Integration of pre- and postharvest treatments for the control of black spot caused by Alternaria alternata in stored persimmon fruit

Postharvest Biology and Technology 59 (2011) 166–171

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Integration of pre- and postharvest treatments for the control of black spot caused by Alternaria alternata in stored persimmon fruit Ilana Kobiler, Miriam Akerman, Leah Huberman, Dov Prusky ∗ Department of Postharvest Science of Fresh Produce, the Volcani Center, Bet Dagan 50250, Israel

a r t i c l e

i n f o

Article history: Received 24 May 2010 Accepted 12 August 2010 Keywords: Disease control Quiescent infections Integrated disease control Induced resistance

a b s t r a c t In Israel, black spot caused by Alternaria alternata is the main postharvest factor that impairs the quality and reduces the storability of persimmon fruit (Diospyros kaki cv. Triumph). The fungus infects the fruit in the orchard and remains quiescent until harvest. After harvest, the pathogen slowly colonizes the fruit during storage at 0 ◦ C, which elicits black spot symptom development 2–3 months after storage entry. A commercial postharvest dip treatment in chlorine at 500 mg L−1 , released from sodium troclosene tablets, effectively controlled black spot in fruit stored for up to 2 months. However, decay incidence increased as the length of storage was extended beyond 2.5 months. The long incubation period that precedes black spot symptom development after harvest enabled the development of a series of integrative approaches for application at the pre- and postharvest stages, in combination with the commercial chlorine dip treatment, to improve the control of black spot disease. Preharvest treatments included treatment with the cytokinin-like N1 -(2-chloro-4-pyridyl)-N3 -phenylurea (CPPU) 30 d after fruit set, or a single spray with the curative fungicide polyoxin B 14 d before harvest, and when one of these was applied in combination with the postharvest chlorine dip treatment, the black spot infected area was reduced by 3 and 60%, respectively, compared with the chlorine dip alone. At the postharvest stage, fogging during storage, or post-storage on-line spraying with sodium troclosene, when applied in combination with the postharvest chlorine dip, improved the percentage of marketable fruit by 2 or 10%, respectively, compared with the chlorine dip alone. The results indicate that postharvest pathogens that show a slow colonization pattern might enable the integration of pre- and postharvest disease control methods to improve quality and reduce postharvest disease development. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Black spot, caused by Alternaria alternata, was initially described as the most economically important postharvest disease of ‘Triumph’ persimmon fruit in all growing regions of Israel (Prusky et al., 1981a, 2001). Since then it has been reported in other persimmongrowing regions and in additional local cultivars (Palou et al., 2009). The primary mode of infection of persimmon fruit by A. alternata is either through small wounds under the sepals of the fruit and/or directly into the fruit cuticle (Prusky et al., 1981a). In most years Alternaria infections remain quiescent until harvest time; the disease develops slightly during storage at 0 ◦ C and expands further during shelf-life. However, in conditions of heavy rain and/or high relative humidity before harvest, the incidence of infection increases and small “active infections” are observed in wounded tissues before harvest. Such conditions lead to significantly increased incidence of decay during storage and, consequently, fruit must be

∗ Corresponding author. E-mail address: [email protected] (D. Prusky). 0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2010.08.009

stored for shorter periods of time to prevent quality reduction that would result in significant losses for growers. Postharvest diseases might be controlled by either pre- or postharvest treatments, or by a combination of the two in cases of high incidence of infection (Janisiewicz and Korsten, 2002). Currently, in order to improve persimmon fruit quality for long-term storage, fruit are treated 2–3 weeks prior to harvest with giberellic acid (GA3 ) at 50 mg L−1 , to prevent softening (Ben Arie et al., 1986, 1996, 1997). After harvest, prior to storage at 0 ◦ C, fruit are subjected to a dip treatment in a chlorine compound, released from sodium troclosene tablets (Prusky et al., 2001). Although the chlorine treatment is effective for the control of black spot in fruit stored at 0 ◦ C for up to 2 months, decay incidence increases significantly as the storage duration is extended beyond 2.5 months. This has stimulated the investment of significant efforts by Prusky et al. (2001, 2006) in the development of new individual and integrated treatments for disease control. Preharvest treatments may affect disease development in stored products via two different mechanisms: (i) by inducing fruit resistance to fungal attack, and/or (ii) by protecting against and/or eradicating pathogenic infections. Induced resistance might result

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from activation of various resistance mechanisms, either natural or inducible, that restrict development of the postharvest pathogen in the host (Terry and Joyce, 2004). However, treatments that induce resistance are not widely used to prevent storage diseases under commercial conditions (Kessmann et al., 1994; Terry and Joyce, 2004). Fungal development can also be restricted by means of growth regulators, such as the GA3 applied to persimmon and celery, which restricts fungal attack by preventing host senescence and improving fruit quality (Afek et al., 1995; Ben Arie et al., 1996; Eshel et al., 2000). Preharvest fungicide applications are usually used if (i) postharvest treatments are not allowed under market regulations, (ii) the quality of the produce is affected by the postharvest treatment, as with grapes (Smilanick et al., 2010), or (iii) the postharvest quality of the produce could be improved by a preharvest treatment that protects against infections in the field or eradicates them there (Adaskaveg et al., 2005). Postharvest treatment forms the main approach to disease control during storage and shipment (Adaskaveg et al., 2005). The type of chemical used depends strongly on the pathogen and the time of application. Whereas fruit infected with fast-colonizing pathogens such as Penicillium rots, must be treated soon after infection with protective fungicides, or later with curative ones (Eckert and Brown, 1986), slow-colonizing ones, such as Alternaria rot, characterized by quiescent infections and slow colonization (Prusky, 1996; Prusky et al., 1981a), may be controlled by longerterm application of treatments after establishment of the infection. Alternaria rots on fruit are controlled by preharvest protectant sprays (Prusky et al., 1981b; Timmer and Zitko, 1994; Zhang and Timmer, 2007), postharvest dips, on-line fungicide sprays (Eckert and Brown, 1986; Prusky et al., 2001), and hormones, such as 2,4D, that delay host senescence and thereby reduce disease incidence (Eckert and Brown, 1986; Kobiler et al., 2001). All of the foregoing suggests that postharvest disease development can be controlled under a variety of application conditions, depending on the speed of pathogen colonization. Our objective in the present study was to demonstrate that postharvest control of the slow-colonizing pathogen A. alternata can be achieved, at the pre- and postharvest stages, through a series of integrative approaches that will further restrict its slow colonization pattern and will complement the efficacy of the commercial postharvest dip treatment with sodium troclosene. The results suggested the importance of the studies that evaluated the dynamics of fungal attack and black spot development (Prusky et al., 1981a) after fruit infection, and that serve as an efficient tool for planning integrated postharvest treatments to improve fruit quality after storage. 2. Materials and methods 2.1. Pathogen and host All the experiments were carried out in persimmon (Diospyros kaki Thunb. cv. Triumph) fruit in 10- to 15-year-old orchards in the center of Israel, the main persimmon-growing region. Fruit used in the experiments were all naturally infected with A. alternata, and the experiments were carried out with fruit from orchards whose fruit usually showed high incidence of natural black spot infection during storage. 2.2. Effects of preharvest treatments of persimmon fruit with growth retardants and fungicides, on black spot development during storage The growth retardant N1 -(2-chloro-4-pyridyl)-N3 -phenylurea (forchlorfenthuron) (CPPU, 10% a.i.; SKW, Germany), which exhibits

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cytokinin-like activity, was tested for its effect on disease development on stored persimmon fruit during storage and shelf-life. The growth regulator was applied 30 d after fruit set, when fruit weighed 20–30 g and were about 18 mm in diameter. The experiments used a randomized block design with five replications, each comprising three or four trees. Each block/treatment was distributed among a single row of trees, and within each block the treated trees were separated by one or two untreated trees. The row of trees chosen for each block of the experiment was separated from other blocks on both sides by untreated rows. The treatment was applied as an aqueous spray from a grove sprayer, at concentrations ranging from 0 to 10 mg L−1 , at about 5–6 L/tree, at a pressure of 120–150 psi (8.4–10.5 kg/cm2 ). At harvest 50 kg of fruit were sampled from the central two or three trees of each replication. The sampled fruit were stored at 0 ◦ C and about 90% RH for about 12 weeks, before being transferred to 20 ◦ C and about 80% RH for 4 d. Control untreated fruit were placed into storage under the same conditions immediately upon arrival from the orchard. Effects of various application patterns of fungicidal sprays were tested: three sprays at 4, 3, and 2 weeks before harvest; two sprays at 3 and 2 weeks before harvest; and a single application at 2 weeks before harvest. The fungicide tested was polyoxin B ([1-{5-N-(5-O-carbomonyl-2-amino-2-deoxy-lxylonyl)-5-amino-5-deoxy-␤-d-allo fur anosyl uronic acid}-5hydroxymethyluracil]) (50 WG; Kaken Pharmaceutical, Japan) at a concentration of 300–1000 mg L−1 . The experiments were arranged in a randomized block design as described above, and trees were sprayed with fungicide at 6–7 L/tree. After harvest, in some experiments, the fruit were subjected to postharvest dip treatments and stored as described above. When a postharvest treatment was applied, the fruit from the field treatment were divided between two boxes, each containing 25 kg (about 175–190 fruit).

2.3. Effects of postharvest, in-storage treatments of persimmon fruit on Alternaria rot development during storage Harvested fruit 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 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 as in the commercial treatment. The commercial treatment used for persimmon fruit was sodium troclosene (1,3-dichloro1,3,5-triazine 2,4,6-trione; Medentech Ltd, Loch Garman, Ireland), a chlorine-based product supplied in tablet form by Concept, Israel. The product contains 62% of chlorine, latently available at 20 ◦ C and pH 5.9. It was applied in an amount that released chlorine to the solution at concentrations of 500 mg L−1 . A kit supplied by the manufacturer was used to measure the exact concentration of available chlorine. The fruit were stored under commercial conditions, subjected to forced-air cooling as described previously (Prusky et al., 2001), and then stored at 0 ◦ C for about 16 weeks, pending transfer to 20 ◦ C for 4 d. Fogging treatments were applied at approximately 13 L/d of sodium troclosene, in two daily applications, at 95% RH, in commercial store rooms measuring 20 m × 20 m fitted with two Optiguide humidity controllers (Optiguide Ltd, Yokneam Illit, Israel) during the 60 d of storage. The rooms contained about 800 bins, each containing 350 kg of fruit. These semicommercial experiments had a fully randomized design, with 10 replications (bins) per treatment, and 250 fruit were sampled from each bin, i.e., a total of 2500 fruit and evaluated after storage. Fruit were regarded as unmarketable when more than 1% of their surface area exhibited black spots. The experiments were performed with fruit from three different orchards.

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2.4. Effects of postharvest, post-storage acid treatments of persimmon fruit on black spot development Persimmon bins were subjected to a dip treatment in sodium troclosene, as used commercially for persimmon fruit, and were stored for 2 months at 0 ◦ C. Bins transferred to the packing line, were unloaded into a water bath and the fruit were partially dried by brushing before being sprayed for 15–20 s with chlorine released from sodium troclosene tablets at 250 mg L−1 or with 50 mM hydrochloric acid, while passing over five transversely oriented, revolving 12-cm-diameter plastic brushes. The compounds were delivered at 100–120 L min−1 at a nozzle pressure of 2 atm and room temperature (Prusky et al., 1999). Fifteen 2.5-kg export boxes (2.5 kg × 15 boxes = 37.5 kg of fruit) containing acid-treated and control fruit from the same orchard were stored for a further 45 d at 0 ◦ C to mimic conditions of export shipment, and infection severity was evaluated and compared at the end of storage. 2.5. Persimmon fruit firmness index and black spot development during storage The severity of infection of fruit that exhibited black spot symptoms was recorded as the percentage of the surface area exhibiting black spot decay. The fruit were assessed initially when they were transferred from storage at 0 ◦ C to shelf-life at 20 ◦ C and again 4 d later (Perez et al., 1994). A fruit was regarded as unmarketable when more than 1% of its surface area exhibited black spot. Firmness was assessed by hand pressure, according to a 10-point firmness scale: firm (10) to soft (1). 2.6. Statistical analysis Experiments were carried out during six consecutive years and repeated two to four times within the 6-year period. The results of one representative experiment are presented. Data were subjected to analysis of variance by means of the Tukey–Kramer HSD Test (P < 0.05). 3. Results 3.1. Effect of preharvest growth-regulator treatment on the incidence of black spot in stored persimmon fruit Sprays of CPPU were initially applied about 30 d after fruit set, at concentrations ranging from 0.25 to 10 mg L−1 . After the first year of experiments, concentrations higher than 2.5 mg L−1 were not used, because they inhibited fruit maturation and prevented color change at harvesting and after a shelf-life period (results not shown). When CPPU was applied at 0.25 and 0.5 mg L−1 the decayed areas were 35 and 60%, respectively, less than those in the controls, but only after shelf-life. However, when CPPU was applied at 1 mg L−1 black-

Fig. 1. Effects of treatments with the growth-regulator N1 -(2-chloro-4-pyridyl)-N3 phenylurea (CPPU) after fruit set on the black-spot-covered area of persimmon cv. Triumph. Growth regulator was applied 30 d after fruit set, and fruit were evaluated after 60 d in storage at 0 ◦ C following harvest, and 4 d of shelf-life. Each value represents the mean ± SD of five replicates, each of 70 fruit.

spot-covered areas were reduced by 41 and 75% after storage and shelf-life, respectively, compared with the controls (Fig. 1). Tests of the effects of CPPU alone and in combination with the sodium troclosene postharvest dip (Table 1) showed that the black-spotcovered areas in both single and combined treatments were similar to one another, but lower than those in the control fruit. However, whereas during shelf-life fruit that had been treated with CPPU or sodium troclosene alone exhibited less black spot incidence than the control, the combined treatment was more effective than either treatment alone. Firmness of fruit treated with CPPU, sodium troclosene or their combination was slightly higher than that of untreated fruit; however, none of the treatments differed in firmness after shelf-life (Table 1). The percentage of marketable fruit at the end of storage was increased by 7–9% by the growth regulator, the chlorine treatment, or their combination. However, after shelf-life, only the combination of the growth regulator and the dip treatment resulted in improved marketability – by about 10% – compared with that of untreated fruit. 3.2. Effect of preharvest fungicide treatment on the incidence of black spot in stored persimmon fruit Polyoxin B fungicide at a concentration of 500 mg g−1 was sprayed according to three programs: three applications at 4, 3, and 2 weeks before harvest; two applications at 3 and 2 weeks before harvest; and a single application 2 weeks before harvest. The three programs achieved similar reductions in the black-spot-covered area compared with the control, i.e., 16–28% reduction (results not shown). This preliminary result, with a single application exhibiting similar efficacy to that of three applications, suggested that the single application had a curative effect, and these findings led us to focus our efforts on single applications 2 weeks before harvest. When a single spray treatment with polyoxin B was applied to naturally infected persimmon fruit the reduction of black spot area improved from 5 to 38% as the concentration of fungicide increased

Table 1 Effect of the growth-regulator N1 -(2-chloro-4-pyridyl)-N3 -phenylurea (CPPU) in combination with postharvest dip treatments with sodium troclosene on black spot area of persimmon cv. Triumph (Gaash Orchard). Growth regulator was applied 30 d after fruit set at a rate of 1 mg L−1 , and chlorine at 500 mg L−1 was released from sodium troclosene tablets during the postharvest dip. The severity of infection (infected tissue %) was recorded as the average percentage of the surface area of the fruit that exhibited black spot. An individual fruit was regarded as unmarketable when this exceeded 1% (marketable % = 100% − unmarketable %). Firmness was assessed by hand pressure, according to a 10-point firmness scale: firm (10) to soft ripe (1). Fruit were evaluated after 12 weeks in storage at 0 ◦ C (O Stor.–Out of storage) and 4 d of shelf-life (Shelf). Infected tissue (%) O Stor. Control CPPU Sodium troclosene CPPU + sodium troclosene

*

1.07a 0.78b 0.78b 0.75b

Firmness index

Marketable (%)

Shelf

O Stor.

Shelf

O Stor.

Shelf

2.57a 2.27b 1.89b 1.67c

8.7a 9.1b 9.6b 9.5b

4.33a 4.46a 5.25a 5.11a

55.0a 62.2b 63.4b 64.0b

19.2a 22.4a 24.5ab 31.5b

* Each value represents the mean of five replicates, each of 80 fruit. Values within columns marked with different letters differ significantly according to the Tukey–Kramer HSD Test at P < 0.05.

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Table 2 Effects of a preharvest spray with polyoxin B and postharvest dip treatment with sodium troclosene, on the incidence of black spot on persimmon fruit cv. Triumph (Bitzaron Orchard). Polyoxin was applied 14 d before harvest and chlorine released from sodium troclosene tablets at 500 mg mL−1 was applied after harvest. The severity of infection (infected tissue %) was recorded as the average percentage of the fruit surface area that exhibited black spot, and individual fruit were regarded as unmarketable when this exceeded 1% (marketable % = 100% − unmarketable %). Fruit were evaluated after 12 weeks of storage at 0 ◦ C. Treatment Preharvest

Infected tissue (%)

Marketable (% ± SD)

0.90a 0.86a 0.59b 0.56b 0.31c 0.21c 0.23c 0.28c

56.5 58.5 71.2 72.1 84.8 91.9 88.5 84.2

Postharvest

Control Polyoxin B 300 mg L−1 Polyoxin B 500 mg L−1 Polyoxin B 1000 mg L−1

– – – Sodium troclosenea Sodium troclosene Sodium troclosene Sodium troclosene

Polyoxin B 300 mg L−1 Polyoxin B 500 mg L−1 Polyoxin B 1000 mg L−1

± ± ± ± ± ± ± ±

6.7 4.2 6.0 6.5 2.8 2.1 1.5 2.2

Each value represents the mean of five replicates, each of 30 fruits. Values within columns marked with different letters differ significantly according to the Tukey–Kramer HSD Test at P < 0.05. a Dipping in a solution containing chlorine released from sodium troclosene tablets at 500 mg L−1 is the current commercial treatment for persimmon fruit.

from 300 to 1000 ␮g g−1 , and this decay reduction was accompanied by an increase of 16% in the percentage of marketable fruit (Table 2). However, when fruit were treated with the sodium troclosene postharvest dip, the reduction of black spot decayed area improved by 56%, and the combination of the preharvest fungicide treatment and the postharvest dip treatment further reduced the black spot by another 17%, i.e., total reductions of 68–77%. Interestingly, analysis of the percentage increase in marketable fruit yielded by either the postharvest treatment alone or the combination of the preharvest fungicide and the postharvest treatment showed an advantage of at least 12% over the best preharvest treatment. Similar results were obtained in experiments carried out in the following year, when the increase in marketable fruit as a result of the combined treatment was 42%, compared with only 16 or 12%, respectively, with the preharvest or the postharvest treatment alone (Table 3). All these findings indicate that the combined treatments exhibited greater efficacy in black spot control than either individual treatment. 3.3. Effect of postharvest in-storage fogging treatment on the incidence of Alternaria rot on stored persimmon fruit Naturally infected fruit in storage were fogged daily with sodium troclosene by means of the Optiguide device (Optiguide Ltd, Yokneam Illit, Israel), which released fogging solution that contained chlorine at 250 mg L−1 . As a result the percentage of marketable fruit among those treated with the combination of pre-

Table 3 Effects of a preharvest spray with polyoxin B and postharvest dip treatment in sodium troclosene on the incidence of black spot on persimmon fruit cv. Triumph (Naot Mordechai Orchard). Polyoxin B was applied 14 d before harvest, at 500 mg L−1 , and chlorine released from sodium troclosene tablets into a dip solution at 500 mg L−1 was applied as a postharvest dip.a The severity of infection (infected tissue %) was recorded as the average percentage of the fruit surface area that exhibited black spot symptoms, and individual fruit were regarded as unmarketable this exceeded 1% (marketable % = 100% – unmarketable %). Fruit were evaluated after 12 weeks of storage at 0 ◦ C. Treatment Preharvest

Postharvest

Control Polyoxin B – Polyoxin B

– Sodium troclosene Sodium troclosene

Infected tissue (%)

Marketable (% ± SD)

2.67A 2.17B 2.10B 1.34C

29.6 42.1 46.0 72.1

± ± ± ±

2.6 1.5 2.8 6.5

Each value represents the mean of five replicates, each of 30 fruits. Values within columns followed by different letters differ significantly according to the Tukey–Kramer HSD Test at P < 0.05. a Chlorine released from sodium troclosene tablets into dip solution at 500 mg L−1 is the current commercial treatment applied to persimmon fruit.

storage dip and in-storage fogging was greater by 2–10% than that among untreated fruit. A combined analysis of fruit from the three orchards showed that whereas the dip treatment increased the marketable fruit yield by 15.4%, the combination of pre-storage and fogging treatments increased it substantially by 20.7%, both compared with untreated fruit (Table 4). These findings indicate that the combined treatments exhibited greater efficacy in black spot control in all the experiments although it did not differ significantly from the individual treatment. 3.4. Effect of post-storage treatment on the incidence of black spot on persimmon fruit In light of the fact that persimmon fruit spend up to 45 d in transit at 0 ◦ C following the normal 2–3 months of storage at 0 ◦ C, the possibility that a post-storage treatment would prevent decay development during the former period was examined. Fruit that had been exposed to the pre-storage sodium troclosene dip were sprayed on line after storage, with either a second chlorine treatment or with 50 mM HCl, before final boxing (Table 5). Post-storage treatment with either sodium troclosene at 250 mg L−1 or 50 mM HCl further reduced the percentage of infected tissue and increased fruit marketability by 4–10 or 6–19%, respectively. 4. Discussion Successful persimmon storage is indicated by the capability to supply firm fruit for extended periods of time, without symptoms of black spot development. No postharvest treatment, apart from the chlorine dip, is used to control disease and to improve persimmon quality (Prusky et al., 2001). Ozone was reported to significantly reduce decay incidence in peaches, table grapes and citrus (Palou et al., 2002), but when it was applied to persimmons at 0.15 ␮L L−1 at 15 ◦ C, the fruit showed flesh softening, which suggests that this treatment cannot be used to preserve fruit firmness during long-term storage (Salvador et al., 2006). In light of the lack of effective single treatments the present study examined combination of other treatments with the commonly used postharvest sodium troclosene dip treatment, in developing new integrated treatment strategies. Application of the growth-regulator CPPU at 0.5–1 mg L−1 30 d after fruit set reduced the incidence of decay development during storage (Table 1) without affecting fruit maturity or the color change from green to orange that occurs close to harvest (Table 1 and Fig. 1). The inhibition of fruit maturing by CPPU at concentrations higher than 2.5 mg L−1 indicates that this early treatment probably affected the fruit physiologically. In mango fruit a combination of CPPU at 10 mg L−1 and GA3 at 100 mg L−1 , applied from the early stage of growth, promoted fruit enlargement and

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Table 4 Effect of sodium troclosene fogging on the incidence of black spot during storage of persimmon fruit cv. Triumph from three different orchards. Sodium troclosene dip-treated fruit (i.e., with chlorine at 500 mg L−1 ) and untreated fruit were fogged during storage by using the Optiguide fogger as described in Section 2. Ten bins of fruit, half dip treated and half untreated, were placed in each of two different store rooms at 0 ◦ C (total of 40 bins per orchard). Fruit from only one room were fogged (F) with chlorine at 250 mg L−1 released from sodium troclosene tablets, twice daily during 2 months of storage, for comparison with those fogged only with water (N). Fruit were regarded as unmarketable when more than 1% of their surface area exhibited black spots. Fruit were evaluated after 12 weeks of storage at 0 ◦ C. Fruit were commercially evaluated for export standard after storage for 10 weeks at 0 ◦ C. Values within columns marked with different letters differ significantly according to the Tukey–Kramer HSD Test at P < 0.05. Fogging treatments (marketable fruits, %) Mor-Ha Sharon

Pre-storage treatment Control Sodium troclosene

Bet Oren

Yakum

Average

N

F

N

F

N

F

N

F

21a 46b

27a 56b

31a 44b

29a 48b

36a 44b

31a 46b

29.3A 44.7B

29.0A 50.0B

Each value represents the mean of a total of 2500 fruits (250 fruit sampled from each of 10 bins, each containing 350 kg of fruit). N – not fogged, F – fogged.

showed potential to increase the commercial value of mango fruit (Sasaki and Utsunomiya, 2002). Fruit growth was also stimulated by the application of CPPU to kiwifruit about 3 weeks after anthesis (Iwahori et al., 1988), by treatments of grape clusters (Zabadal and Bukovac, 2006), and during early development of apples (Percy et al., 2004). In persimmon fruit cv. Matsumoto-Wase-Fuyu, Sugiyama and Yamaki (1995) reported that treatment with CPPU at 5–10 mg L−1 delayed color development during fruit maturation, similarly to our present findings with ‘Triumph’ fruit at concentrations higher than 2.5 mg L−1 . In our experiments, however, CPPU treatment at 1 mg L−1 did not delay fruit maturation, but reduced decay incidence and delayed post-storage fruit softening (Table 1). This result is important because persimmon fruit quality is judged not only by size, but also by firmness and by Alternaria black spot development in stored fruit. The action mechanism of growth retardants in persimmons might be related to their effect on cell-wall growth: GA3 treatment increased the whole cellulose cell-wall content, thereby improving fruit firmness after storage (Ben Arie et al., 1986, 1996). This effect also inhibited A. alternata production of endo-1,4-␤-glucanase by 49% (Eshel et al., 2002); this is an enzyme that contributes to cell-wall maceration in stored persimmons, and thereby promotes decay during fungal colonization of Alternaria infections. Whether CPPU might have a similar effect on fungal growth is not known; however, findings that CPPU increased fruit size (Prusky and Kobiler, unpublished data), and reports of increased yields of cell-wall fractions in enhanced-size apples (Percy et al., 2004) suggest that CPPU treatment might have a similar effect in persimmon fruit. Preharvest treatments of Bartlett and Bosc pear orchards with protective fungicides were used in several cases to control postharvest rots caused by wound pathogens such as Penicillium and Botrytis (Adaskaveg et al., 2005), which is analogous to the present finding that a single preharvest treatment with polyoxin B was effective in controlling postharvest diseases in persimmon fruit. Disease control by a single preharvest application of fungicides sug-

gests that the compound exhibits a curative activity and possibly penetrates into outer cell layers. This may also indicate existence of an “easy-to-reach” location of the infective structures – possibly on open wounds – close to the main period of wound-crack development, i.e., 2–3 weeks before harvest (Prusky et al., 1981a). Under these conditions a single treatment with polyoxin B, a watersoluble, readily absorbed and translocated fungicide that is also persistent, applied 2 weeks before harvest, becomes as efficient as three applications 4, 3, and 2 weeks before harvest (Adaskaveg et al., 2005; Zhang and Timmer, 2007). However, the present finding that the simple surface sanitizer sodium troclosene controlled black spot efficiently suggests that the A. alternata spores occupy rather superficial locations; this finding also further supports the hypothesis that most Alternaria infections enter through open wounds and do not colonize the host tissue immediately. The superficial nature of these A. alternata infections of wounds, together with the stringent storage conditions at 0 ◦ C, delay fungal colonization and raise the possibility that other disease control measures, such as fogging during storage would be effective. In the present study application of sodium troclosene by fogging during storage at 0 ◦ C increased the average percentage of export-quality fruits by 6% compared with that of fruit treated only by dipping. However, the most striking disease control effect was observed when persimmon fruit were treated, 2 months after storage entry, by on-line spraying under pre-shipping conditions in the packing house; this treatment increased the percentage of marketable fruit, which further confirmed the slow rate of colonization by A. alternata during storage. The tested control means were effective in inhibiting fungal growth at different, early stages of development. Both CPPU and polyoxin B inhibited fungal colonization by germinated spores, CPPU probably achieved control by affecting the host cell-wall composition, whereas the fungicide polyoxin B or other fungicides, such as difenoconazole that showed similar control results (Score; Syngenta, Basel, Switzerland) (Prusky, personal communication), did so by direct inhibition of germinated spores. Furthermore, the

Table 5 Effects of post-storage treatments on the incidence of black spot in stored fruit cv. Triumph from two different orchards (Tel Nof and Mesuot Itzchak). Dip in solution containing chlorine at 500 mg L−1 released from sodium troclosene tablets was applied as a pre-storage treatment to all fruit in the experiments.a Fruit were exposed to post-storage treatment with chlorine at 250 mg L−1 or with 50 mM HCl. Fruit were evaluated after 45 d of storage at 0 ◦ C. Severity of infection (infected tissue %) was recorded as the average percentage of the fruit surface area that exhibited black spots, and individual fruit were regarded as unmarketable when this exceeded 1% (marketable % = 100% − unmarketable %). Values within columns marked with different letters differ significantly according to the Tukey–Kramer HSD Test at P < 0.05. Tel Nof

Post-storage treatment Control Sodium troclosenea Hydrochloric acid

Mesuot Itzchak

Infected tissue (%)

Marketable (% ± SD)

Infected tissue (%)

Marketable (% ± SD)

0.61A 0.43B 0.29C

88.5 ± 0.8 92.6 ± 1.5 94.8 ± 1.7

1.08A 0.79B 0.43C

72.3 ± 3.2 81.6 ± 2.6 91.6 ± 1.0

Each value represents the mean of five replicates, each of 70 fruit. a Dip in solution containing chlorine at 500 mg L−1 released from sodium troclosene tablets is the current commercial treatment applied to persimmon fruit.

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possibility of controlling black spot by either fogging during storage or spraying at the late post-storage stage is also indicative of the slow colonization rate of A. alternata in persimmons. In all of these cases, integration of the postharvest dip treatment with any of the other treatments reduced the percentage infected area and improved marketability of the fruit by more than 4%. The efficiency of the combined treatments might reflect the strong temperature dependence of Alternaria colonization: 25 d after infection, at 0 ◦ C the fungus colony measured 1.3 mm in diameter, compared with 11.2 mm at 20 ◦ C (Prusky et al., 1981a). Similar effects were observed in our present study (Table 1) where the colonized area increased by 100% after persimmons were transferred from 0 to 20 ◦ C during shelf-life. Another factor that could affect the efficacy of these integrated disease control treatments is the fungal penetration route. Alternaria spores germinated on the fruit surface, directly penetrated the fruit cuticle, developed between the host cells and produced thin, dark, quiescent infections which renewed their development during fruit ripening. During fruit growth in the orchard, the number of incident latent infections increased from 5 to 17 per fruit after 170 d (Prusky et al., 1981a). This seems to be negligible compared with the infected area concentrated in the wounded tissue beneath the sepals of the fruit, which easily might be 10–15% of the whole fruit surface, which suggested that wounds actually are the main penetration route (Prusky et al., 1981a). This hypothesis was confirmed by analysis of symptom development in infected wound tissue, which showed that at 0 ◦ C colonization was 6.5 times faster in wounded tissue than in unwounded tissue (Prusky et al., 1981a). Furthermore, the presence of colonizing Alternaria in wounded tissue renders them more accessible to the various postharvest treatments and enables these treatments to be used at different periods after infection. The efficacy of post-storage acid treatments in preventing disease development is also interesting. Previous studies revealed the environmental alkalinization by ammonia secretion during A. alternata colonization (Eshel et al., 2002), and suggested that it might form the basis of a new approach to inhibition of disease development (Prusky et al., 2006). The finding that acidic solutions with 1.25 mM HCl almost completely inhibited germination and germtube elongation of A. alternata spores (Prusky et al., 2006) may indicate that germinated spores are still at an early stage of development even after 2 months of storage at 0 ◦ C. The present results suggest that integration of treatments at several different stages after infection could restrict black spot development in persimmon fruit. This possibility rests on the slow colonization rate of Alternaria during most of the life of the fruit and during storage, which enabled the development of combined treatments for reduction of postharvest diseases. Acknowledgments We thank Y. Kaplan, Ruli Cohen and Eran Rotem, of the Mor HaSharon Packing House, for enabling us to perform experiments in their packing house. We also thank Dr. Leo Winner and Timna Zoar for help in various experiments, and Dr. Yoram Fuchs for reviewing the manuscript. This work was financially supported by the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development and the Israel Persimmon Growers’ Association. References Adaskaveg, J.E., Förster, H., Gubler, W.D., Teviotdale, B.L., Thompson, D.F., 2005. Reduced-risk fungicides help manage brown rot and other fungal diseases of stone fruit. Calif. Agric. 59, 109–114.

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