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Antifungal activity of salicylic acid against Penicillium expansum and its possible mechanisms of action
International Journal of Food Microbiology 215 (2015) 64–70
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Antifungal activity of salicylic acid against Penicillium expansum and its possible mechanisms of action Argus Cezar da Rocha Neto 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
i n f o
Article history: Received 12 February 2015 Received in revised form 21 August 2015 Accepted 23 August 2015 Available online 28 August 2015 Chemical compounds studied in this article: Caffeic acid (PubChem CID: 689043) Calcofluor white (PubChem CID: 21065046) Chlorogenic acid (PubChem CID: 1794427) Cinnamic acid (PubChem CID: 444539) Propidium iodide (PubChem CID: 104981) Salicylic acid (PubChem CID: 338) Syringic acid (PubChem CID: 10742) Thiobarbituric acid (PubChem CID: 2723628)
a b s t r a c t Apple is a fruit widely produced and consumed around the world. Blue mold (Penicillium expansum) is one of the main postharvest diseases in apples, leading to a wide use of fungicides and the search for alternative products. The aim of this study was to assess the effect of salicylic acid (SA) against P. expansum, elucidating its mechanisms of action. The antimicrobial effect was determined by exposing conidia to a 2.5 mM SA solution for 0 to 120 min, followed by incubation. The effect of pH on the efficacy of SA against P. expansum was assessed both in vitro and in situ. The action mechanisms were investigated through fluorescence assays, measurement of protein leakage, lipid damage, and transmission electronic microscopy. SA was capable of inhibiting 90% of the fungal germination after 30 min, causing damage to the conidial plasma membrane and leading to protein leakage up to 3.2 μg of soluble protein per g of mycelium. The pH of the SA solution affected the antimicrobial activity of this secondary metabolite, which inhibited the germination of P. expansum and the blue mold incidence in apples in solutions with pH ≤ 3 by 100%, gradually losing its activity at higher pH. © 2015 Elsevier B.V. All rights reserved.
Keywords: Apple Penicillium expansum Phenolic compounds Postharvest Salicylic acid
1. Introduction Apples (Malus domestica Borkh.) are one of the most cultivated and consumed fruits worldwide, reaching a global production above 70 million tons in 2012 (Faostat, 2014). There are several diseases that can affect apples, including blue mold caused by Penicillium expansum, a very aggressive cosmopolitan fungus, tolerant to many adverse environmental conditions. It can spread quickly and produce the mycotoxin patulin, which can be deleterious to human health (da Rocha et al., 2014). From harvesting to storage, apples should be exposed to several procedures that, if well performed, can significantly reduce the incidence of blue mold. This pathogen requires small injuries in the epidermis or natural openings to infect apples (Filonow, 2005; Mondino et al., 2009). In general, disinfection procedures are mostly overlooked, in favor of the use of fungicides as the main way to control postharvest diseases (Calvo et al., 2007; Karabulut et al., 2002). Synthetic fungicides based
⁎ Corresponding author. E-mail address: [email protected] (A.C. da Rocha Neto).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.08.018 0168-1605/© 2015 Elsevier B.V. All rights reserved.
on imidazole and dicarboximide groups are commercially available and recommended by the Ministry of Agriculture, Livestock and Food Supply in Brazil for the control of the blue mold. However, the development of isolates resistant to synthetic chemicals, associated with growing public concerns related to health and environmental pollution, have increased the efforts to research alternative methods of fruit protection against pathogens (Mills and Golding, 2015). Among these, the use of phenolic compounds is of interest, not only due to the important roles of these compounds in plant defenses against pathogens, but also because they have proven to be beneficial to human health exhibiting antioxidant and anti-carcinogenic effects (Sanzani et al., 2010). Salicylic acid (SA), a phenolic acid with an aromatic ring linked to a hydroxyl group or functional derivatives, is an endogenous plant hormone involved in plant growth development and cell signaling (Asghari and Aghdam, 2010; Tian et al., 2007). Many studies have evaluated its antifungal activity against several postharvest pathogens, including P. expansum (Yu and Zheng, 2006), Botrytis cinerea (Wang et al., 2011), and Rhizopus stolonifer (Panahirad et al., 2012). However, the mechanisms of action of SA against those pathogens have not been fully elucidated to date.
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Thus, this study aimed to assess the effect of SA on the germination and growth of P. expansum and elucidate its mechanisms of action. Such approach is assumed to be crucial for the development of efficient alternative strategies to replace the use of fungicides to control blue mold in apple fruits. 2. Materials and methods 2.1. Fungal isolate P. expansum was isolated from an infected apple fruit showing typical symptoms of blue mold, identified and provided by Dr. Rosa Maria Sanhueza, and stored in the mycology collection of the Phytopathology Laboratory (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 prepared in apple juice (4%, v/v) and calibrated to the final concentration with the aid of a Neubauer chamber (hemacytometer). 2.2. Chemicals The caffeic, chlorogenic, cinnamic, salicylic, and syringic phenolic acids were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). They were diluted in sterile distilled water with the aid of a magnetic bar and a stirrer until solutions were homogeneous (Shalmashi and Eliassi, 2008). The concentrations of the phenolic acids varied according to the experiments. Calcofluor White Stain and propidium iodide were also acquired from Sigma-Aldrich Co. (St. Louis, MO, USA) and used at Sections 2.7.1 and 2.7.2 to evaluate the cell wall damage and the plasma membrane integrity of the conidia, respectively. Finally, bovine serum albumin (BSA) was acquired from SigmaAldrich Co. (St. Louis, MO, USA) to quantify total protein and used as standard in Section 2.7.3. 2.3. Screening of phenolic acids The antimicrobial potential of the different phenolic acids was assessed in concave slides. For this, 25 μL of a phenolic acid solution (cinnamic, caffeic, syringic, chlorogenic or salicylic) at 2.5 mM and 25 μL of P. expansum suspension (105 conidia/mL) were added to the slide cavity. Sterile distilled water and sodium hypochlorite (0.5%, v/v) were used as positive and negative controls, respectively. The concave slides were placed inside Petri dishes and incubated for 20 h, at 25 °C ± 1 °C, under high relative humidity. Each treatment was replicated four times and each replicate was represented by a cavity in the concave slide. The germination of 100 conidia was evaluated for each replicate with the aid of an optical microscope (FWL1500 T, Feldmann Wild Leitz). The experiment was conducted twice. 2.4. Minimum contact time between salicylic acid and P. expansum The minimum contact time between SA and P. expansum was determined according to Liu et al. (2007), with modifications. Ten milliliters of a P. expansum suspension (2 × 108 conidia/mL) prepared in apple juice were added to 10 mL of sterile distilled water (negative control), 0.5% sodium hypochlorite (positive control) or 2.5 mM SA, under constant stirring, at room temperature. Aliquots (500 μL) were collected after 0, 5, 15, 30, 60, and 120 minute incubation and being centrifuged (8000 g, 5 min, 4 °C). The conidia were collected, washed twice with a 50 mM phosphate buffer (pH 7.0) and re-suspended in apple juice (4%, v/v) to a final concentration of 105 conidia/mL. Finally, the germination test in concave slides described above was performed with the conidial suspension. The experiment was conduced three times.
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2.5. Effect of acidification or alkalization of the SA solution on P. expansum germination Twenty milliliters of the conidial suspension (105 conidia/mL) prepared in apple juice were added to 20 mL of a 2.5 mM SA solution. Then the pH values of the final mixture were adjusted to 2.0, 2.5, 2.8, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 by the addition of 0.05 N HCl or 2 N NaOH. As control, apple juice (4%, v/v) was used without SA addition at the same pH values described above. The resulting conidial suspensions were submitted to the germination test described above. The experiment was repeated three times. 2.6. Effect of acidification or alkalization of the SA solution on the incidence of P. expansum rot in apples Standardized apple fruits cv. Fuji, acquired from COOPERSERRA (São Joaquim/SC, southern Brazil), were disinfected with a chlorine solution (0.5%, v/v), injured twice in the equatorial zone with a standard needle (1 mm × 5 mm) and immersed into a P. expansum conidial suspension (104 conidia/mL) prepared in sterile distilled water or in salicylic acid solution (both pH ranging from 2 to 6, adjusted with 0.05 N HCl or 2 N NaOH). These suspensions were stirred for 30 min followed by the immersion of apples for 2 min. The fruits were then transferred to plastic containers and kept at 25 °C under high relative humidity, in the dark, throughout the experimental period. Three replicates per treatment were made, where a plastic container containing five fruits represented one replication. 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 3 days (first evaluation was performed after 3 days of incubation). 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. Moreover, the incidence was calculated at the end of the experiment by the division of the number of injuries made in the apples presenting blue mold symptoms by the total number of injuries made in these fruits. The average results were expressed in %. 2.7. Action mechanisms of SA against P. expansum 2.7.1. Damage to the cell wall of P. expansum conidia The evaluation of the SA effects on the pathogen cell wall was performed according to Cerioni et al. (2010), with modifications. Firstly, the conidia of P. expansum were suspended in 2.5 mM SA or sterile distilled water (control) to a final concentration of 108 conidia/mL. The suspensions were maintained at room temperature under constant stirring. Samples (500 μL) were collected at intervals of 0, 5, 15, 30, 60, and 120 min, centrifuged (8000 g, 5 min, 4 °C), double washed with a 50 mM sodium phosphate buffer (pH 7.0) and sterile distilled water, centrifuged (8000 g, 5 min, 4 °C), and re-suspended in apple juice (4%, v/v) to a final concentration of 105 conidia/mL. Aliquots of 50 μL were then transferred to concave slides and incubated as described above. The samples were then transferred to microscope slides, 10 μL of Calcofluor White Stain reagent (Sigma-Aldrich, USA) at 50 μg/mL and 10 μL of 10% (w/v) potassium hydroxide added, and incubated in the dark for 15 min. The conidial morphology and germ tubes were examined with the aid of a fluorescence microscope (Eclipse 50i, Nikon) at the wavelengths of 395 nm (excitation) and 440 nm (emission). The images were recorded with a Nikon digital camera (Coolpix P500) and the experiment conducted twice. 2.7.2. Damage to the plasma membrane of P. expansum conidia Damage to the plasma membrane of P. expansum conidia exposed to SA was determined as described by Liu et al. (2007), with modifications. Conidial suspensions were prepared in 2.5 mM SA or sterile distilled
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water (control), incubated and sampled as described above. After sampling, the P. expansum conidia were stained with propidium iodide (10 μg/mL, Sigma, USA) for 5 min, incubated at 30 °C, centrifuged (8000 g, 5 min, 4 °C), and washed twice with a 50 mM sodium phosphate buffer (pH 7.0), followed by another centrifugation. The supernatant was discarded, the precipitate collected and re-suspended in sterile distilled water (105 conidia/mL). The evaluation was performed with the aid of a fluorescence microscope at the wavelengths of 546 nm (excitation) and 590 nm (emission). Images were taken with a Nikon digital camera and the experiment carried out twice. At least 100 conidia were evaluated under visible light and fluorescence at each time interval, in the different fields, followed by the calculation of the percentage of fluorescent conidia. The damage caused by the exposure of the P. expansum plasma membrane to SA was quantified following the method proposed by Cerioni et al. (2010) and Rice-Evans et al. (1991), with modifications. The conidial suspensions of P. expansum were carried out as described for the determination of the cell wall damage. Aliquots (500 μL) were removed after 0, 5, 15, 30, 60, and 120 min, centrifuged (2000 g, 5 min) and washed twice with a 50 mM sodium phosphate buffer (pH 7.0). The supernatants were discarded, the precipitates re-suspended in sterile distilled water to a final concentration of 5 × 107 conidia/mL, and treated in an ultrasound bath (10 min) to disrupt the conidia. To 1 mL of these suspensions, 1 mL of trichloroacetic acid (TCA) 20% (w/v) was added for precipitation at 4 °C, followed by centrifugation (19,800 g, 5 min). Then, 2 mL of saturated solution containing thiobarbituric acid (TBA) in 0.1 M HCl and 10 mM butylated hydroxytoluene (BHT) were added to the resulting supernatants, followed by incubation in a water bath at 100 °C for 60 min. The collected extracts were cooled in ice bath for 10 min and the absorbance read at 535 nm in a micro plate multi reader (Spectramax Paradigm Molecular Devices). For the blank, the entire procedure was performed, with 1 mL of TCA (centrifuged) added to 2 mL of TBA solution described above, but without the addition of conidial extract. The final concentrations of thiobarbituric acid reactive species (TBARS) were determined using the molar extinction coefficient (ε = 156 mmol/cm) and expressed as pmol/mg of protein. The experiment was conducted twice. 2.7.3. Determination of P. expansum protein leakage Intracellular protein leakage was quantified as proposed by Liu et al. (2010) with modifications. A conidial suspension of P. expansum was transferred to Erlenmeyer flasks (250 mL) containing 100 mL of potato dextrose broth to a final concentration of 5 × 105 conidia/mL. The flasks were incubated at 200 rpm, 25 °C, in the dark. After three days, 3 g of the mycelium (fresh weight) were collected in paper filter (0.2 μm), washed with sterile distilled water and transferred to Erlenmeyer flasks containing 30 mL of sterile distilled water or 2.5 mM SA. After different incubation times (0, 5, 15, 30, 60, and 120 min) at room temperature, aliquots (4 mL) of the mycelium suspension were collected and filtered. The content of total soluble proteins was determined following the Bradford method (1976), using BSA (Sigma-Aldrich, USA) as standard. The filtrates were collected for the determination of the total intracellular protein leakage. Absorbance readings (595 nm) were made with the aid of a micro plate multi reader and the results expressed as μg of soluble protein per g of fresh mycelium. The experiment was conducted three times. 2.7.4. Ultrastructure analysis of P. expansum conidia Suspensions of P. expansum conidia (2 × 108 conidia/mL) were prepared in sterile distilled water (control) or 2.5 mM SA and stirred for 60 min. Then, 500 μL was collected, centrifuged (8000 g, 5 min, 4 °C), washed with a 50 mM sodium phosphate buffer (pH 7.0), centrifuged, and re-suspended in sterile distilled water. For transmission electronic microscopy (TEM), the method described by Schmidt et al. (2009) was performed with modifications. The samples were fixed with a 2.5% glutaraldehyde solution (w/v), 2% sucrose (w/v), and buffered
with 0.1 M cacodylate (pH 7.2), post-fixed with osmium tetroxide for 4 h and dehydrated in a series of aqueous solutions with increasing acetone concentrations. After dehydration, the material was infiltrated with Spurr resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate. The samples were visualized and photographed in a transmission electronic microscope (JEM 1011, Jeol Ltda., 80 kV), in the Central Laboratory of Electronic Microscopy of Federal University of Santa Catarina. 2.8. Statistical analysis The experiments were carried out in a completely randomized design and the experimental design was described above in the relevant sections. 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. The statistical analyses were performed using the software Statistica 10.0 and the graphs were designed by software Prism 5 for Mac OS X. 3. Results and discussion 3.1. Phenolic acids and their antimicrobial potential against P. expansum The antimicrobial activity of phenolic acids was assessed as an alternative to the traditional methods for control of blue mold in apples. These substances are produced in plants mainly in response to stress conditions and have been widely discussed in studies on maintenance of postharvest quality in fruit (Tareen et al., 2012). Salicylic acid was able to completely inhibit the germination of P. expansum conidia at 2.5 mM (Fig. 1). Similar results were found against other pathogens e.g., R. stolonifer, Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolinae, Pythium sp., and Phytophthora sp., where about 70% of conidial germination and growth was inhibited by SA, at a minimum concentration of 2.5 mM (El-Mohamedy et al., 2013; Panahirad et al., 2012). Hypochlorite (0.5% of active chlorine) and SA (2.5 mM) similarly inhibited the germination of P. expansum conidia. The hypochlorite solution in a dose 100 times higher than that commercially used inhibited
Fig. 1. Penicillium expansum conidial germination (%) exposed to phenolic acids at 2.5 mM. Data are the average ± SD of two experiments. Different letters represent significant differences between treatments (Tukey, p ≤ 0.05).
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the germination after just 30 min of exposure of the conidia, whereas SA reduced the germination by up to 90% in the same period (Fig. 2). In many countries, the addition of active chlorine to the fruit disinfection water is a common practice, especially because of its antimicrobial potential and quick action. However, the chlorine used in this process can complex with the natural organic matter of the fruits or with preexisting contaminants in water, forming products that can potentially cause cancer or mutation in animals (Ferraris et al., 2005; Marabini et al., 2006). To validate SA as a new treatment for the management of postharvest disease in apples, analysis of other parameters, such as the pH of the solution, is necessary. pH can be directly associated with the development of the pathogen and its virulence, and can also influence the activities of the antimicrobial compounds by changing chemical structures. Conidia of P. expanum prepared in suspensions with pH adjusted to 2 did not germinate, regardless of the treatment used. However, the conidial germination in the control treatment was partially achieved and fully restarted at pH 2.5 and 3.0, respectively (Fig. 3). Such results were partially discussed by Smilanick et al. (2005) who concluded that the conidia of P. expansum become inactive, but not destroyed, at pH 2, with minor effect in less acidic solutions, e.g., pH 4. Subsequently, Li et al. (2010) demonstrated through biochemical and proteomic assays that the pH value of the medium affects the internal pH of the conidia, leading to decreased levels of ATP and, consequently, conidial inactivation, with no damage to the conidial structure. This may be related to the capacity of P. expansum to acidify the host tissue by the secretion of organic acids, such as D-gluconic acid (pH 3.5) and citric acid (pH 3.1), infecting and colonizing the tissues (Barad et al., 2014). In this sense, P. expansum conidia seem to be well adapted to environmental conditions of pH near 2.5, growing normally at pH above 3. These findings were confirmed with an in situ assay, where the P. expansum conidia in aqueous suspensions with a pH ranging from 2 to 6 were able to germinate and infect the apples, especially in an acidic environment of pH 2 or 3, reaching 100% incidence and an average rate of lesion growth of 0.27 cm per day, leading to a final lesion diameter of 3.59 cm (Fig. 4). At pH 4 to 6, although the in vitro conidial germination rate was high (Fig. 3), a reduced in situ development of the fungus was observed (Fig. 4), probably due to the difficulty of colonizing apple tissues in a less acidic environment. At a standard pH level (2.8), SA completely inhibited the fungus germination, showing a decrease in its antifungal potential at pH 3.5, and completely losing its biological potential at a pH above 5.5 (Fig. 3). This effect was also observed in situ, where the treatment with 2.5 mM SA at pH 2 or 3 inhibited 100% of blue mold incidence in apple fruits, whereas the alkalization of the SA solution to pH ≥ 4 decreased its potential (Fig. 4).
Fig. 2. Minimum contact time between salicylic acid (2.5 mM) and P. expansum for the inhibition of conidial germination (%). Data are the average ± SD of three experiments. Different letters represent significant differences between treatments at the same times (Tukey, p ≤ 0.05).
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Fig. 3. Conidial germination of Penicillium expansum (%) exposed to salicylic acid at 2.5 mM for the studied pH values. SA standard pH was assumed to be 2.8. Data are the average ± SD of three experiments. Different letters represent significant differences between treatments at the same pH (Tukey, p ≤ 0.05).
Fig. 4. Effects of salicylic acid solution with different pHs in apple fruits. (A) Final lesion diameter (cm) of P. expansum. (B) Incidence (%) of blue mold in apple fruits. (c) Lesion growth rate (cm/day) of P. expansum. Data are the average ± SD. Different letters represent significant differences between treatments at different pH's (Tukey, p ≤ 0.05).
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Amborabe et al. (2002) found similar results using SA against the fungus Eutypa lata, at 2 mM and pH value near 4, observing that the phenolic action is specifically due to its molecular arrangement, where a simple displacement of the hydroxyl group on the aromatic ring can reduce the antifungal activity by up to 50%. Other compounds have shown similar patterns where the pH value affects their antimicrobial activity. For example, the antimicrobial effect of several bicarbonates against B. cinerea depends on the pH of the solution, with a predominance of carbonic acids in acidic solutions, which are unstable and show poor antimicrobial potential (Palmer et al., 1997). The commercial fungicide imazalil, commonly used against the citrus green mold, exhibited changes in its biological activity with changes in pH, losing 90% of its antimicrobial effect at pH 4.0 (Smilanick et al., 2005). Thus, increasing the pH of SA solution seems to result in molecular dissociation at neutral pH, with consequent reduction of antifungal and/or antioxidant potential (Amborabe et al., 2002). 3.2. Action mechanisms of SA against P. expansum At 2.5 mM, SA did not induce changes in the morphological characteristics of the cell wall of P. expansum conidia, even after a longer exposure time (data not shown). Similar results were observed against Penicillium digitatum conidia exposed to lethal doses (100 mM) of hydrogen peroxide, which totally inhibited germination, but without apparent damage in the cell wall when examined under fluorescence and transmission electronic microscopy (Cerioni et al., 2010). Damage caused by SA in the plasma membrane of P. expansum conidia were detected through the fluorescence emitted by propidium iodide, which cannot enter undamaged conidia due to the selective membrane permeability but is capable of penetrating damaged cells, intercalating between DNA or RNA strands (Wang et al., 2014). With only 5 minute exposure to 2.5 mM SA, 60% of P. expansum conidia showed fluorescence, reaching 100% of damaged plasma membrane after 30 minute contact with the phenolic compound. In contrast, for the control treatment, the integrity of the conidia plasma membrane was maintained throughout the experiment, with no fluorescence emission (Fig. 5A). These findings were confirmed through the TBA assay, where conidia of P. expansum prepared in distilled water and incubated up to 120 min, did not show any variation in the amounts of reactive species after the TBA reaction. However, when exposed to SA at 2.5 mM for 30 min, about 2000 pmol of TBA reactive species per mg of protein were quantified. These values increased as the time of contact between the conidia and SA increased, reaching a maximum of 6000 pmol of TBA reactive species per mg of protein after 120 minute exposure (Fig. 5B). In addition, after exposure to SA, 2.4 μg of soluble protein per g of fresh mycelium leaked into the medium, reaching a maximum of 3.2 μg of protein, whereas the protein leakage in the control treatment was only 1.8 μg of protein during 120 min of incubation (Fig. 5C). The cell membrane is essential to the cell viability of fungal conidia and mycelium, where damage to the lipid bilayer may result in cellular collapse, leading to the leakage of intracellular compounds (Chen et al., 2014). In comparison, other compounds showed a lesser capacity of inhibiting the pathogen than SA. Zhang et al. (2011) reported the use of 500 mM ß-amino butyric acid inhibiting germination of P. expansum conidia by 90%, causing damage to the plasma membrane in 80% of the conidia after 4 hour exposure, with maximum extravasation of 0.8 mg of protein per g of fresh mycelium to the medium. Liu et al. (2010) found a 100% inhibition of P. digitatum germination in 0.5% silicon. However, the compound caused damage to the plasma membrane in only 50% of the P. digitatum conidia after 4 hour exposure, with a leakage of 1 mg of protein per g of fresh mycelium. In our study, other damage to the plasma membrane was found after the analysis with the aid of a transmission microscope. The P. expansum conidia suspended in sterile distilled water exhibited an organized cell
Fig. 5. Effects of salicylic acid at 2.5 mM on the plasma membrane integrity of the Penicillium expansum conidia according to the exposure times. The integrity of plasma membrane was evaluated by fluorescence method (A), by the quantification of reactive species of thiobarbituric acid — TBARS (B), and by intracellular protein leakage (C). Data are the average ± SD of two (A–B) or three (C) experiments. Different letters represent significant differences between treatments at the same times (Tukey, p ≤ 0.05).
cytoplasm, presenting typical organelles e.g., mitochondria, rough endoplasmic reticulum, and ribosomes, as well as a central vacuole, plasma membrane and a thin cell wall (Fig. 6A–C). In contrast, the conidia suspended in 2.5 mM SA for 60 min exhibited several changes in the intra-cellular morphology, with a massive formation of vacuoles, several invaginations of the plasma membrane, a disorganized cytoplasm, and a significant thickening of the conidia cell walls (Fig. 6D–H). The antimicrobial potential of SA may be related to perturbations caused to cellular respiration, redirecting the electron flow from the cytochrome pathway to an alternative cyanide-related pathway, partially disabling the main cellular respiration pathway and affecting the cell energy generating process (Kapulnik et al., 1992). SA is also capable of binding specifically with proteins that can act on the decomposition of intracellular hydrogen peroxide, leading to the accumulation of this compound into the cell, inactivating the conidia (Chen et al., 1993). Studying the plant pathogen E. lata, Amborabe et al. (2002) observed that 2 mM SA had a direct antimicrobial effect, with several types of damage caused to the cell functions. They found that SA behaves as a dissociating agent causing a change in the trans-membrane pH gradient
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Fig. 6. Transmission electron microscopy of the conidia of P. expansum treated with water (a–c) or with salicylic acid at 2.5 mM (d–h). (a) P. expansum conidia arranged with the presence of a central vacuole (V) and organelles in the cytoplasm (Cy). (b) Detail of the presence of mitochondria (M), rough endoplasmic reticulum (Rer), and a thin cell wall (Cw) on the conidia. (c) Cytoplasm with numerous free ribosomes (R). (d) Irregular morphology of P. expansum conidia after treatment with SA at 2.5 mM. (e) Conidia with formation of large amount of vacuoles. (f–h) Disruption of cellular structures in the cytoplasm of P. expansum conidia and increased thickness of cell wall upon SA exposure.
in membranes of organelles and in the plasma membrane, possibly causing cellular energy loss. A study demonstrated the interaction between the charges of the amino groups present in chitosan and the plasma membrane of P. expansum conidia, resulting in the change of the selective permeability of the conidial plasma membrane, leading to the cell lysis (Wang et al., 2014). SA seems to act directly against P. expansum conidia by penetrating the cell wall and triggering several interactions with the plasma membrane, leading to the disruption of the lipid bilayer and/or causing damage to the proteins responsible for cellular permeability, increasing the concentration of reactive oxygen species. Finally, chemical interactions between the ions formed by SA in solution and the fatty acid constituents of the plasma membrane of P. expansum conidia might determine changes in the lipid model, decreasing its fluidity by modifying the lipid fraction or the bond with Ca++ ions, which are essential in diverse sites of the plasma membrane.
4. Conclusion Salicylic acid solutions with pH ≤ 3 inhibited the in vitro germination of P. expansum and the incidence of blue mold on apples, even at a low concentration, i.e., 2.5 mM. In addition, SA caused leakage of the pathogen's proteins to the medium, measurable lipid damage, and intracellular disorganization. Moreover, SA is a phenolic compound considered safe for the environment and human health and can be an alternative to the fungicides commonly used against the postharvest diseases of apples. However, as the SA molecule was shown to be highly sensitive to pH changes, and once its chemical structure is correlated with its antimicrobial potential, further studies are necessary to completely elucidate its in situ mode of action against P. expansum as well as effects on apple physiology.
Acknowledgments Our special thank you for Dr. Eder Carlos Schmidt from the Laboratory of Seaweed of Federal University of Santa Catarina for all the support given on the ultra-structural analysis and to CAPES for the financial support. The researcher grants from CNPq on behalf of M. Maraschin and R. M. Di Piero are acknowledged.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Antifungal activity of salicylic acid against Penicillium expansum and its possible mechanisms of action Argus Cezar da Rocha Neto 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
i n f o
Article history: Received 12 February 2015 Received in revised form 21 August 2015 Accepted 23 August 2015 Available online 28 August 2015 Chemical compounds studied in this article: Caffeic acid (PubChem CID: 689043) Calcofluor white (PubChem CID: 21065046) Chlorogenic acid (PubChem CID: 1794427) Cinnamic acid (PubChem CID: 444539) Propidium iodide (PubChem CID: 104981) Salicylic acid (PubChem CID: 338) Syringic acid (PubChem CID: 10742) Thiobarbituric acid (PubChem CID: 2723628)
a b s t r a c t Apple is a fruit widely produced and consumed around the world. Blue mold (Penicillium expansum) is one of the main postharvest diseases in apples, leading to a wide use of fungicides and the search for alternative products. The aim of this study was to assess the effect of salicylic acid (SA) against P. expansum, elucidating its mechanisms of action. The antimicrobial effect was determined by exposing conidia to a 2.5 mM SA solution for 0 to 120 min, followed by incubation. The effect of pH on the efficacy of SA against P. expansum was assessed both in vitro and in situ. The action mechanisms were investigated through fluorescence assays, measurement of protein leakage, lipid damage, and transmission electronic microscopy. SA was capable of inhibiting 90% of the fungal germination after 30 min, causing damage to the conidial plasma membrane and leading to protein leakage up to 3.2 μg of soluble protein per g of mycelium. The pH of the SA solution affected the antimicrobial activity of this secondary metabolite, which inhibited the germination of P. expansum and the blue mold incidence in apples in solutions with pH ≤ 3 by 100%, gradually losing its activity at higher pH. © 2015 Elsevier B.V. All rights reserved.
Keywords: Apple Penicillium expansum Phenolic compounds Postharvest Salicylic acid
1. Introduction Apples (Malus domestica Borkh.) are one of the most cultivated and consumed fruits worldwide, reaching a global production above 70 million tons in 2012 (Faostat, 2014). There are several diseases that can affect apples, including blue mold caused by Penicillium expansum, a very aggressive cosmopolitan fungus, tolerant to many adverse environmental conditions. It can spread quickly and produce the mycotoxin patulin, which can be deleterious to human health (da Rocha et al., 2014). From harvesting to storage, apples should be exposed to several procedures that, if well performed, can significantly reduce the incidence of blue mold. This pathogen requires small injuries in the epidermis or natural openings to infect apples (Filonow, 2005; Mondino et al., 2009). In general, disinfection procedures are mostly overlooked, in favor of the use of fungicides as the main way to control postharvest diseases (Calvo et al., 2007; Karabulut et al., 2002). Synthetic fungicides based
⁎ Corresponding author. E-mail address: [email protected] (A.C. da Rocha Neto).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.08.018 0168-1605/© 2015 Elsevier B.V. All rights reserved.
on imidazole and dicarboximide groups are commercially available and recommended by the Ministry of Agriculture, Livestock and Food Supply in Brazil for the control of the blue mold. However, the development of isolates resistant to synthetic chemicals, associated with growing public concerns related to health and environmental pollution, have increased the efforts to research alternative methods of fruit protection against pathogens (Mills and Golding, 2015). Among these, the use of phenolic compounds is of interest, not only due to the important roles of these compounds in plant defenses against pathogens, but also because they have proven to be beneficial to human health exhibiting antioxidant and anti-carcinogenic effects (Sanzani et al., 2010). Salicylic acid (SA), a phenolic acid with an aromatic ring linked to a hydroxyl group or functional derivatives, is an endogenous plant hormone involved in plant growth development and cell signaling (Asghari and Aghdam, 2010; Tian et al., 2007). Many studies have evaluated its antifungal activity against several postharvest pathogens, including P. expansum (Yu and Zheng, 2006), Botrytis cinerea (Wang et al., 2011), and Rhizopus stolonifer (Panahirad et al., 2012). However, the mechanisms of action of SA against those pathogens have not been fully elucidated to date.
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Thus, this study aimed to assess the effect of SA on the germination and growth of P. expansum and elucidate its mechanisms of action. Such approach is assumed to be crucial for the development of efficient alternative strategies to replace the use of fungicides to control blue mold in apple fruits. 2. Materials and methods 2.1. Fungal isolate P. expansum was isolated from an infected apple fruit showing typical symptoms of blue mold, identified and provided by Dr. Rosa Maria Sanhueza, and stored in the mycology collection of the Phytopathology Laboratory (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 prepared in apple juice (4%, v/v) and calibrated to the final concentration with the aid of a Neubauer chamber (hemacytometer). 2.2. Chemicals The caffeic, chlorogenic, cinnamic, salicylic, and syringic phenolic acids were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). They were diluted in sterile distilled water with the aid of a magnetic bar and a stirrer until solutions were homogeneous (Shalmashi and Eliassi, 2008). The concentrations of the phenolic acids varied according to the experiments. Calcofluor White Stain and propidium iodide were also acquired from Sigma-Aldrich Co. (St. Louis, MO, USA) and used at Sections 2.7.1 and 2.7.2 to evaluate the cell wall damage and the plasma membrane integrity of the conidia, respectively. Finally, bovine serum albumin (BSA) was acquired from SigmaAldrich Co. (St. Louis, MO, USA) to quantify total protein and used as standard in Section 2.7.3. 2.3. Screening of phenolic acids The antimicrobial potential of the different phenolic acids was assessed in concave slides. For this, 25 μL of a phenolic acid solution (cinnamic, caffeic, syringic, chlorogenic or salicylic) at 2.5 mM and 25 μL of P. expansum suspension (105 conidia/mL) were added to the slide cavity. Sterile distilled water and sodium hypochlorite (0.5%, v/v) were used as positive and negative controls, respectively. The concave slides were placed inside Petri dishes and incubated for 20 h, at 25 °C ± 1 °C, under high relative humidity. Each treatment was replicated four times and each replicate was represented by a cavity in the concave slide. The germination of 100 conidia was evaluated for each replicate with the aid of an optical microscope (FWL1500 T, Feldmann Wild Leitz). The experiment was conducted twice. 2.4. Minimum contact time between salicylic acid and P. expansum The minimum contact time between SA and P. expansum was determined according to Liu et al. (2007), with modifications. Ten milliliters of a P. expansum suspension (2 × 108 conidia/mL) prepared in apple juice were added to 10 mL of sterile distilled water (negative control), 0.5% sodium hypochlorite (positive control) or 2.5 mM SA, under constant stirring, at room temperature. Aliquots (500 μL) were collected after 0, 5, 15, 30, 60, and 120 minute incubation and being centrifuged (8000 g, 5 min, 4 °C). The conidia were collected, washed twice with a 50 mM phosphate buffer (pH 7.0) and re-suspended in apple juice (4%, v/v) to a final concentration of 105 conidia/mL. Finally, the germination test in concave slides described above was performed with the conidial suspension. The experiment was conduced three times.
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2.5. Effect of acidification or alkalization of the SA solution on P. expansum germination Twenty milliliters of the conidial suspension (105 conidia/mL) prepared in apple juice were added to 20 mL of a 2.5 mM SA solution. Then the pH values of the final mixture were adjusted to 2.0, 2.5, 2.8, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 by the addition of 0.05 N HCl or 2 N NaOH. As control, apple juice (4%, v/v) was used without SA addition at the same pH values described above. The resulting conidial suspensions were submitted to the germination test described above. The experiment was repeated three times. 2.6. Effect of acidification or alkalization of the SA solution on the incidence of P. expansum rot in apples Standardized apple fruits cv. Fuji, acquired from COOPERSERRA (São Joaquim/SC, southern Brazil), were disinfected with a chlorine solution (0.5%, v/v), injured twice in the equatorial zone with a standard needle (1 mm × 5 mm) and immersed into a P. expansum conidial suspension (104 conidia/mL) prepared in sterile distilled water or in salicylic acid solution (both pH ranging from 2 to 6, adjusted with 0.05 N HCl or 2 N NaOH). These suspensions were stirred for 30 min followed by the immersion of apples for 2 min. The fruits were then transferred to plastic containers and kept at 25 °C under high relative humidity, in the dark, throughout the experimental period. Three replicates per treatment were made, where a plastic container containing five fruits represented one replication. 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 3 days (first evaluation was performed after 3 days of incubation). 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. Moreover, the incidence was calculated at the end of the experiment by the division of the number of injuries made in the apples presenting blue mold symptoms by the total number of injuries made in these fruits. The average results were expressed in %. 2.7. Action mechanisms of SA against P. expansum 2.7.1. Damage to the cell wall of P. expansum conidia The evaluation of the SA effects on the pathogen cell wall was performed according to Cerioni et al. (2010), with modifications. Firstly, the conidia of P. expansum were suspended in 2.5 mM SA or sterile distilled water (control) to a final concentration of 108 conidia/mL. The suspensions were maintained at room temperature under constant stirring. Samples (500 μL) were collected at intervals of 0, 5, 15, 30, 60, and 120 min, centrifuged (8000 g, 5 min, 4 °C), double washed with a 50 mM sodium phosphate buffer (pH 7.0) and sterile distilled water, centrifuged (8000 g, 5 min, 4 °C), and re-suspended in apple juice (4%, v/v) to a final concentration of 105 conidia/mL. Aliquots of 50 μL were then transferred to concave slides and incubated as described above. The samples were then transferred to microscope slides, 10 μL of Calcofluor White Stain reagent (Sigma-Aldrich, USA) at 50 μg/mL and 10 μL of 10% (w/v) potassium hydroxide added, and incubated in the dark for 15 min. The conidial morphology and germ tubes were examined with the aid of a fluorescence microscope (Eclipse 50i, Nikon) at the wavelengths of 395 nm (excitation) and 440 nm (emission). The images were recorded with a Nikon digital camera (Coolpix P500) and the experiment conducted twice. 2.7.2. Damage to the plasma membrane of P. expansum conidia Damage to the plasma membrane of P. expansum conidia exposed to SA was determined as described by Liu et al. (2007), with modifications. Conidial suspensions were prepared in 2.5 mM SA or sterile distilled
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water (control), incubated and sampled as described above. After sampling, the P. expansum conidia were stained with propidium iodide (10 μg/mL, Sigma, USA) for 5 min, incubated at 30 °C, centrifuged (8000 g, 5 min, 4 °C), and washed twice with a 50 mM sodium phosphate buffer (pH 7.0), followed by another centrifugation. The supernatant was discarded, the precipitate collected and re-suspended in sterile distilled water (105 conidia/mL). The evaluation was performed with the aid of a fluorescence microscope at the wavelengths of 546 nm (excitation) and 590 nm (emission). Images were taken with a Nikon digital camera and the experiment carried out twice. At least 100 conidia were evaluated under visible light and fluorescence at each time interval, in the different fields, followed by the calculation of the percentage of fluorescent conidia. The damage caused by the exposure of the P. expansum plasma membrane to SA was quantified following the method proposed by Cerioni et al. (2010) and Rice-Evans et al. (1991), with modifications. The conidial suspensions of P. expansum were carried out as described for the determination of the cell wall damage. Aliquots (500 μL) were removed after 0, 5, 15, 30, 60, and 120 min, centrifuged (2000 g, 5 min) and washed twice with a 50 mM sodium phosphate buffer (pH 7.0). The supernatants were discarded, the precipitates re-suspended in sterile distilled water to a final concentration of 5 × 107 conidia/mL, and treated in an ultrasound bath (10 min) to disrupt the conidia. To 1 mL of these suspensions, 1 mL of trichloroacetic acid (TCA) 20% (w/v) was added for precipitation at 4 °C, followed by centrifugation (19,800 g, 5 min). Then, 2 mL of saturated solution containing thiobarbituric acid (TBA) in 0.1 M HCl and 10 mM butylated hydroxytoluene (BHT) were added to the resulting supernatants, followed by incubation in a water bath at 100 °C for 60 min. The collected extracts were cooled in ice bath for 10 min and the absorbance read at 535 nm in a micro plate multi reader (Spectramax Paradigm Molecular Devices). For the blank, the entire procedure was performed, with 1 mL of TCA (centrifuged) added to 2 mL of TBA solution described above, but without the addition of conidial extract. The final concentrations of thiobarbituric acid reactive species (TBARS) were determined using the molar extinction coefficient (ε = 156 mmol/cm) and expressed as pmol/mg of protein. The experiment was conducted twice. 2.7.3. Determination of P. expansum protein leakage Intracellular protein leakage was quantified as proposed by Liu et al. (2010) with modifications. A conidial suspension of P. expansum was transferred to Erlenmeyer flasks (250 mL) containing 100 mL of potato dextrose broth to a final concentration of 5 × 105 conidia/mL. The flasks were incubated at 200 rpm, 25 °C, in the dark. After three days, 3 g of the mycelium (fresh weight) were collected in paper filter (0.2 μm), washed with sterile distilled water and transferred to Erlenmeyer flasks containing 30 mL of sterile distilled water or 2.5 mM SA. After different incubation times (0, 5, 15, 30, 60, and 120 min) at room temperature, aliquots (4 mL) of the mycelium suspension were collected and filtered. The content of total soluble proteins was determined following the Bradford method (1976), using BSA (Sigma-Aldrich, USA) as standard. The filtrates were collected for the determination of the total intracellular protein leakage. Absorbance readings (595 nm) were made with the aid of a micro plate multi reader and the results expressed as μg of soluble protein per g of fresh mycelium. The experiment was conducted three times. 2.7.4. Ultrastructure analysis of P. expansum conidia Suspensions of P. expansum conidia (2 × 108 conidia/mL) were prepared in sterile distilled water (control) or 2.5 mM SA and stirred for 60 min. Then, 500 μL was collected, centrifuged (8000 g, 5 min, 4 °C), washed with a 50 mM sodium phosphate buffer (pH 7.0), centrifuged, and re-suspended in sterile distilled water. For transmission electronic microscopy (TEM), the method described by Schmidt et al. (2009) was performed with modifications. The samples were fixed with a 2.5% glutaraldehyde solution (w/v), 2% sucrose (w/v), and buffered
with 0.1 M cacodylate (pH 7.2), post-fixed with osmium tetroxide for 4 h and dehydrated in a series of aqueous solutions with increasing acetone concentrations. After dehydration, the material was infiltrated with Spurr resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate. The samples were visualized and photographed in a transmission electronic microscope (JEM 1011, Jeol Ltda., 80 kV), in the Central Laboratory of Electronic Microscopy of Federal University of Santa Catarina. 2.8. Statistical analysis The experiments were carried out in a completely randomized design and the experimental design was described above in the relevant sections. 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. The statistical analyses were performed using the software Statistica 10.0 and the graphs were designed by software Prism 5 for Mac OS X. 3. Results and discussion 3.1. Phenolic acids and their antimicrobial potential against P. expansum The antimicrobial activity of phenolic acids was assessed as an alternative to the traditional methods for control of blue mold in apples. These substances are produced in plants mainly in response to stress conditions and have been widely discussed in studies on maintenance of postharvest quality in fruit (Tareen et al., 2012). Salicylic acid was able to completely inhibit the germination of P. expansum conidia at 2.5 mM (Fig. 1). Similar results were found against other pathogens e.g., R. stolonifer, Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolinae, Pythium sp., and Phytophthora sp., where about 70% of conidial germination and growth was inhibited by SA, at a minimum concentration of 2.5 mM (El-Mohamedy et al., 2013; Panahirad et al., 2012). Hypochlorite (0.5% of active chlorine) and SA (2.5 mM) similarly inhibited the germination of P. expansum conidia. The hypochlorite solution in a dose 100 times higher than that commercially used inhibited
Fig. 1. Penicillium expansum conidial germination (%) exposed to phenolic acids at 2.5 mM. Data are the average ± SD of two experiments. Different letters represent significant differences between treatments (Tukey, p ≤ 0.05).
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the germination after just 30 min of exposure of the conidia, whereas SA reduced the germination by up to 90% in the same period (Fig. 2). In many countries, the addition of active chlorine to the fruit disinfection water is a common practice, especially because of its antimicrobial potential and quick action. However, the chlorine used in this process can complex with the natural organic matter of the fruits or with preexisting contaminants in water, forming products that can potentially cause cancer or mutation in animals (Ferraris et al., 2005; Marabini et al., 2006). To validate SA as a new treatment for the management of postharvest disease in apples, analysis of other parameters, such as the pH of the solution, is necessary. pH can be directly associated with the development of the pathogen and its virulence, and can also influence the activities of the antimicrobial compounds by changing chemical structures. Conidia of P. expanum prepared in suspensions with pH adjusted to 2 did not germinate, regardless of the treatment used. However, the conidial germination in the control treatment was partially achieved and fully restarted at pH 2.5 and 3.0, respectively (Fig. 3). Such results were partially discussed by Smilanick et al. (2005) who concluded that the conidia of P. expansum become inactive, but not destroyed, at pH 2, with minor effect in less acidic solutions, e.g., pH 4. Subsequently, Li et al. (2010) demonstrated through biochemical and proteomic assays that the pH value of the medium affects the internal pH of the conidia, leading to decreased levels of ATP and, consequently, conidial inactivation, with no damage to the conidial structure. This may be related to the capacity of P. expansum to acidify the host tissue by the secretion of organic acids, such as D-gluconic acid (pH 3.5) and citric acid (pH 3.1), infecting and colonizing the tissues (Barad et al., 2014). In this sense, P. expansum conidia seem to be well adapted to environmental conditions of pH near 2.5, growing normally at pH above 3. These findings were confirmed with an in situ assay, where the P. expansum conidia in aqueous suspensions with a pH ranging from 2 to 6 were able to germinate and infect the apples, especially in an acidic environment of pH 2 or 3, reaching 100% incidence and an average rate of lesion growth of 0.27 cm per day, leading to a final lesion diameter of 3.59 cm (Fig. 4). At pH 4 to 6, although the in vitro conidial germination rate was high (Fig. 3), a reduced in situ development of the fungus was observed (Fig. 4), probably due to the difficulty of colonizing apple tissues in a less acidic environment. At a standard pH level (2.8), SA completely inhibited the fungus germination, showing a decrease in its antifungal potential at pH 3.5, and completely losing its biological potential at a pH above 5.5 (Fig. 3). This effect was also observed in situ, where the treatment with 2.5 mM SA at pH 2 or 3 inhibited 100% of blue mold incidence in apple fruits, whereas the alkalization of the SA solution to pH ≥ 4 decreased its potential (Fig. 4).
Fig. 2. Minimum contact time between salicylic acid (2.5 mM) and P. expansum for the inhibition of conidial germination (%). Data are the average ± SD of three experiments. Different letters represent significant differences between treatments at the same times (Tukey, p ≤ 0.05).
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Fig. 3. Conidial germination of Penicillium expansum (%) exposed to salicylic acid at 2.5 mM for the studied pH values. SA standard pH was assumed to be 2.8. Data are the average ± SD of three experiments. Different letters represent significant differences between treatments at the same pH (Tukey, p ≤ 0.05).
Fig. 4. Effects of salicylic acid solution with different pHs in apple fruits. (A) Final lesion diameter (cm) of P. expansum. (B) Incidence (%) of blue mold in apple fruits. (c) Lesion growth rate (cm/day) of P. expansum. Data are the average ± SD. Different letters represent significant differences between treatments at different pH's (Tukey, p ≤ 0.05).
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Amborabe et al. (2002) found similar results using SA against the fungus Eutypa lata, at 2 mM and pH value near 4, observing that the phenolic action is specifically due to its molecular arrangement, where a simple displacement of the hydroxyl group on the aromatic ring can reduce the antifungal activity by up to 50%. Other compounds have shown similar patterns where the pH value affects their antimicrobial activity. For example, the antimicrobial effect of several bicarbonates against B. cinerea depends on the pH of the solution, with a predominance of carbonic acids in acidic solutions, which are unstable and show poor antimicrobial potential (Palmer et al., 1997). The commercial fungicide imazalil, commonly used against the citrus green mold, exhibited changes in its biological activity with changes in pH, losing 90% of its antimicrobial effect at pH 4.0 (Smilanick et al., 2005). Thus, increasing the pH of SA solution seems to result in molecular dissociation at neutral pH, with consequent reduction of antifungal and/or antioxidant potential (Amborabe et al., 2002). 3.2. Action mechanisms of SA against P. expansum At 2.5 mM, SA did not induce changes in the morphological characteristics of the cell wall of P. expansum conidia, even after a longer exposure time (data not shown). Similar results were observed against Penicillium digitatum conidia exposed to lethal doses (100 mM) of hydrogen peroxide, which totally inhibited germination, but without apparent damage in the cell wall when examined under fluorescence and transmission electronic microscopy (Cerioni et al., 2010). Damage caused by SA in the plasma membrane of P. expansum conidia were detected through the fluorescence emitted by propidium iodide, which cannot enter undamaged conidia due to the selective membrane permeability but is capable of penetrating damaged cells, intercalating between DNA or RNA strands (Wang et al., 2014). With only 5 minute exposure to 2.5 mM SA, 60% of P. expansum conidia showed fluorescence, reaching 100% of damaged plasma membrane after 30 minute contact with the phenolic compound. In contrast, for the control treatment, the integrity of the conidia plasma membrane was maintained throughout the experiment, with no fluorescence emission (Fig. 5A). These findings were confirmed through the TBA assay, where conidia of P. expansum prepared in distilled water and incubated up to 120 min, did not show any variation in the amounts of reactive species after the TBA reaction. However, when exposed to SA at 2.5 mM for 30 min, about 2000 pmol of TBA reactive species per mg of protein were quantified. These values increased as the time of contact between the conidia and SA increased, reaching a maximum of 6000 pmol of TBA reactive species per mg of protein after 120 minute exposure (Fig. 5B). In addition, after exposure to SA, 2.4 μg of soluble protein per g of fresh mycelium leaked into the medium, reaching a maximum of 3.2 μg of protein, whereas the protein leakage in the control treatment was only 1.8 μg of protein during 120 min of incubation (Fig. 5C). The cell membrane is essential to the cell viability of fungal conidia and mycelium, where damage to the lipid bilayer may result in cellular collapse, leading to the leakage of intracellular compounds (Chen et al., 2014). In comparison, other compounds showed a lesser capacity of inhibiting the pathogen than SA. Zhang et al. (2011) reported the use of 500 mM ß-amino butyric acid inhibiting germination of P. expansum conidia by 90%, causing damage to the plasma membrane in 80% of the conidia after 4 hour exposure, with maximum extravasation of 0.8 mg of protein per g of fresh mycelium to the medium. Liu et al. (2010) found a 100% inhibition of P. digitatum germination in 0.5% silicon. However, the compound caused damage to the plasma membrane in only 50% of the P. digitatum conidia after 4 hour exposure, with a leakage of 1 mg of protein per g of fresh mycelium. In our study, other damage to the plasma membrane was found after the analysis with the aid of a transmission microscope. The P. expansum conidia suspended in sterile distilled water exhibited an organized cell
Fig. 5. Effects of salicylic acid at 2.5 mM on the plasma membrane integrity of the Penicillium expansum conidia according to the exposure times. The integrity of plasma membrane was evaluated by fluorescence method (A), by the quantification of reactive species of thiobarbituric acid — TBARS (B), and by intracellular protein leakage (C). Data are the average ± SD of two (A–B) or three (C) experiments. Different letters represent significant differences between treatments at the same times (Tukey, p ≤ 0.05).
cytoplasm, presenting typical organelles e.g., mitochondria, rough endoplasmic reticulum, and ribosomes, as well as a central vacuole, plasma membrane and a thin cell wall (Fig. 6A–C). In contrast, the conidia suspended in 2.5 mM SA for 60 min exhibited several changes in the intra-cellular morphology, with a massive formation of vacuoles, several invaginations of the plasma membrane, a disorganized cytoplasm, and a significant thickening of the conidia cell walls (Fig. 6D–H). The antimicrobial potential of SA may be related to perturbations caused to cellular respiration, redirecting the electron flow from the cytochrome pathway to an alternative cyanide-related pathway, partially disabling the main cellular respiration pathway and affecting the cell energy generating process (Kapulnik et al., 1992). SA is also capable of binding specifically with proteins that can act on the decomposition of intracellular hydrogen peroxide, leading to the accumulation of this compound into the cell, inactivating the conidia (Chen et al., 1993). Studying the plant pathogen E. lata, Amborabe et al. (2002) observed that 2 mM SA had a direct antimicrobial effect, with several types of damage caused to the cell functions. They found that SA behaves as a dissociating agent causing a change in the trans-membrane pH gradient
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Fig. 6. Transmission electron microscopy of the conidia of P. expansum treated with water (a–c) or with salicylic acid at 2.5 mM (d–h). (a) P. expansum conidia arranged with the presence of a central vacuole (V) and organelles in the cytoplasm (Cy). (b) Detail of the presence of mitochondria (M), rough endoplasmic reticulum (Rer), and a thin cell wall (Cw) on the conidia. (c) Cytoplasm with numerous free ribosomes (R). (d) Irregular morphology of P. expansum conidia after treatment with SA at 2.5 mM. (e) Conidia with formation of large amount of vacuoles. (f–h) Disruption of cellular structures in the cytoplasm of P. expansum conidia and increased thickness of cell wall upon SA exposure.
in membranes of organelles and in the plasma membrane, possibly causing cellular energy loss. A study demonstrated the interaction between the charges of the amino groups present in chitosan and the plasma membrane of P. expansum conidia, resulting in the change of the selective permeability of the conidial plasma membrane, leading to the cell lysis (Wang et al., 2014). SA seems to act directly against P. expansum conidia by penetrating the cell wall and triggering several interactions with the plasma membrane, leading to the disruption of the lipid bilayer and/or causing damage to the proteins responsible for cellular permeability, increasing the concentration of reactive oxygen species. Finally, chemical interactions between the ions formed by SA in solution and the fatty acid constituents of the plasma membrane of P. expansum conidia might determine changes in the lipid model, decreasing its fluidity by modifying the lipid fraction or the bond with Ca++ ions, which are essential in diverse sites of the plasma membrane.
4. Conclusion Salicylic acid solutions with pH ≤ 3 inhibited the in vitro germination of P. expansum and the incidence of blue mold on apples, even at a low concentration, i.e., 2.5 mM. In addition, SA caused leakage of the pathogen's proteins to the medium, measurable lipid damage, and intracellular disorganization. Moreover, SA is a phenolic compound considered safe for the environment and human health and can be an alternative to the fungicides commonly used against the postharvest diseases of apples. However, as the SA molecule was shown to be highly sensitive to pH changes, and once its chemical structure is correlated with its antimicrobial potential, further studies are necessary to completely elucidate its in situ mode of action against P. expansum as well as effects on apple physiology.
Acknowledgments Our special thank you for Dr. Eder Carlos Schmidt from the Laboratory of Seaweed of Federal University of Santa Catarina for all the support given on the ultra-structural analysis and to CAPES for the financial support. The researcher grants from CNPq on behalf of M. Maraschin and R. M. Di Piero are acknowledged.
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