Postharvest Application of Organic and Inorganic Salts To Control

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Postharvest Application of Organic and Inorganic Salts To Control Potato (Solanum tuberosum L.) Storage Soft Rot: Plant Tissue−Salt Physicochemical Interactions E. S. Yaganza,† R. J. Tweddell,‡ and J. Arul*,† †

Department of Food Science and Nutrition and Horticultural Research Centre, Université Laval, Quebec City, Quebec G1V 0A6, Canada ‡ Department of Plant Science and Horticultural Research Centre, Université Laval, Quebec City, Quebec G1V 0A6, Canada ABSTRACT: Soft rot caused by Pectobacterium sp. is a devastating disease affecting stored potato tubers, and there is a lack of effective means of controlling this disease. In this study, 21 organic and inorganic salts were tested for their ability to control soft rot in potato tubers. In the preventive treatment, significant control of soft rot was observed with AlCl3 (≥66%) and Na2S2O3 (≥57%) and to a lesser extent with Al lactate and Na benzoate (≥34%) and K sorbate and Na propionate (≥27%). However, only a moderate control was achieved by curative treatment with AlCl3 and Na2S2O3 (42%) and sodium benzoate (≥33%). Overall, the in vitro inhibitory activity of salts was attenuated in the presence of plant tissue (in vivo) to different degrees. The inhibitory action of the salts in the preventive treatment, whether effective or otherwise, showed an inverse linear relationship with water ionization capacity (pK′) of the salt ions, whereas in the curative treatment, only the effective salts showed this inverse linear relationship. Salt−plant tissue interactions appear to play a central role in the attenuated inhibitory activity of salts in potato tuber through reduction in the availability of the inhibitory ions for salt−bacteria interactions. This study demonstrates that AlCl3, Na2S2O3, and Na benzoate have potential in controlling potato tuber soft rot and provides a general basis for understanding of specific salt−tissue interactions. KEYWORDS: potato tuber soft rot, preservative salts, water ionization, Donnan equilibrium, ionic speciation and diffusion

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potato dry rot,17,18 soft rot,7,19 and Botrytis cinerea mold in grapevines.20 Fumigation of fruits (including tomato, apple, kiwi, grape, and citrus) with acetic acid, propionic acid, formic acid, and sulfur dioxide reduced gray and blue molds caused by B. cinerea, Penicillium sp., Monilinia fructicola, and Rhizopus stolonifer.21−23 The mechanism of inhibitory action of salts has not been well-explored. One study examined the ultrastructural alterations of Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum) caused by exposure to aluminum chloride and sodium metabisulfite.24 Sodium metabisulfite was more effective and faster in killing the bacteria. The mortality caused by aluminum chloride involved membrane damage, but little damage to cellular membranes was observed with sodium metabisulfite. Sodium metabisulfite appeared to exert its effect intracellularly, with bisulfite being transported across the membranes by free diffusion as molecular SO2. Loss of membrane integrity and cytoplasmic leakage were also observed in Fusarium sambucinum and Heterobasidion annosum exposed to these two salts.25 Exposure of fungal plant pathogens containing polyunsaturated fatty acids in their cellular lipid composition to these salts induced lipid peroxidation, and the pathogens were more sensitive to them.26 Because cellular fatty

INTRODUCTION Soft rot is a major bacterial disease affecting potato (Solanum tuberosum L.) tubers. The disease causes significant economic losses worldwide, especially during storage,1,2 where it can spread from a few infected tubers to healthy tubers.3,4 Soft-rotinfected tissues appear wet, cream to tan in color with a soft and slightly granular consistency.2,3 In North America, potato soft rot is mainly caused by the Gram-negative bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica).5,6 Currently, no chemicals are available for an effective control of potato soft rot,2,4,7 and hence, control and management of this disease rely mainly on cultural practices, such as planting disease-free seed tubers and maintaining proper harvesting, handling, and storage conditions.2,3 However, in most of the cases, these practices are insufficient to control the disease effectively. Previous research has shown that the use of the antimicrobial properties of some organic and inorganic salts and their corresponding acids or bases, used typically for food preservation, has potential for controlling plant diseases. Bicarbonate and carbonate salts have been shown to control several diseases affecting roses,8 cucurbit plants,9 postharvest decays of melons,10 and pepper fruits11 as well as citrus green mold12 and potato tuber silver scurf.13−15 Potassium sorbate and calcium propionate successfully controlled potato silver scurf14,15 and carrot black rot.16 Aluminum salts and sodium metabisulfite were reported to inhibit potato silver scurf,15 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 9223

April 14, 2014 August 26, 2014 August 31, 2014 September 1, 2014 dx.doi.org/10.1021/jf5017863 | J. Agric. Food Chem. 2014, 62, 9223−9231

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Table 1. Preventive Effect of Salts in the Control of Potato Soft Rot Caused by P. atrosepticum and P. carotovorum subsp. carotovorum disease inhibition (%)a salt

P. atrosepticum

aluminum dihydroxy acetate aluminum chloride aluminum lactate ammonium acetate ammonium chloride ammonium hydrogen phosphate calcium chloride potassium chloride potassium sorbate sodium acetate sodium benzoate sodium bicarbonate sodium carbonate sodium chloride sodium formate sodium lactate sodium metabisulfite sodium hydrogen phosphate sodium propionate sodium tartrate dibasic trisodium phosphate

10.3 70.1 49.8 18.8 11.0 11.3 18.8 3.8 27.4 14.5 33.8 10.9 10.3 1.0 6.7 10.3 57.3 11.0 27.4 11.0 15.2

efb a bc de ef ef de f cd de c ef ef f ef ef b ef cd ef de

P. carotovorum subsp. carotovorum 9.6 66.0 33.7 29.0 11.6 0.0 25.7 1.4 39.0 5.7 41.3 7.9 6.5 0.0 0.0 0.0 71.9 0.0 28.4 3.8 14.9

g b de e fg i e i cd hi c h h i i i a i e i f

Disease inhibition was calculated as follows: {[disease severity (control tubers) − disease severity (salt-treated tubers)]/disease severity (control tubers)} × 100. bValues of the same column followed by the same letter are not significantly different (p < 0.05) according to Fisher’s protected LSD.

a

acids are highly saturated in bacteria,27 lipid peroxidation by these salts in Pectobacterium sp. is unlikely to occur. Previous work28 showed an inverse sigmoidal relationship between the inhibitory effect of salts on the in vitro growth of Pectobacterium sp. and the water-ionizing capacity or pK′ (apparent acidity or basicity constant) of salt anions or cations primarily and lipophilicity of the salts, suggesting that these two parameters in combination may contribute to the antimicrobial action of the salts in the salt−microbe interactions. However, it is not known whether the observed in vitro antimicrobial action of the salts would persist in vivo in potato tubers. If it did, it raises the question whether those physicochemical characteristics of the salts could also provide a basis in the ternary salt− potato tissue−microbe interactions for the control of soft rot in potato tubers. Thus, the objective of this work was to evaluate the inhibitory activity of several organic and inorganic salts against soft rot in potato tuber and to examine the general physicochemical basis of the inhibitory activity of salts on soft rot development when applied as preventive or curative treatments.

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Effect of Salts on Soft Rot Development. Potato tubers (cv. Norland) were purchased from Propur, Inc. (St. Ambroise, Quebec, Canada) and stored at 4 °C until use. Selected tubers were washed with tap water, surface-sterilized by dipping in 0.525% sodium hypochlorite solution (10 min), rinsed by dipping twice in sterile distilled water for at least 10 min, and dried in ambient air. Four wounds (depth, 1 cm; diameter, 0.5 cm) were performed on each tuber from the apical to the basal ends,29 using a pipet tip. Preventive Application of Salts. Wounded tubers were immersed for 10 min in the different salt solutions (0.2 M) and dried in ambient air. Control tubers were immersed in sterile distilled water. Each wound was then inoculated with 20 μL of a suspension [1 × 107 colony-forming units (CFU)/mL] of either P. atrosepticum or P. carotovorum subsp. carotovorum. Suspensions were prepared from 24 h old cultures grown in 20% tryptic soy broth (Difco). Tubers were subsequently incubated (24 °C) individually in a plastic chamber containing a wet sterile paper towel for 3 days. The disease severity was then assessed by measuring the largest diameter of each rotting lesion,30 and the results were expressed in percentage of disease reduction compared to the control treatment (sterile distilled water; 1.3−2.2 cm lesion diameter). For each tuber, lesion diameter was a mean of the four inoculation sites. A completely randomized experimental design with three replicates was used, and the experiment was repeated twice. Curative Application of Salts. Each wound was inoculated with 20 μL of one of the bacterial suspensions (1 × 107 CFU/mL). After a 12 h incubation period at 24 °C, infected tubers were immersed (10 min) in the different salt solutions (0.2 M) or in distilled water (control). Tubers were then incubated (24 °C) individually in a plastic chamber containing a wet sterile paper towel for 3 days, and the disease severity was evaluated as previously described. A completely randomized experimental design with three replicates was used, and the experiment was repeated twice. Effect of Salt Treatments on Tissue pH. The diffusion of salts in the tuber tissue was evaluated by monitoring the consequent modification of tissue pH for two acidic salts (aluminum chloride and sodium metabisulfite), two alkaline salts (trisodium phosphate and

MATERIALS AND METHODS

Strains of P. carotovorum subsp. carotovorum (ECC 1367) and P. atrosepticum (ECA 709) were provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Quebec City, Quebec, Canada). The bacteria were maintained on nutrient agar (NA, Difco Laboratories, Detroit, MI) slants under mineral oil at 4 °C and served as stock cultures. A total of 21 salts were used in these experiments (Table 1). All salts were purchased from Sigma Chemical Co. (St. Louis, MO), except for ammonium acetate, sodium chloride, and sodium bicarbonate (BDH, Inc., Toronto, Ontario, Canada) and aluminum lactate (Aldrich Chemical, Milwaukee, WI). 9224

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Table 2. Curative Effect of Salts in the Control of Potato Soft Rot Caused by P. atrosepticum and P. carotovorum subsp. carotovorum disease inhibition (%)a salt

P. atrosepticum

aluminum dihydroxy acetate aluminum chloride aluminum lactate ammonium acetate ammonium chloride ammonium hydrogen phosphate calcium chloride potassium chloride potassium sorbate sodium acetate sodium benzoate sodium bicarbonate sodium carbonate sodium chloride sodium formate sodium lactate sodium metabisulfite sodium hydrogen phosphate sodium propionate sodium tartrate dibasic trisodium phosphate

28.0 41.3 30.7 20.0 10.0 14.0 21.3 11.0 18.7 18.7 33.3 14.0 16.0 16.0 15.3 16.7 42.0 17.0 16.0 17.0 15.0

bcb a bc cd e e cd e de de b e de de de de a de de de de

P. carotovorum subsp. carotovorum 7.6 57.6 29.6 6.8 0.0 22.4 0.0 3.6 13.6 3.0 33.5 0.0 0.0 3.8 0.0 7.9 52.8 0.0 0.0 0.0 4.2

cd a b cd e bc e de c de b e e de e cd a e e e de

Disease inhibition was calculated as follows: {[disease severity (control tubers) − disease severity (salt-treated tubers)]/disease severity (control tubers)} × 100. bValues of the same column followed by the same letter are not significantly different (p < 0.05) according to Fisher’s protected LSD.

a

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sodium carbonate), and one lipophilic salt (sodium benzoate). Duplicate tuber slabs (1 cm wide × 3 cm long × 2 cm high) were dipped in 0.2 M of the salt solution and incubated at room temperature for up to 3 h. The slabs were removed from the salt solution at intervals and rinsed thoroughly using deionized water. Three consecutive 1 mm thick slivers (1 mm × 2 cm × 3 cm) were sliced from one end of the potato slab with a sharp knife. The slices were crushed using a garlic press (Atlantic Promotions, Inc., Longueuil, QC, Canada) and suspended in 30 mL of deionized water, and the pH of the slurry was measured. The experiment was repeated 3 times. Minimum Inhibitory Concentration (MIC) of Salts in Vitro. The effect of the concentration of selected salts on the bacterial growth was evaluated. P. carotovorum subsp. carotovorum bacteria was grown in 250 mL flasks containing 50 mL of 20% tryptic soy broth (TSB, Difco) amended with 0−10 mM aluminum chloride, aluminum lactate, sodium metabisulfite, trisodium phosphate, or sodium carbonate or with 0−100 mM sodium benzoate, potassium sorbate, or sodium propionate by incubating at 24 °C with agitation (150 rpm, Lab-Line Instruments, Inc., Melrose Park, IL) for 24 h. The pH of the medium was not adjusted, and it varied with the type of salt. The flasks were inoculated with 100 μL of bacterial suspension (1 × 107 CFU/mL). The bacterial growth was determined by turbidimetry at 600 nm on an ultraviolet/visible (UV/vis) spectrophotometer (Ultrospec 2000, Pharmacia Biotech, Ltd., Cambridge, U.K.), using appropriate blanks (non-inoculated media amended with salt). Results were expressed as the percentage of growth inhibition compared to the control (growth medium not amended with salt). A completely randomized experimental design with three replicates was used, with the experimental unit being a flask. The MIC of the salts was determined from the inhibition salt concentration plots. Statistical Analyses. Analyses of variance were carried out with the GLM procedure of SAS (SAS Institute, Cary, NC). Treatment means were compared using Fisher’s protected least significant difference (LSD) when the analysis of variation (ANOVA) indicated significant differences (p < 0.005).

RESULTS AND DISCUSSION The preventive inhibitory effect of the 21 salts against soft rot development in potato tuber is presented in Table 1. Disease lesion in the control tubers was 1.3−2.2 cm in diameter. Significant reduction of disease caused by P. atrosepticum and P. carotovorum subsp. carotovorum was obtained by the application of aluminum chloride (70.1 and 66.0%, respectively) and sodium metabisulfite (57.3 and 71.9%, respectively) and to a lesser extent by the application of sodium benzoate (33.8 and 41.3%, respectively), aluminum lactate (49.8 and 33.7%, respectively), and potassium sorbate (27.4 and 39.0%, respectively). Calcium chloride, ammonium acetate, and sodium propionate showed only a modest disease reduction. The other salts exhibited either negligible or no effect on disease development. When salts were applied as curative treatment, the highest inhibition of the disease caused by P. atrosepticum and P. carotovorum subsp. carotovorum was obtained with aluminum chloride (41.3 and 57.6%, respectively), sodium metabisulfite (42.0 and 52.8%, respectively), and sodium benzoate (33.3 and 33.5%, respectively) (Table 2). The other salts allowed for a disease reduction ranging from 10.0 to 30.7% on P. atrosepticum and from 0 to 29.0% on P. carotovorum subsp. carotovorum. The dipping treatment of aluminum chloride, sodium metabisulfite, aluminum lactate, and sodium benzoate markedly decreased soft rot severity in infected tubers for both preventive and curative applications, whereas the in vitro test showed that a number of salts (aluminum dihydroxy acetate, aluminum chloride, aluminum lactate, ammonium acetate, potassium sorbate, sodium bicarbonate, sodium carbonate, sodium benzoate, sodium propionate, sodium metabisulfite, and trisodium phosphate) were effective against the causal agents of this disease.28 We observed earlier that the inhibitory effect 9225

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of salts in vitro related primarily to the water ionization capacity of their ions, which is indicated by the acidity constant (pKa) or basicity constant (pKb) of the ions (the lower the pKa or pKb value of the ion, the greater its capacity to ionize water and alter the surrounding pH).28 A greater ionization capacity of water was associated with a higher inhibitory effect in vitro. For higher salt concentrations (>0.1 M), apparent acidity or basicity constants (pK′a or pK′b) of the ions are more appropriate, and hence, the pKa or pKb values were corrected for ionic strength of the salt solutions.28 Figure 1 shows that disease inhibition by the preventive application of salts relates inversely to the pKa′ value of the

activities were attenuated overall for all of the salts tested in the presence of plant tissue compared to the in vitro activities, the decrease in the activity was drastic for salts, such as sodium phosphate, sodium carbonate, and sodium bicarbonate. In addition, both the effective and non-effective ions showed an apparent inverse linear relationship to the pK′ values of the ions in the presence of tissue, in contrast to the general inverse sigmoidal inhibitory pattern observed in in vitro inhibition,28 suggesting that water ionization capacity of the ions should still play a role in their inhibitory activity in the tissue (Figure 1). Disease inhibition by the curative application of salts in relation to the pK′ values of the ions is shown in Figure 2. While most ions exhibited no effect, three of the ions, Al3+,

Figure 1. Relationship between inhibition of soft rot caused by (A) P. atrosepticum and (B) P. carotovorum subsp. carotovorum by preventive application of salts and apparent acidity (pK′a) or basicity (pK′b) constants of the salt ions: (1) PO43−, (2) CO32−, (3) HPO42−, (4) HCO3−, (5) acetate−, (6) tartrate2−, (7) formate−, (8) lactate−, (9) Cl−, (10) NH4+, (11) Na+, and (12) K+. Cations: common anion, chloride. Anions: common cation, sodium, except for sorbate, potassium.

cations (with common chloride anion) and that of the acidic bisulfite anion and the pKb′ values of anions (with common sodium or potassium cation). It is seen clearly that some of the ions, bisulfite, aluminum, benzoate, sorbate, propionate, and calcium, are effective and exhibit a disease inhibition of more than 20%, whereas the other ions (acetate, bicarbonate, carbonate, chloride, formate, lactate, phosphate, potassium, sodium, and tartrate) show little effect. Although the inhibitory

Figure 2. Relationship between inhibition of soft rot caused by (A) P. atrosepticum and (B) P. carotovorum subsp. carotovorum by curative application of salts and apparent acidity (pK′a) or basicity (pK′b) constants of the salt ions: (1) PO43−, (2) CO32−, (3) HPO42−, (4) HCO3−, (5) acetate−, (6) tartrate2, (7) formate−, (8) lactate−, (9) Cl−, (10) NH4+, (11) Na+, (12) K+, (13) propionate−, (14) sorbate−, and (15) Ca2+. Cations: common anion, chloride. Anions: common cation, sodium, except for sorbate, potassium. 9226

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HSO 3−, and benzoate, showed some inhibitory effect, suggesting that these ions may possess certain unique characteristics. The relationship between disease inhibition and the ion pK′ combined with the 1-octanol/water partition coefficient (pK′ − log Po/w) generally followed the same pattern, with a slight improvement in the correlation coefficient for the effective salt group (Figures 3 and 4). Despite the

Figure 4. Relationship between inhibition of soft rot caused by (A) P. atrosepticum and (B) P. carotovorum subsp. carotovorum by curative application of salts and the addition parameter pK′a or pK′b − log Po/w, which combines apparent acidity (pK′a) or basicity (pK′b) constants of the salt ions and their octanol/water partition coefficient (Po/w): (1) PO43−, (2) CO32−, (3) HPO42−, (4) HCO3−, (5) acetate−, (6) tartrate2, (7) formate−, (8) lactate−, (9) Cl−, (10) NH4+, (11) Na+, (12) K+, (13) propionate−, and (14) Ca2+. Cations: common anion, chloride. Anions: common cation, sodium, except for sorbate, potassium.

phosphate, sodium carbonate, sodium benzoate, sodium metabisulfite, and aluminum chloride, respectively, after 2 h of dipping. There was little modification of tissue pH with sodium benzoate (pH 6.3). Whereas both trisodium phosphate and sodium carbonate increased the alkalinity of the tissue, both sodium metabisulfite and aluminum chloride acidified the tissue. At 2 mm depth, there was little change in tissue pH with sodium benzoate and aluminum chloride after 2 h of dipping, but changes in tissue pH were observed for trisodium phosphate (7.7), sodium metabisulfite (5.7), and to a small extent, sodium carbonate (6.7) (Figure 5B). At the depth of 3 mm, small changes in tissue pH were registered after 2 h of dipping only for trisodium phosphate (6.8) and sodium metabisulfite (6.2) (Figure 5C). The penetration of aluminum, a polyvalent cation, appeared to be restricted to the depth of 1 mm, presumably because of its binding to fixed negative charges in the cell wall.31 Among the salts, the in vitro MIC value of aluminum salts, metabisulfite, trisodium phosphate, sodium carbonate, and sodium propionate was ≤10 mM, whereas it was 100 mM for sodium benzoate and potassium sorbate. In effect, the inhibitory effect of salts in vitro did not bear a clear relationship

Figure 3. Relationship between inhibition of soft rot caused by (A) P. atrosepticum and (B) P. carotovorum subsp. carotovorum by preventive application of salts and the addition parameter pK′a or pK′b − log Po/w, which combines apparent acidity (pK′a) or basicity (pK′b) constants of the salt ions and their octanol/water partition coefficient (Po/w): (1) PO43−, (2) CO32−, (3) HPO42−, (4) HCO3−, (5) acetate−, (6) tartrate2−, (7) formate−, (8) lactate−, (9) Cl−, (10) NH4+, (11) Na+, and (12) K+. Cations: common anion, chloride. Anions: common cation, sodium, except for sorbate, potassium.

attenuated inhibitory effect of salts in the preventive and more so in the curative treatments, the water ionization capacity of the ions appears to play a role in the disease inhibition of the salts. Figure 5 shows the time course of the migration of selected salts in the tuber tissue as implied by the changes in tissue pH, where the pH of the tissue dipped in water was 6.4. The pH of tissue dipped in the salt solutions reached a steady state in 2 h at 1 mm depth (Figure 5A) but not at the depths of 2 mm (Figure 5B) or 3 mm (Figure 5C). At the depth of 1 mm, the tissue pH was 11.2, 8.6, 6.2, 5.0, and 4.6 for trisodium 9227

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potassium sorbate (in vitro MIC, 100 mM) still maintained moderate activities in vivo. Overall, the acidic salts, such as aluminum chloride and sodium metabisulfite, and preservative salts, such as sodium benzoate, potassium sorbate, and propionate, still exhibited some significant effect, while alkaline salts, such as trisodium phosphate, sodium carbonate, and bicarbonate, showed little activity in the presence of the tissue compared to their in vitro activity. In addition, the inhibitory activity of all of the salts was attenuated to different degrees when applied as preventive treatment and, more so, when applied as curative treatment in potato tuber compared to their in vitro activity. Several factors intrinsic to potato tubers, such as buffering capacity, or intrinsic to salts, such as water ionization, pH, and ion migration capacity, and salt−plant tissue interactions may be involved in the salt−plant−bacteria interactions, leading to the reduced activity of salts overall, and the near total loss of the activity of alkaline salts. As a result of salt−plant tissue interactions, the ions may undergo speciation, where the resulting ions may be less active against the bacteria. It is also possible that some of the salts that retain moderate activity even in the curative application may possess some unique properties that may have to do with the interaction with the plant tissue or their mobility in the tissue. The buffering capacity of the tuber tissue could dissipate extreme pH conditions, and in the case of multiprotic salts (e.g., phosphates and carbonates), this could lead to their equilibrium favoring the formation of less effective ionic species, thereby dampening their inhibitory capacity in the plant tissue. In fact, the pH of the tuber tissue treated (10 min dipping, 1 mm from the wound surface) with trisodium phosphate (0.2 M solution at pH 12.5) was 10.1, where the predominant species, HPO42−, is less inhibitory on the Pectobacterium bacteria than PO43− present in the salt solution.28 Likewise, the pH of the tissue treated with sodium carbonate (solution pH of 11.1, predominant inhibitory species CO32−) was 8.4, where HCO3− predominates, which was shown in vitro to be slower than CO32− in killing those bacteria.28 Although the buffering role of the tissue was evident when treated with aluminum chloride, sodium metabisulfite, or the organic acid salts, they still showed inhibitory activity in the tuber. For example, the pH of the treated tissue (10 min dipping, 1 mm from the wound surface) with sodium metabisulfite was 5.6 compared to the solution pH of 4.5, and that of aluminum chloride was 4.9 compared to the solution pH of 2.5. A plausible argument can be made that salt−plant tissue interactions play a central role, leading to the attenuated inhibitory activity of salts in potato tuber by reducing the availability of the inhibitory ions for salt−bacteria interactions. The salt−plant interactions may involve alkalinization of plant cell walls and speciation of ions into less or more effective species by Donnan equilibrium32 (ionic equilibrium of charged solutes in the presence of charged membranes or macromolecules such as the cell wall pectin) and electroneutrality, complexation of ions and their reduced migration into the tissue, and the partitioning of diffusible species, generated as a result of the salt−plant interactions, between the plant cells and the bacterial cells, leading to their reduced availability at the infection site. The cell walls behave like a polyanion, owing to the presence of polygalacturonic acids, that sets up Donnan potential (electrostatic potential), resulting in the attraction of cations, including protons, and the repulsion of anions.33−35 This cell

Figure 5. Kinetics of the migration of selected salts in the tuber tissue as indicated by changes in tissue pH. (A) pH change in the millimeter thick tissue, 1 mm from one end of the potato slab; (B) pH change in the millimeter thick tissue, 2 mm from the same end of the potato slab; and (C) pH change in the millimeter thick tissue, 3 mm from the same end of the potato slab, dipped in salt solutions for up to 3 h: (◆) trisodium phosphate, (■) sodium carbonate, (○) control, (∗) sodium benzoate, (▲) sodium metabisulfite, and (×) aluminum chloride. Vertical bars represent standard deviation.

to their activities in the presence of plant tissue. While trisodium phosphate and sodium carbonate (in vitro MIC, ≤10 mM) exhibited little activity in vivo, sodium benzoate and 9228

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cell walls, proton efflux could be expected to increase the concentrations of the non-dissociated and more effective antibacterial species, such as benzoic, sorbic, and propionic acids (by protonation) outside the cell, resulting in the inhibition of bacterial growth and the consequent tissue degradation to some extent. In the presence of highly alkaline salts, such as trisodium phosphate (solution pH of 12.5) and sodium carbonate (solution pH of 11.1), which were effective in the in vitro inhibition of P. atrosepticum and P. carotovorum subsp. carotovorum, the electronegative status of the cell walls would limit the migration (Donnan exclusion) of the alkaline species (HPO42− or PO43− and HCO3− or CO32−) in the cell walls, which will be rather loaded with the sodium cation. In addition, protons would flow out from the cell walls to re-establish electroneutrality and pH gradient. Thus, not only would there be a lack of cell walls strengthening, but also the effective antibacterial alkaline species (e.g., PO43−) will lose their inhibitory capacity because of gradual protonation into less inhibitory species, such as HPO42− and H2PO4−. In addition, the efflux of protons from the cell walls would render them alkaline. Generation of less inhibitory species could therefore explain why these salts were ineffective in controlling soft rot in infected tubers, while they were highly effective inhibitors of bacterial growth in vitro. In contrast to alkaline anions, Donnan exclusion of bisulfite anions results in a different outcome as mentioned above, leading to the observed persistent inhibitory activity of sodium metabisulfite in the presence of plant tissue. Disease inhibition upon preventive salt application was greater than in the curative treatment, indicating that a higher concentration of the salt ions is present at the wound site for salt−pathogen interactions. Presumably, the tissue had a head start in the tissue−pathogen interactions during the preventive treatment through its interactions with the salts as well as the tissue responses to wound stress. In contrast, the pathogen has the head start and advantage in invading the plant tissue in the curative application of salts. In curative salt application, Al3+, HSO3−, and benzoate− maintained some activity in relation to their pK′ values, but the relationship to pK′ failed for those salts that exhibited less than 20% disease inhibition in the preventive treatment (Figure 2). Once the pathogen is well-established in the tissue, it becomes too late to ward off the infection for most of the ions that do not possess any special properties than simple modification of the surrounding pH (acid−base equilibria). The exceptions were the three ions, Al3+, HSO3−, and benzoate−, attributable to their inward diffusion into the tissue as ions or their speciated forms. Effectively, the generation of aqueous molecular SO2 species from sodium metabisulfite, which can diffuse through the cell walls and membranes, may clarify the curative activity of this salt. Aluminum ions do not appear to penetrate far into the tissue (panels B and C of Figure 5), presumably because of complexation by pectins of the cell walls and their presence as complex hydrated ions in an aqueous medium. However, the migration of this ion into the cell walls of the exterior layers of the tissue is still appreciable (Figure 5A), attributable to its high electrochemical potential (electrostatic charge of +3). Because of the efflux of protons from the cell walls, benzoate, sorbate, and propionate ions can all be protonated into their corresponding non-dissociated acid forms, which are cell-wallpermeable and more potent bacterial growth inhibitors than their conjugated bases. In addition, benzoate and sorbate possess a conjugated double bond system that confers to them

wall potential generates a proton gradient across the cell walls, which results in a difference of pH between the inside and outside of the walls34 accompanied by hydrolysis of water. Entry of cations into the cell walls is driven by both chemical potential (higher concentration outside) and electrochemical potential (charges) across the cell walls. Thus, when a salt is present outside the cell walls, its cations would preferentially enter the cell walls, with its anions being excluded by the negative charges of the cell wall (Donnan exclusion), while the protons outflow would re-establish electroneutrality, thus rendering the cell walls more alkaline. However, in the presence of highly acidic salts, such as aluminum chloride (solution pH of 2.5), protons would also enter the cell walls with the cations (Na+ and Al3+) and the cell walls would become more acidic. In addition, water ionization by aluminum ions can generate protons and cause more acidification of the cell walls; otherwise, the cell walls would largely remain alkaline, arising from the Donnan effect, with Na+ and K+ cations, which possess low water ionization capacity. The acidification of cell walls can also affect the activity of the bacterial macerating enzymes, such as the pectate lyases (optimum pH of 8.3−8.5), the cellulases and proteases (optimum pH of 7.0), and the polygalacturonase lyase (optimum pH of 8.5−9.5), which are secreted by the Pectobacterium bacteria.36,37 When a neutral salt (NaCl, KCl, etc.) is present outside, the cell walls would be mainly loaded with the cation (K+ or Na+), to which the cell walls are also more permeable than Cl−. Because the latter anion has low antimicrobial effect against the Pectobacterium bacteria, its presence outside the cell walls may have little effect on the pathogens. The presence of aluminum and calcium salts would result in the cell walls being loaded by these multivalent cations, which can also complex with polygalacturonide residues, reinforcing the cell walls against pathogen attack. Thus, the tissue acidification and additional strengthening of the cell walls by complexation of multivalent aluminum species [Al3+ and Al(OH)2+] by pectins could contribute to the control of soft rot by the aluminum salts, such as AlCl3. However, aluminum dihydroxy acetate exhibited disease inhibition to a lesser extent, probably because this salt solution contains mainly the monovalent Al(OH)2+ species, which has low cell wall complexing ability. The migration of aluminum ions into the plant tissue and the consequent water ionization (pH change) therein may have been limited to the depth of 1 mm (Figure 5A), in part, because of the complexation of aluminum ions to the pectic materials.38 Nevertheless, the complexation of aluminum ions would also reduce their availability against the bacteria to a certain degree. Cell wall complexation of Ca2+ (CaCl2) and its strengthening could also explain the partial inhibitory effect of CaCl2 on soft rot compared to either NaCl or KCl. Although sodium metabisulfite is an acidic salt like aluminum chloride, it yields bisulfite anions which would be excluded by the negative charges of the cell wall (Donnan exclusion). However, proton efflux from the plant cell walls because of the Donnan effect may lead to the protonation of HSO3− and SO32− species and the generation of molecular SO2, which can diffuse freely through the plant cell walls and tissue. Thus, the presence of sulfite species can be expected deeper into the tissue, as seen by the tissue pH change profile (panels A and B of Figure 5). With respect to the organic acid salts (sodium benzoate, potassium sorbate, or sodium propionate) outside the 9229

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a higher degree of hydrophobicity28 and, consequently, a faster migration potential across the cell walls and a higher disease inhibition than propionate. The migration capacity across the cell walls of aliphatic sorbic acid (or propionic acid) can also be expected to be lesser than the aromatic benzoic acid and, hence, appears to be far less effective than benzoic acid in disease inhibition in the situation, where the pathogen has had a head start. Finally, stimulation of defense mechanisms against diseases by certain salts has been shown in several plants. Jeandet et al.20 observed that aluminum chloride stimulated the accumulation of phytoalexins (resveratrol) in grapevine leaves. The elicitation of callose synthesis by both aluminum and calcium has also been reported in several plants.37 However, it is not known whether defense mechanisms are triggered in potato tuber in response to salts, such as aluminum chloride or sodium metabisulfite. It is conceivable that defense reaction may also be triggered in potato tubers, because the inhibitory effect of these salts is stronger in preventive application than in curative treatment, and this aspect deserves further investigation. In conclusion, the results of this study support that aluminum chloride, sodium metabisulfite, and sodium benzoate have potential in controlling effectively soft rot development in potato tubers. This study also provides a general basis for the understanding of specific salt−tissue interactions and for salt selection to control plant diseases. The capacity of salt constituent ions to ionize water molecules appears to relate, in part, to their disease inhibition capacity for some ions, such as aluminum, bisulfite, and organic acid ions. The lack of activity of alkaline salts, such as trisodium phosphate and sodium carbonate, in vivo appears to be due to cell wall alkalinization by the Donnan effect and the speciation of their anions into less effective ions. The persistent, albeit reduced, in vivo activity of aluminum bisulfite, and organic acid salts may be attributed to their specific properties, in addition to the Donnan effect. The acidification of cell walls and complexation of ions by cell wall polymers (aluminum ions), the generation of highly diffusible molecular SO2 (sodium metabisulfite), and generation of a more active and permeable acid form (sodium benzoate) may play a role in the persistent inhibitory activity of these salts in potato tuber, notwithstanding the induction of any biochemical defense mechanisms in the tissue in response to those salts, if any. The possible reduced aluminum ion penetration into the tissue by complexation and partitioning of the diffusible species, molecular SO2 and acid form of benzoate, between the plant cells and the bacterial cells, leading to their reduced availability at the infection site, and the attenuated activity of these salts in the tissue. Further work would be required with respect to the formulation of salts exhibiting synergy and enhancement of disease inhibition in the tubers and the evaluation of potential health and environmental risks associated with salt applications before envisioning industrial application of these salts.

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Notes

The authors declare no competing financial interest.

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REFERENCES

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*Telephone: 418-656-2839. Fax: 418-656-3353. E-mail: joseph. [email protected]. Funding

This study was supported by Conseil de Recherche en Agriculture, Pêche et Alimentation du Québec (CORPAQ), Cultures H. Dolbec, Inc., and Propur, Inc. 9230

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