Efficacy of salicylic acid to reduce Penicillium ...

International Journal of Food Microbiology 221 (2016) 54–60

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Efficacy 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 Florianópolis, Santa Catarina, Brazil Laboratory of Morphogenesis and Plant Biochemistry, Federal University of Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil

a r t i c l e

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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: [email protected] (A.C. da Rocha Neto), [email protected] (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 (Shafiee et al., 2010), the discoloration of peaches (Abassi et al., 2010), as well as maintaining pulp firmness, 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) 54–60

2. Materials and methods 2.1. Apple fruit, pathogen Standardized apple fruit cv. Fuji of three categories (1, 2, and 3), purchased from COOPERSERRA (São 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 classified 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, identified and provided by Dr. Rosa Maria Sanhueza, and stored in the mycology collection of the Laboratory of Plant Pathogen (Federal University of Santa Catarina, Florianópolis, 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 final 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 μL of a SA solution at 0, 1, 2.5, or 5 mM and 25 μL 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

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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 (first 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 = (Σ(θt − θt − 1) / t); where “θ” 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. Influence of an acidic solution on P. expansum in situ The influence 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 acidified 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. Influence 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 quantified as described by Tefera et al. (2007) by weighing each fruit on an analytical balance. Soluble solids (SS) content was quantified by the method of Dong et al. (2001), with modifications. The sampled apples were ground with the aid of a juicer (HL 3235, Walita) and a crude liquid extract (CLE) was obtained. Then, 10 μL 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 modifications. 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 final 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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2.7. Identification and quantification 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, five 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. Identification and quantification of SA present in the apple methanolic extracts were performed according to Schmidt et al. (2012), with modifications. Each sample was placed in an Eppendorf tube, 1 mL 80% acidified methanol (pH 2.0) added, followed by maceration in a Precellys tissue homogenizer (Bertin Corp., Rockville MD, USA). Then 2 mL 80% acidified 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 μL) of each sample were analyzed by a liquid chromatograph (Shimadzu LC — 10 A), fitted with a C18 column (Shim-Pack; 250 mm × 4.6 mm internal θ, 5 μm particle size, 35 °C) and a UV–visible detector operating at 280 nm. The elution used water: acetic acid: etabutanol (350:1:10 v/v/v) as mobile phase at a flow rate of 0.8 mL/min, and the compounds of interest identified 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 quantification of SA was performed based on a standard external curve of gallic acid (0.5–300 μL·mL−1; 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 final 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 significant results, Tukey's test was performed at 5% significance level. When suitable, linear regression analyses were carried out by the software Sisvar 5.0, observing the mathematical model that best fit into the results, based on t-test values at 5% significance 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 efficiency 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 significant 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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Table 1 Germination (%) and germ tube length (μM) 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 significant differences between the doses in germination while different lower letters indicate significant differences between the doses in the germ tube length (Tukey, p ≤ 0.05). Salicylic acid (mM)

Germination (%)

Germ tube length (μm)

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, confirming the results observed here. In addition, the inefficiency 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 insignificant reduction in the severity of gray mold in tomatoes treated with SA curatively. It is suggested that because of its low viscosity, SA quickly flows 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 confirm our previous findings 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 acidified 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 acidified 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 acidified 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 significant differences between treatments in the different categories. Different lower letters indicate significant 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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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 significant differences between beginning (day 0) or end (day 12) in the treatment. Different lower letters indicate significant 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, acidified 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 significant differences between beginning (day 0) or end (day 12) in the treatment. Different lower letters indicate significant 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., αamylases 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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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 significant differences in the column while different lower letter indicate significant 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 acidification 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 acidified 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 confirmed 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 identified 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 profile of SA over the disposal of sewage effluents, 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.

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