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Postharvest Biology and Technology 60 (2011) 192–201

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Postharvest rind breakdown of ‘Nules Clementine’ mandarin is influenced byethylene application, storage temperature and storage duration

Paul J.R. Cronjea,∗, Graham H. Barrya,1, Marius Huysamerb

a Citrus Research International, Department of Horticultural Science, Stellenbosch University, Private Bag X1, Matieland 7602, Stellenbosch, South Africab Department of Horticultural Science, Stellenbosch University, Private Bag X1, Matieland 7602, Stellenbosch, South Africa

a r t i c l e i n f o

Article history:Received 1 November 2010Accepted 13 January 2011

Keywords:CitrusEthylene degreening‘Nules Clementine’ mandarinRind breakdown (RBD)Physiological disorderCanopy position

a b s t r a c t

The progressive postharvest disorder of ‘Nules Clementine’ mandarin (Citrus reticulata Blanco), referred toas rind breakdown (RBD), starts to develop during storage, about 3–5 weeks after harvest. Variation withinthe tree canopy, i.e. inside or outside canopy positions, as well as postharvest handling practices such asethylene degreening, storage temperature and storage duration, were investigated for their influence onRBD incidence. Two experiments were conducted wherein fruit were subjected, in the first experiment, toethylene degreening and a delay in commencement of cold storage (2004), and, in the second experiment,fruit were sampled from the inside and outside of the canopy and cold-stored at either −0.5 ◦C or 7.5 ◦Cduring 2005 and 2007. Rind pigment and carbohydrate contents as well as rind colour and RBD incidencewere recorded during prolonged storage. The senescence-promoting treatments resulted in an increasein RBD incidence, except for storage at −0.5 ◦C, which resulted in a lower occurrence of RBD. Overall,results indicated that the incidence of RBD was aggravated by senescence-promoting factors during thepostharvest handling of fruit and this is thought to lead to the premature senescence of the flavedo.In addition, fruit position in the tree canopy during fruit development contributed significantly to RBDsensitivity, with inside fruit having significantly higher RBD incidence compared to outside fruit.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

‘Nules Clementine’ mandarin (Citrus reticulata Blanco) is widelyplanted in South Africa (Barry and Rabe, 2004). However, the fruitare prone to developing a progressive postharvest physiologicaldisorder with symptoms becoming visible 3–5 weeks after harvest.Symptom development coincides with the commercial shippingperiod and is therefore extremely problematic as the disorder canlead to tremendous financial losses at this stage of the logisticalsupply chain. This disorder is commonly referred to as rind break-down (RBD) of ‘Nules Clementine’ mandarin in the South Africacitrus industry.

Rind breakdown manifests itself in the flavedo of the rind as ran-domly distributed dark/brown spots associated with the collapseof oil glands, locally described as a “leopard spot” pattern (Fig. 1)(Cronje, 2007). Initial industry studies could not elucidate thecausal factors triggering RBD, but identified pre-harvest growingconditions and particularly fruit position within the canopy, lead-ing to a paler/lighter orange-coloured rind, as possibly influencingthe fruit flavedo sensitivity to RBD (Van Rensburg et al., 1995, 2004).

∗ Corresponding author. Tel.: +27 218082689; fax: +27 218082121.E-mail address: [email protected] (P.J.R. Cronje).

1 Present address: XLnT Citrus, Somerset West, South Africa.

Management of postharvest storage temperatures is the primarytool available to reduce development of fruit senescence and phys-iological disorders. Subsequently, postharvest storage temperaturewas shown to influence the incidence of RBD, with fruit stored at7.5 ◦C having a higher propensity to develop RBD compared to stor-age at 4 or 11 ◦C (Khumalo, 2006). In contrast, storage of citrus fruitbelow a cultivar-specific temperature threshold leads to chillinginjury, seen as “pitting” or “scalding” of the citrus rind, and candrastically reduce product quality (Lafuente and Zacarias, 2006).However, the effect on RBD incidence of storage at a temperatureknown to cause chilling damage of the citrus rind has not beendetermined. The recommended storage and shipping temperatureof mandarins, including ‘Nules Clementine’, is 3.5–4 ◦C, to avoidchilling injury (Kays and Paull, 2004; PPECB, 2010).

In addition to storage temperature, storage duration and ethy-lene treatments have been suggested to affect the incidence of RBDin ‘Nules Clementine’ mandarin (Van Rensburg et al., 1995). Ethy-lene degreening is widely used as a postharvest practice in thecitrus industry to ensure adequate rind colour development. How-ever, the efficacy of this senescence-promoting hormone not onlydepends on the physiological stage of development of the fruit rind,but also on the specific cultivar, and its use can lead to the devel-opment of various rind defects (Krajewski and Pittaway, 2002).In contrast, ethylene, may also protect plant tissues against stress(Yang and Hoffmann, 1984), as reported for Penicillium spp. infec-

0925-5214/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.postharvbio.2011.01.009


P.J.R. Cronje et al. / Postharvest Biology and Technology 60 (2011) 192–201 193

Fig. 1. Rind breakdown (RBD) of ‘Nules Clementine’ mandarin fruit. The two topfruit were stored at 7.5 ◦C and had severe (left) and less severe (right) incidence ofRBD. The bottom fruit were stored at −0.5 ◦C and had less severe incidence of RBD.Typical differences in symptom development due to low temperature storage canbe seen between the bottom and the top fruit. The low temperature leads to a morewatery lesion although still randomly situated in the flavedo.

tion (Marcos et al., 2005), rind staining (Lafuente and Sala, 2002),chilling injury (Porat et al., 1999; Lafuente et al., 2001, 2004) andnon-chilling rind pitting (Cajuste and Lafuente, 2007). However, thedirect impact of ethylene as a commercial degreening treatment onRBD incidence has not been documented.

Several rind disorders of citrus fruit have been related tosuboptimal postharvest handling and ambient environmental con-ditions, especially temperature and humidity. These disordersinclude stem-end rind breakdown (SERB), chilling injury, non-chilling postharvest rind pitting and staining, as well as zebra skinof ‘Satsuma’ mandarin (C. unshiu Marc.) fruit (Petracek et al., 2006).Suboptimal temperature and ethylene degreening management, aswell as wax application, prior to or during the development of thesedisorders, have been related to an increase in specific rind disorders.

Several studies have been conducted to determine which fac-tors affect fruit rind sensitivity (Agustí et al., 2001; Van Rensburget al., 2004). However it is only after the recent success of induc-ing rind pitting by alteration of the rind water potential, that thepossibility now exists to elucidate the causal mechanism of sev-eral related rind disorders (Alférez et al., 2003; Alférez and Burns,2004). The significant alteration of rind water status after trans-ferring fruit from 45 to 95% RH, at a constant temperature, wasidentified as a critical factor in the incidence of rind breakdown of‘Navel’ orange [C. sinensis L. (Osb.)] (Alférez et al., 2005) and non-chilling postharvest rind pitting of ‘Marsh’ grapefruit (C. paradisiMacf.) (Alférez and Burns, 2004). The increased electrolyte leakagetriggered by the shift from low to high RH was proposed by Alférezet al. (2008) to be an indication of the loss of membrane functionand organization. They speculated that an increase of phospholi-pase A2 (PLA2) in ‘Fallglo’ mandarin, a hybrid of ‘Bower’ mandarin[C. reticulata × (C. paradisi × C. reticulata)] and ‘Temple’ tangor (C.reticulata × C. sinensis), and ‘Navel’ orange, plays a prominent rolein the development of pitting symptoms.

The aims of this study were, firstly, to investigate the influ-ence of two postharvest practices on RBD incidence, viz. ethylene

treatment (degreening) and time before commencing cold storage.Secondly, to investigate the difference between the sensitivity offruit from the inside (low light) and outside (high light) of the treecanopy on RBD development during prolonged storage at a chilling(−0.5 ◦C) and non-chilling temperature (7.5 ◦C). Thirdly, to supple-ment the recording of the visual RBD symptom development duringcold storage, carbohydrate and pigment contents of the flavedowere determined. Fourthly, a microscopic investigation was con-ducted on mature fruit flavedo with and without RBD symptomsafter 14 weeks of storage.

2. Materials and methods

2.1. Sites, fruit sampling and postharvest handling

Experiments were conducted using fruit from an orchardof ‘Nules Clementine’ mandarin budded on ‘Carrizo’ citrange{[Poncirus trifoliata (L.) Raf.] × [C. sinensis]} rootstock and plantedin 1991 at a spacing of 4.5 m × 2.5 m on the University of Stel-lenbosch experimental farm Welgevallen, Western Cape province,South Africa.

The fruit were picked at commercial harvest maturity on 16 May2004, 14 May 2005 and 16 May 2007. In 2004, no distinction wasmade in this part of the study between inside and outside fruit. Twobins of fruit (approximately 400 kg each) were harvested, one ofwhich received a degreening treatment (3 d at 3 �L L−1 ethylene,>90% RH, 20–22 ◦C), while the other bin remained in the pack-house (ambient conditions ±20 ◦C) prior to receiving the packhousetreatments. In 2005 and 2007, fruit were sampled at a height of1–2 m from the inside (<80% sun, out of direct sunlight) and out-side (full sunlight exposure, east side of trees) of the canopy ofselected trees. In all cases, the harvested fruit were transported to acommercial packhouse where they were drenched (thiabendazole,1000 mg L−1; guazatine, 500 mg L−1; 2.4-D sodium salt, 250 mg L−1;dimethyldidecyl ammonium chloride, 1000 mg L−1) and degreened(except one treatment in 2004) before receiving all standardcommercial packhouse treatments (thiabendazole, 500 mg L−1;imazalil, 500 mg L−1; 2,4-dichlorophenoxyacetic acid, 125 mg L−1,and polyethylene citrus wax application [Citrushine®, Johannes-burg, South Africa]). Thiabendazole and 2,4-dichlorophenoxyaceticacid were incorporated in the wax, whereas imazalil was appliedin the water bath prior to waxing of the fruit.

2.2. Storage conditions and RBD rating

After packing in 2004, the fruit were separated into eight repli-cates of 25 fruit each, and held under ambient conditions (±20 ◦C)in the shade in the packhouse. The fruit were put into 7.5 ◦C coldstorage after 7 d (maximum time permitted for commercial citrusshipments), 11 d or 14 d, with the fruit destined for the 11 and 14 dtreatments being kept at ambient conditions in the packhouse untilthe time they were cold-stored. Temperature and relative humiditywere not controlled during this period. Cumulative RBD incidencewas recorded every second week.

After packing in 2005 and 2007, the inside and outside fruitwere further sub-divided into two lots for storage at −0.5 ◦C or7.5 ◦C. The duration between harvest and cold storage was 6 d,including 3 d degreening. For each treatment (i.e. inside vs. outsidefruit stored at −0.5 ◦C or 7.5 ◦C), eight replicates of 25 fruit eachwere pre-designated for each evaluation date before being put intocold storage and rated for RBD incidence every second week forthe duration of the experiment. The fruit were brought to roomtemperature for 5 d prior to recording RBD incidence, whereafterdestructive sampling took place to remove the flavedo from eachfruit.


194 P.J.R. Cronje et al. / Postharvest Biology and Technology 60 (2011) 192–201

2.3. Rind colour

Rind colour was determined on each sampling date using a chro-mameter (Minolta NR 4000, Osaka, Japan) on the side of the fruitwith the highest colour development. The flavedo was removedfrom the fruit and pooled for each replicate, ensuring adequateflavedo material for the various analyses. The flavedo was frozenin liquid nitrogen whereafter it was freeze-dried (VirTis Freeze-mobile 25ES, The VirTis Company, Gardiner, NY, USA) and storedat −80 ◦C. These samples were milled to a fine powder which wasthen used for rind pigment and carbohydrate analyses.

2.4. Flavedo pigment analysis

To determine the chlorophyll and carotenoid contents, a 0.2 gsub-sample of the freeze-dried and finely milled, powdered flavedowas added to 10 mL of 95% (v/v) aqueous ethanol solvent containingbutylated hydroxytoluene (BHT) (100 mg L−1) and diethyldithio-carbamate (DDC) (200 mg L−1) antioxidants to prevent carotenoiddegradation. The samples were vortexed for two 1-min intervalsand stored at 4 ◦C for 1.5 h to extract the pigments. Thereafter,the solution was poured through ashless filter paper (Schleicher& Schuell, Dassel, Germany) to remove rind particles. The extractswere poured into disposable plastic cuvettes, and absorbancewas measured at 470, 646 and 664 nm in a spectrophotome-ter (Cary 50 conc UV-visible spectrophotometer, Varian Australia(Pty.) Ltd, Mulgrave, Victoria, Australia). A cuvette filled withthe ethanol/antioxidant solvent was used as a calibration stan-dard. From the absorbance readings, chlorophyll a (Ca), chlorophyllb (Cb), total chlorophylls (Ca + b) and total carotenoids (Cx + c)were calculated as �g g−1 DW using the Lichtenthaler equations(Lichtenthaler, 1987).

Ca = 13.36 A664.2 − 5.19 A648.6Cb = 27.43 A648.6 − 8.12 A644.2Ca+b = 5.24 A664.2 + 22.24 A648.36

Cx+c = 1000 A470 − 2.13 Ca − 97.64 Cb

209

2.5. Flavedo carbohydrate content

From each positional treatment (inside and outside fruit) andtreatment date (weeks 0–14), the flavedo of eight replications(n = 8) of 25 fruit each were used for carbohydrate extraction andanalysis. One extraction was performed per replicate.

2.5.1. Extraction of sugarsSucrose, glucose and fructose were extracted from 0.1 g flavedo

with a 5 mL solution of 60% methanol, 25% chloroform and 15%deionised water (MCW) (Koch and Avigne, 1990). Water wasdeionised through a Millipore water filtration system (RiOs/ElixSynergyPakTM system, Millipore SAS, Molsheim, France). The sam-ple and MCW solution were both vortexed for 2 min and left for16 h at ambient temperatures (18 ± 2 ◦C). The extraction mixturewas centrifuged (3000 × gn, 10 min, 20 ± 1 ◦C) and the supernatantwas collected. One mL MCW was added to the residue, which wasvortexed and thereafter centrifuged (3000 × gn, 10 min, 20 ± 1 ◦C).The clear supernatant was again collected and added to the initialsupernatant. To the pooled MCW extract, 1 mL chloroform was firstadded, followed by 1 mL deionised water. The tube was shaken aftereach addition and finally centrifuged (3000 × gn, 10 min, 20 ± 1 ◦C)to separate the layers. The top, aqueous layer, containing thesugars and phenolics, was collected and evaporated to drynessunder a rotary vacuum centrifuge (SC 210 A Speed Vac® Plus,Thermo Savant, Holbrook, NY, USA). An additional step of filter-ing the extract through a C18 column (preparative C18 125 A,

55–105 �m WAT 0250594, Waters corporation, Milford, MA, USA)was deemed necessary to remove phenolic compounds extractedfrom the flavedo and to extend the life of the HPLC column. Thedried residue was dissolved in 5 mL deionised water. The C18 car-tridge (2 g) was conditioned, first with methanol, then with fourportions of 5 mL deionised water under vacuum (VacMaster SampleProcessing Station, International Sorbent Technology Ltd, Glamor-gan, UK). In a preliminary investigation 2 g C18 was found to beadequate to remove the phenolic compounds from the aqueoussugar extract. One mL of the sugar solution was then purified byC18 cartridge under vacuum into a 10-mL volumetric flask. Eachcartridge was furthermore washed with another four aliquots of2 mL deionised water. The eluate was made up to a final volumeof 10 mL by adding deionised water. The eluate was finally filteredthrough a 0.45 �m filter paper (Millex-HV Hydrophilic PVDF, Mil-lipore Corporation, Billerica, MA, USA) into a vial for HPLC analysis.

2.5.2. Carbohydrate analysisCarbohydrate analysis was performed using high performance

liquid chromatography (Agilent 1100 Series HPLC, Agilent, Wald-bronn, Germany) with an auto sampler (110 Series; HewlettPackard, Waldbronn, Germany) operated by HP ChemStationsoftware (LC Rev.A.06.03 [509], Hewlett Packard, Waldbronn,Germany). A TransgenomicTM ion exchange column (stainlesssteel, 3000 mm × 7.8 mm, model ICSep ICE-99-9850, Transge-nomic, Omaha, NE, USA) and TransgenomicTM guard column(model ICSep-ICE-GC-801, Transgenomic Inc., San Jose, CA, USA)maintained at 30 ◦C were used for analysis of sugars and organicacids. Sugars were separated using 17 mM H2SO4 at a flow rate of0.5 mL min−1. A refractive index detector (model G1352A, Agilent,Waldbronn, Germany) was used to detect the separated sugars. Aninjection volume of 30 �L was used per sample. The sugar contentof the freeze-dried flavedo was expressed as mg g−1 DW.

2.6. Fruit respiration rate

Respiration rate (mg CO2 kg−1 h−1) was repeatedly measuredfrom the same eight replicates of five fruit of each of the treat-ments. The fruit for each replicate were put into eight airtight 1-Ljars and closed for 30 min, while still being kept in the respectivecold rooms (−0.5 ◦C and 7.5 ◦C), before a gas sample was taken fromeach jar using an airtight syringe for injection into a gas chromato-graph (Model N6980, Agilent Inc., Wilmington, USA) fitted with aPorapakQ and a Molsieve packed column. The volumes of free spacein the jar as well as the mass of the fruit were used to calculate CO2production rates.

2.7. Microscopic preparation

Rind samples (5 mm × 5 mm) from fruit stored for 14 weeks,with or without RBD symptoms, were fixed in a 1:1 (v/v) solutionof 2.5% glutaraldehyde and 2.5% formaldehyde in 0.075 M phos-phate buffer (NaPO4), pH 7.4 for 1–2 h at room temperature. Thematerial was rinsed three times for 10 min in the 0.075 M bufferbefore being fixed in 0.5% aqueous osmium tetroxide (OsO4) for1–2 h. Thereafter, the samples were rinsed three times with dis-tilled water, before dehydration in an ethanol concentration series[30%, 50%, 70%, 90% and 100% (repeated three times)]. The mate-rial was stored in 100% ethanol before infiltration for transmissionelectron microscopy (TEM) and light microscopy with 30% Quetolin ethanol for 1 h, followed by 60% Quetol for 1 h and pure Quetol for4 h prior to polymerisation at 60 ◦C for 39 h. The material was cutinto 0.5–1 �m sections with a Reichert ultracutE ultra-microtome(Reichert AG., Vienna, Austria) before being transferred onto waterdroplets on a specimen slide and stained with Toluidine blue forlight microscopic images. Light microscope sections were viewed



Fig. 2. The influence of postharvest ethylene degreening (72 h at 3 �L L−1 ethylene,>90% RH and 20–22 ◦C) and a delay in commencement of cold storage on rind break-down incidence during 2004. After being either degreened or not, ‘Nules Clementine’mandarin fruit were held at ambient temperature for 7, 11 or 14 d before cold storageat 7.5 ◦C for 14 weeks. Values are means (n = 8) ± SE.

with a Zeiss Axiovert 200 (Zeiss, Gottingen, Germany) microscopefitted with a Nikon digital camera DXM 1200 (Nikon Insteck 10,Kanagawa, Japan). The TEM ultrathin sections were made using aReichert ultracutE ultra-microtome and contrasted in 4% aqueousuranyl acetate (10 min) before being rinsed with water, followedby 2 min exposure to Reynolds lead citrate and a second rinse inwater (Reynolds, 1963). TEM sections were viewed with a PhillipsEM3001 transmission electron microscope (Phillips, Eindhoven,Nederlands) set at 200 kV.

The material for the scanning electron microscopic (SEM)images was collected directly after the above-mentioned dehydra-tion step and critical point drying with liquid CO2 before beingmounted on a stub and splattered with gold (Biorad E3000, Pobron,West Sussex, UK) (Van der Merwe and Peacock, 1999). The sampleswere viewed with a JSM840 Joel SEM (Joel, Tokyo, Japan) at 5 kV anda working distance of 12 mm.

2.8. Statistical analysis

The data were subjected to analysis of variance using generallinear model (GLM) procedure of Statistical Analysis System (SASv. 6.12, SAS Institute, Cary, NC, USA), and means of eight replicateswere separated by using standard errors.

3. Results

3.1. Rind breakdown incidence

The incidence of RBD (Fig. 1) during 2004 increased as the stor-age period progressed (Fig. 2). A delay in cooling of the fruit resultedin increased RBD, which became evident after only 2 weeks of coldstorage. Furthermore, ethylene treatments accentuated the inci-dence of RBD when storage was delayed by 7 and 11 d. However,no noticeable difference occurred when storage was delayed by

14 d with or without degreening.The RBD incidence recorded during 2005 and 2007 followed

the same pattern as observed in 2004, i.e. the incidence of RBDincreased during storage (Fig. 3A–D). There was a difference in RBDincidence of fruit from the two different canopy positions in boththe 2005 and the 2007 seasons, with the inside fruit being moresusceptible to the development of RBD than outside fruit (Fig. 3Aand B). The possibly mitigating effect of low storage temperature(−0.5 ◦C) on RBD was not as evident in the 2005 season as in 2007,when significantly higher incidences of RBD occurred in the fruitstored at 7.5 ◦C (Fig. 3C and D).

3.2. Rind colour and pigment content

Rind colour, expressed as hue angle, lightness and chroma val-ues, was affected by canopy position as well as subsequent storagetemperature during both seasons (Fig. 4). Fruit borne on the out-side of the tree canopy had a lower hue angle and higher lightnessand chroma. Therefore, rind colour of the outside fruit were notonly more orange, compared to the paler, yellow-coloured rindsof inside fruit, but were also a more vivid colour (lower chroma),which resulted in visibly more orange fruit compared to a pale yel-low rind of the inside fruit (Fig. 4). Although the rind colour ofinside fruit improved during storage, it remained poorer (higherhue angle) than the outside fruit. The outside fruit stored at −0.5 ◦Cdeveloped a better rind colour (lower hue angle) after 8 weekscompared to the inside fruit stored at 7.5 ◦C (Fig. 4).

Following degreening, flavedo chlorophyll content declinedconsiderably, and this variable became inconsequential in deter-mining the colour of the rind during postharvest storage (Fig. 4).However, it is interesting to note that the inside fruit at harvest(week 0) had higher chlorophyll content in the rind than the out-side fruit. In contrast to the chlorophyll content in the flavedo, thecarotenoid content increased from 600 to 800 �g g−1 DW at harvestto as much as 1600 �g g−1 DW after prolonged storage. There wasa noticeable difference of carotenoid content in the rind betweeninside and outside fruit. Although the above-mentioned differencesbetween canopy positions were maintained during 7.5 ◦C storage,the carotenoid content increased until week 14. However, −0.5 ◦Cstorage resulted in a gradual reduction of carotenoid content inboth the inside and the outside fruit flavedo (Fig. 4).

3.3. Rind carbohydrate content and fruit respiration rate

The carbohydrate content, measured as sucrose, fructose andglucose, in the flavedo of the stored fruit did not change drasticallyduring the prolonged storage periods in 2007 (Fig. 5). However,there were noticeable differences and consistent trends of theindividual sugars measured, even though the differences werenot always significant, between storage temperatures (−0.5 ◦C vs.7.5 ◦C), as well as fruit bearing position (inside vs. outside fruit).

Glucose and fructose contents were similar, whereas the canopyposition effect was more evident for sucrose content – the insidefruit at both storage temperatures had lower values than the out-side fruit. Carbohydrate levels were higher in the outside fruit.

Respiration rate measured during 2007 declined from the initialvalues (week 0) after commencing with cold storage (Fig. 6). At har-vest (week 0), the inside fruit had a higher respiration rate than theoutside fruit, although not significantly so. In addition, fruit respi-ration did not differ significantly between inside and outside fruitat either of the two storage temperatures. However, from week 6onwards, respiration rate of the fruit stored at −0.5 ◦C was signifi-cantly lower at week 8, 10 and 12 (±10 mg CO2 kg−1 h−1) than fruitstored at 7.5 ◦C.



Fig. 3. Incidence of rind breakdown of ‘Nules Clementine’ mandarin fruit during 2005 and 2007 of fruit sampled from two positions within the tree’s canopy, viz. inside (A)and outside (B), during cold storage at 7.5 ◦C or −0.5 ◦C (C and D). Values are means (n = 8) ± SE.

3.4. Microscopic cellular structure

In the light microscope images, cellular damage was evident inthe sub-epidermal cell layer of the RBD-affected flavedo (Fig. 7A)as well as in the area adjacent to and above the oil gland (arrows).The unaffected flavedo (Fig. 7D) shows the differences in cell size,shape and cell wall thickness between the various citrus oil glandcell layers as labelled in Fig. 7A, viz. sheath and epithelial cells aswell as the oil filled lumen. The epidermal cell layer was intact inthe affected and unaffected flavedo, however the cellular damageextended from the hypodermis into the thick walled sheath cellsin the RBD-affected tissue.

Recognisable differences were evident between RBD-affectedand unaffected flavedo tissue in the SEM images (Fig. 7B–C andE–F). When comparing Fig. 7B and E, the result of the collapse ofthe hypodermal tissue surrounding the oil glands on the flavedotopography can be seen. This collapse of the hypodermal tissue isthought to be caused by the collapse of an oil gland leading to thecells being damaged by the oil and the eventual collapse in of thiscell layer, as well as to the sheath cells between oil glands (Fig. 7C).The damage caused by the release of the phytotoxic essential oilfrom a collapsed oil gland, can extend in all cell layers of surround-ing flavedo tissue, including the cells around adjacent, but intact oilglands. These lesions are initially restricted to a 2–5 mm area, whichoccurs randomly over the fruit surface. However, in severe cases ofRBD the lesions merge to form larger clusters of dark sunken areas(Fig. 1).

4. Discussion

The incidence of rind breakdown (RBD), a progressive physio-logical disorder of the fruit rind of ‘Nules Clementine’ mandarin,was affected by various postharvest treatments in this study. Of

these treatments, ethylene degreening, a delay in cooling andprolonged storage all increased the occurrence of RBD. Duringpre-harvest fruit development, fruit position within the canopysignificantly affected fruit RBD susceptibility, with higher RBDincidence in inside fruit compared to outside fruit. In addition, dif-ferences in levels of RBD incidence between seasons, shows thatthis disorder cannot be ascribed to one specific factor, e.g. ethy-lene treatment or storage temperature. It is therefore likely that thesensitivity of the fruit flavedo to this disorder is determined dur-ing pre-harvest fruit rind development and that a “trigger/signal”during the postharvest handling period initiates the onset of symp-tom development. Of the factors studied, no single factor is thoughtto act alone as a “trigger/signal”, but it is thought that a com-bination of factors could induce the response leading to RBDsymptoms.

Several rind disorders have been described in the literature,however RBD of ‘Nules Clementine’ mandarin is a different posthar-vest disorder compared to those previously described, e.g. rindpitting, rind staining, chilling injury or oleocellosis, as the posthar-vest conditions under which RBD is expressed, as well as thetime and visual development of the symptoms, vary (Lafuente andZacarias, 2006; Petracek et al., 2006). The first notable difference isthat RBD develops progressively, starting during storage and 3–5weeks after harvest, and is therefore neither indicative of physi-cal damage, as in the case of oleocellosis (Knight et al., 2002), norto exposure to a chilling injury-inducing temperature during stor-age (Lafuente and Zacarias, 2006), since a higher RBD incidence wasobserved at 7.5 ◦C than at −0.5 ◦C storage. Chilling injury symptomsoccurred in fruit stored at −0.5 ◦C, and the incidence increased withduration of cold storage (data not shown). However, the symptomsof chilling injury in ‘Nules Clementine’ mandarin are very differentfrom RBD and manifest as a softening/glazing of the flavedo. There-fore, the RBD symptoms were easily identified and separated fromchilling injury.



Fig. 4. Postharvest changes in rind colour (hue angle, lightness and chroma values), chlorophyll and carotenoid content of ‘Nules Clementine’ mandarin flavedo duringpostharvest in 2005, at a non-chilling (7.5 ◦C) and chilling (−0.5 ◦C) temperature. Values are means (n = 8) ± SE.

Furthermore, no pre-harvest RBD symptoms have been seen inthis study nor have they been reported in the South African cit-rus industry, unlike the case for rind staining of ‘Valencia’ orange(Arpaia et al., 1991), peel pitting of ‘Encore’ mandarin (Maia et al.,2004), rind breakdown of ‘Navelate’ orange (Agustí et al., 2001)or superficial flavedo necrosis (noxan) of ‘Shamouti’ orange (Ben-Yehoshua et al., 2001). The time of symptom development isthought to offer some suggestion as to the cause of a rind disor-der. Therefore, in comparison, rind breakdown/staining of ‘Navel’

orange, which can develop within 1 week after harvest (Alférezet al., 2003), differs appreciably from the 3 to 5 weeks for RBD of‘Nules Clementine’ mandarin. However, superficial flavedo necro-sis (noxan) of ‘Shamouti’ orange and RBD development are similarin that both can occur during commercial shipment. Nonetheless,‘noxan’ is known to occur pre-harvest (Ben-Yehoshua et al., 2001),which could be indicative of a different underlying mechanism.

The symptoms of RBD of ‘Nules Clementine’ mandarin also dif-fer from most other rind disorders. The dark spots associated with a



Fig. 5. Sucrose, glucose and fructose contents of ‘Nules Clementine’ mandarin flavedo during postharvest storage in 2005 of fruit positioned inside and outside the tree’scanopy and at a non-chilling (7.5 ◦C) and chilling (−0.5 ◦C) temperature. Values are means (n = 8) ± SE.

collapsed oil gland (Fig. 1) are distributed randomly over the surfaceof the fruit in a “leopard spot” pattern and are not concentrated ateither the stem-end as in SERB (Albrigo, 1972), or in the equatorialregion as in rind breakdown of ‘Navelina’ and ‘Navelate’ oranges(Alférez et al., 2003). However, RBD shows anatomical similaritiesto other rind disorders in three instances, viz. rind-staining and

Fig. 6. Respiration rate during cold storage of ‘Nules Clementine’ mandarin fruitduring 2007. Values are means (n = 8) ± SE.

breakdown of ‘Navel’ orange and mandarin (Lafuente and Zacarias,2006; Petracek, 2006) and superficial flavedo necrosis (noxan) of‘Shamouti’ orange (Ben-Yehoshua et al., 2001). In these disorders,as in RBD, the hypodermal cells collapse in areas associated withan oil gland (above or between adjacent oil glands). The lesion sub-sequently spreads and becomes a necrotic area. However, in RBDlesions the damaged cells do not spread to the epidermal cell layeras seen in the rind breakdown/staining of ‘Navel’ orange (Agustíet al., 2001).

It is thought that the release of oil from an oil gland could be thecause of the visible cellular damage in RBD, as the symptoms arealways closely associated with a collapsed oil gland, which couldbe due to a physiological breakdown of cellular membranes andorganelles (Alférez et al., 2008). This would lead to the collapseof the complex, multi-cellular structure of the oil gland, therebyleaking phytotoxic oil into the adjacent cellular structures. This oilgland collapse occurs without application of any physical stress, asthe fruit in these experiments were harvested in small quantities toavoid impact during handling and transport and stored without anyweight on them. The anatomical images of RBD lesions show thatthe epidermal cells remain intact, but underlying cell layers adja-cent to the oil gland tend to be severely damaged. In an RBD “pit”it also appears as if only one oil gland collapsed, releasing the phy-totoxic oil which damages the cell layers surrounding the leaky oilgland. The difference in the causal mechanism between oleocellosisand RBD is therefore clear, as the visible oleocellosis symptoms candevelop within 6–12 h after the physical damage of an oil gland or



Fig. 7. Comparative images of different cellular layers of ‘Nules Clementine’ mandarin fruit rind with RBD (A) and without RBD (D) after 14 weeks of storage at 7.5 ◦C. Profileimages with a light microscope (A and D) show the cellular damage of the hypodermal and sheath cells in the RBD tissue (A). SEM images of ‘Nules Clementine’ mandarinrind after 14 weeks of storage at 7.5 ◦C with (B to C) and without RBD (E to F). Topographic changes after the collapse of an oil gland can be compared with (B vs. E).



transfer of rind oil onto the rind (Knight et al., 2002). These differ-ences in time of symptom development and microscopic damageare a clear indication that RBD does not develop from a physicalstress of the rind tissue, which was an initial concern in the SouthAfrican industry.

From a rind morphological point of view, the reticulated man-darin fruit rind has a much thinner albedo than that of an orangeor grapefruit, which could be the reason for the different symp-tomatic development of physiological rind disorders, as well astheir causes. The mechanism of cellular collapse of rind break-down/pitting of ‘Navel’ orange and grapefruit, is proposed to bethe dehydration at low RH (45%) and subsequent re-hydration athigh RH (90%), which leads to water potential differences and ten-sion in the flavedo-albedo interface (Alférez et al., 2003). Pitting of‘Fallglo’ mandarin and grapefruit from Florida were related to waxapplication, especially by the less permeable shellac-based waxes.Exposing fruit to high temperature (≥15 ◦C) after waxing increasedthe incidence of pitting, although fruit were unaffected by ethy-lene exposure during degreening (Petracek et al., 1998). Due to thethin or absent albedo in the reticulated mandarin rind, the fruitrind could differ in its reaction to water stress, however this aspectremains to be tested. It could therefore be argued that due to thesymptom development (time and pattern) of RBD of ‘Nules Clemen-tine’ mandarin this disorder should be seen as a unique postharvestphysiological rind disorder of the citrus fruit flavedo, with a dis-tinct reaction to pre- and postharvest conditions. However, theeffect of sudden fluctuations in relative humidity, and thereforerind turgor and weight loss, between harvest and packing on thedevelopment of this specific rind disorder needs to be elucidated.The progressive developmental pattern of RBD, amplified by ethy-lene treatment and a delay in cold storage, could be interpreted asan indication of a senescence-related physiological process. In sucha process a series of controlled cellular changes results in the deathof cells or tissue and can be attributed to the eventual progres-sive loss of membrane integrity or regulatory control (Legge et al.,1986; Dangl et al., 2000). Senescence, in plant tissue, is responsiveto any sub- or supra-optimal environmental conditions, viz. tem-perature, water deficit and gaseous concentrations (O2, CO2 andethylene) (Dangl et al., 2000), all of which could be mismanagedin the postharvest environment and potentially act as a primarysenescence promoting agent.

Furthermore, since the citrus rind is regarded as a modifiedleaf with the same anatomical features and physiological attributes(Schneider, 1968), it is not surprising that a leaf-like response suchas the chloroplast-to-chromoplast conversion would take placeduring maturation (Gross et al., 1983). This process is commer-cially manipulated during the postharvest period to improve citrusfruit rind colour by exposing fruit for 3 d to 1–3 �L L−1 ethylene gasresulting in chlorophyll breakdown; however, this practice can alsolead to various rind defects, such as zebra skin or horseshoe/greenrings (Krajewski and Pittaway, 2002). Ethylene is thought to benecessary for the progression of senescence in Arabidopsis leaves,acting as either a modulating factor or influencing the timing ofthe process, possibly via the enhanced expression of senescence-associated genes and suppression of photosynthesis-associatedgenes (Grbic and Bleecker, 1995). The increase in RBD after ethy-lene exposure, could therefore have been due to ethylene’s knowninfluence on plant cell senescence. In contrast to this interpreta-tion of the data, it has been shown that the application of ethylenefor 4 d at 2–10 �L L−1 reduced rind staining of ‘Navelina’ and ‘Nave-late’ oranges (Lafuente and Sala, 2002; Cajuste and Lafuente, 2007).However, as discussed above, the time of symptom developmentcould indicate a different underlying causal mechanism.

Temperature management, in the non-chilling range (>4 ◦C fororanges and mandarins), is the primary approach taken duringpostharvest storage of fruit to maintain product quality by decreas-

ing all physiological processes, including the rate of senescence(Kays and Paull, 2004). The increase in RBD due to delays incommencement of cooling of the fruit after packing is thereforeconsistent with basic postharvest principles, and leads to an imple-mentable practice to avoid or reduce the occurrence of this rinddisorder. The higher incidence of RBD of the fruit stored at 7.5 ◦Ccompared with −0.5 ◦C, further substantiates the fact that tempera-ture influences the rate of senescence in the flavedo. Furthermore,the consistent pattern of increased RBD development as storageduration progresses can be seen as the clearest indication thatthis physiological disorder is associated with an accelerated senes-cence process in the more RBD-prone inside fruit, leading to cellularbreakdown, and which could involve water loss from the rind.

Senescence is an energy-requiring process whereby carbohy-drates are catabolised during the various metabolic processesinvolved in senescence. The finite source of carbohydrates in theflavedo, however, does not seem to be a limiting factor duringstorage, as none of the three sugars measured showed a dramaticreduction in quantity during storage. However, it is known thatsugar content affects the growth and development of plant cells.In addition to their role as an energy source, sugars act as a sig-nal to activate various gene-regulated processes, such as pigmentsynthesis and senescence (Graham et al., 1992; Knight and Gray,1994). Therefore, the trend of a lower flavedo carbohydrate contentat onset of postharvest storage of the more RBD-prone inside fruitcompared to outside fruit, could be an indication that resourcesfor developmental respiration were lacking and that those fruit,which developed under deficit light conditions, could not tolerateexposure to stresses during postharvest handling. Fruit respirationrate, indicative of a fruit’s postharvest physiological condition, wasinfluenced not only by the storage temperature, but also by canopyposition in the 7.5 ◦C treatment. The higher respiration rate of theless RBD-prone outside fruit stored at 7.5 ◦C further shows that themitochondria in these fruit are still effective in supplying energyfor maintenance respiration, as there does not seem to be a lack ofrespiratory carbon. The higher levels of carbohydrate and pigmentsin the outside flavedo could be seen as an indicator of the fruit’s ori-gin in the tree canopy and could therefore be used as a potentialscreening tool of fruit with a high or low RBD risk.

The lower carotenoid content of the inside fruit rind comparedto the outside fruit rind in this data set could also be an indica-tion of the reduced anti-oxidative capacity of the flavedo, sincecarotenoids are thought to play an important role in the protec-tion of cellular membranes (Sies and Stahl, 1995; Laurie, 2003).Anti-oxidant species, or the absence thereof, have been implicatedin various postharvest physiological disorders, whereby subopti-mal levels limit shelf-life, product quality and nutritional contentof fresh produce (Hodges, 2003). Temperature and light levels areknown to significantly influence ascorbic acid and vitamin A con-tent in vegetables (Klein and Perry, 1982; Rosales et al., 2007).Ascorbic acid content and light levels have a strong positive cor-relation (although not causally linked), due to the carbohydrates(glucose and fructose) serving as the energy source for ascor-bic acid synthesis, leading to higher levels in more sun-exposedfruit (Harris, 1975; Lee and Kader, 2000; Rosales et al., 2006). Afruit’s position in the canopy could therefore directly influenceits anti-oxidant status, i.e. more sun exposure results in a higheranti-oxidant capacity in the flavedo and a reduced risk of RBDdevelopment, and vice versa.

To conclude, the incidence of RBD of ‘Nules Clementine’ man-darin was increased by postharvest treatments known to promotethe rate of senescence, viz. exposure to ethylene and relativelylong storage at a relatively high temperature (±7.5 ◦C). It is thoughtthat the progressive nature of this physiological disorder is indica-tive of a premature senescence process in those fruit rinds with ahigher propensity for RBD. The higher carbohydrate and carotenoid



contents, and better rind colour development at the onset ofpostharvest storage, are an indication of the importance of thedevelopment of a suitable rind condition during pre-harvest fruitdevelopment. It is possible that this good rind condition (as definedby colour and carbohydrate content in this study) would translateinto a reduction in the occurrence of RBD, even though a lack ofcarbohydrate may not be directly involved in the primary mech-anism of RBD development. The results suggest that this disorderis not the result of any physical stress but rather a lack of cellularfunction followed by a probable disruption of cellular structuresand metabolism. In future studies on RBD, the effect of relativehumidity and water loss in the postharvest environment should beinvestigated, and could add additional information on the mech-anism responsible for this rind disorder. The reaction of the fruitto the postharvest treatments used in the experiments offers someimplementable practices to prevent this disorder, even though thecausative mechanism has not yet been elucidated.

Acknowledgements

This study was made possible by funding from Citrus Growers’Association of Southern Africa and Citrus Research International aspart of the first author’s PhD study. The authors thank Dr. Eliza-beth Rohwer for help with the carbohydrate analysis as well as Mr.Willem van Kerwel for technical assistance in the field.

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Author's personal copy...Author's personal copy P.J.R. Cronje et al. / Postharvest Biology and Technology 60 (2011) 192 201 193 Fig. 1. Rind breakdown (RBD) of Nules Clementine mandarin