POSTHARVEST ETHYLENE CONDITIONING AS A TOOL TO REDUCE QUALITY LOSS

OFSTORED MATURE SWEET ORANGE

ABSTRACT
Ethylene is related to senescence but also induces protective mechanisms against stress in plants.
The citrus industry only applies the hormone to induce fruit degreening. The aim of this work was to
deter-mine the effect of ethylene on the quality of colored citrus fruit stored under commercial
conditions to extend postharvest life, since it protects them from stress causing postharvest
disorders such as chilling injury (CI) and non-chilling peel pitting (NCPP). The effect of conditioning
mature Navelate and Lane Late sweet oranges (Citrus sinensis L. Osbeck) for 4 days with 2 _L
L1ethylene at 12C, rather than at higher temperatures used for degreening, on the quality of fruit
stored at 2 or 12C, was examined. The ethylene conditioning (EC) treatment did not increase color
but reduced calyx abscission and NCPP in fruit of both cultivars stored at 12C, and also CI in
Navelate fruit at 2C. Lane Late fruit did not develop CI but showed a new disorder in EC fruit held
at 2C. This disorder began as scalded areas around the fruit stem end and extended over the fruit
surface during storage. EC had no deleterious effect on the quality of Navelate oranges stored at
either 2 or 12C. Similar results were found in Lane Late fruit although EC slightly increased offflavor perception at 2C and the maturity index at 2 and 12C. Moreover, EC slightly increased the
content of bioactive flavonoids in the pulp of Navelate fruit but significant differences between
control and EC fruit were only found after prolonged storage at 2C. In Lane Late fruit, EC avoided
the initial decrease in flavonoid content found in control samples. Results show, therefore, that EC
at 12C may be a tool to extend postharvest life of NCPP and CI-sensitive oranges, and that the
tolerance of citrus cultivars to the combined effect of EC and non-freezing low temperature (2C)
should be tested to select the proper storage temperature.
Introduction
Several types of postharvest physiological disorders affecting peel quality have been described in
citrus fruit but factors causing them and how they are related to each other is not understood
(Grierson, 1986; Lafuente and Zacaras, 2006; Magwaza et al.,2013). Efforts have been made to
reveal factors responsible fordifferent postharvest citrus fruit disorders and to develop new
strategies to control them. However, currently no proven methodologies exist to reduce their
incidence. Most studies on reduction of physiological peel disorders have been related to
methodologies aiming at reducing chilling injury (CI), and the effect on citrus fruit quality has been
well characterized (Schirra et al., 2005; Mulas and Schirra, 2007).CI and non-chilling peel pitting
(NCPP), also known as rind-staining, are two main postharvest physiological disorders in citrus fruit
(Lafuente and Zacaras, 2006). NCPP develops at temperatures higher than those causing CI,
which in citrus fruit may occur at temperatures ranging between 1 and 12C. Sweet oranges from
the Navel group, may develop these physiological disorders (Lafuente and Sala, 2002; Alfrez et
al., 2003; Holland et al., 2005). Navelate orange (Citrus sinensis (L.) Osbeck) is prone to develop
NCPP as well as CI after prolonged cold storage. In this cultivar, NCPP is characterized by
depressed areas in the peel affecting both the albedo (inner part of the peel) and the flavedo (outer
part of the peel), which eventually turn bronze in color; while CI is manifested as superficial scalding
or flavedo bronze non-depressed areas (Alfrez et al.,2005). The postharvest performance of other
late maturing sweet Navel orange cultivars, which have expanded very quickly in many countries to
extend the citrus season, is less known. This is the case of the late-maturing Lane Late orange (C.
sinensis (L.) Osbeck) that has good fruit quality characteristics. Ethylene is usually related to
senescence but it plays also a roleprotecting plants from stress causing damage (Yang and
Hoffman,1984). Although this hormone may enhance a senescence-related physiological disorder,

rind breakdown in Nules Clementine man-darin (Cronj et al., 2011), or other peel defects such as
zebra skin or horseshoe/green rings in other citrus cultivars (Krajewsky and Pittaway, 2002), it also
protects citrus fruit from stresses causing CI and NCPP (Lafuente et al., 2001; Lafuente and Sala,
2002). The industry only applies this hormone to early-maturing citrus fruit for degreening purposes
although it may reduce physiological disorders in mature fruit harvested after color break
(Lafuenteand Sala, 2002; Lafuente et al., 2004; Cajuste and Lafuente, 2007).The effect on the
quality of citrus fruit of postharvest degreen-ing at 90% RH and 2022C using 24 _L
L1ethylene, has been documented (Carvalho et al., 2008; Mayuoni et al., 2011a; Sdiriet al.,
2012a,b; Moscoso Ramrez and Palou, 2014). Previous data also show that conditioning mature
citrus fruit with ethylene concentrations ranging between 1 and 10 _L L1considerably reduces
NCPP in fruit stored afterwards at 2022C and increases peel phenolic content (Lafuente and
Sala, 2002; Cajuste and Lafuente, 2007).Moreover, it is known that physiological responses of
citrus fruit to ethylene may vary with maturation stage (Lisker et al., 1983; John-Karuppiah and
Burns, 2010). However, there is no information on the effect of ethylene on the quality of fully
mature fruit stored for prolonged periods at lower temperatures under commercial conditions
required to extend postharvest life. Experiments performed to evaluate the effect of degreening
have been limited to examining changes occurring after a single quarantine treatment (1Cfor 16
days) in fruit of citrus cultivars that did not develop physiological disorders (Sdiri et al., 2012a), while
storage duration and temperature affects citrus fruit quality (Mulas and Schirra, 2007;Obenland et
al., 2008). Therefore, the aim of this work was to evaluate the effect of treating full colored mature
sweet Navelate and Lane Late orange cultivars, which are not degreened for commercialization, for
4 days with 2 _L L1ethylene at 9095% relative humidity (RH) on the quality of fruit stored for up
to 60 days at2 or 12C and 9095% RH. This ethylene conditioning (EC) treatment was performed
at 12C rather than at the temperature used for commercial degreening (2021C) (Mayuoni et al.,
2011a; Sdiriet al., 2012a,b). The degreening temperature is recommended to enable color
development after ethylene treatment. Fruit in this study were already fully colored, and therefore a
lower temperature (12C) was used to reduce fruit quality loss. The effects of the treatment on color,
firmness, weight loss and internal quality of the fruit, as well as the incidence of calyx abscission,
decay and physiological disorders were evaluated. Considering that ethylene increases the activity
of the enzyme phenylalanine ammonia-lyase (PAL) in citrus fruit, especially in the more mature fruit
(Liskeret al., 1983), that this is a key enzyme at the entry point of phenyl-propanoid metabolism,
and the health benefits of phenolics (Tripoliet al., 2007), we have also evaluated in the fruit edible
portion the effect of the EC treatment on the accumulation of total phenos and flavonoids, which are
the most abundant phenolics in citrus fruit (Nogata et al., 2006).
2. Materials and methods
2.1. Plant material and postharvest treatments
Fully mature Navelate and Lane Late sweet oranges (C. sinensis (L.) Osbeck) were harvested in
March from the same orchard at Lliria (Valencia, Spain), and immediately delivered to the
laboratory. Fruit were surface-sterilized with a commercial bleach solution (Ballester et al., 2013)
and then randomly divided into 2 groups that were treated with ethylene (group 1) or held in air
(control fruit, group 2). These groups were further subdivided into two subgroups to evaluate the
effect of the storage temperature (2and 12C). Fruit in each subgroup were divided into two lots.
Three replicates of 10 fruit were included in the first lot to estimate periodically the incidence and
severity of physiological disorders and of calyx abscission. The second lot contained 3 replicates of
10 fruit per storage period since they were used to determine changes in peel color but also in other
quality attributes in the pulp requiring destructive methods. Fruit within the first group were
conditioned for 4 days with 2 _L L1ethylene at 9095% RH and 12C (EC fruit), and then stored at

2 (subgroup 1) or 12C (subgroup 2). Control fruit were maintained for 4 days in air at 9095% RH
and 12C (AC fruit) before being stored at 2 or 12C. EC and AC fruit were kept for 8 weeks at 2C
or for 6 weeks at 12C and then transferred to20C for 4 days to simulate a shelf-life (SL) period.
2.2. Estimation of postharvest physiological disorder severity and incidence
Three different physiological disorders were identified under the above mentioned storage
conditions. Since their symptoms were different, severity was evaluated independently during
holding of AC and EC fruit at 2 and 12C, and after the SL period. Depending on the cultivar,
postharvest conditioning treatment or storage condition, fruit showed: (1) NCPP, manifested as
collapsed surface areas that became dark brown with time (Supplemental Fig. S1A); (2) CI,
manifested as bronzed non-depressed areas (scalding) of the fruit surface (Supplemental Fig. S1B);
and/or (3) a physiological disorder, which as far as we know has not been previously described in
citrus fruit, that begins as scalded areas around the stem end of the fruit (Supplemental Fig. S1C)
and extends through the fruit surface during storage (Supplemental Fig. S1D).This disorder, named
SECI (from stem end chilling injury), shows non-depressed areas in the fruit stem end, and
therefore it is different to stem-end-rind-breakdown (SERB). A rating scale from 0(no damage) to 4
(severe damage) (Vicente et al., 2013) was used to determine the average severity damage index
of each physiological disorder. In this scale, only fruit showing a damage score higher than 1 would
be rejected by consumers. The same fruit were used at the various evaluation dates. The severity
indexes were calculated by adding up the products of the number of fruit in each category multiplied
by its score, and then dividing the total obtained by the number of fruit, evaluated as described by
Lafuente et al. (1997). Moreover, the commercial incidence of each physiological disorder was
estimated by calculating the percentage of fruit showing a damage score higher than 1. The results
were the means of 3 replicate samples containing 10 fruit each S.E.M. Supplemental
2.3. Analysis of quality attributes
Changes in fruit quality were assessed by measuring the acidity and SSC in the pulp, peel fruit
color, and fruit firmness and weight loss as described by Holland et al. (1999) and Lafuente et al.
(2011). Soluble solids (Brix) were determined from fruit juice with an Atago/X-1000 digital
refractometer (Atago Co. Ltd., Tokyo,Japan) and acid content was titrated with 0.1 N NaOH using
phenol-phthalein as indicator and expressed as g of anhydrous citric acid in 100 mL of juice. The
maturity index was calculated by dividing the Brix of the extracted juice by its acid content. Peel
color was analyzed by using a Minolta CR-300 Chromameter (Konica MinoltaInc, USA) with a
measuring area of 8 mm at four locations around the equatorial plane of the fruit. The color index
was expressed as the a/b Hunter ratio that is classically used for color measurement in citrus fruit
(Stewart and Wheaton, 1972). This ratio is negative for green fruit and positive for orange fruit,
while zero value corresponds to yellow fruit at the midpoint of color break period. Weight loss was
expressed as the cumulative percentage of fruitweight loss during fruit storage and fruit firmness
determined using a TA-XT2 Texture Analyser (Stable Micro Systems, Godalming, UK)and
expressed as the percentage of fruit deformation resulting from a pressure of 10 N on the fruit
longitudinal axis at a constant speed (Sdiri et al., 2013). Along with these quality attributes, sensory
evaluation was performed by eight semi-trained panelists as described by Sdiri et al. (2012a) using
a rating scale from 1 to 10, where10 = excellent, 5 = fair (commercially acceptable), and 1 =
extremely unpleasant. The average flavor acceptance was calculated following the same procedure
described above to calculate the average index severity of peel disorders.
2.4. Evaluation of calyx abscission and decay incidence

The incidence of calyx abscission and decay was evaluated after conditioning the fruit with ethylene
at 12C, during fruit storage at2 and 12C and after the SL period. Calyx abscission incidence was
expressed as the percentage of fruit without calyx compared to the total number of fruit in each
replicate sample and decay incidence estimated by quantifying the percentage of decayed fruit.
2.5. Analysis of total phenolics and flavonoids
Total phenolic content was determined following a method based on that described by Swain and
Hillis (1959) as reported by Cajuste and Lafuente (2007). Briefly, 500 _L juice were extracted with
1500 _L of ethanol using a Mini Bead beater 8 Cell Disruptor (Biospec Products, Inc.). The extract
was centrifuged at 13,000 g for 5 min at 4C and the supernatant used to estimate phenolic
content. Two sample aliquots of 250 _L of 5-fold diluted supernatant were incubated at room
temperature with 250 _L of 1 N Folin-Ciocalteau and 2500 _L of 2% Na2CO3. After centrifugation
at13,000 g for 5 min at 4C, absorbance was recorded at 724 nm and the total phenolic content
determined by using a standard curve developed with chlorogenic acid. Total flavonoids were
determined in the same supernatant used to analyze total phenolics by following the method
described by Gonzlez-Aguilar et al. (2007) with some modifications. Two supernatant aliquots of
100 _L were diluted with 400 _L of nanopure water and 30 _L of 5% NaNO2 were added to each
aliquot. After5 min incubation at room temperature, 30 _L of 10% AlCl3wereadded and at 6 min the
reaction was stopped by adding 200 _L of 1 N NaOH. Flavonoid contents were determined by
comparing the absorbance at 350 nm with that of prepared blanks, calculated using a standard
curve prepared with hesperidine, which is the major flavonoid in the pulp, and results expressed as
mg 100 g1.The total phenolic and flavonoid contents were determined in the same fruit used to
analyze quality attributes. Results are the means of three biological replicate samples S.E.M.
2.6. Statistical analysis
A mean comparison using the Tukeys test was performed to determine if means values were
significantly different (P 0.05) between EC and AC control fruit for each cultivar and at each
experimental storage temperature and sampling period.
Fig. 1. Non-chilling peel pitting (NCPP) index of Navelate and Lane Late oranges stored at 2 or
12C after being conditioned at 12C for 4 days with 2 _L L1ethylene (EC, white) and of their
respective controls maintained 4 days in air at 12C prior storage (AC, black). Dashed line indicates
transfer of fruit at 20C to simulate shelf-life (SL). Results represent the means of three replicates
samples S.E.M. Asterisks indicate significant differences (P < 0.05) between EC and their
respective control samples (AC) for the same storage period.
3. Results and discussion
3.1. Effect of ethylene conditioning on postharvest physiological disorders of citrus fruit stored at
chilling and non-chilling temperatures
The effects of ethylene degreening on quality attributes of different still-green citrus fruit cultivars
have been reported (Carvalhoet al., 2008; Mayuoni et al., 2011a; Sdiri et al., 2012a,b). However,
these effects were not evaluated after prolonged storage, which favors the development of
physiological disorders. Moreover, still-green citrus fruit are less sensitive to develop CI (Lafuente et
al.,1997) and NCPP (Alfrez and Zacaras, 2013) than mature colored fruit. The efficacy of EC
treatments at 20C reducing NCPP in mature Navelina and Navelate sweet oranges stored under
non-stressful environmental conditions (20C and 9095%RH) has been shown during different
citrus seasons (Lafuente and Sala, 2002; Cajusteet al., 2010; Vicente et al., 2013). Nevertheless,

the effect of EC at12C on the incidence of physiological peel disorders and other quality attributes
of fruit that do not need to be degreened, stored at lower temperatures, remains unknown. Two
storage temperatures were selected in the present study. Fruit were stored at 2C,to maximize the
storage period and to test the combined effect of ethylene and low temperature stress, and at 12C
which might avoid the appearance of CI and delay fruit aging as compared to holding the fruit at
20C.NCPP symptoms were evident by 20 days at 12C in the ACN avelate and Lane Late oranges
and increased thereafter (Fig. 1).While the susceptibility of Navelate fruit to postharvest NCPP is
well known (Alfrez et al., 2003; Sala et al., 2005; Romero et al.,2012), as far as we know, this is
the first study showing that Lane Late orange is also susceptible to the disorder although its
susceptibility is lower (Fig. 1). By day 20, the NCPP index of AC Navelate oranges held at 12C was
higher than 1, indicating the existence of fruit lacking the quality required for commercialization.
Under these conditions, 39% of AC Navelate fruit could be rejected because low external quality
(Table 1). The number of unacceptable LaneLate oranges was much lower by this period (11.5%,
Table 1). This difference between cultivars decreased by 40 days but a lower value was still found
after the SL period in Lane Late fruit. As expected (Lafuente and Sala, 2002; Alfrez et al., 2005),
keeping AC fruit from both cultivars at 2C delayed the appearance of non-commercial NCPP (Table
1) and reduced NCPP severity (Fig. 1) and incidence (Table 1). At this temperature, NCPP index
was lower than 0.8 by40 days storage in Navelate oranges and than 0.6 by 60 days in Lane Late
fruit. Conditioning the fruit with 2 _L L1ethylene at 12C considerably increased the tolerance of
oranges of both cultivars to NCPP (Fig. 1). The treatment was very effective reducing the
development of NCPP at both storage temperatures since the NCPP index of EC fruit was lower
than 1 during fruit storage at 2 or 12C and after simulation of SL (Fig. 1). This is in concordance
with previous findings showing the effect of ethylene reducing NCPP in citrus fruit at20C (Lafuente
and Sala, 2002; Vicente et al., 2013). Furthermore, it shows that despite ethylene-induced
responses being temperature dependent, it is possible to decrease the temperature of the EC
treatment to reduce NCPP incidence and other negative effects on quality attributes occurring at
higher temperatures. As shown in Table 1, the percentage of fruit showing commercially
unacceptable NCPP at 12C was lower than 8% even after the SL period in both cultivars while the
percentage was higher than 50% in the AC fruit. Fruit storage at 2C caused CI in Navelate orange
by 40 days, although CI severity was very low (Fig. 2, upper panel). The effect of conditioning citrus
fruit with ethylene on reducing CI in very chilling sensitive citrus cultivars is low (Lafuente et al.,
2001). However, CI was reduced in the less chilling sensitive citrus cultivar Navelate by the EC
treatment (Fig. 2, upper panel). Thus, EC Navelate oranges did not show commercially relevant CI
for up to 60 days and its severity and incidence were low (data not shown).Results also showed that
AC Lane Late oranges did not develop CI (data not shown) and that fruit from this cultivar
conditioned with ethylene developed SECI when stored at 2C (Fig. 2, lower panel). To the best of
our knowledge, SECI has not been previously described as a postharvest disorder in citrus fruit. It
appears in the fruit stem-end areas as scalded patches and extends superficially along the fruit.
Therefore, SECI differs from SERB, a non-chilling related peel disorder that manifests as depressed
areas in the stem and is associated with senescence and enhanced by dehydration (Grierson,
1986; Romero et al., 2013). Since SECI was not observed at 12C, results indicate that it may be
caused by the combined of fruit lacking the quality required for commercialization. Under these
conditions, 39% of AC Navelate fruit could be rejected because low external quality (Table 1). The
number of unacceptable Lane Late oranges was much lower by this period (11.5%, Table 1). This
difference between cultivars decreased by 40 days but a lower value was still found after the SL
period in Lane Late fruit. As expected (Lafuente and Sala, 2002; Alfrez et al., 2005), keeping AC
fruit from both cultivars at 2C delayed the appearance of non-commercial NCPP (Table 1) and
reduced NCPP severity (Fig. 1) and incidence (Table 1). At this temperature, NCPP index was lower
than 0.8 by 40 days storage in Navelate oranges and than 0.6 by 60 days in Lane Late fruit.

Conditioning the fruit with 2 _L L1ethylene at 12C considerably increased the tolerance of
oranges of both cultivars to NCPP (Fig. 1). The treatment was very effective reducing the
development of NCPP at both storage temperatures since the NCPP index of EC fruit was lower
than 1 during fruit storage at 2 or 12C and after simulation of SL (Fig. 1). This is in concordance
with previous findings showing the effect of ethylene reducing NCPP in citrus fruit at20C (Lafuente
and Sala, 2002; Vicente et al., 2013). Furthermore, it shows that despite ethylene-induced
responses being temperature dependent, it is possible to decrease the temperature of the EC
treatment to reduce NCPP incidence and other negative effects on quality attributes occurring at
higher temperatures. As shown in Table 1, the percentage of fruit showing commercially
unacceptable NCPP at 12C was lower than 8% even after the SL period in both cultivars while the
percentage was higher than 50% in the AC fruit. Fruit storage at 2C caused CI in Navelate orange
by 40 days, although CI severity was very low (Fig. 2, upper panel). The effect of conditioning citrus
fruit with ethylene on reducing CI in very chilling sensitive citrus cultivars is low (Lafuente et al.,
2001). However, CI was reduced in the less chilling sensitive citrus cultivar Navelate by the EC
treatment (Fig. 2, upper panel). Thus, EC Navelate oranges did not show commercially relevant CI
for up to 60 days and its severity and incidence were low (data not shown).Results also showed that
AC Lane Late oranges did not develop CI (data not shown) and that fruit from this cultivar
conditioned with ethylene developed SECI when stored at 2C (Fig. 2, lower panel). To the best of
our knowledge, SECI has not been previously described as a postharvest disorder in citrus fruit. It
appears in the fruit stem-end areas as scalded patches and extends superficially along the fruit.
Therefore, SECI differs from SERB, a non-chilling related peel disorder that manifests as depressed
areas in the stemand is associated with senescence and enhanced by dehydration (Grierson, 1986;
Romero et al., 2013). Since SECI was not observed at 12C, results indicate that it may be caused
by the combined effect of ethylene and chilling in Lane Late oranges, while Navelate oranges are
more tolerant of such a combination in spite of develop-ing some CI. Fruit of both cultivars were
harvested the same date and from the same orchard. Therefore, the different responses of EC
Navelate and Lane Late oranges to cold should be more likely related to genetic factors than to
different preharvest conditions. Overall results indicate that applying ethylene immediately after fruit
harvest at 12C could be a tool to reduce the incidence of physiological disorders in mature citrus
fruit. However, the effect of EC followed by extreme low non-freezing storage temperatures should
be tested in each citrus cultivar. Thus, result from this works how that conditioning the NCPP prone
Navelate orange with ethylene and storing it at 12C could be a feasible method to reduce this peel
disorder. Similar results might be obtained in EC Navelate fruit stored at 2C since CI was very low.
Therefore, storage temperature of Navelate oranges conditioned with ethylene should be selected
also considering changes in other fruit quality attributes. In contrast, conditioning Lane Late fruit
with ethylene could cause SECI if fruit are stored at very low temperatures (2C), although EC fruit
kept at 12C in this cultivar showed very low peel damage. Alternatively, storing AC Lane Late fruit
at 2C would also be effective in reducing NCPP without causing other peel disorders.
3.2. Effect of ethylene conditioning on calyx abscission and disease incidence
Calyx abscission is an important external fruit quality factor since it affects freshness and general
appearance of fruit. The susceptibility to postharvest calyx abscission varies among citrus cultivars
(Carvalho et al., 2008). Results from degreening experiments indicate that the effect of ethylene on
calyx abscission depends on the ethylene doses, the citrus cultivar and the fruit maturity stage (Sdiri
et al., 2012a). Our results showed that calyx abscission was very low at 2C and that the EC
treatment reduced it in fully mature fruit from both cultivars stored at 12C (Fig. 3).In contrast,
treating Clemenules mandarins with 2 L L1ethyl-ene at 21C for degreening purposes favored
calyx abscission in fruit stored for 16 days at 1C (Sdiri et al., 2012a). Results from the same

authors showed that the treatment had a different effect on the early-ripening Navelina orange. In
this cultivar, it induced calyx abscission in deep green fruit but its effect was variable and negligible
in more mature fruit, which is in concordance with the fact that the less mature fruit (deep green),
the more susceptible to abscission (Sdiri et al., 2012a). Further research should be conducted to
elucidate whether the effect of ethylene on reducing calyx abscission in mature Navelate and Lane
Late (Fig. 3) oranges is related to fruit maturity stage and/or the temperature at which ethylene was
applied. The ethylene treatment was performed at12C rather than at 21C since the aim of the EC
treatment was to reduce physiological disorders and minimize fruit aging instead of favoring
degreening at 21C. In this context, it should be noted that PAL activity increases in the calyx
abscission zone of citrus fruit favoring wound healing and thus reducing abscission (Kostenyuket
al., 2002), and that the rise in PAL activity in response to ethylene or other stress factors inducing
the hormone is much less relevant in green than in mature citrus fruit (Lisker et al., 1983; Lafuente
et al., 2003). It is also known that hormonal balances play a role in controlling abscission (Meir et
al., 2010), that indole-3-acetic acid (IAA) may reduce abscission (Yuan et al., 2003) and that
ethylene stimulates the degradation of IAA in citrus tissues (Winer et al., 2000). Since the capacity
of flavedo of citrus fruit for IAA catabolism decreases at the beginning of fruit senescence
(Chamarro et al., 2001), fully mature fruit might be less susceptible to ethylene-induced degradation
of IAA. The complexity of the abscission process is highlighted by results of Sdiri et al.
(2012a).These authors found that abscission was higher in less mature citrus fruit despite young
citrus fruit having higher IAA levels in the abscission zone than mature fruit (Yuan et al., 2003).
Therefore, further research should be conducted to understand factors balanceing citrus fruit
responses to ethylene at 12C and its consequences in calyx abscission. Exogenous application of
ethylene may also affect disease incidence. Degreening with high hormone levels may exacerbate
Diplodia stem-end rot incidence (Smilanick et al., 2006). Conversely, ethylene does not stimulate
Penicillium digitatum grow thand can even reduce green mold incidence in mature citrus fruit
(Marcos et al., 2005). Fruit used in the present work were immediately disinfected after harvest and
held in aseptic cold rooms. Therefore, the incidence of rots, which was mainly caused by P.
digitatum, was negligible both in AC and EC fruit (data not-shown). Diplodia stem-end rot is not
common in citrus fruit growing in Mediterranean climate so it was not surprising the absence of rots
caused by this pathogen. Nevertheless, the incidence of the EC treatment on decay should be
further tested in other citrus producing areas with wet climate and high incidence of Diplodia stemendrot.3.3. Effect of ethylene conditioning on fruit quality attributes Weight loss was very low, and
similar in AC and EC fruit, during the whole storage period in both Navelate (Table 2) and Lane Late
oranges (Table 3) kept either at 2 or 12C. The increase in weight loss occurring after SL simulation
was higher in Lane Late oranges, which were less susceptible than Navelate oranges to NCPP.
Although weight loss was very low, this result reinforces the idea that dehydration is not the only
factor responsible for NCPP development (Cajuste and Lafuente, 2007), and points out the
relevance of genetic factors in the susceptibility of citrus fruit to this physiological disorder. Fruit
firmness slightly decreased after 20 days in fruit held at12C but barely changed at 2C in Navelate
fruit. Moreover, conditioning fruit of this cultivar with ethylene had little effect on this quality attribute
(Table 2). A similar effect was observed in Lane Late oranges (Table 3), which showed higher fruit
firmness. Whether peel ultrastructure and strength are related to the lower susceptibility of this
citrus cultivar to NCPP is unknown but this result agrees with previous findings encouraging
research aimed at identifying cell wall influence on citrus fruit susceptibility to develop NCPP
(Vicente et al., 2013).The EC treatment slightly reduced pulp acidity in Navelate fruit (4 days, Table
2) but such difference was lost during fruit storage. The EC Lane Late oranges had slightly lower
acidity values during storage (Table 3). This effect was lost after SL in fruit stored at 12C.On the
other hand, results show that keeping Navelate and Lane Late oranges at 2C delayed the decline
in pulp acidity with respect to fruit held at 12C (Tables 2 and 3).The storage temperature had little

impact on soluble sugars. The4 day ethylene treatment slightly decreased SSC (4%) in the pulp of
Navelate fruit but not in Lane Late fruit. Slight differences in SSC were found between AC and EC
stored fruit and they were lost after the SL period. The EC treatment slightly increased the maturity
index in Navelate (Table 2) but not in Lane Late oranges (Table 3).After storage, the maturity index
of EC Lane Late oranges was, in general, slightly higher than that of the AC control fruit. However,
the EC treatment did not consistently modify this quality parameter in Navelate fruit. After SL, the
maturity index of ethylene-treated fruit of these cultivars was higher than that of their respective AC
fruit when they were stored at lowest temperature (2C).
For both cultivars, flavor score decreased during fruit storage. Storage at 12 C was finished by 40
days because of deleterious effects on fruit freshness appearance after this period, while storage at
2C could be extended to 60 days. By day 40, flavor scores of Navelate oranges stored at 2 and
12C were about 6.3 and 7.6, respectively (Table 2). Values found in Lane Late fruit were also close
to 7 (Table 3). Negligible differences in fruit flavor were found between AC and EC fruit and any
significant difference was lost after simulation of SL, except in Lane Late fruit kept at 2C. In this
particular sample, EC favored a significant decrease in flavor score, which is coincident with major
fruit SECI damage in this sample. Therefore, the combination of low temperature stress and
ethylene pretreatment appears to affect not only the peel but also the pulp in fruit of this cultivar.
Previous reports indicate that degreening citrus fruit at 21C followed by 16 days cold storage have
little effect on internal fruit quality in early-ripening cultivars, which show still green peel but have
reached commercial pulp maturity index (Sdiriet al., 2012a,b). Results from the present work
confirm this result in more mature sweet oranges and further show that ethylene pre-treatment does
not have a negative effect on internal quality either after prolonged storage at 2C or at a higher
temperature (12C)which might enhance fruit aging and consequent quality loss with respect to fruit
held at 2C.It is well known that the effect of ethylene on citrus fruit color changes is highly
dependent on the peel maturity stage and that still green fruit must reach a threshold color to show
proper commercial color after degreening (Jimnez-Cuesta et al., 1981; Sdiriet al., 2012a). As
expected, the EC treatment barely increased fruit color in fully colored mature Navelate and Lane
Late oranges (Tables 2 and 3). The effect of the hormone was less relevant than that of storage
temperature. Fruit color increased at both 2C and12C and the rate of increase was faster at 12C,
which is in concordance with results reported in Navelina oranges by Carmonaet al. (2012).
Moreover, it has to be mentioned that the effect of the ethylene treatment on the general fruit
freshness appearance was also less relevant than that of the storage temperature. The health
benefits of phenolics are well recognized and it is known that ethylene increases PAL activity and
total phenolics in the peel of mature Navelate oranges (Cajuste and Lafuente, 2007).Therefore, we
examined in the pulp whether the EC treatment was able to increase total phenolic content (Fig. 4)
and also total flavonoids (Fig. 5), which are major phenolics in blond orange cultivars. At harvest,
phenolic content in the edible portion of Navelate fruit was higher than that of Lane Late (Fig. 4). A
low but significant increase in total phenolics was found in response to ethylene (day4) in Lane Late
but not in Navelate fruit. Phenolic changes at 2 or12C were largely irrelevant in AC fruit of both
cultivars (Fig. 4). The more marked effect of the ethylene pretreatment was observed in Navelate
fruit kept at 2C. In this sample, ethylene pretreatment decreased phenolic content with respect to
AC fruit, but signifi-cant differences were only found by 60 days. Results of this work showing little
effect of ethylene increasing phenolics in the pulp of mature fruit agree with those found in other
citrus cultivars harvested before color break (Mayuoni et al., 2011a; Sdiri et al.,2012b). The activity
of PAL, at the entry point of phenylpropanoid biosynthesis, increases in the peel of mature citrus
fruit after ethylene treatment, but not in fruit harvested before color break (Liskeret al., 1983).
Therefore, changes found in phenolics in the pulp of citrus fruit in response to exogenous ethylene
might be more likely related to the fact that the peel is an important physical barrier for gas diffusion

into the fruit than to its maturity stage. Nevertheless, it has to be mentioned that the expression of
genes involved in the synthesis of phenolics may increase in the fruit pulp during ethylene-induced
degreening (Mayuoni et al., 2011b) despite total phenolics not increasing (Mayuoni et al., 2011a).
Similarly, a previous report showed that the hormone had a low and non-consistent effect on the
flavonoid content during degreening of different citrus cultivars (Sdiri et al., 2012b). The ethylene
treatment slightly increased flavonoid content in the pulp of mature Navelate fruit but significant
differences were only found by 40 days at 2C (Fig. 5).In Lane Late oranges, ethylene (4 days in
Fig. 5) did not increase flavonoids, but flavonoid content in EC fruit was higher than in AC fruit for up
to 20 days at 2C. In contrast, no difference was found at 12C after transferring fruit of this cultivar
from ethylene to air (Fig. 5). Total flavonoids remained nearly constant for up to40 days at both
temperatures in AC Navelate fruit. A substantial increase in total flavonoids was only found after this
period at 2C, which may be a consequence of prolonged temperature stress in the pulp of this
chilling susceptible citrus cultivar (Lafuente et al.,2011). In fact, flavonoids did not increase after
prolonged storage at 2C in AC fruit of the chilling tolerant Lane Late fruit (Fig. 5). In this cultivar,
flavonoid levels were significantly lower in AC than in EC fruit at the beginning of cold storage (2C).
Considering the stress imposed by the combination of ethylene plus cold in this citrus cultivar, and
that ethylene by itself did not increase flavonoid levels, further attention should be paid to
understanding whether the higher flavonoid levels in EC fruit might be a defense response to
protect the fruit against such stress. In conclusion, results indicate that EC at 12C had neither a
deleterious effect on internal and external fruit quality nor on the concentration of phenolics or
flavonoids in Navelate orange stored at either 2 or 12C. Therefore, this treatment might be a useful
and feasible tool to extend postharvest life of NCPP and CI-sensitive citrus cultivars. On the other
hand, results from Lane Late orange sindicate that the tolerance of citrus cultivars to the combined
effect of EC and extreme non-freezing low temperature (2C) should be further tested to select the
proper storage temperature. In these cultivars, storage temperatures ranging between 2 and 12C
should be tested to be able to reduce NCPP and loss of fruit freshness while avoiding SECI.

POSTCOSECHA ACONDICIONADO etileno como una herramienta para reducir la prdida

CALIDAD DE ALMACENAR madura dulce NARANJA
ABSTRACTO
El etileno es relacionado con la senescencia, sino tambin induce mecanismos de proteccin
contra el estrs en las plantas. La industria de los ctricos slo se aplica la hormona para inducir
desverdizado fruta. El objetivo de este trabajo fue para disuadir de la mina el efecto del etileno
sobre la calidad de los ctricos de color se almacena en condiciones comerciales para extender la
vida de post-cosecha, ya que los protege de estrs que causa trastornos post-cosecha tales como
dao por fro (IC) y no se enfra picaduras cscara (NCPP). El efecto de condicionamiento maduro
Navelate and Lane Late naranjas dulces (Citrus sinensis L. Osbeck) durante 4 das con 2 L L-1 de
etileno a 12C, ms que a temperaturas ms altas usadas para desverdizado, en la calidad de la
fruta se almacena a 2 o 12C, fue examinado. El tratamiento con etileno acondicionado (CE) no
aument el color pero redujo la abscisin cliz y NCPP en el fruto de ambos cultivares
almacenados a 12C, y tambin en el CI Navelate fruta en 2C. Lane Late fruta no se desarroll IC,
pero mostr un nuevo trastorno en el fruto CE celebrada en 2C. Este trastorno se inici como
reas escaldadas alrededor del extremo del tallo y la fruta se extendi sobre la superficie de la fruta
durante el almacenamiento. CE no tuvo ningn efecto perjudicial sobre la calidad de las naranjas
Navelate almacenados en cualquiera de 2 o 12C. Resultados similares se encontraron en el carril
de la fruta tarda, aunque la CE aument ligeramente la percepcin de sabor desagradable en 2C
y el ndice de madurez a los 2 y 12C. Por otra parte, CE aument ligeramente el contenido de
flavonoides bioactivos en la pulpa de fruta Navelate pero las diferencias significativas entre el
control y la fruta CE slo se encontraron despus de almacenamiento prolongado a 2C. En la
banda de fruta tarda, CE evit la disminucin inicial en el contenido de flavonoides encontrados en
las muestras de control. Los resultados muestran, por tanto, que la CE a 12C puede ser una
herramienta para extender la vida poscosecha del NCPP y CI-sensibles naranjas, y que la
tolerancia de los cultivos de ctricos al efecto combinado de la CE y la baja temperatura de
congelacin no (2C) debera realizarse una prueba para seleccionar la temperatura de
almacenamiento adecuada.
Introduccin
Existen varios tipos de trastornos fisiolgicos que afectan la calidad de post-cosecha cscara se
han descrito en los ctricos, pero factores que las causan y cmo se relacionan entre s no se
entiende (Grierson, 1986; Lafuente y Zacaras, 2006; Magwaza et al, 2013).. Se han hecho
esfuerzos para revelar factores fordifferent postcosecha de ctricos trastornos responsables y
desarrollar nuevas estrategias para controlarlos. Sin embargo, actualmente no existen
metodologas probadas para reducir su incidencia. La mayora de los estudios sobre la reduccin
de los trastornos fisiolgicos cscara se han relacionado con las metodologas destinadas a reducir
el dao por fro (IC), y el efecto sobre la calidad de los ctricos ha sido bien caracterizados (Schirra
et al, 2005;. Schirra y Mulas, 2007) y .CI no se enfra picaduras cscara (NCPP), tambin conocida
como corteza-tincin, son dos principales trastornos fisiolgicos postcosecha en ctricos (Lafuente
y Zacaras, 2006). NCPP desarrolla a temperaturas ms altas que los que causan CI, que en
ctricos puede ocurrir a temperaturas que oscilan entre 1 y 12C. naranjas dulces del grupo Navel,
pueden desarrollar estos trastornos fisiolgicos (Lafuente y Sala, 2002;. Alfrez et al, 2003; Holland
et al., 2005). naranja Navelate (Citrus sinensis (L.) Osbeck) es propenso a desarrollar NCPP, as
como CI despus de almacenamiento en fro prolongado. En este cultivar, NCPP se caracteriza por
zonas deprimidas en la cscara que afectan tanto al albedo (parte interna de la cscara) y la
flavedo (parte exterior de la cscara), que eventualmente se convertir en el color de bronce;

mientras CI se manifiesta como escaldado superficial o flavedo de bronce reas no deprimidos

(Alfrez et al., 2005). El rendimiento poscosecha de otras variedades de naranjo dulce ombligo de
maduracin tarda, que se han expandido muy rpidamente en muchos pases para extender la
temporada de ctricos, es menos conocido. Este es el caso de la naranja de maduracin tarda
Lane Late (C. sinensis (L.) Osbeck) que tiene caractersticas de buena calidad de la fruta. El etileno
se relaciona generalmente con la senescencia, sino que desempea tambin un plantas
roleprotecting del estrs que causa daos (Yang y Hoffman, 1984). A pesar de que esta hormona
puede mejorar un trastorno fisiolgico relacionado con la senescencia, la ruptura corteza en Nules
Clementina hombre-Darin (Cronj et al., 2011), u otros defectos de la cscara como la piel de cebra
o de herradura / anillos verdes en otros cultivares de ctricos (Krajewsky y Pittaway , 2002), sino
que tambin protege los ctricos de las tensiones que causan CI y NCPP (Lafuente et al, 2001;.
Lafuente y Sala, 2002). La industria slo se aplica a esta hormona de maduracin tempranactricos destinados a fines desverdizado aunque puede reducir los trastornos fisiolgicos en las
frutas maduras recolectadas despus de las vacaciones de color (Lafuenteand Sala, 2002;
Lafuente et al., 2004, Cajuste y Lafuente, 2007) .El efecto en la calidad de los ctricos de
postcosecha degreen-cin a 90% de humedad relativa y 20-22C utilizando 2-4 L L-1 etileno, se
ha documentado (Carvalho et al, 2008;. Mayuoni et al, 2011a;. Sdiriet col ., 2012a, b; Moscoso
Ramrez y Palou, 2014). Los datos anteriores muestran tambin que el condicionamiento madura
ctricos con concentraciones de etileno que oscilan entre 1 y 10 L-1 considerablemente reduce
NCPP en la fruta almacenada despus en 20-22C y aumenta el contenido de exfoliacin con fenol
(Lafuente y Sala de 2002 _L; Cajuste y Lafuente, 2007 ) .Por otra parte, se sabe que las
respuestas fisiolgicas de los ctricos a etileno puede variar con el estado de maduracin (Lisker et
al, 1983;. John-Karuppiah y Burns, 2010). Sin embargo, no hay informacin sobre el efecto del
etileno sobre la calidad de la fruta completamente maduro se almacenan durante periodos
prolongados a temperaturas ms bajas y en condiciones comerciales requeridos para extender la
vida de postcosecha. Los experimentos realizados para evaluar el efecto de desverdizado se han
limitado a examinar los cambios que se producen despus de un solo tratamiento de cuarentena
(1C por 16 das) en frutos de los cultivares de ctricos que no desarroll trastornos fisiolgicos
(Sdiri et al., 2012a), mientras que la duracin de almacenamiento y la temperatura afecta a la
calidad de los ctricos (Mulas y Schirra, 2007;. Obenland et al, 2008). Por lo tanto, el objetivo de
este trabajo fue evaluar el efecto del tratamiento completo dulces cultivares de color naranja tarda
maduros Navelate y Lane, que no estn desverdizados para su comercializacin, por 4 das con 2
L L-1 de etileno a 90-95% de humedad relativa (HR ) sobre la calidad de la fruta almacenada
durante un mximo de 60 das o AT2 12C y 90-95% de humedad relativa. Esta etileno
acondicionado tratamiento (CE) se realiz a 12C en lugar de a la temperatura usada para
desverdizado comercial (20-21C) (Mayuoni et al, 2011a;.. Sdiriet al, 2012a, b). Se recomienda que
la temperatura desverdizado para permitir el desarrollo de color despus de tratamiento con
etileno. Fruta en este estudio ya estaban totalmente de color, y por lo tanto se utiliz una
temperatura ms baja (12C) para reducir la prdida de calidad de la fruta. Se evaluaron los
efectos del tratamiento en el color, la firmeza, prdida de peso y la calidad interna de la fruta, as
como la incidencia de la abscisin de cliz, decaimiento y trastornos fisiolgicos. Teniendo en
cuenta que el etileno aumenta la actividad de la enzima fenilalanina amonaco-liasa (PAL) en
ctricos, en especial en el fruto ms maduro (Liskeret al., 1983), que esta es una enzima clave en el
punto de entrada del metabolismo fenil-propanoid, y los beneficios para la salud de los compuestos
fenlicos (Tripoliet al., 2007), tambin hemos evaluado en la parte comestible de frutas el efecto del
tratamiento CE relativa a la acumulacin de fenotipos y flavonoides totales, que son los
compuestos fenlicos ms abundantes en los ctricos (Nogata et al., 2006).
2. Materiales y mtodos

2.1. tratamientos de material vegetal y poscosecha

Navelate and Lane Late naranjas dulces totalmente maduros (C. sinensis (L.) Osbeck) fueron
cosechadas en marzo desde el mismo huerto, en Lliria (Valencia, Espaa), e inmediatamente se
entregan al laboratorio. Los frutos fueron superficie a esterilizar con una solucin de leja comercial
(Ballester et al., 2013) y despus se dividi al azar en 2 grupos que fueron tratados con etileno
(grupo 1) o a cabo en aire (frutos control, grupo 2). Estos grupos se subdividen a su vez en dos
subgrupos para evaluar el efecto de la temperatura de almacenamiento (2 y 12C). Fruto en cada
subgrupo se dividieron en dos lotes. Tres rplicas de 10 frutas fueron incluidas en el primer lote
para estimar peridicamente la incidencia y gravedad de los trastornos fisiolgicos y de abscisin
cliz. El segundo lote contena 3 repeticiones de 10 frutos por perodo de almacenamiento, ya que
se utilizaron para determinar los cambios en color de la piel, sino tambin en otros atributos de
calidad de la pulpa que requiere mtodos destructivos. Fruit dentro del primer grupo se
acondicionaron durante 4 das con 2 L L-1 de etileno a 90-95% de humedad relativa y 12C (CE
fruta), y despus se almacen a 2 (subgrupo 1) o 12C (subgrupo 2). frutas de control se mantuvo
durante 4 das en el aire a 90-95% de humedad relativa y 12C (fruta AC) antes de ser almacenado
a 2 o 12C. CE y la fruta de CA se mantuvieron durante 8 semanas a 2C o durante 6 semanas a
12C y luego se transfieren a 20C durante 4 das para simular un perodo de vida til (SL).
2.2. La estimacin de la severidad del trastorno fisiolgico poscosecha y la incidencia
Tres trastornos fisiolgicos diferentes fueron identificados bajo las condiciones de almacenamiento
arriba mencionadas. Ya que sus sntomas eran diferentes, la gravedad se evalu de forma
independiente durante la celebracin de CA y CE fruta a los 2 y 12C, y despus del perodo SL.
Dependiendo de la variedad, tratamiento de acondicionamiento postcosecha o condicin de
almacenamiento, la fruta mostr: (1) NCPP, que se manifiesta como reas superficiales colapsadas
que se convirti en marrn oscuro con el tiempo (Supplemental Fig S1A.); (2) CI, que se manifiesta
como reas no deprimidos bronceados (escaldado) de la superficie de la fruta (Supplemental Fig
S1B.); y / o (3) un trastorno fisiolgico, que por lo que sabemos no se ha descrito previamente en
los ctricos, que comienza como reas escaldadas de todo el extremo del tallo de la fruta
(Supplemental Fig. S1C) y se extiende a travs de la superficie del fruto durante almacenamiento
(Supplemental Fig. S1D) .Este trastorno, llamado SECI (de dao por fro extremo del tallo), muestra
reas no deprimidos en el extremo del tallo de fruta, y por lo tanto es diferente para detener de
extremo corteza de desintegracin (SERB). Se utiliz una escala de calificacin de 0 (sin dao) a 4
(dao grave) (Vicente et al., 2013) para determinar el ndice de dao gravedad promedio de cada
trastorno fisiolgico. En esta escala, que muestra una nica fruta daos puntuacin superior a 1
sera rechazado por los consumidores. La misma fruta se utiliza en las diversas fechas de
evaluacin. Los ndices de gravedad se calcularon sumando los productos de la serie de fruta en
cada categora multiplicado por su cuenta, y luego dividiendo el total obtenido por el nmero de
frutos, evaluado como se describe por Lafuente et al. (1997). Por otra parte, la incidencia comercial
de cada trastorno fisiolgico se estim mediante el clculo del porcentaje de fruta que muestra un
dao mayor puntuacin que 1. Los resultados fueron los medios de muestras de 3 replicados
contienen 10 frutas cada S.E.M. Hecho suplementario
2.3. Anlisis de los atributos de calidad
Los cambios en la calidad de la fruta se evaluaron mediante la medicin de la acidez y el SSC en la
prdida de pulpa, color de la cscara de frutas y firmeza de la fruta y el peso como se ha descrito
por Holland et al. (1999) y Lafuente et al. (2011). Los slidos solubles (Brix) se determinaron a
partir de zumo de fruta con un / X-1000 refractmetro Atago digitales (Atago Co. Ltd., Tokio, Japn)

y el contenido de cido se titul con NaOH 0,1 N utilizando fenolftaleina como indicador y se
expresan en g de cido ctrico anhidro en 100 ml de jugo. El ndice de madurez se calcul
dividiendo el Brix del jugo extrado por su contenido de cido. color de la piel se analiz usando
un Minolta CR-300 Chromameter (Konica MinoltaInc, EE.UU.) con una zona de medida de 8 mm
en cuatro lugares alrededor del plano ecuatorial de la fruta. El ndice de color se expres como la
relacin de Hunter a / b que se utiliza clsicamente para la medicin del color en los ctricos
(Stewart y Wheaton, 1972). Esta relacin es negativa para la fruta verde y positivo para frutas de
color naranja, mientras que el valor cero corresponde a fruta de color amarillo en el punto medio
del perodo de cambio de color. La prdida de peso se expres como el porcentaje acumulado de
prdida fruitweight durante el almacenamiento de frutas y firmeza de la fruta determinaron usando
un Analizador de Textura TA-XT2 (Stable Micro Systems, Godalming, Reino Unido) y se expres
como el porcentaje de deformacin de fruta resultante de una presin de 10 N en el eje longitudinal
de fruta a una velocidad constante (Sdiri et al., 2013). Junto con estos atributos de calidad,
evaluacin sensorial se realiz por ocho panelistas semientrenados como se describe por Sdiri et
al. (2012a), utilizando una escala de valoracin de 1 a 10, where10 = excelente, 5 = regular
(comercialmente aceptable), y 1 = extremadamente desagradable. La media de aceptacin del
sabor se calcul siguiendo el mismo procedimiento descrito anteriormente para calcular la
gravedad ndice medio de los trastornos de la cscara.
2.4. Evaluacin de la abscisin del cliz y la incidencia de la caries
Se evalu la incidencia de la abscisin del cliz y la decadencia despus de acondicionar la fruta
con etileno a 12C, durante AT2 almacenamiento de frutas y 12C y despus del perodo SL.
incidencia de abscisin del cliz se expres como el porcentaje de frutos sin cliz en comparacin
con el nmero total de fruta en cada muestra repetida y la decadencia incidencia estimada
mediante la cuantificacin del porcentaje de fruta podrida.
2.5. Anlisis de fenoles totales y flavonoides
se determin el contenido de fenoles totales siguiendo un mtodo basado en el descrito por Swain
y Hillis (1.959) segn lo informado por Cajuste y Lafuente (2007). Brevemente, 500 jugo L se
extrajeron con 1.500 L de etanol utilizando un batidor 8 disruptor celular Mini Bead (Biospec
Products, Inc.). El extracto se centrifug a 13.000 g durante 5 min a 4C y el sobrenadante se
utiliza para estimar el contenido fenlico. Dos partes alcuotas de muestra de 250 L de 5 veces
sobrenadante diluido se incubaron a temperatura ambiente con 250 L de 1 N Folin-Ciocalteu y
2500 L de 2% Na2CO3. Despus de centrifugacin at13,000 g durante 5 min a 4C, la
absorbancia se registr a 724 nm y el contenido fenlico total determinado mediante el uso de una
curva estndar desarrollado con cido clorognico. flavonoides totales se determinaron en el
mismo sobrenadante para el anlisis de fenoles totales siguiendo el mtodo descrito por GonzlezAguilar et al. (2007) con algunas modificaciones. Dos alcuotas de sobrenadante de 100 L se
diluyeron con 400 L de agua nanopura y 30 L de 5% NaNO2 se aadieron a cada parte alcuota.
After5 incubacin min a temperatura ambiente, 30 L de 10% AlCl3wereadded y en 6 min, la
reaccin se detuvo mediante la adicin de 200 L de NaOH 1 N. contenido de flavonoides se
determinaron mediante la comparacin de la absorbancia a 350 nm con la de los espacios en
blanco preparadas, calculado utilizando una curva estndar preparada con hesperidina, que es el
principal flavonoide en la pulpa, y los resultados se expresaron como mg fenlico total de 100 g1.El y flavonoide contenido se determinaron de la misma fruta utilizada para analizar los atributos
de calidad. Los resultados son las medias de tres muestras replicadas biolgicos S.E.M.
2.6. anlisis estadstico

Una comparacin de medias mediante la prueba de Tukey se realiz para determinar si los valores
medios fueron significativamente diferentes (P 0,05) entre la CE y AC frutos control para cada
cultivar y en cada perodo de la temperatura de almacenamiento y muestreo experimental.
Fig. 1. La no-refrigeracin ndice de cscara de picaduras (NCPP) de Navelate and Lane naranjas
tardas almacenadas a 2 o 12C despus de ser acondicionada a 12C durante 4 das con 2 _L L1ethylene (CE, blanco) y de sus respectivos controles mantenido 4 das en el aire a 12C
almacenamiento previo (AC, negro). La lnea de puntos indica la transferencia de fruta en 20C
para simular la vida til (SL). Los resultados representan las medias de tres repeticiones muestras
S.E.M. Los asteriscos indican diferencias significativas (P <0,05) entre CE y sus respectivas
muestras de control (AC) para el mismo perodo de almacenamiento.
3. Resultados y discusin
3.1. Efecto de etileno acondicionado en los trastornos fisiolgicos de post-cosecha de ctricos
almacenados a temperaturas de refrigeracin y los no-refrigeracin
Se han reportado los efectos de desverdizado de etileno sobre los atributos de calidad de los
diferentes cultivares de ctricos todava verdes (Carvalhoet al, 2008;.. Mayuoni et al, 2011a;. Sdiri et
al, 2012a, b). Sin embargo, estos efectos no fueron evaluados despus de un almacenamiento
prolongado, lo que favorece el desarrollo de trastornos fisiolgicos. Por otra parte, ci'tricos-verde
son menos sensibles a desarrollar IC (Lafuente et al., 1997) y NCPP (Alfrez y Zacaras, 2013) que
el fruto maduro de color. La eficacia de los tratamientos de la CE en la reduccin de 20C NCPP en
Navelina y Navelate naranjas dulces maduros almacenados bajo condiciones ambientales no
estresantes (20C y 90-95% de humedad relativa) se ha demostrado durante las diferentes
estaciones de ctricos (Lafuente y Sala, 2002; . Cajusteet al, 2010; Vicente et al, 2013).. Sin
embargo, el efecto de la CE at12C sobre la incidencia de trastornos fisiolgicos cscara y otros
atributos de calidad de la fruta que no necesitan ser desverdizadas, almacenado a temperaturas
ms bajas, sigue siendo desconocido. Dos temperaturas de almacenamiento fueron seleccionados
en el presente estudio. Fruit se almacenaron a 2C, para maximizar el periodo de almacenamiento
y para poner a prueba el efecto combinado de etileno y el estrs de baja temperatura, y en 12C
que podra evitar la aparicin de CI y retrasar el envejecimiento de la fruta en comparacin con la
celebracin de la fruta en 20 sntomas C.NCPP fueron evidentes por 20 das a 12C en el avelate
ACN and Lane naranjas tardas y aumentaron a partir de entonces (Fig. 1) .Aunque la
susceptibilidad de Navelate fruta para postcosecha NCPP es bien conocido (Alfrez et al., 2003 ;
Sala et al, 2005;.. Romero et al, 2012), en lo que sabemos, ste es el primer estudio que muestra
que Lane Late naranja tambin es susceptible a la enfermedad aunque su susceptibilidad es menor
(Fig 1).. Por da 20, el ndice de NCPP de naranjas AC Navelate celebradas en 12C fue mayor que
1, lo que indica la existencia de fruta que carece de la calidad requerida para la comercializacin.
En estas condiciones, el 39% de AC Navelate fruta podra ser rechazada debido a la baja calidad
externa (Tabla 1). El nmero de naranjas Lanelate inaceptables fue mucho ms baja por este
perodo (11,5%, Tabla 1). Esta diferencia entre los cultivares se redujo en 40 das, pero un valor
an ms bajo fue encontrado despus del perodo de SL en el carril de fruta tarda. Como era de
esperar (Lafuente y Sala, 2002;. Alfrez et al, 2005), manteniendo la fruta de CA de ambos
cultivares en 2C retras la aparicin de NCPP no comercial (Tabla 1) y redujo la gravedad NCPP
(Fig. 1) y la incidencia (Tabla 1). A esta temperatura, el ndice NCPP fue inferior a 0,8 by40 das de
almacenamiento en naranjas Navelate y de 0,6 x 60 das en Lane Late fruta. Acondicionamiento de
la fruta con 2 _L L-1ethylene a 12C aument considerablemente la tolerancia de las naranjas de
ambos cultivares de NCPP (Fig. 1). El tratamiento fue muy eficaz reducir el desarrollo de NCPP a
ambas temperaturas de almacenamiento ya que el ndice NCPP de fruta CE fue inferior a 1 durante

el almacenamiento de frutas en 2 o 12C y despus de la simulacin de SL (Fig. 1). Esto est en

concordancia con los hallazgos anteriores que muestran el efecto de la reduccin de etileno NCPP
en at20C ctricos (Lafuente y Sala, 2002; Vicente et al, 2013).. Adems, muestra que a pesar de
las respuestas de etileno inducida por ser dependiente de la temperatura, es posible disminuir la
temperatura del tratamiento CE para reducir la incidencia NCPP y otros efectos negativos en los
atributos de calidad que se producen a temperaturas ms altas. Como se muestra en la Tabla 1, el
porcentaje de frutos que presenten comercialmente inaceptable NCPP en 12C fue menor que 8%,
incluso despus del perodo de SL en ambos cultivares mientras que el porcentaje fue mayor que
50% en la fruta AC. almacenamiento de la fruta en la 2C caus CI en naranja Navelate por 40
das, a pesar de la gravedad de CI fue muy baja (Fig. 2, panel superior). El efecto de
acondicionamiento de ctricos con etileno en la reduccin de CI en muy escalofriantes cultivares
ctricos sensibles es baja (Lafuente et al., 2001). Sin embargo, CI se redujo en el menos
enfriamiento sensible Navelate ctricos cultivar por el tratamiento CE (Fig. 2, panel superior). Por lo
tanto, las naranjas Navelate CE no mostraron IC comercialmente relevante para un mximo de 60
das y de su gravedad e incidencia fueron bajos (datos no mostrados) .Results tambin mostr que
el AC carril naranjas tardas no se desarrollaron IC (datos no mostrados) y que a partir de frutas
este cultivar acondicionado con etileno desarroll SECI cuando se almacena a 2C (Fig. 2, panel
inferior). A lo mejor de nuestro conocimiento, SECI no se ha descrito previamente como un
trastorno post-cosecha en ctricos. Aparece en las zonas del extremo del tallo de la fruta como
parches escaldadas y se extiende superficialmente a lo largo de la fruta. Por lo tanto, se diferencia
de la SECI SERB, un trastorno de la cscara no relacionado con la refrigeracin que se manifiesta
como zonas deprimidas en el tallo y est asociada con la senescencia y reforzada por
deshidratacin (Grierson, 1986; Romero et al, 2013).. Desde SECI no se observ a 12C, los
resultados indican que puede ser causada por la combinacin de frutas que carece de la calidad
requerida para la comercializacin. En estas condiciones, el 39% de AC Navelate fruta podra ser
rechazada debido a la baja calidad externa (Tabla 1). El nmero de inaceptables carril naranjas
tardas fue mucho menor por este perodo (11,5%, Tabla 1). Esta diferencia entre los cultivares se
redujo en 40 das, pero un valor an ms bajo fue encontrado despus del perodo de SL en el
carril de fruta tarda. Como era de esperar (Lafuente y Sala, 2002;. Alfrez et al, 2005),
manteniendo la fruta de CA de ambos cultivares en 2C retras la aparicin de NCPP no comercial
(Tabla 1) y redujo la gravedad NCPP (Fig. 1) y la incidencia (Tabla 1). A esta temperatura, el ndice
de NCPP fue menor que 0.8 por 40 das de almacenamiento en naranjas Navelate y de 0,6 por 60
das en Lane fruta Tardo. Acondicionamiento de la fruta con 2 _L L-1ethylene a 12C aument
considerablemente la tolerancia de las naranjas de ambos cultivares de NCPP (Fig. 1). El
tratamiento fue muy eficaz reducir el desarrollo de NCPP a ambas temperaturas de
almacenamiento ya que el ndice NCPP de fruta CE fue inferior a 1 durante el almacenamiento de
frutas en 2 o 12C y despus de la simulacin de SL (Fig. 1). Esto est en concordancia con los
hallazgos anteriores que muestran el efecto de la reduccin de etileno NCPP en at20C ctricos
(Lafuente y Sala, 2002; Vicente et al, 2013).. Adems, muestra que a pesar de las respuestas de
etileno inducida por ser dependiente de la temperatura, es posible disminuir la temperatura del
tratamiento CE para reducir la incidencia NCPP y otros efectos negativos en los atributos de
calidad que se producen a temperaturas ms altas. Como se muestra en la Tabla 1, el porcentaje
de frutos que presenten comercialmente inaceptable NCPP en 12C fue menor que 8%, incluso
despus del perodo de SL en ambos cultivares mientras que el porcentaje fue mayor que 50% en
la fruta AC. almacenamiento de la fruta en la 2C caus CI en naranja Navelate por 40 das, a
pesar de la gravedad de CI fue muy baja (Fig. 2, panel superior). El efecto de acondicionamiento
de ctricos con etileno en la reduccin de CI en muy escalofriantes cultivares ctricos sensibles es
baja (Lafuente et al., 2001). Sin embargo, CI se redujo en el menos enfriamiento sensible Navelate
ctricos cultivar por el tratamiento CE (Fig. 2, panel superior). Por lo tanto, las naranjas Navelate CE

no mostraron IC comercialmente relevante para un mximo de 60 das y de su gravedad e

incidencia fueron bajos (datos no mostrados) .Results tambin mostr que el AC carril naranjas
tardas no se desarrollaron IC (datos no mostrados) y que a partir de frutas este cultivar
acondicionado con etileno desarroll SECI cuando se almacena a 2C (Fig. 2, panel inferior). A lo
mejor de nuestro conocimiento, SECI no se ha descrito previamente como un trastorno postcosecha en ctricos. Aparece en las zonas del extremo del tallo de la fruta como parches
escaldadas y se extiende superficialmente a lo largo de la fruta. Por lo tanto, se diferencia de la
SECI SERB, un trastorno de la cscara no relacionado con la refrigeracin que se manifiesta como
zonas deprimidas de la stemand est asociada con la senescencia y aumentada por deshidratacin
(Grierson, 1986; Romero et al, 2013).. Desde SECI no se observ a 12C, los resultados indican
que puede ser causada por el efecto combinado de etileno y de enfriamiento en el carril naranjas
tardas, mientras que las naranjas Navelate son ms tolerantes de tal combinacin a pesar de
desarrollar-ing algunos CI. Fruto de ambos cultivares fueron cosechadas la misma fecha y en el
mismo huerto. Por lo tanto, las diferentes respuestas de la CE Navelate and Lane naranjas tardas
al fro deben ms probablemente relacionado con factores genticos que a diferentes condiciones
de pre-cosecha. Los resultados globales indican que la aplicacin de etileno inmediatamente
despus de la cosecha de fruta en el 12C podra ser una herramienta para reducir la incidencia de
desrdenes fisiolgicos en los ctricos maduros. Sin embargo, el efecto de la CE seguido por
temperaturas bajas extremas de almacenamiento no-bloqueo no debe ser probado en cada cultivar
ctricos. Por lo tanto, el resultado de esto funciona cmo que el acondicionamiento de la naranja
Navelate propensos NCPP con etileno y almacenarlo en 12C podra ser un mtodo factible reducir
este trastorno cscara. Resultados similares pueden ser obtenidos en la fruta CE Navelate
almacena a 2C desde CI fue muy baja. Por lo tanto, la temperatura de almacenamiento de
naranjas Navelate acondicionado con etileno debe seleccionarse considerando tambin los
cambios en otros atributos de calidad de la fruta. Por el contrario, el acondicionamiento Lane Late
fruta con etileno podra causar SECI si la fruta se almacena a temperaturas muy bajas (2C),
aunque la fruta CE mantiene a 12C en este cultivar mostr daos muy bajo cscara. Como
alternativa, el almacenamiento de AC Lane Late fruta en 2C tambin sera eficaz en la reduccin
de NCPP sin causar otros trastornos pelar.
3.2. Efecto de etileno acondicionado en la abscisin cliz y la incidencia de enfermedades
Cliz abscisin es un importante factor de calidad de la fruta externa ya que afecta a la frescura y
la apariencia general de la fruta. La susceptibilidad a la abscisin del cliz despus de la cosecha
vara entre los cultivares de ctricos (Carvalho et al., 2008). Los resultados de experimentos de
desverdizado indican que el efecto del etileno sobre la abscisin cliz depende de las dosis de
etileno, el cultivar ctricos y la etapa de madurez de la fruta (Sdiri et al., 2012a). Nuestros
resultados mostraron que la abscisin cliz fue muy baja en 2C y que el tratamiento CE redujo en
fruta completamente maduros de ambos cultivares almacenados a 12C (Fig. 3) .En contraste, el
tratamiento de las mandarinas Clemenules con 2 LL-1ethyl- E a 21C con fines de desverdizado
favoreci la abscisin del cliz de la fruta almacenada durante 16 das en 1C (Sdiri et al., 2012a).
Los resultados de los mismos autores demostraron que el tratamiento tuvo un efecto diferente en la
naranja de maduracin temprana Navelina. En este cultivar, se indujo la abscisin cliz en la fruta
verde profundo pero su efecto fue variable e insignificante en la fruta ms madura, que est en
concordancia con el hecho de que la fruta menos maduro (de color verde oscuro), el ms
susceptible a la abscisin (Sdiri et al ., 2012a). Adems se deben realizar investigaciones para
dilucidar si el efecto del etileno en la reduccin de la abscisin del cliz en madura Navelate y Lane
Late (Fig. 3) las naranjas se relaciona con la etapa de madurez de la fruta y / o la temperatura a la
que se aplic etileno. El tratamiento con etileno se realiz at12C en lugar de a 21C ya que el

objetivo del tratamiento CE era reducir los trastornos fisiolgicos y minimizar el envejecimiento de
fruta en lugar de favorecer desverdizado a 21C. En este contexto, hay que sealar que la actividad
PAL aumentos en la zona de cliz abscisin de ctricos que favorece la curacin de heridas y
reduce la abscisin (Kostenyuket al., 2002), y que el aumento de la actividad PAL en respuesta a
etileno o otros factores de estrs la induccin de la hormona es mucho menos relevante en verde
que en ctricos maduros (Lisker et al, 1983;.. Lafuente et al, 2003). Tambin se sabe que los saldos
hormonales juegan un papel en el control de abscisin (Meir et al., 2010), que el cido indol-3actico (IAA) puede reducir la abscisin (Yuan et al., 2003) y que el etileno estimula la degradacin
de IAA en los tejidos de los ctricos (Winer et al., 2000). Puesto que la capacidad de flavedo de los
ctricos para IAA catabolismo disminuye al comienzo de la senescencia de fruta (Chamarro et al.,
2001), totalmente fruta madura podra ser menos susceptibles a la degradacin inducida por etileno
de IAA. La complejidad del proceso de abscisin se pone de relieve por los resultados de Sdiri et
al. (2012a) .Estos autores encontraron que la abscisin fue mayor en los ctricos menos maduro a
pesar de ctricos joven que tiene altos niveles de IAA en la zona de abscisin de frutos maduros
(Yuan et al., 2003). Por lo tanto, an se deben realizar investigaciones para comprender los
factores balanceing respuestas de ctricos de etileno a 12C y sus consecuencias en la abscisin
cliz. la aplicacin exgena de etileno tambin puede afectar la incidencia de la enfermedad.
Desverdizado con niveles elevados de la hormona puede exacerbar Diplodia pednculo incidencia
de pudricin (Smilanick et al., 2006). Por el contrario, el etileno no estimula Penicillium digitatum
crecer thand puede incluso reducir la incidencia del moho verde en ctricos maduros (Marcos et al.,
2005). Fruta utilizada en el presente trabajo fueron desinfectados inmediatamente despus de la
cosecha y se mantiene en cmaras frigorficas aspticas. Por lo tanto, la incidencia de la pudricin,
que fue causada principalmente por P. digitatum, era insignificante, tanto en CA y CE fruta (datos
no-presentados). Diplodia pudricin del pednculo no es comn en los ctricos que crece en clima
mediterrneo por lo que no era sorprendente la ausencia de podredumbres causadas por este
patgeno. Sin embargo, la incidencia de la CE sobre el tratamiento de bajada deben realizar
pruebas ms detalladas en otras zonas productoras de ctricos con clima hmedo y su alta
incidencia de Diplodia tallo-endrot.3.3. Efecto de etileno acondicionado en la calidad de la fruta
atribuye la prdida de peso fue muy baja y similar en CA y CE fruta, durante todo el perodo de
almacenamiento, tanto en Navelate (Tabla 2) y las naranjas tardas Lane (Tabla 3) Se mantuvo bien
a las 2 o 12C . El aumento en la prdida de peso que ocurren despus de la simulacin SL fue
mayor en el carril naranjas tardas, que eran menos susceptibles que las naranjas Navelate a
NCPP. A pesar de la prdida de peso fue muy baja, este resultado refuerza la idea de que la
deshidratacin no es el nico factor responsable del desarrollo del NCPP (Cajuste y Lafuente,
2007), y seala la importancia de los factores genticos en la susceptibilidad de los ctricos a este
desorden fisiolgico. firmeza de la fruta disminuy ligeramente despus de 20 das de la fruta a
cabo at12C pero apenas cambiaron en 2C en Navelate fruta. Por otra parte, el acondicionamiento
fruto de este cultivar con etileno tuvo poco efecto en este atributo de calidad (Tabla 2). Un efecto
similar se observ en el carril naranjas tardas (tabla 3), que mostraron mayor firmeza de la fruta.
Ya sea ultraestructura la cscara y la fuerza estn relacionadas con la menor susceptibilidad de
este ctrico cultivar al NCPP es desconocida, pero este resultado est de acuerdo con los
resultados anteriores de fomentar la investigacin destinada a identificar la influencia de la pared
celular en ctricos susceptibilidad de la fruta para desarrollar NCPP (Vicente et al., 2013). El
tratamiento CE reduce ligeramente la acidez en la pulpa de fruta Navelate (4 das, Tabla 2), pero
esta diferencia se perdi durante el almacenamiento de la fruta. Las naranjas tardas CE carril
tenan valores de acidez ligeramente ms bajos durante el almacenamiento (Tabla 3). Este efecto
se perdi despus de SL en frutos almacenados a 12C.On otra parte, los resultados muestran que
el mantenimiento de Navelate and Lane naranjas tardas en 2C retrasa la disminucin de la acidez
de pulpa con respecto a la fruta celebrada en 12C (Tablas 2 y 3) .La temperatura de

almacenamiento tuvo poco impacto en azcares solubles. The4 tratamiento de da de etileno

disminuy ligeramente SSC (~ 4%) en la pulpa de la fruta Navelate pero no en el carril de fruta
tarda. No se encontraron diferencias leves en SSC entre AC y EC fruta almacenada y que se
perdieron despus del perodo SL.
(2012).