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Effect of acidic solutions and acidic prochloraz on the control of postharvest decay caused by Alternaria alternata in mango and persimmon fruit
Postharvest Biology and Technology 42 (2006) 134–141
Effect of acidic solutions and acidic prochloraz on the control of postharvest decay caused by Alternaria alternata in mango and persimmon fruit D. Prusky ∗ , I. Kobiler, M. Akerman, I. Miyara Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel Received 24 March 2006; accepted 4 June 2006
Abstract The effectiveness of treatments with hydrochloric acid (HCl), alone or in combination with prochloraz, in controlling quiescent infections of Alternaria alternata that cause alternaria rot in mango and persimmon fruit during storage, were compared. Spore germination and germ-tube elongation of A. alternata in vitro were inhibited by 95 and 65%, respectively, by exposure to 1.25 mM HCl, and fungal germination was completely inhibited by 2.50 mM HCl. Application of a combination of hot water spraying and brushing (HWB) for 15–20 s, followed by spraying with 50 mM HCl effectively controlled alternaria rot in stored mango fruit. Similar HWB treatments followed by spraying with increasing concentrations of prochloraz at 45 to 900 g ml−1 in 50 mM HCl, were as effective as treatment with the acid alone, in preventing alternaria rot development. Also in persimmon fruit, dip treatment with 50 mM HCl reduced alternaria rot. However, acidified solutions containing 45 g ml−1 prochloraz inhibited alternaria rot development better than treatment with HCl alone. The enhanced prochloraz activity in acidified solutions was attributed to its enhanced solubility, which resulted in an increase in the fungicide active ingredient in the solution. Present data suggest that the combination of acid solutions, alone or in the presence of reduced prochloraz concentrations, provide a simple treatment for the control of postharvest diseases that alkalinize the host environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Disease control; Quiescent infections; Host alkalinization; Host ammonification; Soluble prochloraz
1. Introduction Transformation of biotrophic-quiescent infection to necrotrophic-active colonization in postharvest rots occurs concurrently with modulation of the host environmental pH (Prusky and Yakoby, 2003). The environmental pH may change either naturally during fruit ripening or through induction by the pathogens (Yakoby et al., 2000). Pathogens may modulate their virulence by local acidification or alkalinization of the host tissue, and this is a factor in the enhancement of pathogenesis and symptom development. Acidification of the ambient pH by the secretion of organic acids, such as gluconic acid by Penicillium expansum (Prusky et al., 2001b), and oxalic acid by Botrytis cinerea (Monteau et al.,
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2000) and Sclerotinia sclerotiorum (Cessna et al., 2000), has been detected. In all these cases, enhanced polyglacturonase gene expression was detected. In contrast alkalinization of the infection court by the secretion of ammonia by Colletotrichum gloeosporioides and Alternaria alternata has been described. Levels of ammonia in decayed tissue infected by Colletotrichum sp. reached concentrations of 3.4 mM in avocado and 1.1 mM in apple, which increased the pH of the infected fruit from 5.9 to 7.2 and from 3.8 to 6.5, respectively (Prusky et al., 2001b). Also, A. alternata accumulated almost 6.0 mmol ammonia per gram of hyphae on persimmon cells, which led to a pH increase (Eshel et al., 2002). In all these cases, alkalinization of the environment increased gene expression of pectate lyase by C. gloeosporioides (Prusky et al., 2001b) and of endoglucanases by A. alternata (Eshel et al., 2002). Thus, by secreting ammonium the pathogen actively modifies the host environment so as to promote the activation of pathogenicity factors and fungal attack.
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The significant changes in environmental pH induced by the pathogens suggested possible use of these finding to modulate fungal colonization and control of postharvest diseases. Control of postharvest diseases of subtropical fruit is vital for maintaining their quality and shelf-life during long periods of storage, under conditions in which the use of postharvest fungicidal treatments is severely limited. Postharvest diseases might be controlled by either pre- or postharvest treatment, or even by a combination of the two in cases of high incidence of disease (Janisiewicz and Korsten, 2002). The new, strict regulations regarding fungicide residues, and reduction of the limits to the legal acceptability of specific fungicides by local authorities make the development of new postharvest decay-control treatments for fruit an important factor in improving their quality, and their safety for humans and the environment. Several aspects of pH regulation are important for the control of postharvest diseases, because the pH (i) directly affects the germination of conidia (Pelser and Eckert, 1977), (ii) influences the virulence exhibited by pathogens through their colonization of host tissue (Prusky et al., 2004), and (iii) affects the toxicity of the fungicides used to control these diseases (Eckert and Eaks, 1989; Smilanick et al., 2005). The influence of pH on the activity of fungicides has been studied in the past for several fungicides, such as sodium o-phenylphenate and imazalil. Proper control of the pH of o-phenylphenate solutions during commercial application is critical in optimizing the effectiveness of the fungicide, controlling fruit residue levels, and minimizing phytotoxicity (Eckert and Eaks, 1989). Imazalil activity, too, is affected by pH; imazalil was found more effective in inhibiting P. italicum growth at pH 7.0 than at pH 5.2 or 5.3 (Siegel et al., 1977; Guan et al., 1989), and that of P. digitatum at pH 5.9 than at pH 5.1 (Daniels et al., 1985). Black spot caused by A. alternata is an economically important postharvest disease of mango and persimmon fruit worldwide, including the subtropical growing regions of Israel (Prusky et al., 1981, 1983, 2002). The primary mode of infection of mango fruit by A. alternata is by direct penetration through the lenticels during fruit growth, followed by quiescence until the fruit is harvested and ripens during long storage periods or shelf-life. In persimmon fruit, A. alternata may penetrate either through small wounds or directly into the fruit cuticle (Prusky et al., 1981). Postharvest development of alternaria rot symptoms in mango fruit during storage is usually prevented by a combination of hot water brushing (HWB) in combination with prochloraz at 225 g ml−1 ; this leads to eradication of latent infections initiated in the orchard, and consequently prevents fungal colonization (Prusky et al., 1999). Postharvest development of A. alternata in persimmons during storage is prevented by a pre-storage chlorine dip treatment (Prusky et al., 2001a). The finding that A. alternata alkalinizes the host environment pH (Eshel et al., 2002) stimulated the examination of the possibility of modulating the environmental pH by acid treatments, as a means of reducing fungal colonization.
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Our objectives in the present study were to determine the influence of external acid treatment on the pathogen and on its symptom development. We also analyzed the effects of acidic solutions on the solubility of prochloraz, and on its biological activity in the control of alternaria rot in subtropical fruit. Our findings indicate that acid treatment may, in itself, reduce symptom development by A. alternata, and it may improve the efficiency of disease control by prochloraz. Acid treatment represents a new approach to disease control and fruit-quality improvement; at the same time it improves safety by reducing the concentration of prochloraz used as a postharvest fungicide application (Prusky et al., 1999).
2. Materials and methods 2.1. Pathogen culture A. alternata was cultured for 1–2 weeks on potato dextrose agar (PDA) (Difco Laboratories, Detroit) at 25 ◦ C. The isolate used was obtained from infected mango or persimmon fruit in Israel. Spores were harvested by adding 3–4 ml of sterile, de-ionized water (diH2 O) to the Petri dish. The spores were then rubbed with a sterile glass rod to free them from the PDA medium, and the spore suspension was passed through two layers of cheese cloth. The suspension was diluted with water to obtain the spore concentrations need in each experiment, according to determination with a haemocytometer. 2.2. Spore germination and colonization assays Sterile microscope slides were used for the germination assays of A. alternata spores. Water solutions containing concentrations of HCl ranging from 0 to 2.5 mM were used as a medium. After 24 h of incubation at 24 ◦ C, the germinated and ungerminated spores in each slide were counted by observation with a (200×) microscope. Within each replicate, 100–150 spores were examined, the percentage of germinated spores was calculated and the lengths of the germinated hyphae were measured. The effect of prochloraz solutions (Sportak, 45% active ingredient, a.i.; AgrEvo UK Limited, Hauxton, England) on colonization of A. alternata was examined on potato dextrose agar (PDA) plates. The plates were seeded with 150 l of a suspension containing 106 spores ml−1 and a 13-mm diameter grade AA filter disc (Whatman International Ltd., Maidstone, England) was placed in the centre of each plate. Fifty microlitres of the appropriate concentrations of prochloraz in 50 mM HCl (Bio Lab, Jerusalem, Israel) or of tap water solutions were placed on the disc. Each treatment included six replicates, and each test was repeated three times. 2.3. pH measurements pH was measured with a flat electrode (Sensorex, Stanton, CA) following an approx. 0.2-mm deep cut with a scalpel
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blade. Mesocarp pH was determined after peeling the pericarp to a depth of 2 mm. pH measurements were taken by placing the flat electrode directly against the exposed tissue. All measurements were repeated on six fruit at three different places (18 measurements) around the equatorial axis of each fruit. The standard error (S.E.) of the mean of pH measurements was never higher than 2.5% (Yakoby et al., 2000). 2.4. Prochloraz determinations Water samples containing the fungicide were extracted five times with 50 ml of methylene chloride, and the organic extracts were pooled. The extracts were dried in a rotary evaporator at 40 ◦ C under vacuum, diluted in acetone to about 10 g ml−1 , and injected into a gas chromatograph GC-HP6890 (Agilent, Palo Alto, CA, USA) fitted with a Restek 35 capillary column (15m × 530 m × 1 m) (Restek, Bellefonte, PA, USA) and detected with a nitrogen phosphorus detector (NPD) (Agilent, USA). The concentration was determined by comparison with a standard amount of pure prochloraz diluted in acetone. 2.5. Postharvest treatments in semi-commercial mango experiments The hot water brushing (HWB) treatment at 55 ◦ C was applied with a machine similar to that described by Prusky et al. (1999). The hot water, alone or in combination with prochloraz, was applied on commercial packing lines. It was sprayed above revolving brushes at a rate of 100–120 l min−1 and at a nozzle pressure of 2 atm (Prusky et al., 1999). The fruit passed over seven transversely oriented, 12-cm diameter plastic brushes for 15–20 s. Acidic prochloraz, at several concentrations, was applied separately as a spray over two revolving brushes, following the HWB treatment, and the effects were compared with those of the commercially used HWB that used prochloraz at 225 g ml−1 . Acidic prochloraz was prepared by dissolving the liquid formulation in solutions containing various concentrations of HCl. After the fruit had been dried, they were waxed by spraying them with polyethylene emulsion wax at a soluble solids concentration of 12% (v/v), over revolving brushes (Safepack, Israel). Mango fruit of ‘Tommy Atkins’ and ‘Keitt’ cultivars were harvested, one from each of two orchards in Israel, transferred to a packing house and treated as described above. The experimental design included complete randomization, with six replications, each comprising a 350-kg bin per treatment. From each of these bins 12 fruit were sub-sampled after waxing, placed in cartons and stored at 12 ◦ C and about 90% RH for 4 weeks, before being transferred to 20 ◦ C and about 80% RH for 4 days. Control untreated fruit were stored under the same conditions, immediately upon arrival from the orchard.
2.6. Mango fruit ripening and alternaria rot development during storage The severity of infection was recorded as the percentage of the surface area of the fruit that exhibited alternaria rot symptoms. The fruit were assessed initially when they were transferred from 12 ◦ C storage to 20 ◦ C storage, and periodically until most of them were ripe. Disease severity was determined on 72 fruit (12 fruit from each of six replicates) by the percent of alternaria decayed area. Fruit were regarded as unmarketable when more than 1% of their surface area exhibited black spots. Experiments were carried out during 2 consecutive years and repeated at least four times at each year. The results of one representative experiment are presented. 2.7. Postharvest treatments in semi-commercial persimmon experiments ‘Triumph’ persimmon fruit harvested from two orchards were transferred to the packing house and dipped in a 3-m3 tank for 30 s. The experiment was conducted in a completely randomized block design in each orchard, with 10 replications, each comprising a 350-kg bin. Each block included control undipped fruit and fruit that were dip-treated for 30 s in the acidic solutions and in the commercial treatment. The commercial treatment used for persimmon fruit was Troclosene-Na (1,3-dichloro-1,3,5-triazine-2,4,6-trione; Medentech Ltd., Loch Garman, Ireland); it is a chlorine-based product supplied in tablet form by Concept, Israel. The product contains 62% latently available chlorine at 20 ◦ C and pH 5.9, and was applied at a concentration of 500 g ml−1 . A kit supplied by the manufacturer measured the exact concentration of available chlorine. Before storage, 50 fruit were sampled from each bin and placed in a separate box in the commercial storehouse. The fruit were stored under commercial conditions, subjected to forced-air cooling as described previously (Prusky et al., 2001a), and then stored at 0 ◦ C for 16 weeks pending transfer to 4 days at 20 ◦ C. The sampled fruit were the only ones evaluated following storage, in these semi-commercial scale experiments. 2.8. Alternaria rot development in persimmons during storage In the semi-commercial experiments, the severity of alternaria rot was evaluated according to the percent of fruit area covered by A. alternata symptoms when the fruit were transferred from storage at 0 to 20 ◦ C, and again 4 days later (Perez et al., 1994). Disease assessment at each stage was applied to 500 fruit (50 fruit from each of 10 replicates). Fruit were regarded as unmarketable when more than 1% of the fruit surface area exhibited black spots. Experiments were carried out during 2 consecutive years and repeated at least four times at each year. The results of one representative experiment are presented.
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2.9. Statistical analysis Data were subjected to analysis of variance by means of the Tukey–Kramer HSD test (P < 0.05).
3. Results 3.1. Effect of acidic solutions on spore germination, germ-tube elongation and colonization of A. alternata A. alternata germination and germ-tube elongation were not affected at HCl concentrations of 0.25 mM, even if the pH of the solution declined from 5.6 to 3.4 (Table 1), but spore germination and germ-tube elongation of A. alternata declined by ca. 95 and 65%, respectively, when the HCl concentration was increased to 1.25 mM and the pH of the solution declined to 2.8. At an HCl concentration of 2.50 mM and pH 2.5, germination and germ-tube elongation were inhibited by 100% (Table 1). 3.2. Effect of acid solutions on the control of alternaria rot in mango and persimmon fruit Spray treatment of naturally A. alternata-infected ‘Tommy Atkins’ mango fruit with 50 mM HCl, following HWB treatment, significantly reduced the incidence of decay after 4 weeks of storage and 4 days of shelf-life, by about 50% (Fig. 1). Lower acid concentrations of 25 mM, or higher ones of 75 mM, within a series of increasing concentrations, did not significantly affect the level of alternaria rot in stored fruit. Dip treatments of ‘Triumph’ persimmon fruit in hydrochloric acid at 50 and 75 mM, prior to 16 weeks of storage, both reduced the incidence of alternaria rot decay by 35–45% similarly to commercial dip treatments with Troclosene-Na (Table 2). Dip treatment with 50 mM hydrochloric acid reduced the peel pH from 5.64 ± 0.1 to 4.38 ± 0.23. 3.3. Effect of concentration of HCl on prochloraz solubility and A. alternata inhibition in vitro Transmittance analysis of a series of dilutions of the fungicide prochloraz in double-distilled water showed reduced transmittance as the fungicide concentration increased from Table 1 Effect of hydrochloric acid on the germination and hyphal length of A. alternata spores after 17 h at 25 ◦ C HCl concentration (mM)
pH value
Germination (%)
Length (m)
0 0.25 1.25 2.50
5.6 3.4 2.8 2.5
100.0 a 100.0 a 5.3 b 0c
13.9 a 14.1 a 4.9 b 0c
Each value represents the mean of five replicates of 100–150 spores each. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test.
Fig. 1. The effect of hot water brushing (HWB) at 55 ◦ C followed by HCl spray and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit. Mango fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and for 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruit. Acid solution pHs ranged between 1.1 and 1.6. Average values at each columns with unlike letters differ significantly according to the Tukey–Kramer HSD test (P < 0.05).
100 to 150 g ml−1 . No transmittance was detected through a 450 g ml−1 prochloraz solution (Fig. 2). However, dilution of prochloraz with increasing concentrations of HCl (1.25–50 mM,) resulted in increased transmittance through the fungicide dilutions. At HCl concentrations ranging from 12 to 50 mM, dilutions of prochloraz to 450 g ml−1 reduced transmittance by only 10% compared with 100% reduction by a water solution (Fig. 2). Determination of the content of soluble prochloraz following dilution of the commercial formulation (calculated concentration) to 250 and 450 g ml−1 active ingredient in water, followed by a 3 min centrifugation (3000 × g), detected the presence of only 10 and 32 g ml−1 , respectively, of active ingredient (Fig. 3). However, if prochloraz dilutions were prepared in acid solutions with HCl concentrations of 2.5, Table 2 The effects of HCl and Troclosene-Na dip treatments on the severity of alternaria rot on ‘Triumph’ persimmon fruit Treatment
Control HCl 50 mM HCl 75 mM Troclosene-Na 500 g ml−1
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.41 a 0.96 b 0.93 b 0.76 b
2.37 a 1.51 b 1.34 b 1.47 b
Persimmon fruit were dip-treated in the packing house 5–10 h after harvest and stored for 16 weeks at 0 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of 10 replicates, each of 50 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. Troclosene-Na at 500 g ml−1 is the currently used commercial treatment.
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Fig. 2. Effect of HCl concentration on the solubility of prochloraz, as determined by transmission at 690 nm.
12.5 or 50 mM, the amount of detectable prochloraz active ingredient in the solution increased two- to four-fold. Inhibition of A. alternata colonization in vitro by acidic prochloraz at concentrations from 22.5 to 90 g ml−1 showed also a 21–25% increase in activity compared with that of prochloraz solutions prepared in distilled water. At higher concentrations of prochloraz (225 and 450 g ml−1 ), the enhancing effect of the acid solution on the prochloraz activity was not seen (Fig. 4). 3.4. Disease-control efficiency of acidic prochloraz in mango and persimmon fruit during storage and shelf-life When the commercial HWB treatment used for mango fruit was followed by a spray of 50 mM HCl, alone or with prochloraz at 45, 225 or 900 g ml−1 , all the acid treatments reduced the incidence of alternaria rot on ‘Tommy Atkins’ fruit compared with that on the controls (Table 3). However, no improvement in disease control was found, even if the concentrations of the fungicide increased 20-fold (Table 3). In a second experiment, the commercial prochloraz treatment, i.e., inclusion in the HWB at 225 g ml−1 , also did not dif-
Fig. 4. Inhibition of A. alternata colonization by acidified and non-acidified prochloraz solutions. Acidified prochloraz was prepared in 50 mM HCl () or, in the controls (), with double-distilled water. Fifty microlitres of acidified or non-acidified prochloraz were placed on a filter disc at the centre of a PDA plate 6 h after inoculation with 150 l of a suspension of 106 A. alternata spores. Inhibition of colonization was measured 5 days later. Each value represents the mean of five replicates. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer (P < 0.05).
fer from HWB treatment followed by a spray with 50 mM HCl, alone or with prochloraz at 45 g ml−1 , in the control of alternaria rot (Table 4). A similar degree of alternaria rot control was obtained when ‘Keitt’ mango fruit were sprayed with acidic prochloraz at 22–132 g ml−1 after the HWB treatment, which suggests that increasing the fungicide concentrations in acidic solution does not improve disease control (data not shown). When alternaria rot was evaluated in persimmon fruit immediately after storage, the treatments with acid alone, acidic prochloraz, and the commercial Troclosene-Na all had similar percent of decayed area levels (Table 5). The acid prochloraz treatment of persimmon fruit was observed to be more effective than the treatment with acid alone only after 4 days of at 20 ◦ C, following storage for 16 weeks at 0 ◦ C. Table 3 The effects of hot water brushing (HWB) at 55 ◦ C followed by a spray of prochloraz in 50 mM HCl solutions and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit Treatment
Fig. 3. Effect of HCl concentration on the amount of actual active ingredient of prochloraz in acid solutions. Solutions containing several concentrations of HCl were used for dilution of the prochloraz fungicide. The calculated prochloraz concentrations obtained by dilution of the commercial fungicide formulation (calculated concentration) were centrifuged at 3000 × g and the supernatant was chemically analyzed for prochloraz concentrations by GC.
HWB HWB + 50 mM HCl HWB + 50 mM HCl + 45 g ml−1 prochloraz HWB + 50 mM HCl + 225 g ml−1 prochloraz HWB + 50 mM HCl + 900 g ml−1 prochloraz
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.28a 1.30a 1.28a
3.02 a 1.53 b 1.39 b
1.55a
1.99 b
1.23a
1.24 b
The fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruits. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test.
D. Prusky et al. / Postharvest Biology and Technology 42 (2006) 134–141 Table 4 The effects of hot water brushing (HWB) at 55 ◦ C followed by 50 mM HCl spray, with or without prochloraz, and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit Treatment
Control HWB HWB + 50 mM HCl HWB + 50 mM HCl + 45 g ml−1 prochloraz HWB + 225 g ml−1 prochloraz*
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
6.51 a 3.23 b 0.60 c 1.51 bc
6.91 a 4.52 b 1.54 c 1.49 c
1.81 bc
1.87 c
The fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. * This combination is currently used as the commercial treatment for mango fruit.
Table 5 The effects of HCl, with or without prochloraz, and Troclonse-Na treatments on the severity of alternaria rot on ‘Triumph’ persimmon fruit Treatment
Control 50 mM HCl 50 mM HCl + 45 g ml−1 prochloraz Troclosen-Na* 500 g ml−1
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.32 a 0.80 b 0.70 b
3.94 a 3.04 b 2.46 c
0.68 b
2.34 c
The fruit were dip-treated in the packing house 5 h after harvest and stored for 16 weeks at 0 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of ten replicates, each of 50 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. * Troclosen-Na at 500 g ml−1 is the current commercial treatment applied to persimmon fruit.
4. Discussion In the complex postharvest handling of mango and persimmon fruit, where control of diseases is necessary to preserve the quality of the produce, the use of safer synthetic fungicides (Kobiler et al., 2001; Prusky et al., 2001a), biological antagonists (Janisiewicz and Korsten, 2002) and physical treatments (Prusky et al., 2001a) are integrated. Studies that revealed host environment alkalinization by ammonia secretion during A. alternata and C. gloeosporioides colonization of fruit have opened a new approach to the modulation of disease development (Prusky et al., 2001a,b, 2004). Postharvest HCl treatment of mango fruit at 50 mM and of persimmon fruit at 50 and 75 mM reduced the incidence of alternaria rot after storage and shelf-life. This result may suggest that acidic solutions may reduce the incidence of alternaria rot by pH modulation. However, acidic solutions were also very efficient in inhibiting spore germination and germ-tube elonga-
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tion, which suggests a direct toxicity effect. Acidic solutions with 1.25 mM HCl almost completely inhibited germination and germ-tube elongation of A. alternata spores in water, but on the rich PDA medium 50 mM HCl did not inhibit fungal colonization by A. alternata spores, which suggests the importance of the nutrient source in determining the direct effect of acid on the fungal response. Griffin (1994) stated that the influence of pH on fungal growth depended upon the ionization of acids or bases in the medium in which the fungus resided. The pH can alter the membrane potentials that control the permeability of fungal membranes to many substances, including the toxic compounds naturally present in the fruit. It is also possible that hydrogen and hydroxyl ion concentrations are important factors for the inhibitory or lethal activity of many compounds (Hwang and Klotz, 1938). The effect of HCl on alternaria rot is not, however, concentration dependent; when mango fruit were treated with HCl at 25 mM or at concentrations higher than 75 mM, alternaria rot incidence did not differ from that of the control. It could be that the acid treatment at 75 mM or higher was already phytotoxic to the tissue. In persimmon fruit, however, increase in HCl concentration from 50 to 75 mM did not reduce the efficiency of the treatment. This might suggest that the host susceptibility to the HCl might affect the final response to the treatment. However, the acidic pH affects not only disease development; acidification of the fungicide prochloraz with HCl improved its biological activity against A. alternata colonization in vitro as observed for Penicillium (Fuchs et al., 1993). The biological activity of prochloraz in acid solutions at concentrations in the range of 22–90 g ml−1 increased by almost 20% compared with that of non-acidified prochloraz. However, at concentrations of 225 g ml−1 and higher, no biological differences in fungal inhibition between acidic and non-acidic solutions were observed. The increase in the biological activity of prochloraz in acidic solutions was probably due to its increased solubility: the acidified prochloraz solutions, i.e., those in 2.5, 12.5 and 50 mM HCl, were found to contain two to four times as much detectable active ingredient. The increase in solubility was also confirmed by the analysis of transmittance through solutions of prochloraz prepared under neutral or acidic conditions; acidified solutions showed improved transmittance, which was considered to be an indicator of fungicide solubility. The increase in the solubility of prochloraz may be attributed to the formation of its hydrochloric salt (pKa ∼ 3.8) in HCl-containing solutions:
Lukens (1971) reported that neutral forms of fungicides penetrated membranes and were more toxic than charged
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forms. In the case of prochloraz in the present study, however, the charged form seems to have been more effective than the neutral form, as indicated by its solubility. A similar but opposite finding was reported for imazalil, which was found more toxic to P. italicum at pH 7.0 than at pH 5.0, and it was observed that less imazalil entered the mycelium at pH 5.0 than at pH 7.0. Siegel et al. (1977) ascribed this difference in potency to the charge present on the molecule, and concluded that the toxicity of imazalil was reduced at lower pH, when the molecule was charged, because the molecule was not incorporated into the mycelium. A slight difference in the pH could have a significant effect on the concentration of the neutral (dissociated) form of imazalil, which is largely responsible for the antifungal activity of that compound (Siegel et al., 1977; Siegel and Ragsdale, 1978; Guan et al., 1989). In practical experiments on the control of alternaria rot on mango and persimmon fruit, acid prochloraz did not reduce the incidence of decay more than the acidic solution alone. Since the mango and persimmon fruit used in the experiment have natural levels of quiescent infections (Prusky et al., 1981, 1983) it is clear that the 50 mM HCl was effective in modulating the environment of the germinated spores that were in a quiescent stage. Interestingly, however, increasing the concentrations of prochloraz from 45 to 900 g ml−1 in the acid solution did not result in enhanced disease control. This may be because of the improved solubility and biological activity at relatively low concentrations of prochloraz and/or because the combined effect of the acid solution and the fungicide on the pathogen resulted in improved disease control. It is possible, however, that under long-term storage conditions, as in the case of persimmon fruit that are stored for 16 weeks at 0 ◦ C, reduced concentrations of the acidified fungicide are needed to reduce the incidence of decay during shelf-life. The present results suggest that modulation of host pH by exogenic treatments provide an efficient approach to the control of postharvest pathogens that alkalinize the host fruit, such as A. alternata. An equivalent approach, using basic treatments, e.g., NaHCO3 , was applied for the control of acidifying pathogens such as P. expansum, P. italicum, and P. digitatum (Prusky et al., 2001b; Porat et al., 2002; Smilanick et al., 1999, 2005; Fallik et al., 1996) as well as for sour rot, caused by Geotrichum citri-aurantii, which cannot be controlled by commercial fungicides (Smilanick and Sorenson, 2001; Smilanick et al., 2005). However, what significantly contributes to the importance of pH-regulating treatments is their ability to reduce the amounts of residues of synthetic fungicides without affecting the efficacy of the treatment. In the present case, the efficacy of the acid treatment and its enhancement through the improved solubility of the fungicide led to improved fruit quality as expressed in reductions in decay development and in the amounts of fungicide residues in the fruit arriving in the market. Random evaluations of fungicide residues in mango before export detected 66-fold lower values in the acidic prochloraz treated fruit.
Acknowledgments We thank A. Keinan (Tzemach Avocado Packing House), M. Shoam (Shoam Packing House), and Y. Kaplan and R. Cohen (Mor HaSharon Packing House) for the possibility of doing experiments in their packing houses. We also thank Dr. P. Goldshlag for the analysis of prochloraz samples. Our thanks are also due to the Israel Mango and Persimmon Growers’ Association and Binational US-Israel Agriculture Research and Development Fund (3363-02) for partial financial support. References Cessna, S.G., Sears, V.E., Dickman, M.B., Low, P.S., 2000. Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12, 2191–2200. Daniels, J.A., Krishnamurthi, R., Rizvi, S.H., 1985. A review of effects of carbon dioxide on microbial growth and food quality. J. Food Prot. 48, 532–537. Eckert, J.W., Eaks, I.L., 1989. Postharvest disorders and diseases of citrus fruits. In: Reuther, W., Calavan, E.C., Carman, G.E. (Eds.), The Citrus Industry, vol. 4. University of California Press, Berkeley, CA, pp. 179–260. Eshel, D., Miyara, I., Ailinng, T., Dinoor, A., Prusky, D., 2002. pH regulates endoglucanase expression and virulence of Alternaria alternata in persimmon fruits. Mol. Plant–Microbe Interact. 15, 774–779. Fallik, E., Grinberg, S., Ziv, O., 1996. Use of bicarbonate salts to reduce decay development in harvested fruits and vegetables. Phytoparasitica 54, 153–154. Fuchs, Y., Zauberman, G., Ronen, R., Rot, I., Weskler, A., Akerman, M., 1993. The physiological basis of litchi fruit pericarp color retention. Acta Hortic. 343, 29–33. Griffin, D.H., 1994. Spore dormancy and germination. In: Griffin, D.H. (Ed.), Fungal Physiology, second ed. Wiley-Liss, New York, pp. 375–398. Guan, J., Kerkenaar, A., de Waard, M.A., 1989. Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum. Neth. J. Plant Pathol. 95, 73–86. Hwang, L., Klotz, L.J., 1938. The toxic effect of certain chemical solutions on spores of Penicillium italicum and P. digitatum. Hilgardia 12, 1–38. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest disease of fruits. Ann. Rev. Phytopathol. 40, 411–441. Kobiler, I., Shalom, Y., Roth, I., Akerman, M., Vinokour, Y., Fuchs, Y., Prusky, D., 2001. Effect of 2,4-dichlorophenoxyacetic acid on the incidence of side and stem-end rots in mango fruits. Postharvest Biol. Technol. 23, 23–32. Lukens, R.J., 1971. Chemistry of Fungicidal Action. Springer-Verlag, New York. Monteau, S., Abouna, S., Lambert, B., Legendre, L., 2000. Differential regulation by ambient pH of putative virulence factor secretion by the phytopathogenic fungus Botrytis cinerea. FEMS Microbiol. Ecol. 43, 359–366. Pelser, P.du.T., Eckert, J.W., 1977. Constituents of orange juice that stimulate the germination of conidia of Penicillium digitatum. Phytopathology 67, 747–754. Perez, A., Ben Arie, R., Dinoor, A., Genizi, D., Prusky, D., 1994. Prevention of Black spot disease in persimmon fruit by giberellic acid and iprodione treatments. Phytopathology 85, 221–232. Porat, R., Daus, A., Weiss, B., Cohen, L., Droby, S., 2002. Effects of combining hot water, sodium bicarbonate and biocontrol on postharvest decay of citrus fruit. J. Hort. Sci. Biotechnol. 7, 441–445. Prusky, D., Yakoby, N., 2003. Pathogenic fungi: leading or led by ambient pH? Mol. Plant Pathol. 4, 509–516.
D. Prusky et al. / Postharvest Biology and Technology 42 (2006) 134–141 Prusky, D., Ben-Arie, R., Guelfat-Reich, S., 1981. Etiology and histology of Alternaria rot of persimmon fruits. Phytopathology 71, 1124–1128. Prusky, D., Fuchs, Y., Yanko, U., 1983. Assessment of latent infections as a basis for the control of postharvest disease of mango. Plant Dis. 67, 816–817. Prusky, D., Fuchs, I., Kobiler, I., Roth, I., Weksler, A., Shalom, Y., Falik, E., Zauberman, G., Pesis, E., Akerman, M., Yekutiely, O., Weisblum, A., Regev, R., Artes, L., 1999. Effect of host water brushing, prochloraz treatment and waxing on the incidence of black spot decay caused by Alternaria alternata in mango fruit. Postharvest Biol. Technol. 15, 165–174. Prusky, D., Eshel, D., Kobiler, I., Yakoby, N., Beno-Moualem, D., Ackerman, M., Zuthji, Y., Ben Arie, R., 2001a. Postharvest chlorine treatments for the control of the persimmon black spot disease caused by Alternaria alternata. Postharvest Biol. Technol. 22, 271–277. Prusky, D., McEvoy, J.L., Leverentz, B., Conway, W.S., 2001b. Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence. Mol. Plant–Microbe Interact. 14, 1105–1113. Prusky, D., Shalom, Y., Kobiler, I., Akerman, M., Fuchs, Y., 2002. The level of quiescent infection of Alternaria alternata in mango fruits at harvest determines the postharvest treatment applied for the control of rots during storage. Postharvest Biol. Technol. 25, 339–347.
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Prusky, D., McEvoy, J.L., Saftner, R., Conway, W.S., Jones, R., 2004. Relationship between host acidification and virulence of Penicillium spp. on apple and citrus fruits. Phytopathology 94, 44–51. Siegel, M.R., Ragsdale, N.N., 1978. Antifungal mode of action of imazalil. Pestic. Biochem. Physiol. 9, 48–56. Siegel, M.R., Kerkenaar, A., Kaars Sijpesteijn, A., 1977. Antifungal activity of the systemic fungicide imazalil. Neth. J. Plant Pathol. 83, 121–133. Smilanick, J.L., Sorenson, D., 2001. Control of postharvest decay of citrus fruit with calcium polysulfide. Postharvest Biol. Technol. 21, 157–168. Smilanick, J.L., Margosan, D., Mlikota, F., Usall, J., Michael, I.F., 1999. Control of citrus green mold by carbonate and bicarbonate salts and the influence of commercial postharvest practices on their efficacy. Plant Dis. 83, 139–145. Smilanick, J.L., Mansour, M.F., Margosan, D.A., Mlikota Gabler, F., 2005. Influence of pH and NaHCO3 on effectiveness of imazalil to inhibit germination of Penicillium digitatum and to control postharvest green mold on citrus fruit. Plant Dis. 89, 640–648. Yakoby, N., Kobiler, I., Dinoor, A., Prusky, D., 2000. pH regulation of pectate lyase secretion modulates the attack of Colletotrichum gloeosporioides on avocado fruits. Appl. Environ. Microbiol. 66, 1026–1030.
Effect of acidic solutions and acidic prochloraz on the control of postharvest decay caused by Alternaria alternata in mango and persimmon fruit D. Prusky ∗ , I. Kobiler, M. Akerman, I. Miyara Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel Received 24 March 2006; accepted 4 June 2006
Abstract The effectiveness of treatments with hydrochloric acid (HCl), alone or in combination with prochloraz, in controlling quiescent infections of Alternaria alternata that cause alternaria rot in mango and persimmon fruit during storage, were compared. Spore germination and germ-tube elongation of A. alternata in vitro were inhibited by 95 and 65%, respectively, by exposure to 1.25 mM HCl, and fungal germination was completely inhibited by 2.50 mM HCl. Application of a combination of hot water spraying and brushing (HWB) for 15–20 s, followed by spraying with 50 mM HCl effectively controlled alternaria rot in stored mango fruit. Similar HWB treatments followed by spraying with increasing concentrations of prochloraz at 45 to 900 g ml−1 in 50 mM HCl, were as effective as treatment with the acid alone, in preventing alternaria rot development. Also in persimmon fruit, dip treatment with 50 mM HCl reduced alternaria rot. However, acidified solutions containing 45 g ml−1 prochloraz inhibited alternaria rot development better than treatment with HCl alone. The enhanced prochloraz activity in acidified solutions was attributed to its enhanced solubility, which resulted in an increase in the fungicide active ingredient in the solution. Present data suggest that the combination of acid solutions, alone or in the presence of reduced prochloraz concentrations, provide a simple treatment for the control of postharvest diseases that alkalinize the host environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Disease control; Quiescent infections; Host alkalinization; Host ammonification; Soluble prochloraz
1. Introduction Transformation of biotrophic-quiescent infection to necrotrophic-active colonization in postharvest rots occurs concurrently with modulation of the host environmental pH (Prusky and Yakoby, 2003). The environmental pH may change either naturally during fruit ripening or through induction by the pathogens (Yakoby et al., 2000). Pathogens may modulate their virulence by local acidification or alkalinization of the host tissue, and this is a factor in the enhancement of pathogenesis and symptom development. Acidification of the ambient pH by the secretion of organic acids, such as gluconic acid by Penicillium expansum (Prusky et al., 2001b), and oxalic acid by Botrytis cinerea (Monteau et al.,
∗
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2000) and Sclerotinia sclerotiorum (Cessna et al., 2000), has been detected. In all these cases, enhanced polyglacturonase gene expression was detected. In contrast alkalinization of the infection court by the secretion of ammonia by Colletotrichum gloeosporioides and Alternaria alternata has been described. Levels of ammonia in decayed tissue infected by Colletotrichum sp. reached concentrations of 3.4 mM in avocado and 1.1 mM in apple, which increased the pH of the infected fruit from 5.9 to 7.2 and from 3.8 to 6.5, respectively (Prusky et al., 2001b). Also, A. alternata accumulated almost 6.0 mmol ammonia per gram of hyphae on persimmon cells, which led to a pH increase (Eshel et al., 2002). In all these cases, alkalinization of the environment increased gene expression of pectate lyase by C. gloeosporioides (Prusky et al., 2001b) and of endoglucanases by A. alternata (Eshel et al., 2002). Thus, by secreting ammonium the pathogen actively modifies the host environment so as to promote the activation of pathogenicity factors and fungal attack.
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The significant changes in environmental pH induced by the pathogens suggested possible use of these finding to modulate fungal colonization and control of postharvest diseases. Control of postharvest diseases of subtropical fruit is vital for maintaining their quality and shelf-life during long periods of storage, under conditions in which the use of postharvest fungicidal treatments is severely limited. Postharvest diseases might be controlled by either pre- or postharvest treatment, or even by a combination of the two in cases of high incidence of disease (Janisiewicz and Korsten, 2002). The new, strict regulations regarding fungicide residues, and reduction of the limits to the legal acceptability of specific fungicides by local authorities make the development of new postharvest decay-control treatments for fruit an important factor in improving their quality, and their safety for humans and the environment. Several aspects of pH regulation are important for the control of postharvest diseases, because the pH (i) directly affects the germination of conidia (Pelser and Eckert, 1977), (ii) influences the virulence exhibited by pathogens through their colonization of host tissue (Prusky et al., 2004), and (iii) affects the toxicity of the fungicides used to control these diseases (Eckert and Eaks, 1989; Smilanick et al., 2005). The influence of pH on the activity of fungicides has been studied in the past for several fungicides, such as sodium o-phenylphenate and imazalil. Proper control of the pH of o-phenylphenate solutions during commercial application is critical in optimizing the effectiveness of the fungicide, controlling fruit residue levels, and minimizing phytotoxicity (Eckert and Eaks, 1989). Imazalil activity, too, is affected by pH; imazalil was found more effective in inhibiting P. italicum growth at pH 7.0 than at pH 5.2 or 5.3 (Siegel et al., 1977; Guan et al., 1989), and that of P. digitatum at pH 5.9 than at pH 5.1 (Daniels et al., 1985). Black spot caused by A. alternata is an economically important postharvest disease of mango and persimmon fruit worldwide, including the subtropical growing regions of Israel (Prusky et al., 1981, 1983, 2002). The primary mode of infection of mango fruit by A. alternata is by direct penetration through the lenticels during fruit growth, followed by quiescence until the fruit is harvested and ripens during long storage periods or shelf-life. In persimmon fruit, A. alternata may penetrate either through small wounds or directly into the fruit cuticle (Prusky et al., 1981). Postharvest development of alternaria rot symptoms in mango fruit during storage is usually prevented by a combination of hot water brushing (HWB) in combination with prochloraz at 225 g ml−1 ; this leads to eradication of latent infections initiated in the orchard, and consequently prevents fungal colonization (Prusky et al., 1999). Postharvest development of A. alternata in persimmons during storage is prevented by a pre-storage chlorine dip treatment (Prusky et al., 2001a). The finding that A. alternata alkalinizes the host environment pH (Eshel et al., 2002) stimulated the examination of the possibility of modulating the environmental pH by acid treatments, as a means of reducing fungal colonization.
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Our objectives in the present study were to determine the influence of external acid treatment on the pathogen and on its symptom development. We also analyzed the effects of acidic solutions on the solubility of prochloraz, and on its biological activity in the control of alternaria rot in subtropical fruit. Our findings indicate that acid treatment may, in itself, reduce symptom development by A. alternata, and it may improve the efficiency of disease control by prochloraz. Acid treatment represents a new approach to disease control and fruit-quality improvement; at the same time it improves safety by reducing the concentration of prochloraz used as a postharvest fungicide application (Prusky et al., 1999).
2. Materials and methods 2.1. Pathogen culture A. alternata was cultured for 1–2 weeks on potato dextrose agar (PDA) (Difco Laboratories, Detroit) at 25 ◦ C. The isolate used was obtained from infected mango or persimmon fruit in Israel. Spores were harvested by adding 3–4 ml of sterile, de-ionized water (diH2 O) to the Petri dish. The spores were then rubbed with a sterile glass rod to free them from the PDA medium, and the spore suspension was passed through two layers of cheese cloth. The suspension was diluted with water to obtain the spore concentrations need in each experiment, according to determination with a haemocytometer. 2.2. Spore germination and colonization assays Sterile microscope slides were used for the germination assays of A. alternata spores. Water solutions containing concentrations of HCl ranging from 0 to 2.5 mM were used as a medium. After 24 h of incubation at 24 ◦ C, the germinated and ungerminated spores in each slide were counted by observation with a (200×) microscope. Within each replicate, 100–150 spores were examined, the percentage of germinated spores was calculated and the lengths of the germinated hyphae were measured. The effect of prochloraz solutions (Sportak, 45% active ingredient, a.i.; AgrEvo UK Limited, Hauxton, England) on colonization of A. alternata was examined on potato dextrose agar (PDA) plates. The plates were seeded with 150 l of a suspension containing 106 spores ml−1 and a 13-mm diameter grade AA filter disc (Whatman International Ltd., Maidstone, England) was placed in the centre of each plate. Fifty microlitres of the appropriate concentrations of prochloraz in 50 mM HCl (Bio Lab, Jerusalem, Israel) or of tap water solutions were placed on the disc. Each treatment included six replicates, and each test was repeated three times. 2.3. pH measurements pH was measured with a flat electrode (Sensorex, Stanton, CA) following an approx. 0.2-mm deep cut with a scalpel
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blade. Mesocarp pH was determined after peeling the pericarp to a depth of 2 mm. pH measurements were taken by placing the flat electrode directly against the exposed tissue. All measurements were repeated on six fruit at three different places (18 measurements) around the equatorial axis of each fruit. The standard error (S.E.) of the mean of pH measurements was never higher than 2.5% (Yakoby et al., 2000). 2.4. Prochloraz determinations Water samples containing the fungicide were extracted five times with 50 ml of methylene chloride, and the organic extracts were pooled. The extracts were dried in a rotary evaporator at 40 ◦ C under vacuum, diluted in acetone to about 10 g ml−1 , and injected into a gas chromatograph GC-HP6890 (Agilent, Palo Alto, CA, USA) fitted with a Restek 35 capillary column (15m × 530 m × 1 m) (Restek, Bellefonte, PA, USA) and detected with a nitrogen phosphorus detector (NPD) (Agilent, USA). The concentration was determined by comparison with a standard amount of pure prochloraz diluted in acetone. 2.5. Postharvest treatments in semi-commercial mango experiments The hot water brushing (HWB) treatment at 55 ◦ C was applied with a machine similar to that described by Prusky et al. (1999). The hot water, alone or in combination with prochloraz, was applied on commercial packing lines. It was sprayed above revolving brushes at a rate of 100–120 l min−1 and at a nozzle pressure of 2 atm (Prusky et al., 1999). The fruit passed over seven transversely oriented, 12-cm diameter plastic brushes for 15–20 s. Acidic prochloraz, at several concentrations, was applied separately as a spray over two revolving brushes, following the HWB treatment, and the effects were compared with those of the commercially used HWB that used prochloraz at 225 g ml−1 . Acidic prochloraz was prepared by dissolving the liquid formulation in solutions containing various concentrations of HCl. After the fruit had been dried, they were waxed by spraying them with polyethylene emulsion wax at a soluble solids concentration of 12% (v/v), over revolving brushes (Safepack, Israel). Mango fruit of ‘Tommy Atkins’ and ‘Keitt’ cultivars were harvested, one from each of two orchards in Israel, transferred to a packing house and treated as described above. The experimental design included complete randomization, with six replications, each comprising a 350-kg bin per treatment. From each of these bins 12 fruit were sub-sampled after waxing, placed in cartons and stored at 12 ◦ C and about 90% RH for 4 weeks, before being transferred to 20 ◦ C and about 80% RH for 4 days. Control untreated fruit were stored under the same conditions, immediately upon arrival from the orchard.
2.6. Mango fruit ripening and alternaria rot development during storage The severity of infection was recorded as the percentage of the surface area of the fruit that exhibited alternaria rot symptoms. The fruit were assessed initially when they were transferred from 12 ◦ C storage to 20 ◦ C storage, and periodically until most of them were ripe. Disease severity was determined on 72 fruit (12 fruit from each of six replicates) by the percent of alternaria decayed area. Fruit were regarded as unmarketable when more than 1% of their surface area exhibited black spots. Experiments were carried out during 2 consecutive years and repeated at least four times at each year. The results of one representative experiment are presented. 2.7. Postharvest treatments in semi-commercial persimmon experiments ‘Triumph’ persimmon fruit harvested from two orchards were transferred to the packing house and dipped in a 3-m3 tank for 30 s. The experiment was conducted in a completely randomized block design in each orchard, with 10 replications, each comprising a 350-kg bin. Each block included control undipped fruit and fruit that were dip-treated for 30 s in the acidic solutions and in the commercial treatment. The commercial treatment used for persimmon fruit was Troclosene-Na (1,3-dichloro-1,3,5-triazine-2,4,6-trione; Medentech Ltd., Loch Garman, Ireland); it is a chlorine-based product supplied in tablet form by Concept, Israel. The product contains 62% latently available chlorine at 20 ◦ C and pH 5.9, and was applied at a concentration of 500 g ml−1 . A kit supplied by the manufacturer measured the exact concentration of available chlorine. Before storage, 50 fruit were sampled from each bin and placed in a separate box in the commercial storehouse. The fruit were stored under commercial conditions, subjected to forced-air cooling as described previously (Prusky et al., 2001a), and then stored at 0 ◦ C for 16 weeks pending transfer to 4 days at 20 ◦ C. The sampled fruit were the only ones evaluated following storage, in these semi-commercial scale experiments. 2.8. Alternaria rot development in persimmons during storage In the semi-commercial experiments, the severity of alternaria rot was evaluated according to the percent of fruit area covered by A. alternata symptoms when the fruit were transferred from storage at 0 to 20 ◦ C, and again 4 days later (Perez et al., 1994). Disease assessment at each stage was applied to 500 fruit (50 fruit from each of 10 replicates). Fruit were regarded as unmarketable when more than 1% of the fruit surface area exhibited black spots. Experiments were carried out during 2 consecutive years and repeated at least four times at each year. The results of one representative experiment are presented.
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2.9. Statistical analysis Data were subjected to analysis of variance by means of the Tukey–Kramer HSD test (P < 0.05).
3. Results 3.1. Effect of acidic solutions on spore germination, germ-tube elongation and colonization of A. alternata A. alternata germination and germ-tube elongation were not affected at HCl concentrations of 0.25 mM, even if the pH of the solution declined from 5.6 to 3.4 (Table 1), but spore germination and germ-tube elongation of A. alternata declined by ca. 95 and 65%, respectively, when the HCl concentration was increased to 1.25 mM and the pH of the solution declined to 2.8. At an HCl concentration of 2.50 mM and pH 2.5, germination and germ-tube elongation were inhibited by 100% (Table 1). 3.2. Effect of acid solutions on the control of alternaria rot in mango and persimmon fruit Spray treatment of naturally A. alternata-infected ‘Tommy Atkins’ mango fruit with 50 mM HCl, following HWB treatment, significantly reduced the incidence of decay after 4 weeks of storage and 4 days of shelf-life, by about 50% (Fig. 1). Lower acid concentrations of 25 mM, or higher ones of 75 mM, within a series of increasing concentrations, did not significantly affect the level of alternaria rot in stored fruit. Dip treatments of ‘Triumph’ persimmon fruit in hydrochloric acid at 50 and 75 mM, prior to 16 weeks of storage, both reduced the incidence of alternaria rot decay by 35–45% similarly to commercial dip treatments with Troclosene-Na (Table 2). Dip treatment with 50 mM hydrochloric acid reduced the peel pH from 5.64 ± 0.1 to 4.38 ± 0.23. 3.3. Effect of concentration of HCl on prochloraz solubility and A. alternata inhibition in vitro Transmittance analysis of a series of dilutions of the fungicide prochloraz in double-distilled water showed reduced transmittance as the fungicide concentration increased from Table 1 Effect of hydrochloric acid on the germination and hyphal length of A. alternata spores after 17 h at 25 ◦ C HCl concentration (mM)
pH value
Germination (%)
Length (m)
0 0.25 1.25 2.50
5.6 3.4 2.8 2.5
100.0 a 100.0 a 5.3 b 0c
13.9 a 14.1 a 4.9 b 0c
Each value represents the mean of five replicates of 100–150 spores each. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test.
Fig. 1. The effect of hot water brushing (HWB) at 55 ◦ C followed by HCl spray and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit. Mango fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and for 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruit. Acid solution pHs ranged between 1.1 and 1.6. Average values at each columns with unlike letters differ significantly according to the Tukey–Kramer HSD test (P < 0.05).
100 to 150 g ml−1 . No transmittance was detected through a 450 g ml−1 prochloraz solution (Fig. 2). However, dilution of prochloraz with increasing concentrations of HCl (1.25–50 mM,) resulted in increased transmittance through the fungicide dilutions. At HCl concentrations ranging from 12 to 50 mM, dilutions of prochloraz to 450 g ml−1 reduced transmittance by only 10% compared with 100% reduction by a water solution (Fig. 2). Determination of the content of soluble prochloraz following dilution of the commercial formulation (calculated concentration) to 250 and 450 g ml−1 active ingredient in water, followed by a 3 min centrifugation (3000 × g), detected the presence of only 10 and 32 g ml−1 , respectively, of active ingredient (Fig. 3). However, if prochloraz dilutions were prepared in acid solutions with HCl concentrations of 2.5, Table 2 The effects of HCl and Troclosene-Na dip treatments on the severity of alternaria rot on ‘Triumph’ persimmon fruit Treatment
Control HCl 50 mM HCl 75 mM Troclosene-Na 500 g ml−1
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.41 a 0.96 b 0.93 b 0.76 b
2.37 a 1.51 b 1.34 b 1.47 b
Persimmon fruit were dip-treated in the packing house 5–10 h after harvest and stored for 16 weeks at 0 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of 10 replicates, each of 50 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. Troclosene-Na at 500 g ml−1 is the currently used commercial treatment.
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Fig. 2. Effect of HCl concentration on the solubility of prochloraz, as determined by transmission at 690 nm.
12.5 or 50 mM, the amount of detectable prochloraz active ingredient in the solution increased two- to four-fold. Inhibition of A. alternata colonization in vitro by acidic prochloraz at concentrations from 22.5 to 90 g ml−1 showed also a 21–25% increase in activity compared with that of prochloraz solutions prepared in distilled water. At higher concentrations of prochloraz (225 and 450 g ml−1 ), the enhancing effect of the acid solution on the prochloraz activity was not seen (Fig. 4). 3.4. Disease-control efficiency of acidic prochloraz in mango and persimmon fruit during storage and shelf-life When the commercial HWB treatment used for mango fruit was followed by a spray of 50 mM HCl, alone or with prochloraz at 45, 225 or 900 g ml−1 , all the acid treatments reduced the incidence of alternaria rot on ‘Tommy Atkins’ fruit compared with that on the controls (Table 3). However, no improvement in disease control was found, even if the concentrations of the fungicide increased 20-fold (Table 3). In a second experiment, the commercial prochloraz treatment, i.e., inclusion in the HWB at 225 g ml−1 , also did not dif-
Fig. 4. Inhibition of A. alternata colonization by acidified and non-acidified prochloraz solutions. Acidified prochloraz was prepared in 50 mM HCl () or, in the controls (), with double-distilled water. Fifty microlitres of acidified or non-acidified prochloraz were placed on a filter disc at the centre of a PDA plate 6 h after inoculation with 150 l of a suspension of 106 A. alternata spores. Inhibition of colonization was measured 5 days later. Each value represents the mean of five replicates. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer (P < 0.05).
fer from HWB treatment followed by a spray with 50 mM HCl, alone or with prochloraz at 45 g ml−1 , in the control of alternaria rot (Table 4). A similar degree of alternaria rot control was obtained when ‘Keitt’ mango fruit were sprayed with acidic prochloraz at 22–132 g ml−1 after the HWB treatment, which suggests that increasing the fungicide concentrations in acidic solution does not improve disease control (data not shown). When alternaria rot was evaluated in persimmon fruit immediately after storage, the treatments with acid alone, acidic prochloraz, and the commercial Troclosene-Na all had similar percent of decayed area levels (Table 5). The acid prochloraz treatment of persimmon fruit was observed to be more effective than the treatment with acid alone only after 4 days of at 20 ◦ C, following storage for 16 weeks at 0 ◦ C. Table 3 The effects of hot water brushing (HWB) at 55 ◦ C followed by a spray of prochloraz in 50 mM HCl solutions and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit Treatment
Fig. 3. Effect of HCl concentration on the amount of actual active ingredient of prochloraz in acid solutions. Solutions containing several concentrations of HCl were used for dilution of the prochloraz fungicide. The calculated prochloraz concentrations obtained by dilution of the commercial fungicide formulation (calculated concentration) were centrifuged at 3000 × g and the supernatant was chemically analyzed for prochloraz concentrations by GC.
HWB HWB + 50 mM HCl HWB + 50 mM HCl + 45 g ml−1 prochloraz HWB + 50 mM HCl + 225 g ml−1 prochloraz HWB + 50 mM HCl + 900 g ml−1 prochloraz
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.28a 1.30a 1.28a
3.02 a 1.53 b 1.39 b
1.55a
1.99 b
1.23a
1.24 b
The fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruits. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test.
D. Prusky et al. / Postharvest Biology and Technology 42 (2006) 134–141 Table 4 The effects of hot water brushing (HWB) at 55 ◦ C followed by 50 mM HCl spray, with or without prochloraz, and waxing on the severity of alternaria rot on ‘Tommy Atkins’ mango fruit Treatment
Control HWB HWB + 50 mM HCl HWB + 50 mM HCl + 45 g ml−1 prochloraz HWB + 225 g ml−1 prochloraz*
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
6.51 a 3.23 b 0.60 c 1.51 bc
6.91 a 4.52 b 1.54 c 1.49 c
1.81 bc
1.87 c
The fruit were treated in the packing house 12–20 h after harvest and stored for 4 weeks at 12 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of five replicates, each of 12 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. * This combination is currently used as the commercial treatment for mango fruit.
Table 5 The effects of HCl, with or without prochloraz, and Troclonse-Na treatments on the severity of alternaria rot on ‘Triumph’ persimmon fruit Treatment
Control 50 mM HCl 50 mM HCl + 45 g ml−1 prochloraz Troclosen-Na* 500 g ml−1
Alternaria decayed area (%) End of storage
Plus 4 days at 20 ◦ C
1.32 a 0.80 b 0.70 b
3.94 a 3.04 b 2.46 c
0.68 b
2.34 c
The fruit were dip-treated in the packing house 5 h after harvest and stored for 16 weeks at 0 ◦ C (end of storage) and 4 days at 20 ◦ C. Each value represents the mean of ten replicates, each of 50 fruit. Values within columns followed by unlike letters differ significantly according to the Tukey–Kramer HSD test. * Troclosen-Na at 500 g ml−1 is the current commercial treatment applied to persimmon fruit.
4. Discussion In the complex postharvest handling of mango and persimmon fruit, where control of diseases is necessary to preserve the quality of the produce, the use of safer synthetic fungicides (Kobiler et al., 2001; Prusky et al., 2001a), biological antagonists (Janisiewicz and Korsten, 2002) and physical treatments (Prusky et al., 2001a) are integrated. Studies that revealed host environment alkalinization by ammonia secretion during A. alternata and C. gloeosporioides colonization of fruit have opened a new approach to the modulation of disease development (Prusky et al., 2001a,b, 2004). Postharvest HCl treatment of mango fruit at 50 mM and of persimmon fruit at 50 and 75 mM reduced the incidence of alternaria rot after storage and shelf-life. This result may suggest that acidic solutions may reduce the incidence of alternaria rot by pH modulation. However, acidic solutions were also very efficient in inhibiting spore germination and germ-tube elonga-
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tion, which suggests a direct toxicity effect. Acidic solutions with 1.25 mM HCl almost completely inhibited germination and germ-tube elongation of A. alternata spores in water, but on the rich PDA medium 50 mM HCl did not inhibit fungal colonization by A. alternata spores, which suggests the importance of the nutrient source in determining the direct effect of acid on the fungal response. Griffin (1994) stated that the influence of pH on fungal growth depended upon the ionization of acids or bases in the medium in which the fungus resided. The pH can alter the membrane potentials that control the permeability of fungal membranes to many substances, including the toxic compounds naturally present in the fruit. It is also possible that hydrogen and hydroxyl ion concentrations are important factors for the inhibitory or lethal activity of many compounds (Hwang and Klotz, 1938). The effect of HCl on alternaria rot is not, however, concentration dependent; when mango fruit were treated with HCl at 25 mM or at concentrations higher than 75 mM, alternaria rot incidence did not differ from that of the control. It could be that the acid treatment at 75 mM or higher was already phytotoxic to the tissue. In persimmon fruit, however, increase in HCl concentration from 50 to 75 mM did not reduce the efficiency of the treatment. This might suggest that the host susceptibility to the HCl might affect the final response to the treatment. However, the acidic pH affects not only disease development; acidification of the fungicide prochloraz with HCl improved its biological activity against A. alternata colonization in vitro as observed for Penicillium (Fuchs et al., 1993). The biological activity of prochloraz in acid solutions at concentrations in the range of 22–90 g ml−1 increased by almost 20% compared with that of non-acidified prochloraz. However, at concentrations of 225 g ml−1 and higher, no biological differences in fungal inhibition between acidic and non-acidic solutions were observed. The increase in the biological activity of prochloraz in acidic solutions was probably due to its increased solubility: the acidified prochloraz solutions, i.e., those in 2.5, 12.5 and 50 mM HCl, were found to contain two to four times as much detectable active ingredient. The increase in solubility was also confirmed by the analysis of transmittance through solutions of prochloraz prepared under neutral or acidic conditions; acidified solutions showed improved transmittance, which was considered to be an indicator of fungicide solubility. The increase in the solubility of prochloraz may be attributed to the formation of its hydrochloric salt (pKa ∼ 3.8) in HCl-containing solutions:
Lukens (1971) reported that neutral forms of fungicides penetrated membranes and were more toxic than charged
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forms. In the case of prochloraz in the present study, however, the charged form seems to have been more effective than the neutral form, as indicated by its solubility. A similar but opposite finding was reported for imazalil, which was found more toxic to P. italicum at pH 7.0 than at pH 5.0, and it was observed that less imazalil entered the mycelium at pH 5.0 than at pH 7.0. Siegel et al. (1977) ascribed this difference in potency to the charge present on the molecule, and concluded that the toxicity of imazalil was reduced at lower pH, when the molecule was charged, because the molecule was not incorporated into the mycelium. A slight difference in the pH could have a significant effect on the concentration of the neutral (dissociated) form of imazalil, which is largely responsible for the antifungal activity of that compound (Siegel et al., 1977; Siegel and Ragsdale, 1978; Guan et al., 1989). In practical experiments on the control of alternaria rot on mango and persimmon fruit, acid prochloraz did not reduce the incidence of decay more than the acidic solution alone. Since the mango and persimmon fruit used in the experiment have natural levels of quiescent infections (Prusky et al., 1981, 1983) it is clear that the 50 mM HCl was effective in modulating the environment of the germinated spores that were in a quiescent stage. Interestingly, however, increasing the concentrations of prochloraz from 45 to 900 g ml−1 in the acid solution did not result in enhanced disease control. This may be because of the improved solubility and biological activity at relatively low concentrations of prochloraz and/or because the combined effect of the acid solution and the fungicide on the pathogen resulted in improved disease control. It is possible, however, that under long-term storage conditions, as in the case of persimmon fruit that are stored for 16 weeks at 0 ◦ C, reduced concentrations of the acidified fungicide are needed to reduce the incidence of decay during shelf-life. The present results suggest that modulation of host pH by exogenic treatments provide an efficient approach to the control of postharvest pathogens that alkalinize the host fruit, such as A. alternata. An equivalent approach, using basic treatments, e.g., NaHCO3 , was applied for the control of acidifying pathogens such as P. expansum, P. italicum, and P. digitatum (Prusky et al., 2001b; Porat et al., 2002; Smilanick et al., 1999, 2005; Fallik et al., 1996) as well as for sour rot, caused by Geotrichum citri-aurantii, which cannot be controlled by commercial fungicides (Smilanick and Sorenson, 2001; Smilanick et al., 2005). However, what significantly contributes to the importance of pH-regulating treatments is their ability to reduce the amounts of residues of synthetic fungicides without affecting the efficacy of the treatment. In the present case, the efficacy of the acid treatment and its enhancement through the improved solubility of the fungicide led to improved fruit quality as expressed in reductions in decay development and in the amounts of fungicide residues in the fruit arriving in the market. Random evaluations of fungicide residues in mango before export detected 66-fold lower values in the acidic prochloraz treated fruit.
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