Cold quarantine responses of ‘Tarocco’ oranges to short hot water and

Postharvest Biology and Technology 78 (2013) 24–33

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Postharvest Biology and Technology

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Cold quarantine responses of ‘Tarocco’ oranges to short hot water andthiabendazole postharvest dip treatments

A. Palmaa, S. D’Aquinoa, S. Vanadiab, A. Angionic, M. Schirraa,!

a C.N.R. Institute of Sciences of Food Production, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italyb C.N.R. Institute of Sciences of Food Production, via Amendola 122/O, 70126 Bari, Italyc 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

Article history:Received 6 September 2012Accepted 9 December 2012

Keywords:CitrusDecayEpicuticular wax morphologyHeat treatmentThiabendazole residuesStorage

a b s t r a c t

This study investigated the effects of brief hot water and thiabendazole (TBZ) postharvest dip treatmentson ultrastructural changes of fruit epicuticular wax (ECW), TBZ residues, decay development and qualitytraits of ‘Tarocco’ oranges [Citrus sinensis (L.) Osbek] subjected to cold quarantine, subsequent simulatedtransport and shelf-life. Commercially mature fruit were submerged in water at 20 "C (control fruit) orTBZ 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 "Cfor 60, 30, and 15 s respectively. Following treatments, fruit were stored for 3 weeks at 1 "C (simulatedquarantine conditions for fruit disinfestations against Mediterranean fruit fly, Medfly), followed by 4 daysat 3 "C (simulated long distance transport), and finally kept at 20 "C for 3 days (shelf-life, SL). Scanningelectron 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 hotwater dips at 53–59 "C for 60–15 s, become visible again after storage for 21 days at 1 "C (quarantineconditions), and changes involving the appearance of rough ultrastructure, presence large curled plates,fissured wax crusts, and areas with ECW deficiencies, 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 producedsimilar TBZ residue levels in fruit, without impairing fruit quality traits such as visual appearance, weightloss, compression test, sensory attributes, juice color parameters (a*, b*, h, L*, and Chroma), and juicechemical characteristics (soluble solids content, titratable acidity, ascorbic acid, glucose, sucrose, citricacid, total phenols, total anthocyanins, and total antioxidant activity).

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Among the orange cultivars, ‘Moro’, ‘Sanguinello’ and ‘Tarocco’are the most cultivated in Italy. Fruit of these cultivars areappreciated by consumers for their typical taste and peculiar char-acteristics of blood-red flesh and rind color, owing to the presenceof phenolic compounds belonging to the class of anthocyanins(Dugo et al., 2003), some of which are known to have several healthpromoting properties (e.g. beneficial effects on capillary fragilityand arteriosclerosis, and antiviral activity, preventing allergies andother inflammatory diseases, anti-cancer activity) (Sajia, 1994).Therefore, from a dietetic viewpoint, anthocyanins represent anadded value for blood oranges due to their important therapeutic

! Corresponding author. Tel.: +39 0783 33224; fax: +39 0783 33959.E-mail address: [email protected] (M. Schirra).

properties, in addition to the well known health benefits of citrusfruit (Attaway and Moore, 1992).

Various citrus-importing countries require quarantine securityprotocols to diminish the risk of accidental introduction of theMediterranean fruit fly (Medfly, Ceratitis capitata), and fruit mustbe certified Medfly free. Therefore Medfly disinfestations shouldbe performed by citrus producing-countries before exportation.Heat treatments (hot water immersion, high temperature forcedair, vapor heat) (Armstrong and Mangan, 2007) and cold quaran-tine treatments (exposure of fruit to near-freezing temperaturesfor 10–16 days) (Armstrong, 1994), are technologies applied ona commercial scale for postharvest insect control of horticulturalcrops as alternatives to chemical fumigation (e.g. methyl bromide).Commodity response to quarantine treatment varies depending onspecies, cultivar, and quarantine treatment conditions.

Quarantine treatments reaching a fruit core temperature of44 "C for 100 min or 46 "C for 50 min did not cause damage orfruit softening in ‘Olinda’ and ‘Campbell’ oranges (Schirra et al.,

0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.postharvbio.2012.12.002

A. Palma et al. / Postharvest Biology and Technology 78 (2013) 24–33 25

2005a) but produced deleterious effects on the quality traits ofblood oranges, such as development of off-flavors and off-tastes,decreased fruit firmness and reduced fruit resistance to decay(Mulas et al., 2001); other methods such as cold quarantine there-fore should be applied to reduce treatment impact on quality(Schirra et al., 2004). On the other hand, cold quarantine can leadto chilling injury (CI) on blood oranges and the application of a hotwater dip at 50 !C for 180 s before cold disinfestation was able toovercome 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 waterdip treatments at 50 !C for 180 s in alleviating CI in citrus fruit,notably improve when applied in combination with the fungicidethiabendazole (TBZ) (Schirra et al., 1998, 2000a). Reduced treat-ment time would be desirable to increase packinghouse output andshorten delays in fruit marketing, therefore treatment tempera-ture should be raised to produce the heat-induced beneficial effectsin terms of physical changes of ECW, host defensive responsesand inhibition of pathogen development (Schirra et al., 2000b,2011).

However, no data are available in the literature on the influ-ence of short hot water treatments used in commercial installationsand its combination with TBZ on ultrastructural changes of fruitsurface, TBZ residues, decay incidence, external and internal fruitquality attributes of blood oranges exposed to a quarantine treat-ment. The present investigation was therefore planned to studythe cold quarantine responses of ‘Tarocco’ blood oranges to short(15–60 s) hot water dip treatments, without or with TBZ onphysical, nutritional and functional properties of fruit. The poten-tial relevance of treatment-induced changes on TBZ residues,CI and decay control after quarantine was also evaluated anddiscussed.

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 Sar-dinia (Italy) and transported to the laboratory immediately afterharvest. Visibly sound fruit free of defect were washed and subdi-vided into 8 groups corresponding to the following dip treatments:(a) water alone at 20 !C (control) or in combination with 1000 mg/LTBZ (TECTO SC, Syngenta Crop Protection S.p.A., Milan, Italy, at42.9% active ingredient, a.i.) for 60 s (standard treatment); (b) wateralone at 53, 56, or 59 !C for 60, 30, and 15 s respectively, or incombination with 300 mg/L TBZ. The concentration of TBZ in thetreatments at 53–59 !C was set based on preliminary trials per-formed in our laboratory.

Following treatments fruit were left to dry at room temper-ature 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 (quar-antine + transport + SL). Each treatment was repeated threefold(replications).

2.2. Scanning Electron Microscopy (SEM) analysis

SEM observations were performed following treatments, afterquarantine and after shelf-life. Samples of five fruit per treatmentwere used for SEM analysis. Two rind samples (2 cm " 2 cm) wereexcised with a razor blade from the equatorial zone of each fruitand immediately fixed in a phosphate buffer (pH 7.4) containing3% glutaraldehyde, and kept at 4 !C until further preparation. Thefixed tissue was rinsed three times in phosphate buffer (pH 7.4)

and then three times in deionized water. They were dried by wash-ing with increasing concentrations of ethanol (20, 50, 70, 80, 95,and 100%), the samples being left for 20 min before each wash. Thedried samples were placed on aluminum stubs using silver con-ductive glue, and a gold–palladium coating was applied with anEdwards S-150 A sputter coater. Until observation, samples werestored in a vacuum dryer. SEM was carried out with a ZEISS DSM962 microscope at 30 kV and 250–2000" magnifications. RelevantSEM micrographs at 250" magnification are shown in the figures.

2.3. Analysis of TBZ residues

TBZ analyses were performed following treatment, after quar-antine, after simulated transport and after shelf-life. Six orangesper replication (4 replications per treatment) were weighed, andthe peel was removed. The peel was weighed and its percentagewith respect to the whole fruit calculated. Then, peel samples werekept frozen at #40 !C until analysis. TBZ analyses were carried outon four replicates of six fruit per treatment, according to Schirraet al. (2008). Briefly, 5 g of homogenized peel were weighed in a40 mL screw-capped flask and 10 mL of ethyl acetate/hexane (1/1)and 2 g of NaCl were added. The mixture was agitated in a rota-tory shaker for 30 min. Subsequently, the phases were allowed toseparate and 1 mL of the organic layer was dried under a gentlenitrogen stream and the residues were dissolved with 1 mL of ace-tone and injected in GC–ITMS for the analysis without any clean-upstep.

2.4. Visual assessment, fruit weight loss, compression test

To determine visual assessment, fruit weight loss and compres-sion test, each treatment group was divided into three subgroupsof three boxes (replications) each. The fruit of the first subgroup (40fruit per replication) were used for overall visual appearance, chil-ling injury (CI), decay and the appearance and absence of the calyx.Fruit of the second subgroup (40 fruit per replication) were used forweight loss determination, whereas fruit of the remaining group(10 fruit per replication) were used for compression test. Overallappearance was evaluated on the basis of the following hedonicscale: 1 = very poor; 3 = poor; 5 = fair; 7 = good; 9 = excellent. CI wasevaluated as pitting or scald of the peel, was rated using a 0–4 sub-jective scale, where 0 = none, 1 = slight, 2 = moderate, and 4 = severeinjury. Finally, a weighted average from a chilling index was calcu-lated. The appearance and absence of the calyx were determinedand the percentage of fruit showing the calyx dried and brownor detached was calculated. Decay incidence was assessed as per-centage of rotten fruit caused by various fungi. To determine fruitweight loss, fruit were individually weighed following treatmentand after selected periods and the percentage of weight loss cal-culated. A compression test was performed by a texture–meterinterfaced with a computerized system (DO-FB0.5TS, Zwick Roell,Germany). The compression curves were obtained by deform-ing the fruits with a metal disk (15 cm diameter) at a speed of100 mm/min. Values were expressed as maximum deformation at3 kg compression force.

2.5. Juice chemical analysis

Chemical analyses of juice were performed following treatmentand after shelf-life. Juice was obtained by squeezing 10 fruit perreplication (3 replications per treatment) with a commercial juicer.Before analyses, the juice was centrifuged (Centurion Scientific Ltd.,West Sussex, England) at 13,000 " g for 15 min and the supernatantfiltered through a 0.45 !m acetate cellulose filter. Titratable acid-ity (TA) of the juice was determined using a potentiometric titrator(Metrom 720 SM Tritino, Swiss); 5 mL of juice diluted in 45 mL

26 A. Palma et al. / Postharvest Biology and Technology 78 (2013) 24–33

distilled water were titrated with NaOH 0.1 N up to pH 8.1. Theresults were expressed as percentage of anhydrous citric acid. Thepercentage of total soluble solids (SSC) was measured by a digitalrefractometer (PR-101, Atago, Japan).

Organic acids and ascorbic acid (AA) were determined with aMerck-Hitachi (Tokyo, Japan) liquid chromatograph with an L-7455photodiode detector (DAD) detector, D-7000 system manager,L7200 autosampler and L-7100 pumps. Simultaneous separa-tion and determination of organic acids and ascorbic acid wereachieved according to the procedure described by Yuan and Chen(1999) and by Chinnici et al. (2005) using a Bio-Rad cationguard column and a Bio-Rad Aminex HPX-87H Hydrogen formcation exchange resin-based column (300 mm ! 7.8 mm i.d.) at40 "C. The mobile phase consisted of 0.005 M sulphuric acidaqueous solution and the samples were isocratically separatedat 0.6 mL/min. Peaks of organic acids and ascorbic acid weremeasured at wavelengths of 210 and 245 nm respectively andwere identified by comparing retention times with those ofstandards and quantification was carried out using externalstandards.

Total phenolic content was analyzed according to the Folin-Ciocalteau colorimetric method (Singleton and Rossi, 1965). Totalphenols 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 amethanol solution of 0.16 mM DPPH was allowed to react for 15 minin a cuvette. The inhibition percentage of the absorbance at 515 nmof DPPH solution added with sample was calculated using the fol-lowing equation: Inhibition % = (Abst=0 # Abst=15 min)/Abst=0 ! 100.

Total anthocyanin contents were determined spectrophotomet-rically 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 aLaChrom Merck-Hitachi liquid chromatograph (Hitachi Ltd., Tokyo,Japan) equipped with a L-7455 photodiode detector (DAD). A LunaC18 column (150 mm ! 4.6 mm, 3 !m, Phenomenex, Castel Mag-giore, 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 thefollowing elution profile: flow rate 0.5 mL/min, t = 0 10% solventB (acetonitrile)/90% solvent A (formic acid 1%, acetic acid 2%),t = 20 20% B linear gradient, t = 38 30% B, post time 12 min. Thechromatogram was monitored simultaneously at 360 and 520 nm.Quantitative analysis of anthocyanins was carried out using theexternal standards method and their concentration was expressedas cyanidin-3-glucoside equivalents. Anthocyanins were identifiedby LC-electrospray ionization (ESI)–MS analysis using an AgilentTechnologies (Palo Alto, CA, USA) 1100 series LC/MSD. Two mLof juice were placed in a 10 mL headspace vial and incubatefor 2 h in a shaking bath at 60 "C, for acetaldehyde and ethanoldetermination. Then a 1 mL headspace gas sample was injectedinto gas chromatograph Agilent 6890 fitted with a flame ionizationdetector (FID) and equipped with a 5% Carbowax 20 M on 80/120Carbograph 1 AW packed column. Run conditions were: N2 ascarrier gas at 30 mL/min; injector at 130 "C; oven 80 "C; detector at150 "C. Ethanol and acetaldehyde were quantified by comparisonof peak area versus concentration of a calibration curve obtainedfrom pure analytical standards subjected to the same analyticalconditions (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 Var-ian Cary 50 spectrophotometer equipped with a Cary Win UVcolor software. CIE L*, a*, b*, Hue angle (h = arctg b*/a*) and Chromavalues (Chroma =

!(a$)2 + (b$)2) were calculated using illumi-

nant D65 and 10" observer, according to the CIE L*, a*, b* 76

Convention (McLaren, 1980). All measurements were done intriplicate.

2.6. Statistical analysis

Statistical analysis was performed by Statgraphics Centurionsoftware (Herndon, VA, USA), version XV Professional statisticalprogram. Analysis of variance (ANOVA) was carried out accordingto a single factor, complete randomized block design with threereplicates for each treatment. Percentages were subjected to theANOVA after transformation in arcsin

%x or

%x before the ANOVA,

depending on the range of variation of data. As fruit treated withwater alone or in combination with TBZ did not reveal significantdifferences (P > 0.05) in color or chemical traits, further statisticalanalyses for these parameters were pooled, Mean comparisons ofthe effects of treatments were calculated, when applicable, by theleast significant difference test at P & 0.05.

3. Results and discussion

3.1. SEM analysis

3.1.1. Following treatmentThe surface of fruit dipped in water at 20 "C (control fruit) dis-

played the typical ultrastructure of mature orange fruit (Cajusteet al., 2010) with areas covered by smooth thin layers of ECW,irregular in shape, raised or folded outwards around edges, andareas free of ECW (Fig. 1a). Cuticular ridges, rough platelets, andwax granules were observed at higher magnification (SEM micro-graph not shown). In contrast, the ECW of fruit treated with TBZ at20 "C (standard treatment) appeared more homogenous than con-trol 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 for-mulation able to enhance the effectiveness of the a.i. increasingwetting, spreading, penetration and retention on the plant cuti-cle (Schönherr and Baur, 1996). Fungicide formulation interactionswith the plant cuticle are complex (Baur, 1998). However, thereis evidence that surfactants can considerably modify the ECW finestructure (Kuzyc and Meggitt, 1983; Noga et al., 1987) presum-ably by selective solubilization of ECW constituents (Tamura et al.,2001). Thus, results of our study suggest that the loss of details ofECW fine structure in fruit treated with TBZ may be ascribed toselective solubilization of ECW by surfactant present in the com-mercial formulation of TBZ fungicide mixture.

Dip treatments with water at 53 "C for 60 s caused the disap-pearance of ECW layers: the fine structures of ECW collapsed andsome 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 "Cfor 15 s (Fig. 4a) showed the loss of the ultrastructural features offruit surface, the platelets tended to ‘relax’ and melt, covering theareas free of ECW, and creating a smoother appearance.

TBZ application at 53 "C resulted in a partial melting of ECW, butwax layers were hardly visible (Fig. 2b), and no difference could bedetected with the treatments at 20 "C. When treatments with TBZwere performed at 56 or 59 "C (Figs. 3b and 4b) changes in the ECWmicromorphology were more evident, and a higher level of waxmelting could be assessed at 59 "C (Fig. 3b versus Fig. 4b). The differ-ences in ECW fine structure of fruit subjected to hot water at 59 "Cwithout or with TBZ were negligible. A similar behavior involvingchanges in ECW ultrastructure of fruit treated with hot water wasreported previously (Schirra and D’hallewin, 1997; Schirra et al.,2005b; Dore et al., 2009). These features were ascribed to the partialmelting and redistribution of ECW layer along fruit surface.


Fig. 1. SEM micrographs (250! magnifications) 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 quarantineThe wax layer of control fruit became rough, and the lifting

around edges of wax plates was increased (Fig. 1c). Build up ofcrystalline wax granules was also observed and areas free of ECWenlarged. Wax platelets of an indefinite design and stomata uncov-ered by wax were visible at higher magnification (SEM micrographnot shown).

Fruit treated with TBZ at 20 "C displayed similar features as con-trol fruit but the areas uncovered by ECW were less extensive in size(Fig. 1d versus Fig. 1c). The micromorphology of the cuticular sur-face of fruit treated with water at 53–59 "C was similar, but wasdifferent as compared to control fruit (Figs. 2c, 3c and 4c versusFig. 1c).

Fruit treated with TBZ at 53 "C (Fig. 2d) showed very irregularwax scabs, partially detached from cuticle, with large edges com-pletely curled outwards, whereas ultrastructural changes of fruittreated with TBZ at 56 "C (Fig. 3d) were only small. By contrast,fruit treated with TBZ at 59 "C exhibited large irregular ECW lay-ers, 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 andextension of wax deprived zones.

3.1.3. After shelf-lifeThe control fruit and fruit treated with water at 53 or 59 "C

showed changes involving the appearance of rough structures withwax deposits, a network of large curled plates, and large areas withECW along with skin surface, and ECW deficiencies became muchmore pronounced than those observed after quarantine, whereastreatments at 56 "C showed less differences compared with quar-antined fruits (Figs. 1–4e). Surface details of fine structure of TBZtreated fruit (Figs. 1–4f) were generally less visible than control andfruit treated with water at 53–59 "C. In addition, only fruit treatedwith TBZ at 56 "C (Fig. 3f) maintained a homogeneous shape, with-out the appearance of ECW free zones. The appearance of fissuredwax crusts along with large areas without ECW, may be relatedto loss of elasticity of waxy layer and the subsequent detachmentof wax plates, factors that may be ascribed to fruit senescence.


Fig. 2. SEM micrographs (250! magnifications) 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 beneficial effect of hot water treatment in terms ofpartial melting and redistribution of ECW layer along fruit surfaceand subsequent closure of microwounds which represent potentialgaps for conidia of wound pathogens (Schirra et al., 2000b, 2011),are only transitory and are lost during fruit storage. The differenteffect on fruit surface of TBZ treatment at 56 "C is consistent in allpostharvest steps and indicates that this was the best temperaturefor the treatment.

3.2. Influence of treatment and storage conditions on TBZresidues in fruit

Immediately after TBZ treatments at 1000 mg/L and 20 "C or at300 mg/L and 53–59 "C, residue levels in fruit were similar (Table 1).Previous studies on ‘Tarocco’ oranges (Schirra et al., 1998) showedthat fruit treated with TBZ at 50 "C experienced a greater persis-tence of TBZ with respect to fruit treated at room temperatureand this occurrence was related to the better encapsulation andcoverage of the a.i. by ECW, thus providing better protection to

the fungicide. Results of the present study displayed a reversetrend, TBZ persistence being higher in fruit treated at 20 "C witha decline after shelf-life of ca 12% versus ca 39% in fruit treated at53–59 "C. However, treatment duration used in this study was short(15–60 s), being the same as that employed commercially in manylocations in the US, whereas much longer periods (180 s) used inprevious studies (Schirra et al., 1998) resulted in a greater impactof heat on ‘rearrangement’ of ECW structure and TBZ persistence.

3.3. Influence of treatments on CI, decay, and fruit qualityattributes

The incidence of fruit affected by CI was very small after quar-antine and subsequent simulated transport (data not shown) andwas relatively low also after shelf life (Table 2). CI incidencewas reduced in fruit treated at 53 "C and remarkably reduced inthe other fruit samples. It is recognized that cold quarantinedblood oranges are susceptible to CI, especially when fruits arereturned to warm temperature (Schirra et al., 2004). The lack of


Fig. 3. SEM micrographs (250! magnifications) 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 (a–d and f).

CI found in our study was related to the relatively short period ofsimulated transport and shelf-life. The percentage of fruit show-ing the calyx dried and brown or detached was not significantlyaffected by treatments (data not shown). Pooled data (mean val-ues ± standard deviation) of this percentage after shelf life averaged34.4 ± 5.4.

Overall appearance was rated as very good up to quaran-tine + storage (rate 8–8.5), without significant differences amongtreatments (data not shown), whereas after shelf-life all treatedfruit were judged as good to very good (rate 7.1–7.5) and fairly good(rate 6) for the control fruit (Table 2). The lower score received bycontrol fruit was related to the higher incidence of CI. There was no

Table 1Thiabendazole (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 additionaldays 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 Quarantine Transport Shelf life

1000 mg/LTBZ 20 "C 60 6.65 a(a) 6.65 a(a) 6.37 ab(ab) 5.87 a(b)300 mg/L TBZ 53 "C 60 6.96 a(a) 5.78 b(b) 5.36 b(b) 4.26 b(c)300 mg/L TBZ 56 "C 30 6.56 a(a) 5.63 b(b) 5.72 b(b) 4.04 b(c)300 mg/L TBZ 59 "C 15 6.31 ab(a) 5.59 b(a) 5.64 b(a) 3.89 b(b)

a Values within columns (without brackets) or rows (in brackets) followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure,P # 0.05. Letters without brackets relate to comparisons of the influence of treatments within each storage time. Letters in brackets relate to comparisons of the effect ofstorage time within each treatment.


Fig. 4. SEM micrographs (250! magnifications) 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 subsequenttransport decay incidence averaged about 8% (data not shown),reaching approximately 30% after shelf-life (Table 1). The causalagents of decay were green mold and, to a lesser extent, blue moldcaused by Penicillium digitatum and P. italicum respectively (data

not shown). Treatment with 300 mg/L TBZ at 53 or 56 "C signifi-cantly reduced decay development with respect to control fruit,whereas the effect of the other treatments was not significant.After shelf-life, treatments with hot water alone at 53–59 "C sig-nificantly reduced the incidence of decay with respect to control

Table 2Influence 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 Dip time (s) Chilling injury Overall appearance Decay (%)

Water, 20 "C 60 0.60 a 6.1 b 28.9 aWater, 53 "C 60 0.30 b 7.2 a 13.3 bWater, 56 "C 30 0.08 c 7.1 a 15.6 bWater, 59 "C 15 0.09 c 7.2 a 16.7 b1000 mg/L TBZ, 20 "C 60 0.07 c 7.2 a 23.6 ab300 mg/L TBZ, 53 "C 60 0.00 c 7.5 a 3.3 cd300 mg/L TBZ, 56 "C 30 0.02 c 7.1 a 1.1 d300 mg/L TBZ, 59 "C 15 0.05 c 7.3 a 4.4 c

a Chilling injury index, rating scale 0–4; overall appearance, rating scale 0–9. Values within columns followed by unlike letters differ significantly by Fisher’s least significantdifference (LSD) procedure, P # 0.05.


Table 3Influence 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 Dip time (s) L* a* b* a*/b* h Chroma*

Harvest 98.79 1.23 1.19 1.03 44.14 1.71

Quarantine + transport + SLWater, 20 !C 60 98.92ns 1.68 a 1.04 b 1.61 a 31.71 b 1.97ns

Water, 53 !C 60 98.62 1.60 a 1.03 b 1.59 a 32.14 b 2.14Water, 56 !C 30 98.71 1.37 ab 1.04 b 1.31 ab 37.98 ab 1.90Water, 59 !C 15 98.42 1.15 b 1.17 a 0.98 b 45.50 a 1.64

a Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure, P " 0.05.

fruit (Table 2). Best results in controlling decay were achieved withTBZ at 1000 mg/L and 20 !C (2.5% decay), and with 300 mg/L TBZ at53–56 !C (0.8–3.3% decay). Conversely the influence of TBZ at 59 !Con decay control was not significant. The lack of efficacy of TBZ at59 !C in controlling decay may be ascribed to physiological stressof the peel, which might weaken the natural defense propertiesof fruit, although visible symptoms of treatment damage were notdetected.

Fruit weight loss and fruit compression test values were notaffected by treatments (data not shown). Pooled data (mean val-ues ± standard deviation) of weight loss averaged 2.31 ± 0.71 and3.11 ± 0.75 after transport and shelf-life, respectively. At harvestcompression test values averaged 4.81 ± 0.21, whereas after trans-port and shelf-life were 7.11 ± 0.77 and 7.43 ± 0.66, respectively.

L*, and Chroma parameters were not significantly affected bytreatments, whereas treatment at 59 !C caused a decrease in redpigmentation of juice as supported by lower values of a*, highervalues of b*, and consequently lower values of the a*/b* ratio, witha higher h, with respect to control fruit (Table 3).

Treatments at 53 and 56 !C did not affect SSC, TA, or the SSC-TAratio, according to previous studies on ‘Fortune’ mandarins sub-merged in water at 50–58 !C for 180 s (Schirra and D’hallewin,1997). However, when water was applied on ‘Tarocco’ oranges at59 !C for 15 s, TA values were lower than control fruit and SSC-TAratio increased accordingly (Table 4). Acetaldehyde levels increasedin fruit treated at 59 !C. Fruit treated at 56 and 59 !C registeredrespectively 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 citrusfruit was related to the accumulation of ethanol in the juice (Cohenet al., 1990), which may cause the development of off-flavors(Cohen et al., 1990; Hagenmaier, 2002). However, the present studyshowed that the increases of ethanol found in ‘Tarocco’ orangestreated at 56 and 59 !C did not adversely affect fruit sensory qualityand no visible treatment damage occurred. By contrast, a dramaticaccumulation of ethanol was found in previous studies on ‘For-tune’ mandarins subjected to hot water dips at 58 !C for 180 s andstored for 30 days at 6 !C plus three days of simulated shelf-life at20 !C (Schirra and D’hallewin, 1997). After shelf-life, fruit appeared

brown and aged and the taste was judged poor due to the off-flavordevelopment. Such adverse effects on fruit quality observed in ‘For-tune’ mandarins were ascribed to phytotoxic effects caused bythe excessive temperature (58 !C) and prolonged exposure of fruitto heat (180 s) (Schirra and D’hallewin, 1997). Treatment damageand off-flavor development were reported in ‘Satsuma’ mandarinswhen submerged in water for 120 s at temperatures higher than50 !C (Ghasemnezhad et al., 2008). It is recognized that mandarinsare much more prone to develop off-flavors than other citrus vari-eties (Shi et al., 2005). This occurrence was ascribed to the lowpermeability of mandarin peel to gas which favors the build-up ofvolatiles, such as ethanol and acetaldehyde in the juice, and theconsequent perception of off-flavors (Shi et al., 2007). However,compounds other than ethanol have been related with the off-flavor development in citrus fruit. In blood oranges variations offree and bound hydroxycinnamic acids levels during storage wereproven to be a reliable index of detrimental off-flavors development(Fallico et al., 1996).

The concentrations of sucrose, glucose, and citric acid were notsignificantly affected by treatments, whereas lower values of fruc-tose and malic acid were recorded in fruit treated at 56 or 59 !C(Table 5).

Investigations on blood oranges revealed that hot water dippingfor 180 s at 50 !C did not significantly affect the content of AA dur-ing or after quarantine (Schirra et al., 2004). Accordingly, resultsof this study showed that differences in AA content between fruittreated at 53–56 !C and control fruit were not significant (Table 6).Various reports have shown that vitamin C loss in citrus fruit duringstorage at different temperatures is slight (Nagy, 1980). However,significantly lower AA values were recorded in fruit treated at 59 !Cindicating the stress conditions caused by this treatment. Simi-lar adverse effects on AA levels were observed in blood orangesexposed to a fruit core temperature of 44 !C for 100 min or 46 !C for50 min (Mulas et al., 2001) and in mature green tomatoes (Lycop-ersicum esculentum Mill.) after heat treatment at 38 !C and 50% RHfor 24 h (Yahia et al., 2001).

After shelf-life there was a relatively small decline (ca 16–22%)in AA with respect to fresh fruit, supporting previous studies onblood oranges (Schirra et al., 2004). The low storage temperatures

Table 4Influence 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 Dip time (s) SST TA SST/TA MeCHO EtOH

Harvest 11.2 1.38 8.07 0.37 21.0

Quarantine + transport + SLWater, 20 !C 60 10.94ns 1.20 ab 9.12 b 0.83 b 55.9 cWater, 53 !C 60 10.87 1.23 a 8.84 b 0.81 b 62.8 bcWater, 56 !C 30 10.76 1.18 b 9.12 b 0.86 b 77.1 bWater, 59 !C 15 10.80 1.13 c 9.56 a 0.94 a 100.6 a

a Values within columns followed by unlike letters differ significantly by Fisher’s least significant difference (LSD) procedure, P " 0.05; ns = not significant.


Table 5Influence 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 additionaldays of simulated transport at 3 !C (quarantine + transport) and subsequent 3 days of simulated shelf life (quarantine + transport + SL). All parameters are expressed asg/100 mL).a

Treatments Dip time (s) Sucrose Fructose Glucose Malic acid Citric acid Ascorbic acid Total phenols Total anthocyanins Antioxidant activity

Harvest 5.22 2.16 1.93 0.06 1.29 72.3 105.6 0.82 31.4

Quarantine + transport + SLWater, 20 !C 60 4.82ns 2.52 a 2.15ns 0.038 a 1.025 ab 60.4 a 102.4ns 1.16 a 22.5ns

Water, 53 !C 60 4.79 2.41ab 2.12 0.031ab 1.081a 58.7 ab 102.2 1.15 a 23.5Water, 56 !C 30 4.79 2.33 b 2.03 0.024 bc 1.014 ab 58.1 ab 101.9 0.99 ab 23.3Water, 59 !C 15 4.72 2.29 b 2.03 0.020 c 0.967 b 56.6 b 100.8 0.90 b 23.1


Table 6Influence 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 ofsimulated transport at 3 !C and subsequent 3 days of simulated shelf life (quarantine + transport + SL).a

Treatments Dip time (s) Cyanidin 3-5diglucoside

Cyanidin3-glucoside

Delphinidin 3-6malonyl glucoside

Cyanidin-3-malonylglycoside

Cyanidin 3-dioxalonilglucoside

Peonydin 3-6-maloniylglucoside

Harvest 0.032 0.244 0.042 0.318 0.057 0.097

Quarantine + transport + SLWater, 20 !C 60 0.045 a 0.284ns 0.068 b 0.403 a 0.077ns 0.088ns

Water, 53 !C 60 0.043 a 0.308 0.097 a 0.404 a 0.080 0.097Water, 56 !C 30 0.041 a 0.258 0.058 b 0.367 ab 0.086 0.092Water, 59 !C 15 0.036 b 0.269 0.064 b 0.326 b 0.077 0.088


during quarantine and transport, the short shelf-life period, alongwith the low levels of anthocyanins and the high content of totalacids, may account for the relatively small degradation rate of AAin the juice (Nagy, 1980; Poei-Langston and Wrolstad, 1981).

Differences in total phenols and antioxidant activity amongtreatments after shelf-life were not significant. After shelf-life, totalanthocyanin levels were higher than in freshly harvested fruit, inagreement with previous studies on red oranges stored at lowtemperatures (Rapisarda et al., 2001; Lo Piero et al., 2005). Fruittreated at 59 !C had lower values of total anthocyanins than con-trol fruit and fruit treated at 53 !C. Individual anthocyanins werenot affected by treatments, with the exception of delphinidin-3-6-malonyl-glucoside that showed significantly higher values infruit treated at 53 !C and cyanidin-3-5-diglucoside and cyanidin-3-malonyl-glycoside with lower values in fruit treated at 59 !C. Thehighest pigmentation in juice of fruit treated at 53 !C and controlwas also confirmed by the higher values of a*/b* ratio (Table 3).

Various factors are known to affect the effectiveness of heattherapy against decay-causing agents. These include the moisturecontent of spores, age of the inoculum and inoculum concentra-tion and host (Fallik and Lurie, 2007). In addition, the sensitivity offungal spores to heat treatments considerable depends by species(Sommer et al., 1967; Castejon-Munoz and Bollen, 1993). Previ-ous studies investigated the beneficial effects of hot water dipsfor several minutes to control green mold (Houck, 1967; Smootand Melvin, 1965), blue mold (Palou et al., 2001), and brown rotcaused by Phytophtora spp. in citrus fruit (Feld et al., 1979; KIotzand DeWolfe, 1961). Schirra et al. (2004) showed the potentialof postharvest hot water dips for 180 s at 50 !C to control CI anddecay of blood oranges after quarantine. The beneficial effects of hotwater treatment on decay control were related to heat impact onpathogens and on redistribution of ECW layer which may improvephysical barriers to wound pathogen penetration and reduce dis-ease incidence caused by green and blue mold decay in citrusfruit (Schirra et al., 2000a, 2011). However, that immersion time(180 s) was longer than that used in most commercial installations(15–60 s). Yet, when short (15–60 s) treatment times are applied,the water temperature should be increased to achieve the beneficialeffects of heat in terms of inhibition of pathogens, activation of host

defense responses, enhanced diffusion and penetration of a.i. intocuticular wax, and increased permeation of a.i. into rind wounds(Schirra et al., 2000a, 2011). Present results have shown that hotwater treatments at 53–56 !C for 60–30 s effectively reduced decaydevelopment in ‘Tarocco’ oranges, without impairing fruit qual-ity. However, better results were obtained when hot water at53–56 !C was combined with 300 mL TBZ. In addition, treatmentswith 300 mL TBZ at 53–56 !C for 60–30 s showed a better perfor-mance than unheated TBZ as less than 1/3 of active ingredient wasrequired in the treatment bath to produce similar TBZ residues infruit and to achieve a similar control of decay, as a result of heatin reducing the diffusion barrier of fruit cuticle to active ingredientpenetration. Conversely, treatment at 59 !C cannot be applied to‘Tarocco’ oranges being close to threshold for treatment damage,as revealed by the reduced efficacy of TBZ in controlling decay.

Acknowledgements

The authors gratefully acknowledge Mr. Salvatore Marceddu fortechnical assistance in SEM analysis.

Research supported by CNR-MiUR, ‘Sviluppo delle Esportazionidi Prodotti Agroalimentari del Mezzogiorno’.

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Cold quarantine responses of ‘Tarocco’ oranges to short hot water and