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Postharvest rind staining in Navel oranges is aggravated by changes in storage relative humidity: effect on respiration, ethylene production and water potential
Postharvest Biology and Technology 28 (2003) 143 /152 www.elsevier.com/locate/postharvbio
Postharvest rind staining in Navel oranges is aggravated by changes in storage relative humidity: effect on respiration, ethylene production and water potential Fernando Alfe´rez a, Manuel Agusti b, Lorenzo Zacarı´as a,* a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos, CSIC, Apartado de Correos 73, Burjassot, Valencia 46100, Spain Instituto Agroforestal Mediterraneo, Universidad Polite´cnica de Valencia, Camino de Vera s/n, Valencia 46022, Spain
b
Received 10 April 2002; accepted 3 July 2002
Abstract The influence of altering storage temperature and relative humidity (RH) on the incidence of postharvest rind staining and on the physiological behaviour of Navel sweet oranges (Citrus sinensis L. Osbeck) was examined. Fruit of ‘Navelina’ orange stored at low (45%) or high (95%) RH did not develop rind staining upon transfer from 30 to 20 8C or to 12 8C. By contrast, in fruit stored at constant temperature (20 8C), transfer from 45 to 95% RH increased the incidence of rind staining, despite the rate of weight loss being similar to that of fruit for which the storage temperature was reduced. This effect was more rapid in fruit exposed for prolonged periods at low RH. A marked and transient stimulation in the respiration rate and ethylene production was observed within 12 /24 h of fruit being transferred from low to high RH. Water potential (Cw) in flavedo and albedo tissue was reduced by storage of the fruit at 45% RH, and the capacity for it to recover after transfer to 95% RH was more rapid in the flavedo than in the albedo and dependent on the time of storage at low RH. The influence of preharvest susceptibility on the development of the disorder during postharvest storage in ‘Navelate’ sweet orange was also studied. Fruit harvested from orchards with a high incidence of rind breakdown on the tree were more prone to develop rind staining after storage, had higher ethylene production and low flavedo and albedo Cw at harvest than fruit harvested from orchards not affected by natural rind breakdown. Collectively, these results indicate that in Navel oranges, alteration of peel water status is a critical factor in the incidence of postharvest rind breakdown, which is also highly influenced by fruit conditions at harvest. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Ethylene; Physiological disorder; Relative humidity; Respiration; Water potential
1. Introduction
* Corresponding author. Tel.: /34-96-3900022; fax: /3496-3636301 E-mail address: [email protected] (L. Zacarı´as).
Physiological peel disorders or peel blemishes are important factors affecting external citrus fruit quality for fresh consumption. The causes responsible for many of these disorders are not well understood, their incidence being erratic, showing
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high variability from year to year and among orchards. Besides, during the postharvest life of fruit it is difficult to ascribe a particular symptom of damage to a specific cause, and different factors may stimulate similar responses (Grierson, 1986). Fruit of Navel sweet oranges are prone to develop rind breakdown and staining (Eaks, 1964; Zaragoza and Alonso, 1975; Casas and Garcı´a-Bataller, 1986), the incidence depending upon the cultivar. Fruit of ‘Navelate’ oranges are sensitive to stain on the tree (Agusti et al., 2001) and under postharvest storage (Alfe´rez and Zacar´ıas, 2001), whereas ‘Navelina’ oranges only stain during postharvest storage (Casas and Garcı´aBataller, 1986; Lafuente and Sala, 2002). In the last few years the impact of these disorders has increased and, depending on the year, up to 60% of fruit per tree have been found to be affected for ‘Navelate’ oranges (Agusti et al., 2001), and considerable losses during postharvest handling and transport have also resulted. Rind breakdown of ‘Navelate’ sweet orange on the tree has been characterised as irregular colourless depressed areas on the peel in which oil glands appear to be intact. Scanning-electron microscopic sections reveal that cells of the flavedo /albedo transitional zone are first affected, showing reduced cytoplasm, and twisted and squashed walls. As the disorder progresses, collapsed cells extend to the outer flavedo and inner albedo layers. Wax morphology and cuticle permeability are however unaffected (Agusti et al., 2001). Initial symptoms of the disorder occur after a few days of low temperature and high relative humidity (RH) following days of high temperature and low RH, suggesting that under field conditions, sudden changes in the loss of water may be responsible for the disorder (Agusti et al., 2001). Under postharvest conditions, Lafuente and Sala (2002) have reported that rind staining in ‘Navelina’ oranges was reduced by a pre-treatment with 10 ml l 1 ethylene for 4 days but enhanced by increasing the RH under storage at 22 8C. In ‘Shamouti’ sweet orange, the peel blemish known as noxan has been shown to be reduced by different postharvest treatments at high RH and by reducing storage temperature to 5 8C (BenYehoshua et al., 2001). However the specific pre-
or post-harvest conditions inducing rind breakdown are still unclear. Morphologically similar disorders at non-chilling temperatures have been also described in fruit of ‘Marsh’ grapefruit (Petracek et al., 1995), ‘Temple’ orange (Petracek et al., 1997) and ‘Fallglo’ tangerine (Petracek et al., 1998b). These disorders, however, are only produced on waxed fruit and by alteration of the internal gas concentrations (Petracek et al., 1997, 1998a,b), suggesting that similar symptoms may result from different causes. Despite different studies suggesting that most of these blemishes may be due to water stress (Grierson, 1986; Ben-Yehoshua et al., 2001; Lafuente and Sala, 2002), it is also well documented that dehydration of orange fruit produces a reduction in albedo thickness, loss of peel turgor and softening of the fruit, yet the fruit does not stain (Ben-Yehoshua, 1987). Therefore, water stress itself appears not to be the only factor responsible for rind breakdown and staining of Navel sweet oranges. The objective of this study was to examine whether alterations of the RH or temperature during postharvest storage may be responsible for rind staining in Navel sweet oranges and how they affect respiration rate, ethylene production and flavedo and albedo water potential. The involvement of preharvest susceptibility on the incidence of postharvest rind staining was also evaluated and is discussed.
2. Materials and methods 2.1. Plant material Mature fruit of ‘Navelina’ and ‘Navelate’ sweet oranges (Citrus sinensis, L. Osbeck) were used in the experiments. ‘Navelina’ fruit were harvested between December and January from adult trees grafted onto citrange Carrizo (Poncirus trifoliata /Citrus sinensis , L. Osbeck) rootstock and grown in a commercial orchard in Lliria (Valencia, Spain). ‘Navelate’ fruit were harvested from December to March from adult trees also
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grafted onto citrange Carrizo rootstock and grown in different orchards of the Valencia citrus area. To examine the influence of environmental preharvest conditions on the incidence of postharvest rind staining, fruit of ‘Navelate’ sweet orange were selected from two orchards (Godelleta, Valencia, Spain) with trees of the same age and subjected to the same management. The first orchard was selected for its high incidence of natural rind breakdown over several years whereas the second one was selected for its low levels of affected fruit. In both cases, fruit were randomly harvested from 12 trees (a design of three-tree plots and four replications used for evaluation of rind breakdown of fruit on the tree; Agusti et al., 2001), from the exterior of the canopy, uniform in size and free of any damage or visual defects on the peel. After delivery to the laboratory, fruit were stored in plastic containers without any previous treatment. 2.2. Storage conditions To study the effects of storage temperature on rind staining, ‘Navelina’ fruit were stored for up to 21 days at 30, 20 and 12 8C and at both 45 and 95% RH. After 4 days storage at 30 8C, two replicates of 30 fruit each were transferred to 20 or to 12 8C, maintaining the RH constant. Mature ‘Navelina’ and ‘Navelate’ fruit were used to examine the effect on rind staining of altering the storage RH at constant temperature. Fruit were randomly divided into 4 replicates of 60 fruit each. Three replicates were stored at 209/ 1 8C and 459/2% RH and the fourth replicate was held at 209/1 8C and 959/2% RH. After 7 and 14 days of storage at 45% RH, a replicate of 60 fruit was transferred to 95% RH for the remainder of the storage period. To ensure a high RH regime during the storage period, fruit were placed in plastic boxes under a continuous flow of humidified air. In the different experiments, 30 fruit per storage condition were used to determine rind staining index (RSI) and weight loss throughout the whole storage period. To examine how the environmental growing conditions at harvest may affect postharvest development of rind breakdown, ‘Navelate’ fruit
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were harvested in January from 12 trees from selected orchards with marked differences in the proportion of affected fruit. Fruit were divided at random into 3 groups that were stored under the following conditions: (a) 20 8C and 45% for up to 21 days, (b) 20 8C and 95% RH for up to 21 days and (c) 20 8C and 45% RH for 4 days and then transferred to 20 8C and 95% RH for the remainder of the experiment. Each group contained 3 replicates of 20 fruit. 2.3. Carbon dioxide and ethylene production Respiration rate and ethylene production from the whole fruit was determined by incubating four replicates of 3 /4 fruit in 1.7-l jars. After 3 h of incubation at 20 8C, 1-ml air samples from the headspace of the jar were withdraw with a hypodermic syringe and injected into a gas chromatograph (Perkin Elmer Autosample). Carbon dioxide concentration was analysed by a thermal conductivity detector coupled to a Chromosorb column at 60 8C. Ethylene production was determined using a flame ionization detector and an activated alumina column kept at 140 8C. 2.4. Water potential measurement To measure flavedo and albedo water potential, 5 mm disks from the equatorial area of the fruit were excised by a cork borer. Disks of 1/2 mm thick flavedo and of 3/4 mm thick albedo were sliced with a blade and placed in a sample chamber (C-52, Wescor Inc. Logan, UT) connected to a psychrometer switchbox (PS-10) and to a dew point microvoltimeter (HT-33T). The dew point hygrometer was previously calibrated with NaCl solutions of known concentrations. To ensure initial water vapor equilibrium, water potential measurements were done at least 2 h after setting the sample into the chamber. For each water potential measurement 2/3 fruit were used. 2.5. Estimation of rind staining index Fruit were inspected and rated on a scale from 0 (no stain) to 3 (severe), based on extent of browning and injury. The results are expressed as
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RSI, calculated according to: S (Rind stain scale (0 /3) /number of fruit within each class)/total number of fruit. Experiments were repeated at least twice a season and the study was conducted over 3/4 consecutive seasons. Results reported here correspond to representative experiments but the same pattern of changes was consistently observed.
3. Results To determine if changes in the rate of water loss by alteration of storage temperature and RH may be responsible for rind breakdown in Navel sweet orange, two different experimental procedures were used. First, ‘Navelina’ fruit were stored at 30 8C under constant low (45%) and high (95%) RH, and after a weight lost of 3/4% (4 days) they were transferred to 12 and 20 8C. Figs. 1A and B show that weight loss was very high in fruit stored at 30 8C and 45% RH and that transfer of fruit from 30 to 12 8C or 20 8C reduced water loss to a rate similar to that of fruit continuously stored at
12 or 20 8C. No rind staining were observed in fruit stored under these temperature regimes and at both 45 and 95% RH (Fig. 1C and D). In a second experiment, the effect of altering RH in ‘Navelina’ fruit stored at constant temperature (20 8C) was also examined. After 3 weeks, weight loss of fruit stored at 45% RH was about 4 times higher than that of those kept at 95% (Fig. 2A). Transfer of fruit to 95% RH after 7 (2.2% of weight lost) and 14 (4.2%) days at 45% RH reduced dehydration to a rate simiar to that of fruit continuously stored at 95% RH. (Fig. 2A). At constant RH, the RSI remained low (0.5) for up to 3 weeks of storage, but upon transfer from 45 to 95% RH, a marked increase in rind breakdown was produced. By 4 days after transfer, the RSI increased from 0.2 to 1.2 in fruit stored 7 days at 45% RH and from 0.4 to 2.0 in fruit stored 14 days at 45% RH (Fig. 1B). Sensitivity to rind staining by transfer of the fruit from low to high RH was consistently observed in mature fruit of both ‘Navelina’ (Fig. 3A and B) and ‘Navelate’ (Fig. 3C /G) sweet oranges and increased with fruit maturation (data not shown).
Fig. 1. Weight loss (A and B) and RSI (C and D) in ‘Navelina’ oranges stored at 30 (k), 20 (^) and 12 8C (\) and 45% (left panels) and 95% RH (right panels) and of fruit transferred after 4 days storage from 30 to 20 8C (') or to 12 8C (%). Arrows indicate the day of transfer. Values are the means9/S.D. of 30 fruit.
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Fig. 2. Weight loss (A) and RSI (B) of ‘Navelina’ oranges stored at 20 8C and 45% (k) or 95% (I) RH, and of fruit transferred to 95% RH after 7 (m) and 14 (j) days storage at 45% RH. Arrows indicate the time of transfer. Values are the mean9/S.D. of 30 fruit.
Initial symptoms of postharvest rind breakdown in ‘Navelina’ and ‘Navelate’ oranges were the depression or collapse of irregular areas usually scattered over the equatorial region of the fruit. Depressed flavedo areas became apparent within 3 /7 days after transfer of fruit from 45 to 95% RH, and were irregular in size, from small spots clustering as they spread (Fig. 3A, D and E) to big depressions covering a large surface area of the fruit (Fig. 3C). Several days after the appearance of flavedo depression, affected areas became dried, discoloured and turned progressively brown and black (Fig. 3B and E), forming sunken clustered areas with necrotic epidermal cells (Fig. 3F and G).
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Fig. 3. Postharvest rind staining in ‘Navelina’ (A and B) and ‘Navelate’ (C /G) oranges. The disorder first appears as irregular depressed flavedo areas (A, C and D), becoming progressively dry and brown (B and E). Severe symptoms (F and G), forming sunken clusters with necrotic epidermal cells.
Transfer of ‘Navelina’ fruit from 45 to 95% RH also induced a significant increase in the respiration rate and ethylene production (Fig. 4). In mature fruit, the respiration rate declined after storage at 20 8C regardless of the RH, but after 7 days storage at 95% it was slightly higher than in fruit maintained under 45% RH. Transfer of fruit from 45 to 95% RH, irrespective of the time, almost doubled the rate of respiration, which reached a maximum within 12/24 h after the shift in RH (Fig. 4A). During fruit storage, ethylene production was very low (/0.03 nl g 1 h 1) and remained nearly constant under both 45 and 95% RH for up to 21 days. Parallel with respiration,
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Fig. 4. Respiration rate (A) and ethylene production (B) of ‘Navelina’ oranges stored at 20 8C and 45% (k) or 95% (I) RH, and of fruit transferred to 95% RH after 7 (m) and 14 (j) days storage at 45% RH. Arrows indicate the time of transfer. Values are the mean9/S.D. of 3 replicates of 4 fruit.
transfer of fruit to 95% RH after 7 or 14 days storage at 45%, induced a 5- and a 7-fold increase in ethylene production, respectively (Fig. 4B). The increases in respiration and ethylene were transient, since 3 /5 days after storage at 95% RH the rates of both processes were similar to those of fruit kept at constant RH. Water potential (Cw) of flavedo and albedo tissue 24 and 48 h after transfer was also determined (Fig. 5A and B). In freshly harvested fruit, Cw was slightly lower in the albedo than in the flavedo. After 7 days storage at 45% RH, Cw was reduced by 30 and 15% in the flavedo and albedo, respectively, whereas under high RH it remained constant in the flavedo and slightly higher in the
Fig. 5. Water potential of flavedo (A) and albedo (B) of ‘Navelina’ fruit stored for 7 and 14 days at 20 8C and 45% (open bars) and 95% RH (hatched bars), and 24 and 48 h after transfer from 45 to 95% RH (filled bars). Values are the mean9/ S.D. of 6 measurements.
albedo. Recovery of Cw by transfer to 95% RH was faster in the flavedo than in the albedo and dependent on the time of storage under low RH. After 48 h of transfer of fruit stored for 7 days at 45% RH, Cw in the flavedo increased by 25% and only by 11% in the albedo. However, prolonged storage at low RH reduced the ability of Cw to recover by transfer to 95% RH (Fig. 5). To evaluate the influence of environmental preharvest conditions on the susceptibility of the fruit to postharvest rind staining, the effect of altering RH during storage on ‘Navelate’ fruit harvested from orchards prone (319/2.5% of affected fruit at commercial maturity) and not prone (B/3%) to natural rind breakdown, was examined. After 16 days storage at 20 8C and at both 45 and 95% RH, the proportion and severity of rind breakdown were higher in fruit from the
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Table 1 Percentage and index of rind staining in ‘Navelate’ fruit harvested from orchards affected and not-affected by natural rind staining and stored for up to 16 days at 20 8C under different RH conditions Storage conditions
16 days 45% RH 16 days 95% RH 4 days 45%/12 days 95%
Affected orchard
Not-affected orchard
%
RS index
%
RS index
54.3a 80.4b 90.8b
0.64a 1.21b 1.92c
28.2a 43.1b 66.7c
0.36a 0.43a 1.42b
Fruit were harvested in January. Within each column, values followed by the same letter are not significant at P B/0.05% using Student’s t -test.
affected orchard stored at 95% RH (Table 1). Transfer of the fruit to 95% after 4 days of storage at 45% RH increased 3- and 4-fold the RSI for fruit from affected and not-affected orchards, respectively, the percentage and severity of damage being higher in the former. Interestingly, ethylene production was 6 times higher in freshly harvested fruit from the affected orchard and became almost constant after storage at 45 or 95% RH for up to 4 days. Transfer of the fruit to 95% RH sustained the high rate of ethylene production up to day 5 and then declined (Fig. 6B). In fruit from the not-affected orchard, ethylene production remained low during storage at 45% RH whereas at 95% RH a significant
increase after 4 days was observed. The transfer to 95% RH increased 12-fold ethylene production after 12 h, declining thereafter (Fig. 6A). Fig. 7 shows Cw of flavedo and albedo of fruit harvested from the two orchards differing in rind breakdown severity after 7 days of storage at 45 and 95% RH. In both tissues, Cw was about 10% lower in fruit from the affected orchard. After 7 days of storage, flavedo and albedo Cw of fruit harvested from the affected orchards slightly increased but there were no significant differences between 45 and 95% RH. By contrast, in the tissues of fruit from the not-affected orchard, Cw substantially recovered with storage at high RH (Fig. 7).
Fig. 6. Ethylene production of ‘Navelate’ oranges harvested from an orchard not-affected (A) or affected (B) by natural rind staining and stored for up to 7 days at 20 8C and 45% (I) and 95% (k) RH or transferred to 95% RH after 4 days storage at 45% (m). Arrows indicate the time of transfer. Values are the mean9/S.D. of 3 replicates of 4 fruit.
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Fig. 7. Water potential of flavedo (A) and albedo (B) of ‘Navelate’ oranges harvested from an orchard affected (filled bars) or not-affected (open bars) by natural rind staining and stored for 7 days at 20 8C and 45 and 95% RH. Values are the mean9/S.D. of 6 measurements.
4. Discussion The results reported in this study indicate that changes in RH during storage, particularly transfer to high RH of fruit dehydrated at low RH provoke rind breakdown in Navel oranges. Under field conditions we have previously observed that rind breakdown of ‘Navelate’ oranges occurred after several days of high RH and low transpiration following a period of low RH and high transpiration (Agusti et al., 2001). These observations suggest that sudden increases in the environmental RH after episodes of fruit dehydration appear to be responsible for rind staining in Navel sweet orange either under field conditions or during postharvest storage. Water stress has been related to other disorders morphologically similar to Navel rind breakdown, such as stem end rind breakdown in ‘Valencia’
oranges (Albrigo, 1972) and noxan in ‘Shamouti’ oranges (Ben-Yehoshua et al., 2001). In ‘Shamouti’ fruit, the incidence of noxan was reduced by different postharvest treatments that maintained a high RH and reduced weight loss (BenYehoshua et al., 2001). Lafuente and Sala (2002), however, found that the storage of ‘Navelina’ oranges at high RH was deleterious to the incidence of rind staining. Our results showed that water stress or fruit dehydration itself appear not to be the main cause inducing rind staining in ‘Navelina’ and ‘Navelate’ sweet oranges. By manipulating the storage temperature, at constant RH regimes, we were able to induce water loss even higher than that of fruit stored under 45% RH at 20 8C and transferred to 95% RH. Under these conditions, the peel of the fruit lost turgidity and became soft, characteristic symptoms of citrus fruit dehydration (Ben-Yehoshua, 1987), but it did not pit or stain, indicating that dehydration alone does not provoke rind staining in Navel oranges. Transfer of fruit previously dehydrated at 45 / 95% RH provoked a substantial increase in the RSI, that was higher in fruit stored at 45% RH for longer periods (Fig. 2B). Using these experimental conditions we were able to reproduce rind breakdown in ‘Navelina’ and ‘Navelate’ sweet oranges and thus study the physiological responses of the fruit to these postharvest conditions. The magnitude of water loss appears to be an important factor in the development of rind staining upon transfer to high RH. In fact, we observed that fruit with less than 2% weight loss did not develop peel blemish (data not shown) and that the disorder appeared faster in fruit maintained for 14 days at 45% than in those stored for 7 days at the same RH. This fruit response was also dependent on fruit maturity, as development of rind breakdown was faster in more mature fruit (data not shown). These observations raise the question of the nature of the responses of dehydrated Navel fruit to sudden changes in RH and how these changes may be related to rind staining development. In fruit stored under a high water vapor pressure deficit (45% RH), water is driven from the epidermal cell to the surrounding atmosphere and, consequently, reduces flavedo and albedo Cw (Fig. 4). Transfer of the fruit to a high RH
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atmosphere would reduce the water vapor pressure deficit and change the water movement from the air phase to the inner cells of the peel. Our results show that flavedo cells, as expected, recovered Cw faster than the inner albedo cells, and this might create a suction force demanding water from the outer layers of hydrated cells. This process may explain the initial appearance of the blemish as a depressed flavedo area compressed to the adjacent albedo, in accordance with our results showing that the disorder is initiated at the transitional layer of cells between flavedo and albedo (Agusti et al., 2001). In the flavedo and albedo of fruit exposed for a prolonged period at low RH, Cw were recovered more slowly after transfer to 95% RH, probably due to cellular damage and membrane deterioration of water-stressed cells (BenYehoshua, 1969; Ben-Yehoshua et al., 1983). Maintaining albedo cells under low Cw for extended periods would increase their demand of water from the flavedo cells and may explain the rapid increase in the RSI observed in fruit transferred to 95% RH after 14 days at 45% RH or even in fruit harvested from orchards susceptible to rind staining on the tree. Mantell et al. (1980) using tritiated water showed that the turnover of water was extremely low in the peel of citrus fruit, even in fruit with a high transpiration rate, corroborating our suggestions. These earlier responses of dehydrated fruit to sudden changes in the surrounding RH may explain the twisted and flattened morphology of affected cells of the albedo (Agusti et al., 2001). The characteristic staining and browning of the flavedo observed after several days (Fig. 3) would be a late response, probably due to oxidative processes (Agusti et al., 2001; Lafuente and Sala, 2002). Stimulation of the respiration rate and ethylene production was also an early and transient response to the transfer of dehydrated fruit to high RH, before the appearance of initial symptoms of depression in the flavedo. Since adjustment of flavedo and albedo Cw occurred within few days after the transfer, the increases in respiration and ethylene production appear to be related to sudden changes in cell wall turgor or to cellular damage induced by the shift in the environmental RH, rather than to the development of rind staining.
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Stimulation of the respiration by different stress conditions, including water stress, has been previously reported in citrus and other fruit (Vines et al., 1968; Ben-Yehoshua et al., 1983; Kader, 1987), and water stress-induced ethylene production is a well documented phenomena (Apelbaum and Yang, 1981; Morgan and Drew, 1997). Hydration of wheat leaves at full turgor after water stress increased ethylene production (Beltrano et al. 1997). Felix et al. (2000) have found that hyperosmotic shock in tomato cell cultures induced an increase in ACC synthase activity, in the extracellular pH and in the release of K ions. These observations suggest that the changes induced by hydration of water-stressed Navel fruit may induce membrane and cellular damage responsible for the stimulation of respiration and ethylene production. It is interesting to note that ethylene production of ‘Navelate’ fruit harvested from a high rind stain affected orchard was significantly higher than that of fruit harvested from a not-affected orchard and that flavedo and albedo Cw of these fruit did not recovered after storage at 95% RH. Thus, ethylene production at harvest may be a physiological marker of the susceptibility of the fruit to the disorder. The reduced capability of Cw adjustment of the flavedo and albedo tissue of fruit from affected orchards after storage at different RH and their higher development of rind staining (Table 1) agree with the results obtained in fruit stored at low RH for prolonged periods and indicate that the water status of fruit at harvest is a critical factor for the incidence and development of rind staining during postharvest storage. These observations may explain the erratic incidence of rind staining observed during postharvest handling and storage of Navel oranges. In conclusion, sudden changes in the environmental RH are characteristic of the climatic conditions in the citrus growing areas, and diurnal changes in water (Albrigo, 1977) and osmotic (Kaufman, 1970) potential have been demonstrated in citrus fruit. Harvesting the fruit after a period of water stress, and postharvest handling and storage conditions that allow excessive water loss, followed by washing, waxing and storage at high RH, may be deleterious for the incidence of
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rind breakdown in Navel oranges. Although the experimental systems and the storage conditions used in this study do not correspond with the standard postharvest procedures for Navel oranges, they were effective in inducing fruit rind staining and may be indicative of the factors responsible for the disorder.
Acknowledgements This study was supported by grants from Generalitat Valenciana, Conselleria d’Agricultura, Pesca i Alimentacio´ (GV-CAPA 97-02 and GCCAPA 00-15). We thank Reva S.A. for the use of their commercial ‘Navelate’ orchards and Miguel Sabater for providing us with ‘Navelina’ oranges.
References Agusti, M., Almela, V., Juan, M., Alfe´rez, F., Tadeo, F.R., Zacarı´as, L., 2001. Histological and physiological characterization of rind breakdown of Navelate sweet orange. Ann. Bot. 88, 415 /422. Albrigo, L.G., 1972. Distribution of stomatal and epicuticular wax on oranges as related to stem end rind breakdown and water loss. J. Am. Soc. Hort. Sci. 97, 220 /223. Albrigo, L.G., 1977. Rootstocks affect ‘Valencia’ oranges fruit quality and water balance. Proc. Int. Soc. Citric. 1, 62 /65. Alferez, F., Zacarı´as, L., 2001. Postharvest pitting in Navel oranges at non-chilling temperature: influence of relative humidity. Acta Hortic. 553, 307 /308. Apelbaum, A., Yang, S.F., 1981. Biosynthesis of stress ethylene induced by water deficit. Plant Physiol. 68, 594 /596. Beltrano, J., Montaldi, E., Ba´rtoli, C., Carbone, A., 1997. Emission of water stress ethylene in wheat (Triticum aestivum L.) ears: effects of rewatering. Plant Growth Regul. 21, 121 /126. Ben-Yehoshua, S., 1969. Gas exchange, transpiration, and the commercial deterioration of stored citrus fruits. J. Am. Soc. Hortic. Sci. 94, 524 /528. Ben-Yehoshua, S., 1987. Transpiration, water stress and gas exchange. In: Weichmann, J. (Ed.), Postharvest Biology of Vegetables. Marcel Dekker, Inc, New York, pp. 113 /170. Ben-Yehoshua, S., Peretz, J., Moran, R., Lavie, B., Kim, J.J., 2001. Reducing the incidence of superficial flavedo necrosis
(noxan) of Shamouti oranges (Citrus sinensis , Osbeck). Postharvest Biol. Technol. 22, 19 /27. Ben-Yehoshua, S., Shapiro, B., Even Chen, Z., Lurie, S., 1983. Mode of action of plastic film in extending life of lemon and bell pepper fruits by alleviation of water stress. Plant Physiol. 73, 87 /93. Casas, A., Garcı´a-Bataller, L., 1986. Manchas en las naranjas Navelina. Rev. Agroquim. Tecnol. Aliment 26, 309 /317. Eaks, I.L., 1964. The effect of harvesting and packinghouse procedures on rind staining of Central California Washington navel oranges. Proc. Am. Soc. Hortic. Sci. 58, 245 /256. Felix, G., Regenass, M., Boller, T., 2000. Sensing of osmotic pressure changes in tomato cells. Plant Physiol. 124, 1169 / 1174. Grierson, W., 1986. Physiological disorders. In: Wardowsky, W.F., Nagy, S., Grierson, W. (Eds.), Fresh Citrus Fruits. Avi Publishing Company, Wesport, CN, pp. 361 /378. Kader, A.A., 1987. Respiration and gas exchange of vegetables. In: Weichmann, J. (Ed.), Postharvest Biology of Vegetables. Marcel Dekker, Inc, New York, pp. 25 /43. Kaufman, M.R., 1970. Water potential components in growing citrus fruits. Plant Physiol. 46, 143 /149. Lafuente, M.T., Sala, J.M., 2002. Abscisic acid and the influence of ethylene, humidity and temperature on the incidence of postharvest rindstaining of Navelina oranges (Citrus sinensis L. Osbeck) fruits. Postharvest Biol. Technol. 25, 49 /57. Mantell, A., Goldschmidt, E.E., Monselise, S.P., 1980. Turnover of tritiated water in calamondin plants. J. Am. Soc. Hort. Sci. 105, 741 /744. Morgan, P.W., Drew, M.C., 1997. Ethylene and plant responses to stress. Plant Physiol. 100, 620 /630. Petracek, P.D., Wardowski, W.F., Brown, G.E., 1995. Pitting of grapefruit that resembles chilling injury. HortScience 30, 1422 /1426. Petracek, P.D., Dou, H., Malik, I., 1997. A postharvest pitting of temple oranges stimulated by high temperature storage and wax application. Proc. Fla. State Hort. Soc. 110, 211 / 214. Petracek, P.D., Dou, H., Pao, S., 1998a. The influence of applied waxes on postharvest physiological behavior and pitting of grapefruit. Postharvest Biol. Technol. 14, 99 /106. Petracek, P.D., Montalvo, L., Dou, H., Davis, C., 1998b. Postharvest pitting of ‘Fallglo’ tangerine. J. Am. Soc. Hort. Sci. 123, 130 /135. Vines, H.M., Grierson, G., Edwards, G.J., 1968. Respiration, internal atmosphere and ethylene evolution of citrus fruits. Proc. Am. Soc. Hortic. Sci. 92, 227 /234. Zaragoza, S., Alonso, E., 1975. El manchado de la corteza de los agrios: estudio preliminar de la variedad Navelate. Manchas pre-recoleccio´n. INIA Ser. Prot. Veg. 4, 31 /32.
Postharvest rind staining in Navel oranges is aggravated by changes in storage relative humidity: effect on respiration, ethylene production and water potential Fernando Alfe´rez a, Manuel Agusti b, Lorenzo Zacarı´as a,* a
Instituto de Agroquı´mica y Tecnologı´a de Alimentos, CSIC, Apartado de Correos 73, Burjassot, Valencia 46100, Spain Instituto Agroforestal Mediterraneo, Universidad Polite´cnica de Valencia, Camino de Vera s/n, Valencia 46022, Spain
b
Received 10 April 2002; accepted 3 July 2002
Abstract The influence of altering storage temperature and relative humidity (RH) on the incidence of postharvest rind staining and on the physiological behaviour of Navel sweet oranges (Citrus sinensis L. Osbeck) was examined. Fruit of ‘Navelina’ orange stored at low (45%) or high (95%) RH did not develop rind staining upon transfer from 30 to 20 8C or to 12 8C. By contrast, in fruit stored at constant temperature (20 8C), transfer from 45 to 95% RH increased the incidence of rind staining, despite the rate of weight loss being similar to that of fruit for which the storage temperature was reduced. This effect was more rapid in fruit exposed for prolonged periods at low RH. A marked and transient stimulation in the respiration rate and ethylene production was observed within 12 /24 h of fruit being transferred from low to high RH. Water potential (Cw) in flavedo and albedo tissue was reduced by storage of the fruit at 45% RH, and the capacity for it to recover after transfer to 95% RH was more rapid in the flavedo than in the albedo and dependent on the time of storage at low RH. The influence of preharvest susceptibility on the development of the disorder during postharvest storage in ‘Navelate’ sweet orange was also studied. Fruit harvested from orchards with a high incidence of rind breakdown on the tree were more prone to develop rind staining after storage, had higher ethylene production and low flavedo and albedo Cw at harvest than fruit harvested from orchards not affected by natural rind breakdown. Collectively, these results indicate that in Navel oranges, alteration of peel water status is a critical factor in the incidence of postharvest rind breakdown, which is also highly influenced by fruit conditions at harvest. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Ethylene; Physiological disorder; Relative humidity; Respiration; Water potential
1. Introduction
* Corresponding author. Tel.: /34-96-3900022; fax: /3496-3636301 E-mail address: [email protected] (L. Zacarı´as).
Physiological peel disorders or peel blemishes are important factors affecting external citrus fruit quality for fresh consumption. The causes responsible for many of these disorders are not well understood, their incidence being erratic, showing
0925-5214/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 0 2 ) 0 0 1 2 0 - 5
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high variability from year to year and among orchards. Besides, during the postharvest life of fruit it is difficult to ascribe a particular symptom of damage to a specific cause, and different factors may stimulate similar responses (Grierson, 1986). Fruit of Navel sweet oranges are prone to develop rind breakdown and staining (Eaks, 1964; Zaragoza and Alonso, 1975; Casas and Garcı´a-Bataller, 1986), the incidence depending upon the cultivar. Fruit of ‘Navelate’ oranges are sensitive to stain on the tree (Agusti et al., 2001) and under postharvest storage (Alfe´rez and Zacar´ıas, 2001), whereas ‘Navelina’ oranges only stain during postharvest storage (Casas and Garcı´aBataller, 1986; Lafuente and Sala, 2002). In the last few years the impact of these disorders has increased and, depending on the year, up to 60% of fruit per tree have been found to be affected for ‘Navelate’ oranges (Agusti et al., 2001), and considerable losses during postharvest handling and transport have also resulted. Rind breakdown of ‘Navelate’ sweet orange on the tree has been characterised as irregular colourless depressed areas on the peel in which oil glands appear to be intact. Scanning-electron microscopic sections reveal that cells of the flavedo /albedo transitional zone are first affected, showing reduced cytoplasm, and twisted and squashed walls. As the disorder progresses, collapsed cells extend to the outer flavedo and inner albedo layers. Wax morphology and cuticle permeability are however unaffected (Agusti et al., 2001). Initial symptoms of the disorder occur after a few days of low temperature and high relative humidity (RH) following days of high temperature and low RH, suggesting that under field conditions, sudden changes in the loss of water may be responsible for the disorder (Agusti et al., 2001). Under postharvest conditions, Lafuente and Sala (2002) have reported that rind staining in ‘Navelina’ oranges was reduced by a pre-treatment with 10 ml l 1 ethylene for 4 days but enhanced by increasing the RH under storage at 22 8C. In ‘Shamouti’ sweet orange, the peel blemish known as noxan has been shown to be reduced by different postharvest treatments at high RH and by reducing storage temperature to 5 8C (BenYehoshua et al., 2001). However the specific pre-
or post-harvest conditions inducing rind breakdown are still unclear. Morphologically similar disorders at non-chilling temperatures have been also described in fruit of ‘Marsh’ grapefruit (Petracek et al., 1995), ‘Temple’ orange (Petracek et al., 1997) and ‘Fallglo’ tangerine (Petracek et al., 1998b). These disorders, however, are only produced on waxed fruit and by alteration of the internal gas concentrations (Petracek et al., 1997, 1998a,b), suggesting that similar symptoms may result from different causes. Despite different studies suggesting that most of these blemishes may be due to water stress (Grierson, 1986; Ben-Yehoshua et al., 2001; Lafuente and Sala, 2002), it is also well documented that dehydration of orange fruit produces a reduction in albedo thickness, loss of peel turgor and softening of the fruit, yet the fruit does not stain (Ben-Yehoshua, 1987). Therefore, water stress itself appears not to be the only factor responsible for rind breakdown and staining of Navel sweet oranges. The objective of this study was to examine whether alterations of the RH or temperature during postharvest storage may be responsible for rind staining in Navel sweet oranges and how they affect respiration rate, ethylene production and flavedo and albedo water potential. The involvement of preharvest susceptibility on the incidence of postharvest rind staining was also evaluated and is discussed.
2. Materials and methods 2.1. Plant material Mature fruit of ‘Navelina’ and ‘Navelate’ sweet oranges (Citrus sinensis, L. Osbeck) were used in the experiments. ‘Navelina’ fruit were harvested between December and January from adult trees grafted onto citrange Carrizo (Poncirus trifoliata /Citrus sinensis , L. Osbeck) rootstock and grown in a commercial orchard in Lliria (Valencia, Spain). ‘Navelate’ fruit were harvested from December to March from adult trees also
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grafted onto citrange Carrizo rootstock and grown in different orchards of the Valencia citrus area. To examine the influence of environmental preharvest conditions on the incidence of postharvest rind staining, fruit of ‘Navelate’ sweet orange were selected from two orchards (Godelleta, Valencia, Spain) with trees of the same age and subjected to the same management. The first orchard was selected for its high incidence of natural rind breakdown over several years whereas the second one was selected for its low levels of affected fruit. In both cases, fruit were randomly harvested from 12 trees (a design of three-tree plots and four replications used for evaluation of rind breakdown of fruit on the tree; Agusti et al., 2001), from the exterior of the canopy, uniform in size and free of any damage or visual defects on the peel. After delivery to the laboratory, fruit were stored in plastic containers without any previous treatment. 2.2. Storage conditions To study the effects of storage temperature on rind staining, ‘Navelina’ fruit were stored for up to 21 days at 30, 20 and 12 8C and at both 45 and 95% RH. After 4 days storage at 30 8C, two replicates of 30 fruit each were transferred to 20 or to 12 8C, maintaining the RH constant. Mature ‘Navelina’ and ‘Navelate’ fruit were used to examine the effect on rind staining of altering the storage RH at constant temperature. Fruit were randomly divided into 4 replicates of 60 fruit each. Three replicates were stored at 209/ 1 8C and 459/2% RH and the fourth replicate was held at 209/1 8C and 959/2% RH. After 7 and 14 days of storage at 45% RH, a replicate of 60 fruit was transferred to 95% RH for the remainder of the storage period. To ensure a high RH regime during the storage period, fruit were placed in plastic boxes under a continuous flow of humidified air. In the different experiments, 30 fruit per storage condition were used to determine rind staining index (RSI) and weight loss throughout the whole storage period. To examine how the environmental growing conditions at harvest may affect postharvest development of rind breakdown, ‘Navelate’ fruit
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were harvested in January from 12 trees from selected orchards with marked differences in the proportion of affected fruit. Fruit were divided at random into 3 groups that were stored under the following conditions: (a) 20 8C and 45% for up to 21 days, (b) 20 8C and 95% RH for up to 21 days and (c) 20 8C and 45% RH for 4 days and then transferred to 20 8C and 95% RH for the remainder of the experiment. Each group contained 3 replicates of 20 fruit. 2.3. Carbon dioxide and ethylene production Respiration rate and ethylene production from the whole fruit was determined by incubating four replicates of 3 /4 fruit in 1.7-l jars. After 3 h of incubation at 20 8C, 1-ml air samples from the headspace of the jar were withdraw with a hypodermic syringe and injected into a gas chromatograph (Perkin Elmer Autosample). Carbon dioxide concentration was analysed by a thermal conductivity detector coupled to a Chromosorb column at 60 8C. Ethylene production was determined using a flame ionization detector and an activated alumina column kept at 140 8C. 2.4. Water potential measurement To measure flavedo and albedo water potential, 5 mm disks from the equatorial area of the fruit were excised by a cork borer. Disks of 1/2 mm thick flavedo and of 3/4 mm thick albedo were sliced with a blade and placed in a sample chamber (C-52, Wescor Inc. Logan, UT) connected to a psychrometer switchbox (PS-10) and to a dew point microvoltimeter (HT-33T). The dew point hygrometer was previously calibrated with NaCl solutions of known concentrations. To ensure initial water vapor equilibrium, water potential measurements were done at least 2 h after setting the sample into the chamber. For each water potential measurement 2/3 fruit were used. 2.5. Estimation of rind staining index Fruit were inspected and rated on a scale from 0 (no stain) to 3 (severe), based on extent of browning and injury. The results are expressed as
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RSI, calculated according to: S (Rind stain scale (0 /3) /number of fruit within each class)/total number of fruit. Experiments were repeated at least twice a season and the study was conducted over 3/4 consecutive seasons. Results reported here correspond to representative experiments but the same pattern of changes was consistently observed.
3. Results To determine if changes in the rate of water loss by alteration of storage temperature and RH may be responsible for rind breakdown in Navel sweet orange, two different experimental procedures were used. First, ‘Navelina’ fruit were stored at 30 8C under constant low (45%) and high (95%) RH, and after a weight lost of 3/4% (4 days) they were transferred to 12 and 20 8C. Figs. 1A and B show that weight loss was very high in fruit stored at 30 8C and 45% RH and that transfer of fruit from 30 to 12 8C or 20 8C reduced water loss to a rate similar to that of fruit continuously stored at
12 or 20 8C. No rind staining were observed in fruit stored under these temperature regimes and at both 45 and 95% RH (Fig. 1C and D). In a second experiment, the effect of altering RH in ‘Navelina’ fruit stored at constant temperature (20 8C) was also examined. After 3 weeks, weight loss of fruit stored at 45% RH was about 4 times higher than that of those kept at 95% (Fig. 2A). Transfer of fruit to 95% RH after 7 (2.2% of weight lost) and 14 (4.2%) days at 45% RH reduced dehydration to a rate simiar to that of fruit continuously stored at 95% RH. (Fig. 2A). At constant RH, the RSI remained low (0.5) for up to 3 weeks of storage, but upon transfer from 45 to 95% RH, a marked increase in rind breakdown was produced. By 4 days after transfer, the RSI increased from 0.2 to 1.2 in fruit stored 7 days at 45% RH and from 0.4 to 2.0 in fruit stored 14 days at 45% RH (Fig. 1B). Sensitivity to rind staining by transfer of the fruit from low to high RH was consistently observed in mature fruit of both ‘Navelina’ (Fig. 3A and B) and ‘Navelate’ (Fig. 3C /G) sweet oranges and increased with fruit maturation (data not shown).
Fig. 1. Weight loss (A and B) and RSI (C and D) in ‘Navelina’ oranges stored at 30 (k), 20 (^) and 12 8C (\) and 45% (left panels) and 95% RH (right panels) and of fruit transferred after 4 days storage from 30 to 20 8C (') or to 12 8C (%). Arrows indicate the day of transfer. Values are the means9/S.D. of 30 fruit.
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Fig. 2. Weight loss (A) and RSI (B) of ‘Navelina’ oranges stored at 20 8C and 45% (k) or 95% (I) RH, and of fruit transferred to 95% RH after 7 (m) and 14 (j) days storage at 45% RH. Arrows indicate the time of transfer. Values are the mean9/S.D. of 30 fruit.
Initial symptoms of postharvest rind breakdown in ‘Navelina’ and ‘Navelate’ oranges were the depression or collapse of irregular areas usually scattered over the equatorial region of the fruit. Depressed flavedo areas became apparent within 3 /7 days after transfer of fruit from 45 to 95% RH, and were irregular in size, from small spots clustering as they spread (Fig. 3A, D and E) to big depressions covering a large surface area of the fruit (Fig. 3C). Several days after the appearance of flavedo depression, affected areas became dried, discoloured and turned progressively brown and black (Fig. 3B and E), forming sunken clustered areas with necrotic epidermal cells (Fig. 3F and G).
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Fig. 3. Postharvest rind staining in ‘Navelina’ (A and B) and ‘Navelate’ (C /G) oranges. The disorder first appears as irregular depressed flavedo areas (A, C and D), becoming progressively dry and brown (B and E). Severe symptoms (F and G), forming sunken clusters with necrotic epidermal cells.
Transfer of ‘Navelina’ fruit from 45 to 95% RH also induced a significant increase in the respiration rate and ethylene production (Fig. 4). In mature fruit, the respiration rate declined after storage at 20 8C regardless of the RH, but after 7 days storage at 95% it was slightly higher than in fruit maintained under 45% RH. Transfer of fruit from 45 to 95% RH, irrespective of the time, almost doubled the rate of respiration, which reached a maximum within 12/24 h after the shift in RH (Fig. 4A). During fruit storage, ethylene production was very low (/0.03 nl g 1 h 1) and remained nearly constant under both 45 and 95% RH for up to 21 days. Parallel with respiration,
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Fig. 4. Respiration rate (A) and ethylene production (B) of ‘Navelina’ oranges stored at 20 8C and 45% (k) or 95% (I) RH, and of fruit transferred to 95% RH after 7 (m) and 14 (j) days storage at 45% RH. Arrows indicate the time of transfer. Values are the mean9/S.D. of 3 replicates of 4 fruit.
transfer of fruit to 95% RH after 7 or 14 days storage at 45%, induced a 5- and a 7-fold increase in ethylene production, respectively (Fig. 4B). The increases in respiration and ethylene were transient, since 3 /5 days after storage at 95% RH the rates of both processes were similar to those of fruit kept at constant RH. Water potential (Cw) of flavedo and albedo tissue 24 and 48 h after transfer was also determined (Fig. 5A and B). In freshly harvested fruit, Cw was slightly lower in the albedo than in the flavedo. After 7 days storage at 45% RH, Cw was reduced by 30 and 15% in the flavedo and albedo, respectively, whereas under high RH it remained constant in the flavedo and slightly higher in the
Fig. 5. Water potential of flavedo (A) and albedo (B) of ‘Navelina’ fruit stored for 7 and 14 days at 20 8C and 45% (open bars) and 95% RH (hatched bars), and 24 and 48 h after transfer from 45 to 95% RH (filled bars). Values are the mean9/ S.D. of 6 measurements.
albedo. Recovery of Cw by transfer to 95% RH was faster in the flavedo than in the albedo and dependent on the time of storage under low RH. After 48 h of transfer of fruit stored for 7 days at 45% RH, Cw in the flavedo increased by 25% and only by 11% in the albedo. However, prolonged storage at low RH reduced the ability of Cw to recover by transfer to 95% RH (Fig. 5). To evaluate the influence of environmental preharvest conditions on the susceptibility of the fruit to postharvest rind staining, the effect of altering RH during storage on ‘Navelate’ fruit harvested from orchards prone (319/2.5% of affected fruit at commercial maturity) and not prone (B/3%) to natural rind breakdown, was examined. After 16 days storage at 20 8C and at both 45 and 95% RH, the proportion and severity of rind breakdown were higher in fruit from the
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Table 1 Percentage and index of rind staining in ‘Navelate’ fruit harvested from orchards affected and not-affected by natural rind staining and stored for up to 16 days at 20 8C under different RH conditions Storage conditions
16 days 45% RH 16 days 95% RH 4 days 45%/12 days 95%
Affected orchard
Not-affected orchard
%
RS index
%
RS index
54.3a 80.4b 90.8b
0.64a 1.21b 1.92c
28.2a 43.1b 66.7c
0.36a 0.43a 1.42b
Fruit were harvested in January. Within each column, values followed by the same letter are not significant at P B/0.05% using Student’s t -test.
affected orchard stored at 95% RH (Table 1). Transfer of the fruit to 95% after 4 days of storage at 45% RH increased 3- and 4-fold the RSI for fruit from affected and not-affected orchards, respectively, the percentage and severity of damage being higher in the former. Interestingly, ethylene production was 6 times higher in freshly harvested fruit from the affected orchard and became almost constant after storage at 45 or 95% RH for up to 4 days. Transfer of the fruit to 95% RH sustained the high rate of ethylene production up to day 5 and then declined (Fig. 6B). In fruit from the not-affected orchard, ethylene production remained low during storage at 45% RH whereas at 95% RH a significant
increase after 4 days was observed. The transfer to 95% RH increased 12-fold ethylene production after 12 h, declining thereafter (Fig. 6A). Fig. 7 shows Cw of flavedo and albedo of fruit harvested from the two orchards differing in rind breakdown severity after 7 days of storage at 45 and 95% RH. In both tissues, Cw was about 10% lower in fruit from the affected orchard. After 7 days of storage, flavedo and albedo Cw of fruit harvested from the affected orchards slightly increased but there were no significant differences between 45 and 95% RH. By contrast, in the tissues of fruit from the not-affected orchard, Cw substantially recovered with storage at high RH (Fig. 7).
Fig. 6. Ethylene production of ‘Navelate’ oranges harvested from an orchard not-affected (A) or affected (B) by natural rind staining and stored for up to 7 days at 20 8C and 45% (I) and 95% (k) RH or transferred to 95% RH after 4 days storage at 45% (m). Arrows indicate the time of transfer. Values are the mean9/S.D. of 3 replicates of 4 fruit.
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Fig. 7. Water potential of flavedo (A) and albedo (B) of ‘Navelate’ oranges harvested from an orchard affected (filled bars) or not-affected (open bars) by natural rind staining and stored for 7 days at 20 8C and 45 and 95% RH. Values are the mean9/S.D. of 6 measurements.
4. Discussion The results reported in this study indicate that changes in RH during storage, particularly transfer to high RH of fruit dehydrated at low RH provoke rind breakdown in Navel oranges. Under field conditions we have previously observed that rind breakdown of ‘Navelate’ oranges occurred after several days of high RH and low transpiration following a period of low RH and high transpiration (Agusti et al., 2001). These observations suggest that sudden increases in the environmental RH after episodes of fruit dehydration appear to be responsible for rind staining in Navel sweet orange either under field conditions or during postharvest storage. Water stress has been related to other disorders morphologically similar to Navel rind breakdown, such as stem end rind breakdown in ‘Valencia’
oranges (Albrigo, 1972) and noxan in ‘Shamouti’ oranges (Ben-Yehoshua et al., 2001). In ‘Shamouti’ fruit, the incidence of noxan was reduced by different postharvest treatments that maintained a high RH and reduced weight loss (BenYehoshua et al., 2001). Lafuente and Sala (2002), however, found that the storage of ‘Navelina’ oranges at high RH was deleterious to the incidence of rind staining. Our results showed that water stress or fruit dehydration itself appear not to be the main cause inducing rind staining in ‘Navelina’ and ‘Navelate’ sweet oranges. By manipulating the storage temperature, at constant RH regimes, we were able to induce water loss even higher than that of fruit stored under 45% RH at 20 8C and transferred to 95% RH. Under these conditions, the peel of the fruit lost turgidity and became soft, characteristic symptoms of citrus fruit dehydration (Ben-Yehoshua, 1987), but it did not pit or stain, indicating that dehydration alone does not provoke rind staining in Navel oranges. Transfer of fruit previously dehydrated at 45 / 95% RH provoked a substantial increase in the RSI, that was higher in fruit stored at 45% RH for longer periods (Fig. 2B). Using these experimental conditions we were able to reproduce rind breakdown in ‘Navelina’ and ‘Navelate’ sweet oranges and thus study the physiological responses of the fruit to these postharvest conditions. The magnitude of water loss appears to be an important factor in the development of rind staining upon transfer to high RH. In fact, we observed that fruit with less than 2% weight loss did not develop peel blemish (data not shown) and that the disorder appeared faster in fruit maintained for 14 days at 45% than in those stored for 7 days at the same RH. This fruit response was also dependent on fruit maturity, as development of rind breakdown was faster in more mature fruit (data not shown). These observations raise the question of the nature of the responses of dehydrated Navel fruit to sudden changes in RH and how these changes may be related to rind staining development. In fruit stored under a high water vapor pressure deficit (45% RH), water is driven from the epidermal cell to the surrounding atmosphere and, consequently, reduces flavedo and albedo Cw (Fig. 4). Transfer of the fruit to a high RH
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atmosphere would reduce the water vapor pressure deficit and change the water movement from the air phase to the inner cells of the peel. Our results show that flavedo cells, as expected, recovered Cw faster than the inner albedo cells, and this might create a suction force demanding water from the outer layers of hydrated cells. This process may explain the initial appearance of the blemish as a depressed flavedo area compressed to the adjacent albedo, in accordance with our results showing that the disorder is initiated at the transitional layer of cells between flavedo and albedo (Agusti et al., 2001). In the flavedo and albedo of fruit exposed for a prolonged period at low RH, Cw were recovered more slowly after transfer to 95% RH, probably due to cellular damage and membrane deterioration of water-stressed cells (BenYehoshua, 1969; Ben-Yehoshua et al., 1983). Maintaining albedo cells under low Cw for extended periods would increase their demand of water from the flavedo cells and may explain the rapid increase in the RSI observed in fruit transferred to 95% RH after 14 days at 45% RH or even in fruit harvested from orchards susceptible to rind staining on the tree. Mantell et al. (1980) using tritiated water showed that the turnover of water was extremely low in the peel of citrus fruit, even in fruit with a high transpiration rate, corroborating our suggestions. These earlier responses of dehydrated fruit to sudden changes in the surrounding RH may explain the twisted and flattened morphology of affected cells of the albedo (Agusti et al., 2001). The characteristic staining and browning of the flavedo observed after several days (Fig. 3) would be a late response, probably due to oxidative processes (Agusti et al., 2001; Lafuente and Sala, 2002). Stimulation of the respiration rate and ethylene production was also an early and transient response to the transfer of dehydrated fruit to high RH, before the appearance of initial symptoms of depression in the flavedo. Since adjustment of flavedo and albedo Cw occurred within few days after the transfer, the increases in respiration and ethylene production appear to be related to sudden changes in cell wall turgor or to cellular damage induced by the shift in the environmental RH, rather than to the development of rind staining.
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Stimulation of the respiration by different stress conditions, including water stress, has been previously reported in citrus and other fruit (Vines et al., 1968; Ben-Yehoshua et al., 1983; Kader, 1987), and water stress-induced ethylene production is a well documented phenomena (Apelbaum and Yang, 1981; Morgan and Drew, 1997). Hydration of wheat leaves at full turgor after water stress increased ethylene production (Beltrano et al. 1997). Felix et al. (2000) have found that hyperosmotic shock in tomato cell cultures induced an increase in ACC synthase activity, in the extracellular pH and in the release of K ions. These observations suggest that the changes induced by hydration of water-stressed Navel fruit may induce membrane and cellular damage responsible for the stimulation of respiration and ethylene production. It is interesting to note that ethylene production of ‘Navelate’ fruit harvested from a high rind stain affected orchard was significantly higher than that of fruit harvested from a not-affected orchard and that flavedo and albedo Cw of these fruit did not recovered after storage at 95% RH. Thus, ethylene production at harvest may be a physiological marker of the susceptibility of the fruit to the disorder. The reduced capability of Cw adjustment of the flavedo and albedo tissue of fruit from affected orchards after storage at different RH and their higher development of rind staining (Table 1) agree with the results obtained in fruit stored at low RH for prolonged periods and indicate that the water status of fruit at harvest is a critical factor for the incidence and development of rind staining during postharvest storage. These observations may explain the erratic incidence of rind staining observed during postharvest handling and storage of Navel oranges. In conclusion, sudden changes in the environmental RH are characteristic of the climatic conditions in the citrus growing areas, and diurnal changes in water (Albrigo, 1977) and osmotic (Kaufman, 1970) potential have been demonstrated in citrus fruit. Harvesting the fruit after a period of water stress, and postharvest handling and storage conditions that allow excessive water loss, followed by washing, waxing and storage at high RH, may be deleterious for the incidence of
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rind breakdown in Navel oranges. Although the experimental systems and the storage conditions used in this study do not correspond with the standard postharvest procedures for Navel oranges, they were effective in inducing fruit rind staining and may be indicative of the factors responsible for the disorder.
Acknowledgements This study was supported by grants from Generalitat Valenciana, Conselleria d’Agricultura, Pesca i Alimentacio´ (GV-CAPA 97-02 and GCCAPA 00-15). We thank Reva S.A. for the use of their commercial ‘Navelate’ orchards and Miguel Sabater for providing us with ‘Navelina’ oranges.
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