Postharvest Biology and Technology 78 (2013) 2433

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Postharvest Biology and Technology

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Cold quarantine responses of Tarocco oranges to short hot water and thiabendazole postharvest dip treatments
A. Palma a , S. DAquino a , S. Vanadia b , A. Angioni c , M. Schirra a,
a b c

C.N.R. Institute of Sciences of Food Production, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy C.N.R. Institute of Sciences of Food Production, via Amendola 122/O, 70126 Bari, Italy Department of Science of Life and Environment, University of Cagliari, Via Fiorelli 1, 09126 Cagliari, Italy

a r t i c l e

i n f o

a b s t r a c t
This study investigated the effects of brief hot water and thiabendazole (TBZ) postharvest dip treatments on ultrastructural changes of fruit epicuticular wax (ECW), TBZ residues, decay development and quality traits of Tarocco oranges [Citrus sinensis (L.) Osbek] subjected to cold quarantine, subsequent simulated transport and shelf-life. Commercially mature fruit were submerged in water at 20 C (control fruit) or TBZ at 1000 mg/L and 20 C for 60 s, or in hot water without or with TBZ at 300 mg/L and 53, 56, or 59 C for 60, 30, and 15 s respectively. Following treatments, fruit were stored for 3 weeks at 1 C (simulated quarantine conditions for fruit disinfestations against Mediterranean fruit y, Medy), followed by 4 days at 3 C (simulated long distance transport), and nally kept at 20 C for 3 days (shelf-life, SL). Scanning electron microscopy (SEM) analysis of Tarocco orange surface showed that the typical wax platelets, lifting around edges of wax plates and areas free of epicuticular wax (ECW), that disappeared after hot water dips at 5359 C for 6015 s, become visible again after storage for 21 days at 1 C (quarantine conditions), and changes involving the appearance of rough ultrastructure, presence large curled plates, ssured wax crusts, and areas with ECW deciencies, became much more pronounced after shelf-life. These occurrences were related to the transient effect of hot water treatment in decay control. Conversely, treatments with 300 mg/L TBZ 53 C for 60 s or 56 C for 30 s effectively reduced decay after quarantine. These treatments were as effective as standard treatment with 1000 mg/L TBZ at 20 C and produced similar TBZ residue levels in fruit, without impairing fruit quality traits such as visual appearance, weight loss, compression test, sensory attributes, juice color parameters (a*, b*, h, L*, and Chroma), and juice chemical characteristics (soluble solids content, titratable acidity, ascorbic acid, glucose, sucrose, citric acid, total phenols, total anthocyanins, and total antioxidant activity). 2012 Elsevier B.V. All rights reserved.

Article history: Received 6 September 2012 Accepted 9 December 2012 Keywords: Citrus Decay Epicuticular wax morphology Heat treatment Thiabendazole residues Storage

1. Introduction Among the orange cultivars, Moro, Sanguinello and Tarocco are the most cultivated in Italy. Fruit of these cultivars are appreciated by consumers for their typical taste and peculiar characteristics of blood-red esh and rind color, owing to the presence of phenolic compounds belonging to the class of anthocyanins (Dugo et al., 2003), some of which are known to have several health promoting properties (e.g. benecial effects on capillary fragility and arteriosclerosis, and antiviral activity, preventing allergies and other inammatory diseases, anti-cancer activity) (Sajia, 1994). Therefore, from a dietetic viewpoint, anthocyanins represent an added value for blood oranges due to their important therapeutic

Corresponding author. Tel.: +39 0783 33224; fax: +39 0783 33959. E-mail address: mario.schirra@ispa.cnr.it (M. Schirra). 0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.002

properties, in addition to the well known health benets of citrus fruit (Attaway and Moore, 1992). Various citrus-importing countries require quarantine security protocols to diminish the risk of accidental introduction of the Mediterranean fruit y (Medy, Ceratitis capitata), and fruit must be certied Medy free. Therefore Medy disinfestations should be performed by citrus producing-countries before exportation. Heat treatments (hot water immersion, high temperature forced air, vapor heat) (Armstrong and Mangan, 2007) and cold quarantine treatments (exposure of fruit to near-freezing temperatures for 1016 days) (Armstrong, 1994), are technologies applied on a commercial scale for postharvest insect control of horticultural crops as alternatives to chemical fumigation (e.g. methyl bromide). Commodity response to quarantine treatment varies depending on species, cultivar, and quarantine treatment conditions. Quarantine treatments reaching a fruit core temperature of 44 C for 100 min or 46 C for 50 min did not cause damage or fruit softening in Olinda and Campbell oranges (Schirra et al.,

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2005a) but produced deleterious effects on the quality traits of blood oranges, such as development of off-avors and off-tastes, decreased fruit rmness and reduced fruit resistance to decay (Mulas et al., 2001); other methods such as cold quarantine therefore should be applied to reduce treatment impact on quality (Schirra et al., 2004). On the other hand, cold quarantine can lead to chilling injury (CI) on blood oranges and the application of a hot water dip at 50 C for 180 s before cold disinfestation was able to overcome the risk of CI and decay development after quarantine, without impairing fruit quality (Schirra et al., 2004). It has been shown that the effectiveness of postharvest hot water dip treatments at 50 C for 180 s in alleviating CI in citrus fruit, notably improve when applied in combination with the fungicide thiabendazole (TBZ) (Schirra et al., 1998, 2000a). Reduced treatment time would be desirable to increase packinghouse output and shorten delays in fruit marketing, therefore treatment temperature should be raised to produce the heat-induced benecial effects in terms of physical changes of ECW, host defensive responses and inhibition of pathogen development (Schirra et al., 2000b, 2011). However, no data are available in the literature on the inuence of short hot water treatments used in commercial installations and its combination with TBZ on ultrastructural changes of fruit surface, TBZ residues, decay incidence, external and internal fruit quality attributes of blood oranges exposed to a quarantine treatment. The present investigation was therefore planned to study the cold quarantine responses of Tarocco blood oranges to short (1560 s) hot water dip treatments, without or with TBZ on physical, nutritional and functional properties of fruit. The potential relevance of treatment-induced changes on TBZ residues, CI and decay control after quarantine was also evaluated and discussed. 2. Materials and methods 2.1. Plant material, treatments and storage conditions Commercially mature blood oranges [Citrus sinensis (L.) Osbek] cv. Tarocco were harvested from a farm located in southern Sardinia (Italy) and transported to the laboratory immediately after harvest. Visibly sound fruit free of defect were washed and subdivided into 8 groups corresponding to the following dip treatments: (a) water alone at 20 C (control) or in combination with 1000 mg/L TBZ (TECTO SC, Syngenta Crop Protection S.p.A., Milan, Italy, at 42.9% active ingredient, a.i.) for 60 s (standard treatment); (b) water alone at 53, 56, or 59 C for 60, 30, and 15 s respectively, or in combination with 300 mg/L TBZ. The concentration of TBZ in the treatments at 5359 C was set based on preliminary trials performed in our laboratory. Following treatments fruit were left to dry at room temperature and stored for 3 weeks at 1 C (quarantine conditions) plus 4 days of simulated transport at 3 C (quarantine + transport) and subsequent 3 days of simulated shelf-life at 20 C (quarantine + transport + SL). Each treatment was repeated threefold (replications). 2.2. Scanning Electron Microscopy (SEM) analysis SEM observations were performed following treatments, after quarantine and after shelf-life. Samples of ve fruit per treatment were used for SEM analysis. Two rind samples (2 cm 2 cm) were excised with a razor blade from the equatorial zone of each fruit and immediately xed in a phosphate buffer (pH 7.4) containing 3% glutaraldehyde, and kept at 4 C until further preparation. The xed tissue was rinsed three times in phosphate buffer (pH 7.4)

and then three times in deionized water. They were dried by washing with increasing concentrations of ethanol (20, 50, 70, 80, 95, and 100%), the samples being left for 20 min before each wash. The dried samples were placed on aluminum stubs using silver conductive glue, and a goldpalladium coating was applied with an Edwards S-150 A sputter coater. Until observation, samples were stored in a vacuum dryer. SEM was carried out with a ZEISS DSM 962 microscope at 30 kV and 2502000 magnications. Relevant SEM micrographs at 250 magnication are shown in the gures. 2.3. Analysis of TBZ residues TBZ analyses were performed following treatment, after quarantine, after simulated transport and after shelf-life. Six oranges per replication (4 replications per treatment) were weighed, and the peel was removed. The peel was weighed and its percentage with respect to the whole fruit calculated. Then, peel samples were kept frozen at 40 C until analysis. TBZ analyses were carried out on four replicates of six fruit per treatment, according to Schirra et al. (2008). Briey, 5 g of homogenized peel were weighed in a 40 mL screw-capped ask and 10 mL of ethyl acetate/hexane (1/1) and 2 g of NaCl were added. The mixture was agitated in a rotatory shaker for 30 min. Subsequently, the phases were allowed to separate and 1 mL of the organic layer was dried under a gentle nitrogen stream and the residues were dissolved with 1 mL of acetone and injected in GCITMS for the analysis without any clean-up step. 2.4. Visual assessment, fruit weight loss, compression test To determine visual assessment, fruit weight loss and compression test, each treatment group was divided into three subgroups of three boxes (replications) each. The fruit of the rst subgroup (40 fruit per replication) were used for overall visual appearance, chilling injury (CI), decay and the appearance and absence of the calyx. Fruit of the second subgroup (40 fruit per replication) were used for weight loss determination, whereas fruit of the remaining group (10 fruit per replication) were used for compression test. Overall appearance was evaluated on the basis of the following hedonic scale: 1 = very poor; 3 = poor; 5 = fair; 7 = good; 9 = excellent. CI was evaluated as pitting or scald of the peel, was rated using a 04 subjective scale, where 0 = none, 1 = slight, 2 = moderate, and 4 = severe injury. Finally, a weighted average from a chilling index was calculated. The appearance and absence of the calyx were determined and the percentage of fruit showing the calyx dried and brown or detached was calculated. Decay incidence was assessed as percentage of rotten fruit caused by various fungi. To determine fruit weight loss, fruit were individually weighed following treatment and after selected periods and the percentage of weight loss calculated. A compression test was performed by a texturemeter interfaced with a computerized system (DO-FB0.5TS, Zwick Roell, Germany). The compression curves were obtained by deforming the fruits with a metal disk (15 cm diameter) at a speed of 100 mm/min. Values were expressed as maximum deformation at 3 kg compression force. 2.5. Juice chemical analysis Chemical analyses of juice were performed following treatment and after shelf-life. Juice was obtained by squeezing 10 fruit per replication (3 replications per treatment) with a commercial juicer. Before analyses, the juice was centrifuged (Centurion Scientic Ltd., West Sussex, England) at 13,000 g for 15 min and the supernatant ltered through a 0.45 m acetate cellulose lter. Titratable acidity (TA) of the juice was determined using a potentiometric titrator (Metrom 720 SM Tritino, Swiss); 5 mL of juice diluted in 45 mL

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distilled water were titrated with NaOH 0.1 N up to pH 8.1. The results were expressed as percentage of anhydrous citric acid. The percentage of total soluble solids (SSC) was measured by a digital refractometer (PR-101, Atago, Japan). Organic acids and ascorbic acid (AA) were determined with a Merck-Hitachi (Tokyo, Japan) liquid chromatograph with an L-7455 photodiode detector (DAD) detector, D-7000 system manager, L7200 autosampler and L-7100 pumps. Simultaneous separation and determination of organic acids and ascorbic acid were achieved according to the procedure described by Yuan and Chen (1999) and by Chinnici et al. (2005) using a Bio-Rad cation guard column and a Bio-Rad Aminex HPX-87H Hydrogen form cation exchange resin-based column (300 mm 7.8 mm i.d.) at 40 C. The mobile phase consisted of 0.005 M sulphuric acid aqueous solution and the samples were isocratically separated at 0.6 mL/min. Peaks of organic acids and ascorbic acid were measured at wavelengths of 210 and 245 nm respectively and were identied by comparing retention times with those of standards and quantication was carried out using external standards. Total phenolic content was analyzed according to the FolinCiocalteau colorimetric method (Singleton and Rossi, 1965). Total phenols were expressed as gallic acid equivalents. Antioxidant activity was assessed using the free radical DPPH, according to Bonded et al. (1997). The mixture containing 3 mL of a methanol solution of 0.16 mM DPPH was allowed to react for 15 min in a cuvette. The inhibition percentage of the absorbance at 515 nm of DPPH solution added with sample was calculated using the following equation: Inhibition % = (Abst=0 Abst=15 min )/Abst=0 100. Total anthocyanin contents were determined spectrophotometrically using the pH differential method (Rapisarda et al., 2000). The individual anthocyanins (cyanidin-3-5-diglucoside, cyanidin-3-glucoside, delphinidin-3-6-malonyl-glucoside, cyanidin-3-malonyl-glycoside, cyanidin-3-dioxolane-glucoside, and peonidin-3-6-malonyl-glucoside) were determined with a LaChrom Merck-Hitachi liquid chromatograph (Hitachi Ltd., Tokyo, Japan) equipped with a L-7455 photodiode detector (DAD). A Luna C18 column (150 mm 4.6 mm, 3 m, Phenomenex, Castel Maggiore, BO, Italy), equipped with a precolumn (7.5 mm 4.6 mm i.d.) was employed. HPLC elution was carried out at 35 C using the following elution prole: ow rate 0.5 mL/min, t = 0 10% solvent B (acetonitrile)/90% solvent A (formic acid 1%, acetic acid 2%), t = 20 20% B linear gradient, t = 38 30% B, post time 12 min. The chromatogram was monitored simultaneously at 360 and 520 nm. Quantitative analysis of anthocyanins was carried out using the external standards method and their concentration was expressed as cyanidin-3-glucoside equivalents. Anthocyanins were identied by LC-electrospray ionization (ESI)MS analysis using an Agilent Technologies (Palo Alto, CA, USA) 1100 series LC/MSD. Two mL of juice were placed in a 10 mL headspace vial and incubate for 2 h in a shaking bath at 60 C, for acetaldehyde and ethanol determination. Then a 1 mL headspace gas sample was injected into gas chromatograph Agilent 6890 tted with a ame ionization detector (FID) and equipped with a 5% Carbowax 20 M on 80/120 Carbograph 1 AW packed column. Run conditions were: N2 as carrier gas at 30 mL/min; injector at 130 C; oven 80 C; detector at 150 C. Ethanol and acetaldehyde were quantied by comparison of peak area versus concentration of a calibration curve obtained from pure analytical standards subjected to the same analytical conditions (Schirra et al., 2004). Color parameters of juices solution diluted 1/10 with water, were measured in glass cells of 10 mm path length using a Varian Cary 50 spectrophotometer equipped with a Cary Win UV color software. CIE L*, a*, b*, Hue angle (h = arctg b*/a*) and Chroma values (Chroma = (a )2 + (b ) ) were calculated using illumi observer, according to the CIE L*, a*, b* 76 nant D65 and 10
2

Convention (McLaren, 1980). All measurements were done in triplicate. 2.6. Statistical analysis Statistical analysis was performed by Statgraphics Centurion software (Herndon, VA, USA), version XV Professional statistical program. Analysis of variance (ANOVA) was carried out according to a single factor, complete randomized block design with three replicates for each treatment. Percentages were subjected to the ANOVA after transformation in arcsin x or x before the ANOVA, depending on the range of variation of data. As fruit treated with water alone or in combination with TBZ did not reveal signicant differences (P > 0.05) in color or chemical traits, further statistical analyses for these parameters were pooled, Mean comparisons of the effects of treatments were calculated, when applicable, by the least signicant difference test at P 0.05. 3. Results and discussion 3.1. SEM analysis 3.1.1. Following treatment The surface of fruit dipped in water at 20 C (control fruit) displayed the typical ultrastructure of mature orange fruit (Cajuste et al., 2010) with areas covered by smooth thin layers of ECW, irregular in shape, raised or folded outwards around edges, and areas free of ECW (Fig. 1a). Cuticular ridges, rough platelets, and wax granules were observed at higher magnication (SEM micrograph not shown). In contrast, the ECW of fruit treated with TBZ at 20 C (standard treatment) appeared more homogenous than control fruit; layers of ECW, raised or folded outwards around edges, were hardly visible and the ECW platelets and granules relaxed (Fig. 1a). Surfactants are often added to agrochemicals to design a formulation able to enhance the effectiveness of the a.i. increasing wetting, spreading, penetration and retention on the plant cuticle (Schnherr and Baur, 1996). Fungicide formulation interactions with the plant cuticle are complex (Baur, 1998). However, there is evidence that surfactants can considerably modify the ECW ne structure (Kuzyc and Meggitt, 1983; Noga et al., 1987) presumably by selective solubilization of ECW constituents (Tamura et al., 2001). Thus, results of our study suggest that the loss of details of ECW ne structure in fruit treated with TBZ may be ascribed to selective solubilization of ECW by surfactant present in the commercial formulation of TBZ fungicide mixture. Dip treatments with water at 53 C for 60 s caused the disappearance of ECW layers: the ne structures of ECW collapsed and some stomatal openings were occluded with wax (Fig. 2a and e). Similarly, fruit treated with water at 56 C for 30 s (Fig. 3a) or 59 C for 15 s (Fig. 4a) showed the loss of the ultrastructural features of fruit surface, the platelets tended to relax and melt, covering the areas free of ECW, and creating a smoother appearance. TBZ application at 53 C resulted in a partial melting of ECW, but wax layers were hardly visible (Fig. 2b), and no difference could be detected with the treatments at 20 C. When treatments with TBZ were performed at 56 or 59 C (Figs. 3b and 4b) changes in the ECW micromorphology were more evident, and a higher level of wax melting could be assessed at 59 C (Fig. 3b versus Fig. 4b). The differences in ECW ne structure of fruit subjected to hot water at 59 C without or with TBZ were negligible. A similar behavior involving changes in ECW ultrastructure of fruit treated with hot water was reported previously (Schirra and Dhallewin, 1997; Schirra et al., 2005b; Dore et al., 2009). These features were ascribed to the partial melting and redistribution of ECW layer along fruit surface.

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Fig. 1. SEM micrographs (250 magnications) of cuticle surface of Tarocco oranges after dip treatment at 20 C for 60 s in water (a) or thiabendazole (TBZ) at 1000 mg/L (b), after subsequent quarantine for 3 weeks at 1 C (c, water; d, TBZ), and next subjected to 4 days of simulated transport at 3 C plus 3 days of shelf-life at 17 C (e, water; f, TBZ). The arrows (a and c) indicate stomata.

3.1.2. After quarantine The wax layer of control fruit became rough, and the lifting around edges of wax plates was increased (Fig. 1c). Build up of crystalline wax granules was also observed and areas free of ECW enlarged. Wax platelets of an indenite design and stomata uncovered by wax were visible at higher magnication (SEM micrograph not shown). Fruit treated with TBZ at 20 C displayed similar features as control fruit but the areas uncovered by ECW were less extensive in size (Fig. 1d versus Fig. 1c). The micromorphology of the cuticular surface of fruit treated with water at 5359 C was similar, but was different as compared to control fruit (Figs. 2c, 3c and 4c versus Fig. 1c). Fruit treated with TBZ at 53 C (Fig. 2d) showed very irregular wax scabs, partially detached from cuticle, with large edges completely curled outwards, whereas ultrastructural changes of fruit treated with TBZ at 56 C (Fig. 3d) were only small. By contrast, fruit treated with TBZ at 59 C exhibited large irregular ECW layers, partially detached edges and areas without ECW (Fig. 4d).

Comparison with standard treatments (TBZ at 1000 mg/L and 20 C) showed marked differences in the shape of the wax aggregates and extension of wax deprived zones. 3.1.3. After shelf-life The control fruit and fruit treated with water at 53 or 59 C showed changes involving the appearance of rough structures with wax deposits, a network of large curled plates, and large areas with ECW along with skin surface, and ECW deciencies became much more pronounced than those observed after quarantine, whereas treatments at 56 C showed less differences compared with quarantined fruits (Figs. 14e). Surface details of ne structure of TBZ treated fruit (Figs. 14f) were generally less visible than control and fruit treated with water at 5359 C. In addition, only fruit treated with TBZ at 56 C (Fig. 3f) maintained a homogeneous shape, without the appearance of ECW free zones. The appearance of ssured wax crusts along with large areas without ECW, may be related to loss of elasticity of waxy layer and the subsequent detachment of wax plates, factors that may be ascribed to fruit senescence.

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Fig. 2. SEM micrographs (250 magnications) of cuticle surface of Tarocco oranges after dip treatment at 53 C for 60 s in water (a) or thiabendazole (TBZ) at 300 mg/L (b), after subsequent quarantine for 3 weeks at 1 C (c, water; d, TBZ), and next subjected to 4 days of simulated transport at 3 C plus 3 days of shelf-life at 17 C (e, water; f, TBZ). The arrows indicate stomata (a, b, d, and e).

Therefore, the benecial effect of hot water treatment in terms of partial melting and redistribution of ECW layer along fruit surface and subsequent closure of microwounds which represent potential gaps for conidia of wound pathogens (Schirra et al., 2000b, 2011), are only transitory and are lost during fruit storage. The different effect on fruit surface of TBZ treatment at 56 C is consistent in all postharvest steps and indicates that this was the best temperature for the treatment. 3.2. Inuence of treatment and storage conditions on TBZ residues in fruit Immediately after TBZ treatments at 1000 mg/L and 20 C or at 300 mg/L and 5359 C, residue levels in fruit were similar (Table 1). Previous studies on Tarocco oranges (Schirra et al., 1998) showed that fruit treated with TBZ at 50 C experienced a greater persistence of TBZ with respect to fruit treated at room temperature and this occurrence was related to the better encapsulation and coverage of the a.i. by ECW, thus providing better protection to

the fungicide. Results of the present study displayed a reverse trend, TBZ persistence being higher in fruit treated at 20 C with a decline after shelf-life of ca 12% versus ca 39% in fruit treated at 5359 C. However, treatment duration used in this study was short (1560 s), being the same as that employed commercially in many locations in the US, whereas much longer periods (180 s) used in previous studies (Schirra et al., 1998) resulted in a greater impact of heat on rearrangement of ECW structure and TBZ persistence. 3.3. Inuence of treatments on CI, decay, and fruit quality attributes The incidence of fruit affected by CI was very small after quarantine and subsequent simulated transport (data not shown) and was relatively low also after shelf life (Table 2). CI incidence was reduced in fruit treated at 53 C and remarkably reduced in the other fruit samples. It is recognized that cold quarantined blood oranges are susceptible to CI, especially when fruits are returned to warm temperature (Schirra et al., 2004). The lack of

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Fig. 3. SEM micrographs (250 magnications) of cuticle surface of Tarocco oranges after dip treatment at 56 C for 30 s in water (3a) or thiabendazole (TBZ) at 300 mg/L (b), after subsequent quarantine for 3 weeks at 1 C (c, water; d, TBZ), and next subjected to 4 days of simulated transport at 3 C plus 3 days of shelf-life at 17 C (e, water; f, TBZ). The arrows indicate stomata (ad and f).

CI found in our study was related to the relatively short period of simulated transport and shelf-life. The percentage of fruit showing the calyx dried and brown or detached was not signicantly affected by treatments (data not shown). Pooled data (mean values standard deviation) of this percentage after shelf life averaged 34.4 5.4.

Overall appearance was rated as very good up to quarantine + storage (rate 88.5), without signicant differences among treatments (data not shown), whereas after shelf-life all treated fruit were judged as good to very good (rate 7.17.5) and fairly good (rate 6) for the control fruit (Table 2). The lower score received by control fruit was related to the higher incidence of CI. There was no

Table 1 Thiabendazole (TBZ) residues (mg/kg, on a whole fruit basis), in Tarocco oranges immediately after treatment (time 0) and after 3 weeks at 1 C (quarantine), 4 additional days of simulated transport at 3 C (transport), and subsequent 3 days of simulated shelf life.a Treatments Dip time (s) TBZ residues (mg/kg) Time 0 1000 mg/LTBZ 20 C 300 mg/L TBZ 53 C 300 mg/L TBZ 56 C 300 mg/L TBZ 59 C 60 60 30 15 6.65 a(a) 6.96 a(a) 6.56 a(a) 6.31 ab(a) Quarantine 6.65 a(a) 5.78 b(b) 5.63 b(b) 5.59 b(a) Transport 6.37 ab(ab) 5.36 b(b) 5.72 b(b) 5.64 b(a) Shelf life 5.87 a(b) 4.26 b(c) 4.04 b(c) 3.89 b(b)

a Values within columns (without brackets) or rows (in brackets) followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05. Letters without brackets relate to comparisons of the inuence of treatments within each storage time. Letters in brackets relate to comparisons of the effect of storage time within each treatment.

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Fig. 4. SEM micrographs (250 magnications) of cuticle surface of Tarocco oranges after dip treatment at 59 C for 15 s in water (a) or thiabendazole (TBZ) at 300 mg/L (b), after subsequent quarantine for 3 weeks at 1 C (c, water; d, TBZ), and next subjected to 4 days of simulated transport at 3 C plus 3 days of shelf-life at 17 C (e, water; f, TBZ). The arrows indicate stomata (d and e).

decay in untreated fruit after quarantine while after subsequent transport decay incidence averaged about 8% (data not shown), reaching approximately 30% after shelf-life (Table 1). The causal agents of decay were green mold and, to a lesser extent, blue mold caused by Penicillium digitatum and P. italicum respectively (data

not shown). Treatment with 300 mg/L TBZ at 53 or 56 C signicantly reduced decay development with respect to control fruit, whereas the effect of the other treatments was not signicant. After shelf-life, treatments with hot water alone at 5359 C signicantly reduced the incidence of decay with respect to control

Table 2 Inuence of postharvest treatments on chilling injury index, overall appearance, and decay percentage in Tarocco oranges subjected to cold quarantine for 3 weeks at 1 C, 4 additional days of simulated transport at 3 C and subsequent 3 days of simulated shelf-life (quarantine + transport + SL).a Treatments Water, 20 C Water, 53 C Water, 56 C Water, 59 C 1000 mg/L TBZ, 20 C 300 mg/L TBZ, 53 C 300 mg/L TBZ, 56 C 300 mg/L TBZ, 59 C Dip time (s) 60 60 30 15 60 60 30 15 Chilling injury 0.60 a 0.30 b 0.08 c 0.09 c 0.07 c 0.00 c 0.02 c 0.05 c Overall appearance 6.1 b 7.2 a 7.1 a 7.2 a 7.2 a 7.5 a 7.1 a 7.3 a Decay (%) 28.9 a 13.3 b 15.6 b 16.7 b 23.6 ab 3.3 cd 1.1 d 4.4 c

a Chilling injury index, rating scale 04; overall appearance, rating scale 09. Values within columns followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05.

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Table 3 Inuence of postharvest treatments on color parameters L* (brightness), a* (redness) and b* (yellowness), a*/b* ratio, h (hue angle) and Chroma (saturation) in Tarocco oranges subjected to cold quarantine for 3 weeks at 1 C, plus 4 days of simulated transport at 3 C (quarantine + transport) and subsequent 3 days of simulated shelf-life (quarantine + transport + SL).a Treatments Harvest Quarantine + transport + SL Water, 20 C Water, 53 C Water, 56 C Water, 59 C
a

Dip time (s)

L* 98.79

a* 1.23 1.68 a 1.60 a 1.37 ab 1.15 b

b* 1.19 1.04 b 1.03 b 1.04 b 1.17 a

a*/b* 1.03 1.61 a 1.59 a 1.31 ab 0.98 b

h 44.14 31.71 b 32.14 b 37.98 ab 45.50 a

Chroma* 1.71 1.97ns 2.14 1.90 1.64

60 60 30 15

98.92ns 98.62 98.71 98.42

Values within columns followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05.

fruit (Table 2). Best results in controlling decay were achieved with TBZ at 1000 mg/L and 20 C (2.5% decay), and with 300 mg/L TBZ at 5356 C (0.83.3% decay). Conversely the inuence of TBZ at 59 C on decay control was not signicant. The lack of efcacy of TBZ at 59 C in controlling decay may be ascribed to physiological stress of the peel, which might weaken the natural defense properties of fruit, although visible symptoms of treatment damage were not detected. Fruit weight loss and fruit compression test values were not affected by treatments (data not shown). Pooled data (mean values standard deviation) of weight loss averaged 2.31 0.71 and 3.11 0.75 after transport and shelf-life, respectively. At harvest compression test values averaged 4.81 0.21, whereas after transport and shelf-life were 7.11 0.77 and 7.43 0.66, respectively. L*, and Chroma parameters were not signicantly affected by treatments, whereas treatment at 59 C caused a decrease in red pigmentation of juice as supported by lower values of a*, higher values of b*, and consequently lower values of the a*/b* ratio, with a higher h, with respect to control fruit (Table 3). Treatments at 53 and 56 C did not affect SSC, TA, or the SSC-TA ratio, according to previous studies on Fortune mandarins submerged in water at 5058 C for 180 s (Schirra and Dhallewin, 1997). However, when water was applied on Tarocco oranges at 59 C for 15 s, TA values were lower than control fruit and SSC-TA ratio increased accordingly (Table 4). Acetaldehyde levels increased in fruit treated at 59 C. Fruit treated at 56 and 59 C registered respectively higher and notably higher values of ethanol (Table 4) and lower values of fructose and malic acid than control fruit (Table 5). An increase in the TSS/TA ratio during storage of citrus fruit was related to the accumulation of ethanol in the juice (Cohen et al., 1990), which may cause the development of off-avors (Cohen et al., 1990; Hagenmaier, 2002). However, the present study showed that the increases of ethanol found in Tarocco oranges treated at 56 and 59 C did not adversely affect fruit sensory quality and no visible treatment damage occurred. By contrast, a dramatic accumulation of ethanol was found in previous studies on Fortune mandarins subjected to hot water dips at 58 C for 180 s and stored for 30 days at 6 C plus three days of simulated shelf-life at 20 C (Schirra and Dhallewin, 1997). After shelf-life, fruit appeared

brown and aged and the taste was judged poor due to the off-avor development. Such adverse effects on fruit quality observed in Fortune mandarins were ascribed to phytotoxic effects caused by the excessive temperature (58 C) and prolonged exposure of fruit to heat (180 s) (Schirra and Dhallewin, 1997). Treatment damage and off-avor development were reported in Satsuma mandarins when submerged in water for 120 s at temperatures higher than 50 C (Ghasemnezhad et al., 2008). It is recognized that mandarins are much more prone to develop off-avors than other citrus varieties (Shi et al., 2005). This occurrence was ascribed to the low permeability of mandarin peel to gas which favors the build-up of volatiles, such as ethanol and acetaldehyde in the juice, and the consequent perception of off-avors (Shi et al., 2007). However, compounds other than ethanol have been related with the offavor development in citrus fruit. In blood oranges variations of free and bound hydroxycinnamic acids levels during storage were proven to be a reliable index of detrimental off-avors development (Fallico et al., 1996). The concentrations of sucrose, glucose, and citric acid were not signicantly affected by treatments, whereas lower values of fructose and malic acid were recorded in fruit treated at 56 or 59 C (Table 5). Investigations on blood oranges revealed that hot water dipping for 180 s at 50 C did not signicantly affect the content of AA during or after quarantine (Schirra et al., 2004). Accordingly, results of this study showed that differences in AA content between fruit treated at 5356 C and control fruit were not signicant (Table 6). Various reports have shown that vitamin C loss in citrus fruit during storage at different temperatures is slight (Nagy, 1980). However, signicantly lower AA values were recorded in fruit treated at 59 C indicating the stress conditions caused by this treatment. Similar adverse effects on AA levels were observed in blood oranges exposed to a fruit core temperature of 44 C for 100 min or 46 C for 50 min (Mulas et al., 2001) and in mature green tomatoes (Lycopersicum esculentum Mill.) after heat treatment at 38 C and 50% RH for 24 h (Yahia et al., 2001). After shelf-life there was a relatively small decline (ca 1622%) in AA with respect to fresh fruit, supporting previous studies on blood oranges (Schirra et al., 2004). The low storage temperatures

Table 4 Inuence of postharvest treatments on soluble solids content (SSC, %), titratable acidity (% anhydrous citric acid), maturity index (SST:TA ratio, SST/TA), acetaldehyde (MeCHO, mg/100 mL) and ethanol (EtOH, mg/100 mL) content in Tarocco oranges subjected to cold quarantine for 3 weeks at 1 C, 4 additional days of simulated transport at 3 C (quarantine + transport) and subsequent 3 days of simulated shelf-life (quarantine + transport + SL).a Treatments Harvest Quarantine + transport + SL Water, 20 C Water, 53 C Water, 56 C Water, 59 C
a

Dip time (s)

SST 11.2

TA 1.38 1.20 ab 1.23 a 1.18 b 1.13 c

SST/TA 8.07 9.12 b 8.84 b 9.12 b 9.56 a

MeCHO 0.37 0.83 b 0.81 b 0.86 b 0.94 a

EtOH 21.0 55.9 c 62.8 bc 77.1 b 100.6 a

60 60 30 15

10.94ns 10.87 10.76 10.80

Values within columns followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05; ns = not signicant.

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A. Palma et al. / Postharvest Biology and Technology 78 (2013) 2433

Table 5 Inuence of postharvest treatments on sucrose, fructose, glucose, malic acid, and citric acid in Tarocco oranges subjected to cold quarantine for 3 weeks at 1 C, 4 additional days of simulated transport at 3 C (quarantine + transport) and subsequent 3 days of simulated shelf life (quarantine + transport + SL). All parameters are expressed as g/100 mL).a Treatments Harvest Quarantine + transport + SL 60 Water, 20 C Water, 53 C 60 Water, 56 C 30 Water, 59 C 15
a

Dip time (s)

Sucrose 5.22 4.82ns 4.79 4.79 4.72

Fructose 2.16 2.52 a 2.41ab 2.33 b 2.29 b

Glucose 1.93 2.15ns 2.12 2.03 2.03

Malic acid 0.06 0.038 a 0.031ab 0.024 bc 0.020 c

Citric acid 1.29 1.025 ab 1.081a 1.014 ab 0.967 b

Ascorbic acid 72.3 60.4 a 58.7 ab 58.1 ab 56.6 b

Total phenols 105.6 102.4ns 102.2 101.9 100.8

Total anthocyanins 0.82 1.16 a 1.15 a 0.99 ab 0.90 b

Antioxidant activity 31.4 22.5ns 23.5 23.3 23.1

Values within columns followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05; ns = not signicant.

Table 6 Inuence of postharvest treatments on individual anthocyanins (mg/100 mL) in Tarocco oranges subjected to cold quarantine for 3 weeks at 1 C, 4 additional days of simulated transport at 3 C and subsequent 3 days of simulated shelf life (quarantine + transport + SL).a Treatments Harvest Quarantine + transport + SL 60 Water, 20 C Water, 53 C 60 Water, 56 C 30 15 Water, 59 C
a

Dip time (s)

Cyanidin 3-5 diglucoside 0.032 0.045 a 0.043 a 0.041 a 0.036 b

Cyanidin 3-glucoside 0.244 0.284ns 0.308 0.258 0.269

Delphinidin 3-6 malonyl glucoside 0.042 0.068 b 0.097 a 0.058 b 0.064 b

Cyanidin-3-malonyl glycoside 0.318 0.403 a 0.404 a 0.367 ab 0.326 b

Cyanidin 3-dioxalonil glucoside 0.057 0.077ns 0.080 0.086 0.077

Peonydin 3-6-maloniyl glucoside 0.097 0.088ns 0.097 0.092 0.088

Values within columns followed by unlike letters differ signicantly by Fishers least signicant difference (LSD) procedure, P 0.05; ns = not signicant.

during quarantine and transport, the short shelf-life period, along with the low levels of anthocyanins and the high content of total acids, may account for the relatively small degradation rate of AA in the juice (Nagy, 1980; Poei-Langston and Wrolstad, 1981). Differences in total phenols and antioxidant activity among treatments after shelf-life were not signicant. After shelf-life, total anthocyanin levels were higher than in freshly harvested fruit, in agreement with previous studies on red oranges stored at low temperatures (Rapisarda et al., 2001; Lo Piero et al., 2005). Fruit treated at 59 C had lower values of total anthocyanins than control fruit and fruit treated at 53 C. Individual anthocyanins were not affected by treatments, with the exception of delphinidin-36-malonyl-glucoside that showed signicantly higher values in fruit treated at 53 C and cyanidin-3-5-diglucoside and cyanidin-3malonyl-glycoside with lower values in fruit treated at 59 C. The highest pigmentation in juice of fruit treated at 53 C and control was also conrmed by the higher values of a*/b* ratio (Table 3). Various factors are known to affect the effectiveness of heat therapy against decay-causing agents. These include the moisture content of spores, age of the inoculum and inoculum concentration and host (Fallik and Lurie, 2007). In addition, the sensitivity of fungal spores to heat treatments considerable depends by species (Sommer et al., 1967; Castejon-Munoz and Bollen, 1993). Previous studies investigated the benecial effects of hot water dips for several minutes to control green mold (Houck, 1967; Smoot and Melvin, 1965), blue mold (Palou et al., 2001), and brown rot caused by Phytophtora spp. in citrus fruit (Feld et al., 1979; KIotz and DeWolfe, 1961). Schirra et al. (2004) showed the potential of postharvest hot water dips for 180 s at 50 C to control CI and decay of blood oranges after quarantine. The benecial effects of hot water treatment on decay control were related to heat impact on pathogens and on redistribution of ECW layer which may improve physical barriers to wound pathogen penetration and reduce disease incidence caused by green and blue mold decay in citrus fruit (Schirra et al., 2000a, 2011). However, that immersion time (180 s) was longer than that used in most commercial installations (1560 s). Yet, when short (1560 s) treatment times are applied, the water temperature should be increased to achieve the benecial effects of heat in terms of inhibition of pathogens, activation of host

defense responses, enhanced diffusion and penetration of a.i. into cuticular wax, and increased permeation of a.i. into rind wounds (Schirra et al., 2000a, 2011). Present results have shown that hot water treatments at 5356 C for 6030 s effectively reduced decay development in Tarocco oranges, without impairing fruit quality. However, better results were obtained when hot water at 5356 C was combined with 300 mL TBZ. In addition, treatments with 300 mL TBZ at 5356 C for 6030 s showed a better performance than unheated TBZ as less than 1/3 of active ingredient was required in the treatment bath to produce similar TBZ residues in fruit and to achieve a similar control of decay, as a result of heat in reducing the diffusion barrier of fruit cuticle to active ingredient penetration. Conversely, treatment at 59 C cannot be applied to Tarocco oranges being close to threshold for treatment damage, as revealed by the reduced efcacy of TBZ in controlling decay. Acknowledgements The authors gratefully acknowledge Mr. Salvatore Marceddu for technical assistance in SEM analysis. Research supported by CNR-MiUR, Sviluppo delle Esportazioni di Prodotti Agroalimentari del Mezzogiorno. References
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