Effect of harvest date and 1-MCP (SmartFresh™) treatment on ‘Golden Delicious’ apple cold storage physiological disorders

Postharvest Biology and Technology 110 (2015) 77–85

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Effect of harvest date and 1-MCP (SmartFreshTM) treatment on ‘Golden Delicious’ apple cold storage physiological disorders Custódia M.L. Gagoa , Adriana C. Guerreiroa , Graça Miguela , Thomas Panagopoulosb , Claudia Sánchezc , Maria D.C. Antunesa,* a b c

Faculdade de Ciências e Tecnologia, Universidade do Algarve, MeditBio, Ed. 8, Campus de Gambelas, 8005-139 Faro, Portugal Faculdade de Ciências e Tecnologia, Universidade do Algarve, CIEO, Ed. 8, Campus de Gambelas, 8005-139 Faro, Portugal Instituto Nacional de Investigação Agrária e Veterinária, INIAV, IP. Quinta do Marquês, 2784-505 Oeiras, Portugal

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 November 2014 Received in revised form 15 July 2015 Accepted 17 July 2015 Available online xxx

It is recognized that 1-methylcyclopropene (1-MCP) is able to influence fruit ripening, reduce superficial scald and improve post-storage quality in apples. However, 1-MCP may also increase disorders as bitter pit and diffuse skin browning. The objective of this work was to investigate the effect of 1-MCP (625 nL L 1) and 3 different maturity stages (early, middle and late harvest date) on the incidence and development of physiological disorders and ripening delay during storage at 0.5  C and subsequent shelflife at room temperature  22  C, in ‘Golden Delicious’ apples. Fruit were collected in 6 orchards at a weekly interval, and half were treated with 1-MCP after 3 d cold storage, and the other half left as control. Quality parameters were evaluated at harvest, after 1-MCP treatment and after 6 months storage as well as after 7 d shelf-life. 1-MCP treatment of ‘Golden Delicious’ was effective for slowing softening, reduce electrolyte leakage and color changes associated with ripening (lightness and hue parameters), did not affect soluble solids content and had no clear effect on fruit phenols and antioxidant activity. 1-MCP inhibited superficial scald and significantly reduced rot; however, this treatment enhanced the development of bitter pit in some orchards. Harvest date did not influence scald, bitter pit and firmness but decreased weight loss, total phenols, soluble solid content and antioxidant activity from 2nd to 3rd harvest. The application of 1-MCP, 3 d after cold storage, to ‘Golden Delicious’ apples, reduced ripening and superficial scald, did not induce diffuse skin browning, but increased bitter pit incidence. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Bitter pit Superficial scald Shelf life Diffuse skin browning Ripening

1. Introduction It is widely recognized that 1-methylcyclopropene (1-MCP) is able to influence fruit ripening and improve post-storage quality in most climacteric fruits (Blankenship and Dole, 2003; Watkins, 2008). 1-MCP blocks ethylene receptors and prevents ethylene effects in plant tissues for extended periods (Sisler and Serek, 1997). The beneficial effects of 1-MCP on respiration and ethylene production inhibition, delay of fruit ripening, and alleviation of certain ethylene-induced postharvest physiological disorders have been well recognized. However, studies focused on the effects of 1-MCP on phenolic and antioxidant activity presented inconsistent results (MacLean et al., 2003, 2006; Fawbush et al., 2009; Hoang et al., 2011). 1-MCP treatments improve the storage life of apples and inhibit or delay the development of some disorders such as superficial scald (Watkins, 2008). ‘Golden Delicious’ clones are

* corresponding author:. E-mail address: [email protected] (M.D.C. Antunes). http://dx.doi.org/10.1016/j.postharvbio.2015.07.018 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.

relatively resistant to superficial scald and 1-MCP treatment is mainly used to reduce softening rate of the fruit and thus improve its storability. However, 1-MCP treatment may also cause disorders such as bitter pit (Calvo and Candan, 2010) and diffuse skin browning (DSB), especially in ‘Golden Delicious’ apple cultivars (Larrigaudière et al., 2008, 2010). According to Larrigaudière et al. (2010) DSB arose only in a few countries, especially those that have very hot summers and little rainfall. This disorder affects only the fruit skin and is manifest as diffuse browning and roughening of the skin. The lenticels are not affected and the skin is not depressed. This disorder is prevented by placing the fruit, just after harvest, at around 6  C and then slowly decreasing the temperature after 1-MCP treatment. The same authors suggested that DSB and superficial scald are two different disorders involving different oxidative processes. Bitter pit is characterized by brown corky spots of dead tissue confined to areas just under the skin. Susceptibility of fruit to bitter pit has three components—genetic, climatic and orchard management. Even within susceptible varieties, seasonal differences are

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common, with hot dry summers much more associated with higher disorder incidence than cooler summers (Watkins, 2009). Bitter pit is a physiological disorder of apples (Malus domestica Borkh) that has been related to calcium deficiency in the fruit (Ferguson and Watkins, 1989). Few studies about the effects of physiological ripening stage at the time of 1-MCP treatment within a single cultivar are available (Watkins et al., 2000; Mir et al., 2001; Watkins and Nock, 2005; Calvo and Candan, 2010), although Mir et al. (2001) found that its effectiveness declined slightly with advancing harvest date of ‘Redchief Delicious’ apple. Moreover, no studies were found reporting the effect of ripening stage when 1-MCP was applied on the development of physiological disorders as DSB or bitter pit in ‘Golden Delicious’ apple cultivars. Consequently, the main goal of this work was to investigate the effect of SmartFreshTM (625 nL L 1 1-MCP) on the incidence and development of physiological disorders and ripening during cold storage (at 0.5  C in normal atmosphere) and subsequent shelf-life at room temperature 22  C in ‘Golden Delicious’ apples harvested at different maturity stages. 2. Material and methods 2.1. Orchards Six ‘Golden Delicious’ apple orchards were selected in the Centre-West Region of Portugal, all being managed under commercial conditions. Thirty days before the first harvest date, data loggers were placed in the orchards for temperature measurements. The maximum temperatures were between 25.3 and 36.4  C and the minimum between 8.8 and 18.4  C. 2.2. Fruit harvest, postharvest treatments and sampling dates ‘Golden Delicious’ apples (M. domestica Borkh.) were harvested from six selected orchards in three consecutive weeks from the same randomly selected trees, with a week interval, respectively at 5, 12 and 19 September (1st, 2nd and 3rd harvests). The 1st harvest corresponds to the normal harvest date by producers in the region. Apples were harvested in the morning and were transported to the University of Algarve, in the afternoon. Immediately after arrival at University of Algarve (on harvest day) fruit were stored in a cold room at 0.5  C. After 3 d of storage, half of the fruit (4 crates with 70 fruit each), from each orchard, were treated with 625 nL L 1 1-MCP by using SmartFreshTM (Agrofresh Inc., Rohm and Haas, Spring House, PA, USA) according to the manufacturer’s recommendations. The 1MCP treatment was done at 0.5  C and overnight for 20 h. Then, fruit were stored at 0.5  C in normal atmosphere and relative humidity 90–95% for 6 months (180 d). The sampling dates were the day following each harvest, the day after SmartFreshTM (625 nL L 1 1-MCP) treatment and after 6 months storage. On the last two dates, fruit were evaluated also after an additional 7 d of shelf-life at room temperature (22  C). At each sampling date, quality evaluation parameters such as color (CIEL  Hue and Chroma), DSB, superficial scald, internal browning, bitter pit, firmness and soluble solids content were measured. Then, peel segment samples from fruit of each treatment and replication were collected and immediately frozen in liquid N2 and stored at 80  C until the respective assay. 2.3. Ethylene measurements Three ‘Golden Delicious’ apples from each orchard and each harvest date were analyzed immediately after 1-MCP treatment

and again after 7 d shelf-life of room temperature storage (22  C). The same measurements were made after 6 months cold storage. The fruit were placed in 1 l bottles (1 or 2 fruit/bottle) and sealed. After 1 h at room temperature, headspace gas samples were withdrawn and injected (1 mL) into a gas chromatograph (PYE UNICAM 204) equipped with a flame ionization detector (FID) and a Porapack Q column (4.0 mm internal diameter, 1.5 m length). The carrier gas was nitrogen at a flow rate of 0.5 mL s 1 and the injector, detector and column temperatures were 25  C, 150  C and 100  C, respectively. Ethylene concentrations (ECs) in the headspaces were expressed as ng kg 1 s 1. 2.4. Assessment of starch index, diffuse skin browning, superficial scald and bitter pit Starch index was assessed on 15 fruit from each orchard, on the day following each harvest time. Iodine–starch index (1–10 scale) was evaluated by dipping an equatorial slice of the fruit into Lugol’s iodine solution (15 g KI + 6 g I2 + 1 L H2O) for 1 min and comparing visually the color with starch conversion chart for apples (Ctifl, 2002). DSB, superficial scald and bitter pit were visually evaluated on 100 fruit per orchard, harvest date, and treatment, after 6 months storage and consecutive shelf-life. The incidence of each disorder was calculated as percentage of the total number of fruit. 2.5. Skin color, flesh firmness, soluble solids content, electrolyte leakage and weight loss A total of 20 fruit per orchard and treatment were evaluated per sampling date (total 120 fruit per treatment). Skin color was measured in the CIE L* a* b* color space with a CR-300 colorimeter (CE Minolta, Japan) with a D65 light source and the observer at 10 . All measurements were performed on the widest part of the fruit, as a mean of 3 points. The a* and b* readings were converted to the vectorial coordinates hue angle (h ) and chroma (C*) using the equations h = arc tan b*/a* and C* = (a*2 + b*2)1/2, respectively (Mcguire, 1992). Flesh firmness was measured on two opposite sides of each fruit, after peel removal, with a Chatillon Force TCD 200 and Digital Force Gauge DFIS 50 (Jonh Chatillon & Sons, Inc., U.S.A.), fitted with a convex probe of 11 mm diameter mounted in a standard drill press. Results are presented as the mean maximum force required to push the probe 11 mm into the fruit flesh. Soluble solids content (SSC) was measured in juice of each fruit using a digital refractometer (Model PR-100, Atago Co., Tokyo, Japan). Electrolyte leakage was assessed as described by Antunes and Sfakiotakis (2008) with modifications. From each orchard, each treatment and sampling date were taken 2 samples, composed by 5 peel discs with 18 mm diameter, from 5 different fruits. Peel discs were washed with deionized water to eliminate the electrolyte leakage at the cut surface and placed in 20 mL of deionized water. After incubation at 22–25  C for 6 h, the conductivity of the suspension was measured with an Orion 011007 conductivity meter (Thermo Scientific Orion StarTM, Beverly, USA). The suspension was then placed for 20 min at 120  C into an autoclave and allowed to cool till room temperature, then conductivity was measured again and taken as total leakage. The percentage of electrolyte leakage was taken as the ratio (100) of the conductivity measurements before and after boiling. Weight loss was calculated by weighing always the same sample of fruit (two samples with 20 fruit from each orchard, harvest date and treatment), and expressed as percentage of the initial weight.

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2.6. Extraction and assay of total phenols (Folin–Ciocalteau) and antioxidant activity The total phenols of ‘Golden Delicious’ apples were extracted from frozen peel segments. From every orchard for each treatment and in each sampling date were taken 3 samples. Extraction was done in each sample (1 g) using the procedure described by Coseteng and Lee (1987), with modifications. Briefly, the tissues were extracted twice with 80% ethanol (ethanol: water 80:20, v/v) using a water-bath at 100  C (10 + 5 min). After being centrifuged at 2370  g for 5 min, samples were adjusted to 10 mL with 80% ethanol and kept at 20  C until use. The total phenolic content was determined using the Folin– Ciocalteau reagent and Gallic acid as standard as described by Slinkard and Singleton (1977). Each fruit extract (0.2 mL) or gallic acid concentration was mixed with 0.8 mL of an aqueous sodium carbonate solution (75 g L 1) and were added 1 mL of 10% (v/v) Folin–Ciocalteau reagent. After 30 min of reaction at room temperature, the absorbance was measured at 765 nm in a Shimadzu, UV–vis recording spectrophotometer model UV-160A (Shimadzu Corporation, Kyoto, Japan). In the same fruit extracts was determined the Trolox equivalent antioxidant capacity (TEAC), an improved assay for antioxidant capacity measurement based on the discoloration of the monocation radical 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic  acid) (ABTS +) (Re et al., 1999). Trolox was used as an antioxidant standard and the activity was expressed in terms of molar content of Trolox per mass of fruit peel, mmol kg 1. 2.7. Mineral analyses After 6 months of cold storage, samples of skin and pulp were collected from each fruit used for evaluation of quality parameters (from the six orchards and only from the first harvest date) and dried at 60  C for 48 h. Then fruit material was ground, incinerated at 450  C, and digested in 10 mL of 1 M HCl. Nutrient concentrations were determined according to standard procedures (AOAC, 1990). Potassium was measured by flame photometry (Jenway PFP 7) and Mg and Ca by atomic absorption spectrometry at 422.7 nm and 285.3 nm, respectively. 2.8. Statistical analysis Data were subjected to one-way analyses of variance (ANOVA) using orchard as a replicate and the postharvest treatment as a fixed factor with two levels: control and 1-MCP treatment. Pearson correlations were used to quantify the relationships among factors assessed. The statistical package SPSS version 20 (IBM, Inc.) was used for statistical analyses. Principal component analysis (PCA) was performed using the free statistical software Chemoface version 1.5 (Nunes et al., 2012). 3. Results 3.1. Effect of harvest date on maturity indices, phenols content, antioxidant activity and electrolyte leakage As expected, the starch index increased from the first to the third harvest, whereas firmness, phenols content, antioxidant activity and electrolyte leakage decreased during the same time period (Table 1). The soluble solids (%) and color parameters did not change significantly among the three harvest dates.

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Table 1 Effect of harvest date on maturity indices, phenols content, antioxidant activity and electrolyte leakage (EL). Parameters

1st harvest

2nd harvest

3rd harvest

Starch index Soluble solids content (%) L* Chroma Hue Firmness (N) Total phenolsa Antioxidant activityb Electrolyte leakage (%)

4.99  0.199b 14.58  0.056a 74.69  0.185b 42.91  0.376a 108.08  0.562a 70.32  0.488a 11.46  0.376a 10.11  0.572a 58.85  2.400a

6.57  0.239ab 14.71  0.249a 75.79  0.212a 43.15  0.409a 108.39  0.433a 65.89  0.633b 9.87  0.364b 10.91  0.384a 57.48  0.599a

7.87  0.155a 14.37  0.042a 75.48  0.223ab 43.45  0.324a 109.51  0.464a 64.5v0.570b 8.52  0.311c 8.10  0.3.53b 54.42  0.735b

a

Gallic acid equivalents per mass of fresh peel, g kg 1. Trolox equivalents per mass of fresh peel, mmol kg 1; The values followed by the same letter in the same row are not significantly different by Duncan’s multiple range test, at P < 0.05. b

3.2. Ethylene Ethylene measurements were carried out to confirm the effect of 1-MCP on inhibiting ethylene production (Table 2). Fruit treated with 1-MCP showed an extremely low ethylene production after application of postharvest treatments, and remained low during 7 d of shelf-life. However, control fruit had initially a low ethylene production which greatly increased during shelf-life (7 d). These results confirm that the 1-MCP treatment was well applied and efficiently inhibited ethylene production, independently on the harvest date. After 6 months cold storage and in the following shelf-life the apples treated with 1-MCP showed low values of ethylene production, without differences among harvest dates. At the same time, untreated fruit showed high values of ethylene production, which were increasing from the first to the third harvest date. 3.3. Ripening behavior through storage and shelf-life 3.3.1. Color, soluble solids content, firmness and weight loss Lightness increased and  hue decreased during the 6 months of storage but L* remained constant during the subsequent shelf-life, while  hue decreased in all treatments (data not shown). The time of harvest did not influence the L*, while, after storage, 1-MCP treated fruit from the 3rd harvest showed higher values of  hue than fruit from the 1st and 2nd harvest dates. SSC had similar values, at harvest, for the 3 harvest dates (Fig. 1A). However, it increased during shelf-life and storage, except in the 3rd harvest where no significant changes were observed, for both treatments. Firmness decreased significantly for all treatments after 6 months of storage. As expected, 1-MCP significantly reduced softening for all harvest dates either at harvest or after storage as compared to controls (Fig. 1B). This behavior was alike for the 3 harvest dates. The firmness of the control at harvest decreased significantly from the first to the second harvest, remaining constant thereafter. For 1-MCP treated fruit the decrease was significant only from the 2nd to the 3rd harvest. However, there was no significant effect of harvest time in firmness after 6 months storage in both treatments (Fig. 1B). Weight loss increased significantly during storage similarly for control and 1-MCP treatments, except in the 3rd harvest where 1MCP showed lower values (Fig. 1C). After 7 d shelf-life, weight loss continued to increase except for the 1st harvest date in which 1MCP treated fruit, weight loss was not significant after cold storage. In shelf-life just after harvest, no treatments showed differences in weight loss among the harvest dates (Fig 1C). However, 1-MCP treated fruit showed after storage, lower weight loss in the 3rd harvest than in fruit from the first 2 harvests.

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Table 2 Effect of 1-MCP postharvest treatment (625 nL L 1) in ethylene production of ‘Golden Delicious’ apples from the 3 harvest dates, before storage- and after 6 months of cold storage. Measurements were made after postharvest treatments application or cold storage (0 d) and plus one week shelf life (7 d). Harvest

Postharvest

Ethylene production (ng kg

Date

Treatment

Before storage

1

s

1

) After storage

0d

7d

0d

7d

1st

Control 1-MCP

0.242  0.05 0.057  0.02

bcC cB

23.573  4.46 0.024  0.01

aA cB

16.058  0.864 3.360  0.556

bB cA

23.588  1.28 4.696  0.70

cA dA

2nd

Control 1-MCP

1.937  0.73 0.160  0.01

aD bcC

24.568  3.10 0.020  0.00

aB cC

18.263  0.71 1.483  0.12

bC cB

28.739  1.31 3.612  0.31

bA dA

3rd

Control 1-MCP

0.721  0.23 0.152  0.02

bC bcBC

14.960  4.57 0.030  0.01

bB cC

35.788  2.82 0.742  0.14

aA cB

40.755  1.91 3.536  0.54

aA dA

The values followed by the same lower case letter in the same column and by the same upper letter in the same row are not significantly different by Duncan’s multiple range test, at P < 0.05.

3.3.2. Electrolyte leakage, phenol content and antioxidant activity During cold storage, electrolyte leakage increased for all treatments (Fig. 2A). The increase was higher in untreated than 1-MCP treated fruit. Interestingly, electrolyte leakage was maintained through shelf life for all treatments, except at the beginning of the experiment in control fruit of the 1st and 2nd harvest, in which it decreased. The 1-MCP treated fruit had lower electrolyte leakage than control fruit after storage in all three harvests, with the lowest occurring for the first harvest (Fig. 2A). In the 1st harvest date, phenols behaved similarly in both 1MCP treated fruit and control, showing a significant increase in cold storage (Fig. 2B). However, in 2nd and 3rd harvest dates 1MCP treated fruit had significantly higher phenolic than control just after treatment, which decreased significantly during storage to values close to the ones of control. After shelf-life before storage, control fruit from the 1st harvest had increased phenol content, while in 2nd and 3rd harvest dates phenolic values remained constant. Apples treated with 1-MCP maintained phenolic content throughout shelf-life when harvested at the 1st or 3rd dates, but decreased when harvested at the 2nd harvest date. It is visibly noticeable, with the exception of 1st harvest, that phenolic content is higher in 1-MCP treated fruit than in control. Antioxidant activity of control fruit increased during storage for fruit from the 1st harvest, decreased for the 2nd harvest and remained similar for the 3rd harvest (Fig. 2C). 1-MCP treated fruit maintained similar antioxidant activity during cold storage for all harvest dates. The antioxidant activity of control fruit after shelflife and before storage increased for the 1st harvest time and maintained similar levels for the 2nd and 3rd harvests. For 1-MCP treated fruit, 1st and 2nd harvest did not exhibit any change in antioxidant activity, while in 3rd harvest it decreased. During shelf-life after storage, both treatments decreased antioxidant activity for the 3rd harvest time, but for the 1st and 2nd harvest dates antioxidant activity values remained similar for 1-MCP treated fruit and increased for control fruit. Generally there was a decrease in antioxidant activity with harvest date; this decrease being attenuated by 1-MCP treatment. 3.4. Physiological disorders and rot DSB is a disorder reported by Larrigaudière et al. (2010) that only affects fruit in regions with very hot summers and little rainfall. Usually, the risk of DSB is present when in the 30 d before harvesting orchards had 15 d with minimum temperatures above 15  C. According to temperature data obtained in the orchards of the experiment reported here, the DSB risk was present in all of them for the three harvest dates, with days of minimum temperature above 15  C ranging from 12 to 18 d (data not

shown). However, no fruit were found with DSB symptoms for any orchard or treatment. No symptoms of superficial scald were observed in 1-MCP treated fruit, at the end of cold storage plus 7 d shelf-life (Fig. 3A). In control fruit the symptoms of scald appeared only during shelflife after storage and, as an average of all orchards, the percentage of fruit affected increased from the 1st to the 3rd harvest date. Also, producer 6 showed the highest values for scald symptoms followed by producer 4 but this last only in the third harvest (Fig. 3A). Superficial scald was correlated negatively with K and positively with Ca/K contents in apple flesh (P < 0.01), as well as negatively with Mg content in apple peel (P < 0.05) (Table 3). As a mean of all orchards, the bitter pit percentage was higher in 1-MCP than in control fruit (Fig. 3B). Also there were no significant differences in bitter pit incidence among harvest dates. However, there were great differences among orchards reinforcing the importance of orchard management in bitter pit development, mainly nutrition. Orchard 4 had the lowest percentage of bitter pit fruit followed by orchard 5 and 1 (Fig. 3B). Orchards 2, 3 and 6 had the higher values being the 1-MCP treatment which clearly more affected the bitter pit fruit percentage. The symptoms of bitter pit have been related to the concentrations of Ca, K, Mg and some ratios of these nutrients in apple peel. In this work, negative correlations were found between bitter pit percentage and Ca content (P < 0.01) or the ratio Ca/Mg (P < 0.01) in ‘Golden Delicious’ apple peel, while for flesh there was only a positive correlation between Mg and BP (P < 0.05) (Table 3). Fruit rot after storage plus 7 d shelf-life was lower in 1-MCP treated fruit than in control as an average of all orchards (Fig. 3C). For all orchards 1-MCP treated fruit showed almost no rotten fruit. Orchards 5 and 4 showed the lowest% of rot, while orchard 3 had a very high% of rotten fruit in control with the values decreasing from the 1st to 2nd harvest (Fig. 3C). Rot was not significantly correlated with any of the nutrients (Ca, Mg, K) either in the flesh or peel (Table 3). 3.5. Principal component analysis (PCA) The data processed by PCA showed that the two principal components explained 86.98% of the total variation. The component loadings demonstrate how the original variables were related to each other (Fig. 4). The first and second components explained, respectively 55.30% and 31.68% of total variation. The PCA analysis defined clearly two groups: control group and 1-MCP treated group, which are discriminated mainly by the variables firmness, electrolyte leakage, lightness, bitter pit, superficial scald and hue parameter (Fig. 4). Within the two

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Fig. 1. Effect of harvest time and 1-MCP postharvest treatment (625 nL L 1) on soluble solids content (A), firmness (B) and weight loss (C), during storage at 0.5  C, and shelf-life at 22  C in ‘Golden Delicious’ apples: measurements were made on the day after postharvest treatment (0 d) and after shelf-life (7 d); and after cold storage (6 months) and plus shelf-life (7 d). Bars with the same lower case letter in the same harvest date, and each bar (treatment, storage time (0 or 6 months) and shelf-life day (0 or 7 d)) with the same upper case letter in different harvest date are not significantly different by Duncan’s multiple range test, at P < 0.05.

groups, variables such as weight loss, b* value, antioxidants and SSC influence essentially the separation of the 3rd harvest date. 4. Discussion 4.1. Ripening behavior before storage Maturity at harvest is the most important factor that determines storage life and final fruit quality. Immature or overripe fruits are more susceptible to physiological disorders and have a shorter storage life than those fruit harvested at the proper maturity stage (Kader, 1999).

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Fig. 2. Effect of harvest time and1-MCP postharvest treatment (625 nL L 1) on electrolyte leakage (A), phenols content (B) and antioxidant activity (C), during storage at 0.5  C, and shelf-life at 22  C in ‘Golden Delicious’ apple peel. Measurements were made on the day after postharvest treatment (0 d) and after shelf-life (7 d); and after cold storage (6 months) and plus shelf-life (7 d). Bars with the same lower case letter in the same harvest date, and each bar (treatment, storage time (0 or 6 months) and shelf-life day (0 or 7 d)) with the same upper case letter in different harvest dates are not significantly different by Duncan’s multiple range test, at P < 0.05.

The loss of fruit firmness during storage is a serious concern as it results in quality losses leading to soft and mealy fruit (Kader, 1999). The decrease of firmness observed in the present research from 1st to 3rd harvest, was coincident with the increase of starch index as expected due to advanced ripening (Table 1). An increase of SSC would be expected according to the results of starch index and firmness obtained, nevertheless there is no alteration in that parameter (Table 1). Such results may indicate that fruit were harvested at mid or late mature stages, in which less conversion of starch in soluble sugars had occurred (Jan et al.,

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authors have demonstrated that the fluctuations of phenols content during apple ripening are dependent on the cultivar (Del Campo et al., 2006). The levels of total phenols in the present work were within the ranges reported by Carbone et al. (2011). The antioxidant activity accompanied the phenol content pattern, being directly correlated in the three harvest dates (data not shown). Such results may indicate that this property is highly related with the phenols content, as also reported by Carbone et al. (2011). Electrolyte leakage can be used as the indicator of cellular membrane integrity. As reported in other fruit, electrolyte leakage gradually increased during ripening (Wade, 1995; Antunes and Sfakiotakis, 2008). However, in our study electrolyte leakage of apple peel decreased from first to third harvest, while the ripening process proceeded. The 1-MCP interacts with ethylene receptors and thereby prevents ethylene-dependent responses (Sisler and Serek, 1997; Blankenship and Dole, 2003). Our data are consistent with this statement; the ethylene production of fruit treated with 1-MCP was negligible as compared to untreated fruit (Table 2). Effectively, 1-MCP treatment inhibited ethylene production in ‘Golden Delicious’ apples as well as in others cultivars or species, but the persistence of the inhibition can be variable (Fan et al., 1999; Jeong et al., 2002; Lu et al., 2013). Cultivar, harvest maturity and storage treatments affect the response of apples to 1-MCP, probably reflecting ethylene production by the fruit at the time of treatment (Watkins, 2008; Watkins and Nock, 2012). Cultivars such as ‘Delicious’ and ‘Granny Smith’ maintain low ethylene production and high flesh firmness over extended storage periods after 1-MCP treatment, whereas others such as ‘Cortland’ and ‘McIntosh’ tend to recover from 1-MCP induced inhibition of ripening during storage (Fan et al., 1999; Rupasinghe et al., 2000; Tsantili et al., 2007; Magazin et al., 2010). 4.2. Ripening behavior after storage

Fig. 3. Development of superficial scald (A), bitter pit (B) and rot (C), during 6 months storage at 0.5  C plus 7 d shelf life at 22  C in ‘Golden Delicious’ apple of control and 1-MCP postharvest treated (625 nL L 1) fruit.

2012). In fact, the first harvest corresponded to normal harvest time by farmers. Total phenolic compounds decreased from the first to the third harvest. Bizjak et al. (2013), also reported a diminution of some phenol compounds, while others, particularly anthocyanins increased in peal of ‘Braebum’ apple till ripening, nevertheless without changes during advanced ripening. However, other

Neither 1-MCP treatment nor harvest date had a significant effect on fruit lightness, nevertheless L* values increased with cold storage. Saftner et al. (2003) also reported that 1-MCP had no effect in skin lightness of ‘Golden Delicious’ apples. The SSC in 1-MCP treated fruit can be higher, lower or the same as those in untreated fruit (Fan et al., 1999; Saftner et al., 2003; Moran and Mcmanus, 2005). Our data confirm that 1-MCP had little effect on fruit SSC and that fruit from late harvest date had significantly lower SSC than fruit from the two first harvests (Fig. 1A). This may be because SSC values were from the first harvest, correspondent to that of eating ripe SSC values. It has been widely reported that 1-MCP prevents or delays fruit softening (Fan et al., 1999; Rupasinghe et al., 2000; Mir et al., 2001; Saftner et al., 2003; Moran and Mcmanus, 2005; Lu et al., 2013). This is confirmed in our work by the clearly higher firmness of the fruit treated with 1-MCP after cold storage. Although, firmness values were significantly lower in late harvest date fruit (Fig. 1B). This is probably due to reduced effect of 1-MCP when fruit are softer, being more effective for early harvested fruit.

Table 3 Pearson’s correlations between physiological disorders (bitter pit, superficial scald), rot and nutrient level (Ca, Mg, K) in apple flesh and peel. Flesh Parameters BP (%) S. Scald Rots * **

Peel Ca 0.301 0.160 0.381

K

Mg

Ca/K

0.494 0.774** 0.359

0.670* 0.097 0.357

0.284 0.938** 0.075

Correlation is significant at the 0.05 level (2-tailed). Correlation is significant at the 0.01 level (2-tailed).

Ca/Mg 0.424 0.180 0.408

Ca 0.767** 0.044 0.437

K

Mg 0.231 0.072 0.024

0.128 0.626* 0.164

Ca/K 0.457 0.128 0.296

Ca/Mg 0.748** 0.209 0.398

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Fig. 4. Scores and loading plot for PC1 and PC2 for Control and 1-MCP treated fruits at 3 harvest dates (1, 2 and 3). The percentage of total variance explained from each principal component is shown in parentheses. Cont1- untreated fruit from 1st harvest, Cont2-untreated fruit from 2nd harvest, Cont3- untreated fruit from 3rd harvest, MCP1- 1-MCP treated fruit from 1st harvest, MCP2- 1-MCP treated fruit from 2nd harvest, MCP3- 1-MCP treated fruit from 3rd harvest, BP- bitter pit, Fmfirmness, Hue- hue angle; Scald- superficial scald, EL- electrolyte leakage, Llightness, Rot- rot, a- a value (color), WL- weight loss, b- b value (color), Antantioxidant activity, Phenols- total phenols content.

As shown in Fig. 2C both treatments lost less weight in late harvested fruit in accordance with that reported by Jan et al. (2012). In the 3rd harvest, 1-MCP was effective in reducing the weight loss. Such behavior was also reported for avocado fruit (Jeong et al., 2002) and Chinese kale (Sun et al., 2012). The changes in electrolyte leakage clearly reflected changes in membrane permeability during storage and shelf-life. In the first two harvests, and at the beginning of the experiment, electrolyte leakage decreased during the shelf-life period for both control and 1-MCP-treated fruit. This transient variation in electrolyte leakage probably reflects structural changes and/or the reorganization of membrane components in response to a short period of three days in a cold room (Fig. 2A). Similar behavior was found in pineapples after 21 d cold storage (Hu et al., 2011). Probably, the most mature fruit (3rd harvest) have lost this ability. During storage, electrolyte leakage increased in both treatments, nevertheless 1-MCP treated fruit had a higher degree of membrane structure maintenance and permeability. Similar behavior was reported in ‘Branquilla’ pears (Larrigaudière et al., 2004). This may be because of better cell wall maintenance promoted by 1-MCP. Total phenolic content in fruit peel increased during storage from early harvested fruit in both untreated and 1-MCP treated fruit; the opposite happened in the other two harvest dates. Several articles in the literature report little or no effect of storage on phenolic compounds and total antioxidant capacity of climacteric fruit, while other studies have detected minor fluctuations in phenolic concentrations (Awad and de Jager, 2003; Łata, 2008). The variability in these reports may be due to various factors including apple cultivar, storage conditions and/or postharvest treatments (Van der Sluis et al., 2001). According to Hoang et al. (2011), 1-MCP treatment significantly reduced the total antioxidant activity in the peel tissue of ‘Cripps Pink’ apple. The effect of 1-MCP content has been investigated by Larrigaudière et al. (2004) who reported an increase in the enzymatic antioxidant capacity following 1-MCP treatment in ‘Blanquilla’

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pears, and interpreted it as a sign of a general metabolic change influenced by ethylene. In contrast, Shaham et al. (2003) observed low activities of most antioxidant enzymes in 1-MCP treated ‘Granny Smith’ apples. In this study, no clear effect of treatment on phenols or antioxidant activity can be attributed to 1-MCP. It is widely known that 1-MCP treatment prevents development of superficial scald on ‘Golden Delicious’ apples (Fig. 3A) as on other apple and pears cultivars (Fan et al., 1999; Rupasinghe et al., 2000; Watkins et al., 2000; Gago et al., 2013). However, in ‘Golden Delicious’ 1-MCP treatment may also cause disorders such as DSB and bitter pit (Calvo and Candan, 2010; Larrigaudière et al., 2008, 2010). The reason for the appearance of DSB disorder remains unknown but it is possibly related with ethylene metabolism and senescence-related processes (Larrigaudière et al., 2008, 2010). And, it is thought that the risk of DSB may be present if during the 30 d before harvest, orchards have 15 d with minimum temperatures above 15  C, as happened in this study (Table 3). The incidence of DSB, under these conditions, can be totally prevented by storing apples at 6  C just after harvest and slowly decreasing the temperature to 1  C after 1-MCP application (Dupille, personal communication). Indeed, Warner (2005) reported that a quick cooling of fruit immediately after harvest markedly enhances the appearance of DSB. However, these do not seem to be the main factors for the emergence of the DSB since both conditions were present in this experiment, and there were no signs of fruit with DSB, even in the less mature fruit considered more sensitive (Larrigaudière et al., 2010). Warner (2005) also reported that 1-MCP appears to exacerbate DSB, particularly when fruit is treated immediately after being put into low-temperature storage. So, hypothetically the three days of cold before 1-MCP application may have avoided the appearance of DSB in fruit of this study. In fact, Gamrasni et al., (2010) concluded that gradual cooling following treatment, by itself, was not enough to prevent DSB; but, a delay of 10 d in the application of 1-MCP avoided it. Bitter pit incidence, on average, was higher in 1-MCP treated fruit than in control fruit (Fig. 4B). Similar results were reported by Calvo and Candan (2010) in ‘Granny Smith’ apples. However, there was a high variability among orchards, including orchards 4 and 5 where the 1-MCP did not enhance bitter pit incidence. All this non-uniformity is likely due to differences in cultural techniques used. Effectively, Saure (2005) reported that occurrence of bitter pit in apples is related to factors of orchard management, especially the excessive thinning of fruits, high plant vigor, excessive N fertilization, and water stress. Low Ca concentrations in fruit, promotes damage that progresses to tissue death, i.e., bitter pit (Amarante et al., 2006). According to Ferguson and Watkins (1989) low Ca and high levels of Mg, K and N in apples are the main factors that predispose fruit to the occurrence of this physiological disorder. In our experiment, bitter pit percentage was negatively correlated with Ca and Ca/Mg peel content and positively correlated with flesh Mg content (Table 3). Differences in cultural techniques used in the orchards, may be also the cause of different rot percentages after cold storage in fruit from different orchards. Nevertheless, 1-MCP significantly reduced rot percentage in all orchards. Saftner et al. (2003) and Jeziorek et al. (2010) found reduced rot after cold storage in 1-MCP treated ‘Golden Delicous’ apples and after the shelf-life period (Jeziorek et al., 2010). The PCA analysis defined clearly two groups: Control group (untreated fruit) characterized by higher rot, scald incidence, lightness and electrolyte leakage; and the 1-MCP treated fruit group, with higher bitter pit, firmness and hue angle. Total phenols, chroma, soluble solid content, antioxidant activity, b* values and weight loss marked a clear decrease from the first two harvests to the 3rd harvest.

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