanzani et al., 2010). It can also synthesize the mycotoxin patulin,
Corresponding authors.
E-mail addresses: neto.acrn@gmail.com (A.C. da Rocha Neto), robson.piero@ufsc.br
(R.M. Di Piero).

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.01.007
0168-1605/ 2016 Elsevier B.V. All rights reserved.

which can be deleterious to human health (da Rocha et al., 2014;

Wright et al., 2014).
For these reasons, the use of synthetic fungicides continues to be the
main defense against this pathogen worldwide (Zhang et al., 2011),
leaving residues in the fruits and also in industrial wash-water
(Poulsen et al., 2009; Maxin et al., 2012).
With the increase of social concerns about environment and public
health (Droby et al., 2009) and the selection of resistant isolates to the
active ingredients of fungicides (Weber and Palm, 2010; Lima et al.,
2011), new strategies against P. expansum are required.
The use of salicylic acid (SA) is thought to be a good alternative to
control diseases. This molecule is involved in the biosynthesis of defensive compounds in plants and exhibited antimicrobial effects against
various fungi that cause rot (Panahirad et al., 2012), e.g., Botrytis cinerea
in pears (Zhang et al., 2008), Fusarium oxysporum in tomatoes (Mandal
et al., 2009), and P. expansum in apples (da Rocha Neto et al., 2015).
In addition, SA can help to preserve the physical and chemical characteristics of fruits by preventing, for example, the weight loss of stored
strawberries (Shaee et al., 2010), the discoloration of peaches (Abassi
et al., 2010), as well as maintaining pulp rmness, total soluble solids
content, and titratable acidity of grapes (Qin et al., 2015).
Thus, this study aimed to evaluate the potential of salicylic acid to
protect apple fruit cv. Fuji of different quality categories against
P. expansum and to preserve the physicochemical characteristics of the
fruit.

A.C. da Rocha Neto et al. / International Journal of Food Microbiology 221 (2016) 5460

2. Materials and methods

2.1. Apple fruit, pathogen
Standardized apple fruit cv. Fuji of three categories (1, 2, and 3), purchased from COOPERSERRA (So Joaquim, Santa Catarina, Brazil) and
stored in a cold-chamber (temperature: 4 C 2 C; humidity:
85% 1%; 2 months) prior to use, were disinfected with 0.5% (v/v) hypochlorite solution for 2 min, rinsed in tap water and air-dried.
The fruit were classied as category 1 when at least 40% of their epidermal area presented red-coloration and had neither sunburn nor
open lesions, whereas for fruit categorized as 2 these values were 20%
red-colored epidermal area, a maximum of 20% sunburn and 20 mm2
of open lesions. Finally, category-3 fruit presented less than 10% of
their epidermis with red-coloration, more than 20% with sunburn and
up to 70 mm2 of open lesions.
P. expansum was isolated from an infected apple fruit exhibiting typical symptoms of blue mold, identied and provided by Dr. Rosa Maria
Sanhueza, and stored in the mycology collection of the Laboratory of
Plant Pathogen (Federal University of Santa Catarina, Florianpolis,
Brazil) with the code MANE 138.
The isolate was grown and maintained in potato dextrose agar
(PDA) culture medium, at 25 C for two weeks prior to use. The conidial
suspension was calibrated to the nal concentration for each experiment with the aid of a Neubauer chamber (hemocytometer).
2.2. Salicylic acid
Salicylic acid (2-Hydroxybenzoic acid) was acquired from SigmaAldrich Co. (St. Louis, MO, USA; Sigma product code 247588) and diluted in sterile distilled water with the aid of a magnetic bar and a stirrer
(Shalmashi and Eliassi, 2008; da Rocha Neto et al., 2015). The concentration of SA varied according to the experiments.
2.3. Antifungal potential of salicylic acid against P. expansum
The antimicrobial potential of SA was assessed in concave microscope slides. For this, 25 L of a SA solution at 0, 1, 2.5, or 5 mM and
25 L of P. expansum conidial suspension (105 conidia/mL) were
added to the slide cavity. Sterile distilled water was used as positive
control. The concave slides were placed inside Petri dishes and incubated for 20 h at 25 C 1 C under high relative humidity. Four replicates
were performed per treatment and each replicate was represented by a
cavity in the concave slide. The germination of 100 conidia and the
length of germ the tube of 20 conidia were evaluated for each replicate
with the aid of an optical microscope (FWL1500 T, Feldmann Wild
Leitz). The experiment was conducted three times.
2.4. Protective, curative, and eradicative effects of salicylic acid in apples
under two storage conditions
Disinfected apples of the three categories were distributed in plastic
trays, injured twice in the equatorial region with the aid of a standardized needle (5 mm deep 1 mm wide) and treated. There were four
replicates per treatment; a tray with 4 fruits represented one replicate.
The protective potential of SA was evaluated immersing the injured
apples from the three categories in sterile distilled water or in 2.5 mM
SA solution for 2 min. After drying, the fruit were immersed into a
P. expansum conidial suspension (104 conidia/mL) for 2 min. The curative potential was evaluated changing the order of the applications,
i.e., fruit were inoculated and then immersed in distilled water or SA
solution.
Finally, the eradicative potential was evaluated through the
immersion of injured apples in a P. expansum conidial suspension
(10 4 conidia/mL) previously prepared in sterile distilled water
(A) or 2.5 mM SA solution (B). These suspensions were stirred for

55

30 min then apples were immersed in the suspensions (A or B) for

2 min.
In a second experiment, the eradicative potential of SA was
assessed through the immersion of injured apples (categories 1, 2,
and 3) in a conidial suspension of P. expansum (10 4 conidia/mL)
prepared in sterile distilled water or 2.5 mM SA, for 2 min. Two
more treatments were added at this time: the immersion of fruits
only in sterile distilled water or only in 2.5 mM SA, without the
presence of P. expansum conidia. All these suspensions were stirred
for 30 min before immersion of fruit (2 min).
The trays containing the fruit (under high relative humidity)
were incubated at room temperature (25 C 1 C) or in a coldchamber (4 C 1 C), in the dark, throughout the experimental
period.
The rate of growth of the lesions was determined by measuring
the diameter of the lesion (cm) of each injury made in every single
fruit, with a standard ruler, every 4 or 10 days, depending on the
conditions of the incubation (rst evaluation was performed after 4
and 10 days of incubation at 25 C and 4 C, respectively). Based on
the average value of the lesion diameters over time, the lesion growth
rate was estimated in each tray as follows: LGR = ((t t 1) / t);
where represents the average diameter of the lesion at time t. The
results were expressed in cm/day (da Rocha Neto et al., 2015).
The disease incidence was calculated at the end of the experiment
by the division of the number of injuries made in the apples
presenting characteristic symptoms of blue mold by the total
number of injuries made in these fruits. The average results were
expressed in %.

2.5. Inuence of an acidic solution on P. expansum in situ

The inuence of an acidic solution against blue mold incidence
and severity in different apples categories were also evaluated.
Standardized apples (categories 1, 2 or 3) were injured twice in the
equatorial zone as describe above, and immersed into a
P. expansum conidial suspension (104 conidia/mL) prepared in sterile
distilled water (pH 7.0), distilled water acidied with 0.05 N HCl
(pH 3.0) or 2.5 mM SA. These suspensions were stirred for 30 min,
followed by the immersion of apples for 2 min. The apples were
incubated at 25 C 1 C, under high relative humidity, in the
dark, throughout the experimental period. Incidence and severity
of the rot were evaluated as described above.

2.6. Inuence of SA on the physicochemical characteristics of apples

From the experiments described in Section 2.4 (second eradicative
experiment) and Section 2.5, 10 apples (categories 1, 2 or 3) were
sampled at the beginning (day 0) and at the end (day 12) of the
experiment for physicochemical assays.
The weight loss of the fruit (WLF) was quantied as described by
Tefera et al. (2007) by weighing each fruit on an analytical balance.
Soluble solids (SS) content was quantied by the method of Dong
et al. (2001), with modications. The sampled apples were ground
with the aid of a juicer (HL 3235, Walita) and a crude liquid extract
(CLE) was obtained. Then, 10 L of CLE was added to the refractometer
(RT-30 ATC, Instrutherm) and the SS contents were determined. The
results were expressed in Brix.
Titratable acidity (TA) was determined according to the method
proposed by Tefera et al. (2007), with modications. TA levels were
measured by the titration with 0.1 N sodium hydroxide of CLE diluted
in sterile distilled water (10%). The relative calculation of TA was carried
out from the nal volume of the sodium hydroxide solution necessary to
alkalinize the pH of apple juice to 8.2. The obtained results were
expressed in % of malic acid present in apples.

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A.C. da Rocha Neto et al. / International Journal of Food Microbiology 221 (2016) 5460

2.7. Identication and quantication of residual SA in apples

Firstly, apples from the three different categories were
injured then immersed for 2 min in a P. expansum conidial suspension (10 4 conidia/mL) prepared in sterile distilled water or in
2.5 mM SA.
Then, ve disks of the fruit pulp with epidermis were sampled
(6 mm diameter 4 mm depth, totaling approximately 300 mg, for
each replicate) at the beginning (0 h) and at the end (80 h) of the experiment. Five replicates for each treatment were used.
Identication and quantication of SA present in the apple methanolic extracts were performed according to Schmidt et al. (2012), with
modications. Each sample was placed in an Eppendorf tube, 1 mL
80% acidied methanol (pH 2.0) added, followed by maceration in a
Precellys tissue homogenizer (Bertin Corp., Rockville MD, USA). Then
2 mL 80% acidied methanol was added to the macerate and incubated
for 1 h in darkness and ice bath. After incubation, the mixture was centrifuged for 5 min (6000 rpm, 4 C) and the supernatant collected.
Aliquots (10 L) of each sample were analyzed by a liquid chromatograph (Shimadzu LC 10 A), tted with a C18 column (Shim-Pack;
250 mm 4.6 mm internal , 5 m particle size, 35 C) and a UVvisible
detector operating at 280 nm. The elution used water: acetic acid: etabutanol (350:1:10 v/v/v) as mobile phase at a ow rate of 0.8 mL/min,
and the compounds of interest identied by co-chromatography; the
retention times of standard compounds (gallic acid and salicylic
acid Sigma Aldrich, USA) were compared under the same experimental conditions. The quantication of SA was performed based on a standard external curve of gallic acid (0.5300 LmL1; y = 35158; r2 =
0.99) taking into consideration the peak areas of interest for the calculation of concentration, where the resulting values represent the average

of 3 injections per sample per treatment. The nal concentration of SA

was expressed in mM.
2.8. Statistical analysis
The experiments were carried out in a completely randomized design and the experimental plots were described above for each item.
The data were subjected to Levene's or Cochran's tests to check the homogeneity of the variances of the treatments (factorial analysis and
one-way ANOVA), followed by analysis of variance and the respective
F-test (5%). When the F-test showed signicant results, Tukey's test
was performed at 5% signicance level.
When suitable, linear regression analyses were carried out by the
software Sisvar 5.0, observing the mathematical model that best t
into the results, based on t-test values at 5% signicance level.
All other statistical analyses were performed using the software
Statistica 10.0 and the graphs were designed by software Prism 5 for
Mac OS X.
Moreover, in Fig. 1, as the control samples for all experiments
showed similar results for incidence, severity, and lesion growth rate,
not differing statistically regardless the form of application (curatively,
eradicatively or protectively), they were grouped as one single result
to reduce the amount of information.
3. Results and discussion
When applied in vitro SA (2.5 mM) completely inhibited conidial
germination and germ tube development of P. expansum. However as
the SA concentration decreased, its efciency was also reduced,
inhibiting phytopathogen germination by up to 85% at 1 mM

Fig. 1. Curative, eradicative or preventive application of 2.5 mM SA solution in apples of different quality categories (1, 2, and 3) against P. expansum incidence (%), severity (cm) and
growth (cm/day). The apples were stored at 25 C (12 days) or at 4 C (40 days) throughout the experimental period. Data represent the average SD. Different upper letters indicate
signicant differences between the forms of application in the fruit's different quality categories (Tukey, p 0.05). No differences were observed between categories in a form of
application.

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57

Table 1
Germination (%) and germ tube length (M) of P. expansum conidia exposed to different
doses of SA. Data are shown as the average SD of three independent experiments. Different upper letters indicate signicant differences between the doses in germination
while different lower letters indicate signicant differences between the doses in the germ
tube length (Tukey, p 0.05).
Salicylic acid (mM)

Germination (%)

Germ tube length (m)

0
1
2.5
5

92.25 3.4 A
13.5 3.11 B
0C
0C

118.25 7.14 a
23.75 3.86 b
0c
0c

(Table 1). A correlation between the antimicrobial activity of SA and its

concentration was found by Yu and Zheng (2006) who observed that
the compound inhibited more than 50% of the germination of
P. expansum only at concentrations above 0.7 mM.
Not only the concentration but also the mode of application of SA on
apples affected SA antimicrobial activity against P. expansum. Applied
eradicatively, SA (2.5 mM) inhibited 100% of blue mold incidence,
both at 25 C and at 4 C, in all three apple categories; however, the compound did not have a preventive and curative effect, regardless the incubation temperature and the quality category of the fruit (Fig. 1).
According to Barreira et al. (2010), the blue mold rot caused by
P. expansum is reduced but not inhibited by low temperatures,
conrming the results observed here. In addition, the inefciency of
SA preventively or curatively to control decay has already been reported. Zhang et al. (2008) showed that 0.5 mM SA preventively applied
against the gray mold (B. cinerea) in peach fruits reduced rot incidence
by only 20%. Wang et al. (2011) also observed an insignicant reduction
in the severity of gray mold in tomatoes treated with SA curatively.
It is suggested that because of its low viscosity, SA quickly ows off
the surface of apples without forming a physical barrier able to protect
the wounded fruit against the pathogen. Moreover, SA was probably
not able to activate the defense mechanisms of the fruit in order to prevent pathogen colonization, probably due to the lack of time between
the exposure of the fruit to the SA solution, thus failing to control the
disease curatively.
As noted, SA could greatly reduce the amount of viable conidia when
applied in an eradicative way. These results conrm our previous ndings that after 1 h of contact between SA and conidia, no viable conidia
were detected (da Rocha Neto et al., 2015).
It is well known that the higher the concentration of P. expansum conidia in water, the higher the percentage of infection in apples (Spotts,
1986). Thus, the eradicative methodology could be used in packinghouses, adding SA to the water used to clean the apples that arrive
from orchard. Thus conidia brought from the orchard on infected fruit
that accumulated in the packinghouse water would have their viability
compromised by SA activity, preventing the infection of new apples
during postharvest procedures.
Only apples immersed in SA eradicatively did not show incidence of
blue mold, whereas in fruits immersed in acidied distilled water
(pH 3) blue mold reached 100% incidence at the end of the experimental
period. Thus the incidence and the lesion growth rate in apples immersed in acidied water were similar to those found in fruits immersed in distilled water at pH 7 control (Fig. 2).
These results suggest that the antimicrobial effect of SA is due mainly
to its chemical structure rather than its capacity to acidify the solution,
an assumption in agreement with the results of Amborabe et al.
(2002), where the antimicrobial effect of SA against Eutypa lata in
grapes was shown to be related to its molecular structure.
In addition to its antimicrobial activity, which appears to be the main
effect for the control of blue mold in apple fruits treated eradicatively,
SA maintained the main physicochemical characteristics of the fruits,
regardless the category analyzed (data not shown). Apples from category 3, initially weighing 151.1 g, maintained their average weight
throughout the experiment after treatment with SA (150 g), whereas

Fig. 2. Eradicative application of 2.5 mM SA solution or acidied water (pH 3) in apples of

different quality categories (1, 2, and 3) against P. expansum incidence (%), severity (cm)
and growth (cm/day). Data are shown as the average SD. Different upper letters
indicate signicant differences between treatments in the different categories. Different
lower letters indicate signicant differences between categories with the same
treatment (Tukey, p 0.05).

fruits immersed in distilled water weighed 147 g. A lower average

weight was observed (144 g) in the fruit challenged with the phytopathogen (Fig. 3A).
Total soluble solids content in apples immersed in SA solution, with
or without conidia, did not differ statistically throughout the experimental period. However, there was an increase from 11.2 to 13.2 Brix
in fruits immersed only in water. These values were similar to those
fruit challenged with P. expansum (Fig. 3B).
Kazemi et al. (2011) also observed an inverse relation for the SA concentration and the weight loss and content of soluble solids in apples cv.
Jonagold treated with SA at 1.5 mM or 3 mM and stored at 5 C. However, in their study the fruit were not challenged with P. expansum. This
demonstrated that SA was able to maintain the soluble solids content
and other physicochemical characteristics of apples, preventing degradation over time.
SA is an antagonist of ethylene, able to reduce its biosynthesis and
action in plants and fruit by reducing the respiration rate of fruit or

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A.C. da Rocha Neto et al. / International Journal of Food Microbiology 221 (2016) 5460

Fig. 3. Physicochemical characteristics of apples (category 3) exposed to water, 2.5 mM SA,

P. expansum conidia or to conidia of P. expansum prepared in 2.5 mM SA. Average fruit
mass in grams (A), soluble solids content in Brix (B) and titratable acidity in % (C). Data
are the average SD. Different upper letters represent signicant differences between
beginning (day 0) or end (day 12) in the treatment. Different lower letters indicate
signicant differences between treatments (Tukey, p 0.05).

even stomata closure (Norman et al., 2004). Being an uncoupler and an

inhibitor of mitochondrial electron transport, SA probably decreases the
availability of substrate to catabolic reactions, contributing to maintenance of soluble solids content and the weight of fruit.

Fig. 4. Physicochemical characteristics of apples (category 3) exposed to P. expansum

conidia prepared in water, acidied water (pH 3) or SA. Average fruit mass in grams (A),
soluble solids content in Brix (B) and titratable acidity in % (C). Data are the average
SD. Different upper letters indicate signicant differences between beginning (day 0) or
end (day 12) in the treatment. Different lower letters indicate signicant differences
between treatments (Tukey, p 0.05).

The maintenance of soluble solids content in apples when SA was

applied eradicatively also occurred, probably due to its antimicrobial activity, preventing the development of the fungus and consequently, the
catabolism of sugars present in the fruit by the pathogen, through the
action of secreted enzymes that can degenerate the tissues, e.g., amylases and pectinases (Balkan and Ertan, 2005).
Regarding titratable acidity, apples immersed in SA solution showed
a similar metabolic response to apples immersed only in water (Fig. 3C).

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59

Table 2
Salicylic acid concentration (mM) of apples of different quality categories (1, 2, and 3) immersed in a suspension of P. expansum conidia prepared in water or in 2.5 mM SA solution. Data
are shown as the average SD. The samples were collected in the beginning (0 h) or in the end (80 h) of experimental period. Different upper letters indicate signicant differences in the
column while different lower letter indicate signicant differences in the lines (Tukey, p 0.05).
Category

Retention time (min)

Conidia in water (0 h)

Conidia in water (80 h)

Conidia in SA (0 h)

Conidia in SA (80 h)

1
2
3

4.1
4.1
4.1

0.012 0.003 B ab
0.019 0.002 A
0.012 0.001 B b

0.015 0.002 ab
0.013 0.003
0.012 0.001 b

0.011 0.001 B b
0.016 0.002 A
0.017 0.003 A a

0.017 0.004 a
0.015 0.003
0.016 0.002 a

However, the immersion of the apples in the conidial suspension of

P. expansum led to an acidication of the fruit tissues at the end of the
experiment (Fig. 3C), probably due to secretion of citric and gluconic
acids during the process of colonization by P. expansum (Prusky and
Lichter, 2008),
Even the eradicative treatment with acidied distilled water
(pH 3.0) did not prevent the deterioration of the apples, regardless of
their quality category. A weight loss of up to 9% and an increase of
total soluble solids content by up to 13% in apples of category 3 were observed (Fig. 4), not differing from control (sterile distilled water,
pH 7.0). However, when applied eradicatively, SA conrmed the previous results, preserving the physicochemical parameters (weight and
soluble solids content) of apples throughout the experimental period
(Fig. 4A and B).
The presence of secondary metabolites, such as phenolic acids, in
fruit is commonly reported in the literature (Sun et al., 2002; Boyer
and Liu, 2004). Biosynthesis of these compounds usually depends on
the environmental conditions under which fruit was grown, varying
not only between species but also within the species. Along with several
other phenolic compounds, SA is usually found in fruit in various quantities (Russel et al., 2009). In our study, regardless of the quality categories of the fruit, the SA amounts detected were not high, with a
maximum of 0.019 mM of SA at time 0 h (Table 2).
These results are in agreement with Russel et al. (2009), who detected only 0.047 mM of conjugated SA in apple fruit and also with Schieber
et al. (2001) who identied chlorogenic acid, caffeic acid and epicatechin as the major compounds in apples cv Jonagold and Elstar.
Finally, apples of the three quality categories eradicatively treated
with SA presented a total content of SA similar to control group, revealing a non-persistence trait of that phenolic acid in apple tissue, even
after 80 h of the treatment (0.016 mM of SA), indicating its rapid degradation (Table 2).
This rapid degradation was reported in some studies of environmental toxicology. Heberer (2002), for example, studying the concentration
prole of SA over the disposal of sewage efuents, observed that SA
has a fast degradation in surface waters, contrarily to other polar
compounds.
4. Conclusion
Salicylic acid inhibited 100% of the germination of P. expansum and
prevented the development of conidia in apples of categories 1, 2, and
3 when applied eradicatively at 2.5 mM, under storage temperatures
of 25 C and 4 C. In addition, the use of SA preserved the weight and
soluble solids content of the fruit. Considering the high antimicrobial
activity of SA and its ability to preserve important physiological
characteristics of fruit, regardless the presence or absence of pathogens,
the use of SA solution in an eradicative way could be an alternative for
the postharvest treatment of apples, not only because it can reduce
the pathogen inoculum, but also because it can extend the shelf life of
the treated fruit.
Acknowledgments
The researcher grants from CNPq on behalf of M. Maraschin and R.
M. Di Piero are acknowledged.

References
Abassi, N.A., Hafeez, S., Tareen, M.J., 2010. Salicylic acid prolongs shelf life and improves
quality of Maria Delicia peach fruit. Acta Horticult. 880, 191197.
Amborabe, B., Fleurat-Lessard, P., Chollet, J.F., Roblin, G., 2002. Antifungal effects of
salicylic acid and other benzoic acid derivatives towards Eutypa lata: structureactivity relationship. Plant Physiol. Biochem. 40, 10511060.
Balkan, B., Ertan, F., 2005. Production and properties of -amylase from Penicillium
chrysogenum and its application in starch hydrolysis. Prep. Biochem. Biotechnol. 35,
169178.
Barreira, M.J., Alvito, P.C., Almeida, C.M.M., 2010. Occurrence of patulin in apple-basedfoods in Portugal. Food Chem. 121, 653658.
Boyer, J., Liu, R.H., 2004. Apple phytochemicals and their health benets. Nutr. J. 3, 115.
Buron-Moles, G., Lopez-Perez, M., Gonzlez-Candelas, L., Vias, I., Teixid, N., Usall, J.,
Torres, R., 2012. Use of GFP-tagged strains of Penicillium digitatum and Penicillium
expansum to study hostpathogen interactions in oranges and apples. Int. J. Food
Microbiol. 160, 162170.
Chitarra, M.I.F., Chitarra, A.B., 2005. Ps-colheita de frutas e hortalias: siologia e
manuseio. UFLA, Lavras.
da Rocha Neto, A.C., Maraschin, M., Di Piero, R.M., 2015. Antifungal activity of salicylic acid
against Penicillium expansum and its possible mechanisms of action. Int. J. Food
Microbiol. 215, 6470.
Daniel, C.K., Lennox, C.L., Vries, F.A., 2015. In vivo application of garlic extracts in combination with clove oil to prevent postharvest decay caused by Botrytis cinerea, Penicillium expansum and Neofabraea alba on apples. Postharvest Biol. Technol. 99, 8892.
Dong, L., Zhou, H.W., Sonego, L., Lers, A., Lurie, S., 2001. Ripening of red Rosa plums: effect
of ethylene and 1-methycyclopropene. Aust. J. Plant Physiol. 28, 10391045.
Droby, S., Wisniewski, M., Macarisin, D., Wilson, C., 2009. Twenty years of postharvest
biocontrol research: is it time for a new paradigm? Postharvest Biol. Technol. 52,
137145.
FAOSTAT (Food and Agriculture Organization of the United Nations Statistical Database),
2012a. Apple (Malus domestica) production: top production. URL http://faostat.fao.
org/site/339/default.aspx (Accessed 29.12.14).
Heberer, T., 2002. Tracking persistent pharmaceutical residues from municipal sewage to
drinking water. J. Hydrol. 266, 175189.
Kazemi, M., Aran, M., Zamani, S., 2011. Effect of salicylic acid treatments on the quality
characteristics of apple fruits during storage. Am. J. Plant Physiol. 6, 113119.
Lima, G., Castoria, R., de Curtis, F., Raiola, A., Ritieni, A., de Cicco, V., 2011. Integrated control of
blue mold using new fungicides and biocontrol yeasts lowers levels of fungicide residues and patulin contamination in apples. Postharvest Biol. Technol. 60, 164172.
Mandal, S., Mallick, N., Mitra, A., 2009. Salicylic acid-induced resistance to Fusarium
oxysporum f. sp. Lycopersici in tomato. Plant Physiol. Biochem. 47, 642649.
Maxin, P., Weber, R.W.S., Pedersen, H.L., Williams, M., 2012. Control of a wide range of
storage rots in naturally infected apples by hot-water dipping and rinsing. Postharvest Biol. Technol. 70, 2531.
Norman, C., Howell, K.A., Millar, A.H., Whelan, J.M., Day, D.A., 2004. Salicylic acid is an uncoupler and inhibitor of mitochondrial electron transport. Plant Physiol. 134,
492501.
Panahirad, S., Nahandi, F.Z., Safaralizadeh, R., Alizadeh-Salteh, S., 2012. Postharvest control of Rhizopus stolonifer in peach (Prunus persica L. Batsch) fruits using salicylic
acid. J. Food Saf. 32, 502507.
Poulsen, M.E., Naef, A., Gasse, S., Christen, D., Rasmussen, P.H., 2009. Inuences of different
disease control pesticide strategies on multiple pesticide residue levels in apple.
J. Hortic. Sci. Biotechnol. 84, 5861.
Prusky, D., Lichter, A., 2008. Mechanisms modulating fungal attack in post-harvest pathogen interactions and their control. Eur. J. Plant Pathol. 121, 281289.
Qin, X., Xiao, H., Xue, C., Yu, Z., Yang, R., Cai, Z., Si, L., 2015. Biocontrol of gray mold in
grapes with the yeast Hanseniaspora uvarum alone and in combination with salicylic
acid or sodium bicarbonate. Postharvest Biol. Technol. 100, 160167.
Rocha, M.E.B., Freire, F.C.O., Maia, F.E.F., Guedes, M.I.F., Rondina, D., 2014. Mycotoxins and
their effects on human and animal health. Food Control 36, 159165.
Russel, W.R., Labat, A., Scobbie, L., Duncan, G.J., Duthie, G.G., 2009. Phenolic acid content of
fruits commonly consumed and locally produced in Scotland. Food Chem. 115,
100104.
Sanzani, S.M., Schena, L., Girolamo, A., Ippolito, A., Gonzalez-Candela, L., 2010. Characterization of genes associated with induced resistance against Penicillium expansum in
apple fruit treated with quercetin. Postharvest Biol. Technol. 56, 111.
Schieber, A., Keller, P., Carle, R., 2001. Determination of phenolic acid and avonoids of apple
and pear by high-performance liquid chromatography. J. Chromatogr. A 910, 265273.
Schmidt, E.C., Pereira, B., Santos, R.W., Gouveia, C., Costa, G.B., Faria, G.S.M., Scherner, F.,
Horta, P.A., Martins, R.P., Latini, A., Ramlov, F., Maraschin, M., Bouzon, Z.L., 2012. Responses of the macroalgae Hypnea musciformis after in vitro exposure to UV-B.
Aquat. Bot. 100, 817.

60

A.C. da Rocha Neto et al. / International Journal of Food Microbiology 221 (2016) 5460

Shaee, M., Taghavi, T.S., Babalar, M., 2010. Addition of salicylic acid to nutrient solution
combined with postharvest treatments (hot water, salicylic acid, and calcium dipping) improved postharvest fruit quality of strawberry. Sci. Hortic. 124, 4045.
Shalmashi, A., Eliassi, A., 2008. Solubility of salicylic acid in water, ethanol, carbon tetrachloride, ethyl acetate, and xylene. J. Chem. Eng. Data 53, 199200.
Spadaro, D., Vola, R., Piano, S., Gullino, M.L., 2002. Mechanisms of action and efcacy of
four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Postharvest Biol. Technol. 24, 123134.
Spotts, R.A., 1986. Relationships between inoculum concentrations of three decay fungi
and pear fruit decay. Plant Dis. 70, 386389.
Sun, J., Chu, Y.F., Wu, X., Liu, R.H., 2002. Antioxidant and antiproliferative activities of common fruits. J. Agric. Food Chem. 50, 74497454.
Tefera, A., Seyoum, T., Woldetsadik, K., 2007. Effect of disinfection, packaging, and storage
environment on the shelf life of mango. Biosyst. Eng. 96, 201212.
Vilanova, L., Vias, I., Torres, R., Usall, J., Buron-Moles, G., Teixid, N., 2014a. Increasing
maturity reduces wound response and lignication processes against Penicillium
expansum (pathogen) and Penicillium digitatum (non-host pathogen) infection in apples. Postharvest Biol. Technol. 88, 5460.
Vilanova, L., Vias, I., Torres, R., Usall, J., Buron-Moles, G., Teixid, N., 2014b. Acidication
of apple and orange hosts by Penicillium digitatum and Penicillium expansum. Int.
J. Food Microbiol. 178, 3949.

Wang, Y.Y., Li, B.Q., Qin, G.Z., Tian, S.P., 2011. Defense response of tomato fruit at different
maturity stages to salicylic acid and ethephon. Sci. Hortic. 129, 183188.
Weber, R.W.S., Palm, G., 2010. Resistance of storage rot fungi Neofabrae perennans, N. alba,
Glomerella acutata and Neonectria galligena against thiophanate-methyl in Northern
German apple production. J. Plant Dis. Prot. 117, 185191.
Wright, S.A.I., de Felice, D.V., Ianiri, G., Pinedo-Rivilla, C., de Curtis, F., Castoria, R., 2014.
Two rapid assays for screening of patulin biodegradation. Int. J. Environ. Sci. Technol.
11, 13871398.
Yu, T., Zheng, X.D., 2006. Salicylic acid enhances biocontrol efcacy of the antagonist Cryptococcus laurentii in apple fruit. J. Plant Growth Regul. 25, 166174.
Zhang, H., Ma, L., Wang, L., Jiang, S., Dong, Y., Zheng, X., 2008. Biocontrol of gray mold
decay in peach fruit by integration of antagonistic yeast with salicylic acid and their
effects on postharvest quality parameters. Biol. Control 47, 6065.
Zhang, C., Wang, J., Zhang, J., Hou, C., Wang, G., 2011. Effects of -aminobutyruc acid on
control of postharvest blue mold of apple fruit and its possible mechanisms of action.
Postharvest Biol. Technol. 61, 145151.