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Physiological responses of tomato fruit to cyclic intermittent temperature regimes
Postharvest Biology and Technology 14 (1998) 283 – 296
Physiological responses of tomato fruit to cyclic intermittent temperature regimes Francisco Arte´s *, Francisco Garcı´a, Jose´ Marquina, Antonio Cano 1, J. Pablo Ferna´ndez-Trujillo 2 Posthar6est and Refrigeration Laboratory, Food Science and Technology Department, CEBAS-CSIC, PO Box 4195, E-30080, Murcia, Spain Received 18 July 1997; accepted 5 August 1998
Abstract Long life tomatoes (Lycopersicon esculentum Mill. cultivar ‘Durinta’) at breaker stage, treated with 0.5 g l − 1 iprodione or washed in water, were stored at 9, 12 or 20°C for up to 28 days. Fruit stored at 9°C were warmed to 20°C for 1 day every week, whereas fruit stored at 12°C were cooled to 2°C for 1 day every week. Fungicide treatment reduced decay and pitting on fruit stored more than 3 weeks. At 9°C, a slight synergistic effect on fruit pitting was observed from fungicide alone or fungicide plus intermittent warming. Compared with fruit stored at a constant 9°C, intermittently warmed tomatoes had better surface colour and flavour, were slightly less firm with less severe pitting, and were in better condition both at the end of the storage period and after a 3-day shelf-life. In fruit held at constant 20°C, 9°C or intermittently warmed, increased pectolytic enzyme activity accompanied a fall in respiration rate and ethylene production. In fruit held at a continuous 9°C, polygalacturonase activity was reduced slightly during the third week of storage. Intermittently cooled fruit showed enhanced taste and appearance compared with fruit held at a constant 12°C, but had more decay and pitting after the second cooling treatment. After cooling, slight reductions in L*a*b* Colour Space lightness were detected. During the shelf-life period, tomatoes previously stored continuously for 2 or 3 weeks at 9 or 12°C produced more ethylene and had higher respiration rates. By the third week, ethylene production was severely reduced by cooling at 2°C, but a possible relationship between pitting and non-ripening-dependent ethylene production was indicated. The respiration rate was not affected by the disorder incidence. We conclude that intermittent warming is more beneficial than intermittent cooling because of pitting development at 2°C in intermittently cooled fruit. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Ethylene; Iprodione; Colour; Pectolytic enzymes; Pitting; Respiration
* Corresponding author. E-mail: [email protected] 1 Present address: Department of Plant Biology (Plant Physiology), University of Murcia, E-30100, Murcia, Spain. 2 Present address: Department of Fruit and Vegetable Science, Cornell University, Ithaca, NY 14853, USA. 0925-5214/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0925-5214(98)00055-6
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1. Introduction Tomato fruit are susceptible to chilling injury (CI) at storage temperatures below 12°C. Typical symptoms of CI in tomato include surface pitting, uneven ripening or failure to ripen, increased fungal development, and a less acceptable flavour profile (Hobson, 1981; Cheng and Shewfelt, 1988). Ripening development in conventional cultivars of tomato is characterised by chlorophyll degradation and lycopene synthesis, an increase in respiration rate (RR) and ethylene production (EP), and increased endopolygalacturonase (endoPG) activity associated with softening (Giovannoni et al., 1992). Physiological changes during ripening that are induced by CI stress include a decreased rate of red colour development and a delayed rise in endoPG activity (Palma et al., 1995), high pectinmethylesterase (PME) activity with a previous loss of pericarp or whole fruit firmness (Marangoni et al., 1995), transient stimulation of EP, and modifications in RR (Cheng and Shewfelt, 1988). CI therefore limits quality and storage life in tomato. Chilling injury of tomatoes has been reduced by intermittent warming (IW) treatments, effectiveness being dependent on cultivar and time-temperature regimes (Hobson, 1981; Arte´s and Escriche, 1994). Arte´s and Escriche (1994) found that storage of fruit at 9°C with cycles of IW of 1 day at 20°C every 6 days reduced chilling injury in two cultivars, although the same cycles in fruit stored at 6°C did not. We have developed a technique in which cycles of cooling are applied to chilling-sensitive fruit between temperatures that would normally induce CI after a previous non-chilling temperature (intermittent cooling or IC). The advantages of IC include a delay in senescence (Arte´s et al., 1993; Sankat and Mohammed, 1993), but sometimes increased fruit susceptibility to CI and decay associated with water condensation also occurs (Sankat and Mohammed, 1993). Other treatments using temperature manipulations, such as gradual cooling prior to or during storage, have reduced CI in tomatoes (Marangoni et al., 1996; Lurie and Sabehat, 1997).
The aim of the present work was to study the effectiveness of cycles of IW or original cycles of IC, with or without a fungicide pretreatment to reduce disorders and particularly surface pitting, on the storage and physiological behaviour of a long life tomato cultivar.
2. Materials and methods
2.1. Plant material and experimental design The vine tomato (Lycopersicon esculentum Mill.) cultivar Durinta (W-424 F1 line from Western Seed, Las Palmas de Gran Canaria, Spain) is the product of two long life tomato parents (red fruits, long shelf life (3 weeks) gene independent from softness, without the ripening inhibitor (rin) characters; van Vliet, 1996, Western Seed, personal communication). The tomatoes were grown in a greenhouse in Mazarro´n (Murcia), on the Mediterranean southeast coast of Spain. Plants were grown in a mixture of sand and gravel with the following characteristics: pH5 8.5; electrical conductivity 7.85 dS m − 1; 63.5 mmol Na + ; 2.22 mmol K + ; 1.34 mmol Ca2 + ; 4.94 mmol Mg2 + ; 66.4 mmol Cl − ; 1.12 mmol CO23 − ; 4.82 mmol SO24 − . Localised fertigation was employed using 1.5 l water day − 1. Electrical water conductivity at 3 dS m − 1 was increased to 1.5 dS m − 1 by the application of 1N-1P-3K soluble fertiliser as needed. Fruit were harvested at stage 2 (breaker) according to the USDA tomato colour chart, transported 45 km by ventilated car on the day of harvest to the laboratory at Murcia, and sorted for uniform size and freedom from defects and blemishes. Calyces were carefully removed to avoid possible CO2 accumulation in the internal atmosphere of the chilled fruit after transfer to 20°C (Hobson, 1981; Bergevin et al., 1993). Mean fruit weights (9 S.E.) at harvest (n= 24) were 1179 4.7 g, equatorial diameter 63.191 mm and longitudinal diameter 50.29 0.7. Quality parameters at harvest (n= 6 replicates of four fruit each) were: pH 4.19 0.1 (Crison pH-meter); total soluble solids= 4.379 0.04° Brix (Atago
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N1 hand refractometer, Tokyo, Japan); titratable acidity= 92.693.3 mmol H + l − 1 (titration with 0.1 N NaOH up to pH 8.1; 5 ml samples). Fruit were then washed with water and randomised into seven lots of 24 fruits. Two lots were used for flesh firmness evaluation and hedonic tests, one for colour development measurements and three for quality evaluation. For measurement of pectolytic enzyme activities, three additional replicates of 24 fruits each were used in the three treatments. Half of the fruit lots were then dipped in an aqueous solution of iprodione [3-(3,5-dichlorophenyl)-N-(1-methylethyl)2-4-dioxo-1-imidazolidinecarboxamide (Rovral Aquaflo), Rhoˆne-Poulenc Agro, Madrid, Spain] at 0.5 g l − 1 active ingredient, at 15°C for 2 min. The fruit were air-dried at 15°C for about 30 min and any remaining liquid removed by blotting. Treatments were: Continuous storage at 20, 12 or 9°C for 28 days; storage at 9°C with three cycles of IW of 1 day at 20°C every 6 days (IW); storage at 12°C with three cycles of IC of 1 day at 2°C every 6 days (IC). For all treatments, an additional shelf-life period of 3 days at 20°C was used. Following the method previously described by Arte´s and Escriche (1994) and Arte´s et al. (1993), plastic boxes with moulded plastic trays containing the fruit were placed inside 360-l gas-tight chambers, with an air flow of 360 l h − 1, located in cold rooms at 9, 12 or 20°C and 95% RH. Warming or cooling was applied by removing the corresponding boxes from the cold room to another room at 20 or 2°C (95% RH) respectively. After 1 day the fruit were transferred back to continuous storage.
2.2. Temperature recording Temperatures were recorded automatically at 2-min intervals on three fruit used only for this purpose with a datalogger (Multitracker system, Datapaq Inc., Cambridge, UK) connected to six thermocouples equipped with needles inserted under the peel or in the central part of the columella.
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2.3. Quality losses Losses due to dehydration, wilting, pitting, decay, microcracking (also known as russeting) and senescence were evaluated weekly, after removal from cold storage and on the same fruit after 3 days of shelf-life at 20°C and 75–80% RH. Microcracking was observed as single, lignified radial, or in some cases lateral, fractures in the cuticle or skin (0.5–2 mm width, usually 10–30 mm length) without any necrotic tissues. All fruit were visually examined and disorders recorded using four classes as previously reported (Ferna´ndez-Trujillo et al., 1998). In the very slight to slight disorder class, less than 5% of the tomato skin was affected, and pitted areas were less than 3 mm diameter. Only moderate and severe levels were considered in total losses, when disorders covered between 5 and 15% of the surface (areas of 3–10 mm diameter in the case of pitting). Fungi were identified according to conventional methodology (Arte´s and Escriche, 1994). Losses from storage disorders, decay and dehydration were recorded as percentages on a fresh weight basis. Each fruit sample was inspected weekly for presence of physiological disorders and a disorder index calculated according to Ferna´ndez-Trujillo et al. (1998). The index represents the degree of the disorder on a 0–100 scale, while total losses represent the severity of injuries.
2.4. Colour, flesh firmness and hedonic tests Tomato colour and flesh firmness were measured according to Arte´s and Escriche (1994) and Ferna´ndez-Trujillo et al. (1998). The Minolta CR300 tristimulus colorimeter was calibrated with a white reference plate, C illuminant and 2° observer of standard C.I.E. L*a*b* Colour Space coordinates. The hue angle [tan − 1(b* · a* − 1)] was calculated. Hedonic tests (flavour, taste, visual quality and overall quality) were carried out (n= 6 fruit per treatment) by a panel of six trained people on fruit after 4 weeks of storage and additional subsequent ripening. Evaluation was on a 0 to 5 point scale: 0, extremely poor; 1, poor; 2, fair
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(fairly fresh, limit of edibility); 3, good (fresh, limit of marketability); 4, very good; 5, excellent. An average score plus standard error (n = 6 judges) was calculated for each treatment.
2.5. Respiration rate and ethylene production CO2 and C2H4 were measured with a Hewlett Packard 5730A gas chromatograph fitted with TCD and FID detectors. Measurements were made on individual fruit in hermetically sealed jars over given times according to Ferna´ndez-Trujillo et al. (1998), using five fruits for each treatment, except in continuous 2°C storage or in intermediate transfer to ripening after 2 or 3 weeks of continuous storage at 9 or 12°C when four and three fruit were used, respectively.
2.6. Pectolytic enzyme acti6ity Pectolytic enzymes were extracted by homogenizing outer pericarp tissue combined from three tomato fruit per replicate, in an aqueous solution containing 0.5 M NaCl and 1% polyvinylpyrrolidone (Arte´s et al., 1996). Homogenates were filtered through four layers of cheesecloth and centrifuged at 10000×g for 10 min at 4°C. Enzyme assays for PG (EC 3.2.1.15) and PME (EC 3.1.1.11) were conducted according to Arte´s et al. (1996). PG activity was expressed as mmol of reducing groups (ml extract) − 1 h − 1. PME was expressed as mequiv. H + (ml extract) − 1 h − 1.
2.7. Statistical analysis Data for flesh firmness and hedonic tests of iprodione-treated fruit were subjected to fully factorial multivariate analysis of variance (M)ANOVA. Colour analysis data obtained on the 5th, 12th, 19th, 26th and 29th day after harvest were used for statistical analysis of repeated measurements on each fruit (Ato and Lo´pez, 1994). Percent loss data, except weight loss and indices, were transformed to arcsin for statistical analysis. A similar (M)ANOVA design of the repeated measurements was also performed for losses and indices using data from the first or the
second week to the end of storage. Data from variables related to quality losses after shelf-life at 20°C were not included in the inspection time factor. In the experimental design used for losses, iprodione and storage regime were the ‘betweensubject’ factors and inspection time was the ‘within-subject’ factor. Mean comparisons were performed using LSDs except for pitting losses and indices, when the Bonferroni test (Ato and Lo´pez, 1994) was used. 3. Results
3.1. Fruit temperatures In the IC treatment, the time to acquire a constant temperature regime (differences lower than 0.2°C between air and fruit temperatures, or between peel and columella) was 6.67 h (from 12 to 2°C) or 8.77 h (from 2 to 12°C). In the IW treatment, these times were 7.70 h (from 9 to 20°C) or 6.73 h (from 20 to 9°C). In both treatments, with warming or cooling, differences between air and columella tissue were less than 1°C within 4.5 h.
3.2. Flesh firmness, colour and hedonic sensory tests Fruit ripened after harvest softened from 61 to 16 N after 28 days at 20°C. Firmness of fruit from 12°C and IC regimes averaged 26 N (data not shown). Firmness of fruit at 9°C (38 N) was greater than with IW or other treatments after storage (P 50.01), but not after 3 days of shelflife at 20°C. Fruit kept at 20°C became increasingly red as indicated by decreasing hue angles (Fig. 1A). Hue angles indicated that fruit kept at 12°C ripened more slowly and were not affected by IC. Fruit kept at 9°C also ripened more slowly, but IW fruit tended to ripen faster after the first warming. However, by the time of the third IW, no difference between IW fruit held at 9 and 12°C treatments could be detected. After 4 weeks of continuous storage at 9°C, fruit did not ripen as rapidly as those from any other treatment, even after a shelf-life period.
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Fig. 1. Changes in hue angle and lightness (L*) and during storage and after 3 days of shelf-life at 20°C. Bars are 2 ×S.E. (n = 24). Treatments were: continuous storage at 9, 12 and 20°C; IW, intermittent warming of 1 day at 20°C every 6 days of storage at 9°C; IC, intermittent cooling of 1 day at 2°C every 6 days of storage at 12°C. Dashed lines indicate shelf-life. Arrows indicate transfer times of fruit to intermittent warming or cooling periods (bold solid line).
A slight reduction in L* was detected during the cooling periods in IC fruit. This effect was not related to the induction of pitting in IC because 9°C untreated fruit exhibited similar onset of pitting but not a decrease in L* values
(Fig. 1BFig. 2AFig. 3A). Fruit colour was blotchy orange to red after the first cooling, but to a lesser extent thereafter, because the IC effect on red colour was reversible at 12°C (Fig. 1B).
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Fig. 2. Mean losses, on a percentage fresh weight basis, during storage of ‘Durinta’ tomatoes washed with water (A) or additionally treated with 0.5 g l − 1 of iprodione (B). Thin or bold arrows indicate transfer times of fruit to intermittent warming or cooling (IW and IC, respectively), or shelf-life at 20°C respectively. Bars are 2 ×S.E. (n = 3). For treatment details, see Fig. 1.
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Fig. 3. Physiological disorder indices (0 to 100 scale) during storage of ‘Durinta’ tomatoes washed with water (A) or additionally treated with 0.5 g l − 1 of iprodione (B). Thin or bold arrows indicate transfer times of fruit to intermittent warming or cooling, or shelf-life at 20°C, respectively. Bars are 2 × S.E. (n=3). See Fig. 1 for treatment details.
The shelf-life of fruit kept at 20°C was 2 weeks (taste of 2.9 9 0.3 and overall quality 3.8 9 0.2 U). After 4 weeks of storage, no significant differences
in visual and overall quality of fruit among continuous and cyclical treatments were observed, except reduced flavour in fruit kept continuously
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at 9°C compared with IW fruit (2.3 9 0.4 and 3.5 90.6 U, respectively, P B0.05). After subsequent ripening, fruit from continuous and cyclical treatments were similar, although the best overall quality was attained by IC fruit, particularly due to taste and visual quality (3.890.4 and 4.49 0.2 U, respectively).
3.3. Quality losses 3.3.1. Weight losses and decay After the shelf-life period, iprodione was effective in reducing weight losses only in fruit from 9°C and IW treatments (iprodione ×treatment interaction was significant, P 5 0.001). This fact was probably associated with a reduction in CI in both treatments, because 9°C and IW treatments showed the highest weight losses when untreated (three-way interaction significant at P 50.001; Fig. 2). Untreated fruit developed fungal decay after 3 weeks (Fig. 2). After a shelf-life period, IW fruit were the least susceptible to decay, but at 9°C fruit developed Alternaria spp. on pitted areas. Increased losses after shelf-life occurred in IC fruit (pitting with or without Alternaria or Cladosporium spp. growth) and in fruit at 12°C (shrivelling and senescence). Minimum decay occurred in the IW treatment even when fruit were not treated with iprodione (Fig. 2). Compared with continuous storage at 12°C, IC did not exacerbate the decay that appeared after 3 weeks of storage (Fig. 2). Iprodione reduced decay and total losses, especially at 12°C (Fig. 2). Iprodione was also effective in reducing and delaying Alternaria spp. growth on senescent 20°C fruit (1.4% in treated fruit after 4 weeks at 20°C; 10.9% in untreated fruit after the 3rd week at 20°C). With IW, only 0.7% of fruit ripened unevenly during storage and 1.4% after shelf-life. After 28 days at 20°C, high levels of senescence were detected (see below). 3.3.2. Pitting Slight pitting occurred in iprodione-treated fruits stored for 28 days at 9 or 12°C but its incidence increased in both treated and untreated
IW fruits, and in 9°C and IC untreated fruit (Fig. 2). Slight symptoms of surface pits appeared after 1 week of storage in all treatments except in continuous 12°C storage (Fig. 3). However, pitted skin was rapidly colonised by fungi in 9°C fruit, probably masking the real incidence of pitting. After the shelf-life period, Alternaria spp. colonised most of the pitted areas (except 0.7% (w/w) in IW and 1.4% in 9°C but untreated, and 1.4% of IC treated). IC fruit showed pitting after the second cooling, with fungi colonising pitted areas after the third cooling. Slight pitting developed after the first exposure to IW, the location of pitting mainly corresponding to the fruit zone that was last to ripen. Pitting losses and CI index only slightly increased after the second warming period, which induced partial ripening (Fig. 1). In untreated fruit after 4 weeks of storage plus shelf-life at 20°C, about 20% fruit suffered pitting without significant differences among 9°C, IW and IC treatments, while 12°C fruit was slightly affected (6%). However, in fungicide-treated fruit subjected to these treatments, pitting was detected (10, 8 and 15%, respectively), 12°C fruit being again slightly affected (4%). Iprodione reduced fruits affected by moderate to severe pitting (by 5%) and also had a synergistic effect when combined with 9°C or IW storage (an additional 5%). However, the increased pitting indicated by the pitting index was only reduced by storage at 12°C with or without iprodione pretreatment (Fig. 3).
3.3.3. Senescence, annular breakdown and microcracking After the second week of storage, senescence symptoms appeared particularly in 20°C fruit (2, 50, 65 and 85% losses in the 2nd, 3rd and 4th week, and subsequent ripening inspections). Other treatments were practically free from advanced senescence at the end of subsequent ripening (9°C, IW treated, 12°C untreated) or slightly affected (2.8% in 12°C treated fruit and 1.4% in other treatments). A form of wilting was identified, manifested as dehydration around the calyx zone associated with browning of the tissues affected. This disor-
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der, which to the best of our knowledge has not been previously reported, was called ‘annular breakdown’. Its occurrence may be related to the removal of sepals prior to storage. Symptoms of this disorder resembled the ‘stem end rind breakdown’ in citrus fruit. After subsequent ripening, less than 1% moderate annular breakdown appeared in both IW and IC untreated fruit or in 12°C treated fruit. Annular breakdown in untreated fruit was more frequent than in treated fruit but only slight severity (Fig. 3). Occasional increases in this disorder occurred after 3 weeks of storage. Microcracking symptoms were observed mainly in fruit held continuously at 9°C without fungicide treatment (Fig. 3). In treated fruit, annular breakdown and microcracking indices after shelf-life were lower than 3.8 and 2.5, respectively in cold treatments and 5.6 and 2.8 in control fruit (Fig. 3). At 9°C, iprodione reduced microcracking after the second week of storage (fungicide × storage time× storage regime interaction significant at P B0.01), but at 20°C exacerbated the increase of annular breakdown during storage (P B 0.001) and to a lesser extent microcracking (Fig. 2).
3.4. Respiration and ethylene production In ‘Durinta’ tomatoes, RR and EP decreases progressively, without a clear respiratory climacteric onset or EP peak during the shelf-life period (Figs. 4 and 5). During storage at 2°C, EP declined but RR remained constant (Figs. 4 and 5). During the shelf-life of this fruit, EP increased continuously, indicating a possible response to low temperatures and failure to ripen (data not shown). Even after the 4th day of shelf-life, RR and EP in these fruits remained higher than in other treatments (Figs. 4 and 5). In fruit kept at 9 or 12°C, transfer to 20°C after 2 or 3 weeks of storage resulted in increased RR and EP compared with that in fruit kept continuously at 20°C (Figs. 4 and 5). In the fourth week of storage, a peak of EP was slightly delayed in fruit from the treatments compared to that in fruit at 20°C. In IW fruit, EP was stimulated to similar degrees during successive warmings periods, rates ranging from 3 to 4.5 ml C2H4 kg − 1 h − 1 (Fig. 5).
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A slight EP stimulation after return to 9°C after the third warming period could be related to a slight increase in pitting index in this case (Fig. 3 section B, Fig. 5). EP was transiently stimulated after the second and third cooling periods when fruit returned to 12°C storage (Fig. 5). This stimulation was in parallel with the first pitting symptoms (Fig. 2BFig. 3B). In the third cooling period, EP reached the lower levels attained at continuous 2°C (0.23 9 0.03 ml C2H4 kg − 1 h − 1), and more than three times lower than in the previous cooling periods (Fig. 5).
3.5. Pectolytic enzyme acti6ities Activities of pectolytic enzymes increased rapidly during ripening at 20°C, but were delayed in fruit from 9°C or IW treatments (Fig. 6). PME activities were similar. Slight uneven ripening in fruit from the 9°C treatment between the second and the third weeks of storage (Fig. 1) was associated with slightly lower PG activity compared with that in IW fruit (Fig. 6). PG or PME activities in fruit from 9°C or IW treatments did not reach those found in fruit kept continuously at 20°C (Fig. 6).
4. Discussion IW reduced pitting and decay the most, and resulted in good fruit firmness and colour, and in the lowest total losses after storage and shelf-life, without detrimental sensory effects. Overall fruit quality in the IW treatment was better than that reported for a conventional cultivar (Arte´s and Escriche, 1994). Pitting incidence and severity developed to a similar extent after the second warming period (more advanced fruit ripening stage). Variation in chilling sensitivity during tomato ripening has been reported as biphasic, with a decline at the onset of ripening followed by a senescence-related increase (Autio and Bramlage, 1986). IC resulted in increased pitting. Also, water condensation that occurs at 12°C on the fruit subjected to IC probably accelerated senescence
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Fig. 4. Respiration rate during 26 days of storage and additional shelf-life at 20°C of ‘Durinta’ tomatoes. See Fig. 1 for treatment details (a continuous storage at 2°C is included). Vertical arrows represent transfer to intermittent warming (bold dashed line) or cooling periods (bold solid line). All fruits were treated with 0.5 g l − 1 of iprodione. Horizontal arrows ( ) represent transfer to ripening at 20°C (dashed lines). Bars are S.E. (n= 5 except at 2°C or in intermediate transfer to ripening when n =4 and n =3, respectively).
and/or skin deterioration resulting in stimulated fungal attacks, as shown in mangoes (Sankat and Mohammed, 1993). Clearly IC treatment conditions require adjustment to reduce pitting and
water condensation, while retaining the positive effect on taste. The differences in ripening-dependent storage time for the application of 1 day of IC or IW without stimulating pitting is evident,
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Fig. 5. Ethylene production during 26 days of storage and additional shelf-life at 20°C of ‘Durinta’ tomatoes. See Fig. 1 for treatment details (a continuous storage at 2°C is included). Vertical arrows represent transfer to intermittent warming (bold dashed line) or cooling periods (bold solid line). All fruits were treated with 0.5 g l − 1 of iprodione. Horizontal arrows ( ) represent transfer to ripening at 20°C (dashed lines). Bars are S.E. (n= 5 except at 2°C or in intermediate transfer to ripening when n = 4 and n= 3, respectively).
perhaps related to differences in membrane repair after cold storage (Marangoni et al., 1996). The relatively small effect on ripening at low temperatures could be related to low sensitivity to CI of this cultivar (Brown et al., 1989). The effect
of cooling at 2°C on lycopene biosynthesis was reversible but the onset of pitting was not. Reversibility of chilling sensitivity, evaluated as the ability to develop red colour (Saltveit, 1991), was strongly dependent on the length of the warming and cooling periods.
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Beneficial effects of IW in comparison with continuous 9°C storage on development of Alternaria spp. and Cladosporium spp. were remarkable, confirming previous studies on Alternaria spp. (Arte´s et al., 1987; Arte´s and Escriche, 1994). Iprodione was effective in reducing pitting and weight losses particularly during storage at 9°C alone or combined with IW. A single or synergistic iprodione effect does not appear to have been previously reported. Colour and flesh firmness differences in ‘Durinta’ fruit do not appear to be directly associated with differences in EP, RR or pectolytic enzyme activities. The decrease in RR and EP during ripening is typical behaviour similar to that found in ‘Alcobaca’ (alc) tomato fruits with 95% red colour developed, although the PG activity is not as low for the hybrids (Mutschler, 1984; Watkins et al., 1988). The delayed EP peaks found in all
Fig. 6. Enzyme activities assayed in fruit washed with water during continuous storage at 20°C (), 9°C ( –), or IW () (intermittent warming of 1 day at 20°C every 6 days at 9°C). (A) PME activity: PMEu is defined as mequiv. H + released (ml of extract) − 1 h − 1. (B) PG activity: PGu is defined as mmol of reducing groups (ml of extract) − 1 h − 1. Arrows indicate intermittent warming (dashed lines). Bars are S.E. (n= 3).
the treatments during the fourth week of storage (Fig. 5) was rather a sign of senescence (microcracking, annular breakdown, shrivelling) appearing at this time than a delayed response to the climacteric. Typically, in chilling sensitive plants, RR increases initially, but as chilling proceeds it decreases. When the burst of RR does not return to those found at nonchilling temperatures, CI symptoms usually become apparent (Lyons and Breidenbach, 1990). As reported in other cultivars (Arte´s et al., 1987; Cheng and Shewfelt, 1988), the onset of RR in ‘Durinta’ occurred as a late response to CI (Fig. 4). The stimulation of EP during shelf-life after continuous 2°C storage, or during subsequent ripening for 2 or 3 weeks at continuous 9 or 12°C fruit compared with 20°C fruit, agrees with results from Cheng and Shewfelt (1988). These treatments resulted in different ripening stages during storage (Fig. 1), but treatment differences on EP were evident only during shelf-life when pitting developed (Fig. 5). Increased EP following chilling has also been shown in cucumbers to be the result of an increased capacity of the tissue to synthesize ACC and/or a change in compartmentalisation of the reactants (Wang and Adams, 1982; Cabrera and Saltveit, 1990). However, this chilling-induced EP stimulation did not occur in tomato with different degrees of inhibition of lycopene synthesis (Watkins et al., 1990). This suggests that chilling-induced EP shown in our experiment (Fig. 5) was mostly a response to pitting or eventually other biochemical processes in spite of uneven ripening. Other evidence for the relationship between EP and skin disorders is found in the different rates of EP during normal ripening and the general trend for EP to decrease to a constant level (Fig. 5). The stem scar is the main channel for carbon dioxide and ethylene release (Bergevin et al., 1993; de Vries et al., 1996). ‘Durinta’ tomatoes were only affected in the skin adjacent to this zone by storage (Fig. 3B, annular breakdown disorder). Therefore, a possible explanation of a high upsurge or increase in EP is the onset of skin disorders as a consequence of chilling or senescence, since uneven ripening was in general unimportant.
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Slight chilling stress during continuous storage at 12°C was indicated by quite similar and even higher RR and lower EP than 20°C fruit, as previously described (Brown et al., 1989). When IC was applied, an increase in pitting was accompanied by an impairment of EP after the second cooling period (Figs. 2, 3 and 5). This also occurred in the continuous 2°C treatment (Fig. 5). The effect of chilling on EP could be explained by the stimulation of ACC oxidase activity after short exposures to chilling (the case of shelf-life after 9 or 12°C continuous storage) (Watkins et al., 1990) and the loss of ACC oxidase activity during longer exposures (Cabrera and Saltveit, 1990). In tomato, transcripts for the gene encoding the oxidase increased during cold storage (Watkins et al., 1990). The relationship between pitting and EP after cooling or warming periods may be associated with the alleviation of CI due to ethylene-stimulated metabolism in some IW cycles, associated with proposed effects of IW in conditioning fruit tissues against cell damage (Watkins et al., 1995). Fruit from both IC and continuous 12°C, and to a lesser extent from IW and continuous 9°C, were at similar ripening stages during storage (Fig. 1). This ensured that comparisons of physiological effects were not confounded by differences in ripening stages. The slight stimulation of EP by warming only partly agrees with previous results in a conventional cultivar of tomato (Arte´s et al., 1987), in peaches (Ferna´ndez-Trujillo et al., 1998) and in apples (Watkins et al., 1995), probably due to different ripening behaviour and CI symptoms in these latter fruit. PG activity increased during ripening and was lower in fruit kept at 9°C, but patterns of change were not directly related to EP (e.g. see from the first to the second week of storage, Figs. 5 and 6). Temperatures lower than 9°C (e.g. 4°C, see Palma et al., 1995) or different IW conditions than the regime assayed are required for a higher inhibition of this activity. For commercial purposes, IW or IC regimes involve moving produce from one room to another, requiring the availability of specific rooms and methods to control condensation on the fruits. For IW, energy costs are compensated for
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by reduction of pitting, Alternaria spp. infection and chemical usage.
Acknowledgements Financial support was provided by the Spanish CICYT (ALI-95/530 Project). J.P. Ferna´ndezTrujillo is indebted to CSIC for a PhD grant. A. Cano is indebted to Caja de Ahorros del Mediterra´neo for a grant. Thanks are due to J.A. Martı´nez for technical help, Dura´n S.A.T. for supplying tomatoes and other facilities, and Dr C.B. Watkins for a critical review of the manuscript.
References Arte´s, F., Escriche, A.J., 1994. Intermittent warming reduces chilling injury and decay of tomato fruit. J. Food Sci. 59, 1053 – 1056. Arte´s, F., Toma´s, M., Escriche, A.J., Marı´n, J.G., 1987. Application of programmed and modulated conditions in the cold storage of tomatoes. Respiratory activity, ethylene emission, and alterations by fungi. In: Primo, E., Fito, P. (Eds.), Proc. II World Cong. Food Technol., 2. Barcelona, pp. 1561 – 1573. Arte´s, F., Escriche, A.J., Marı´n, J.G., 1993. Treating Primofiori lemons in cold storage with intermittent warming and carbon dioxide. HortScience 28, 819 – 821. Arte´s, F., Cano, A., Ferna´ndez-Trujillo, J.P., 1996. Pectolytic enzyme activity during intermittent warming storage of peaches. J. Food Sci. 61, 311 – 313, 321. Ato, M., Lo´pez, J.J., 1994. Modelos de disen˜o experimental [Experimental design models]. In: Fundamentos de estadı´stica con SYSTAT. Ra-ma, Madrid, Spain, pp. 316 – 343. Autio, W.R., Bramlage, W.J., 1986. Chilling sensitivity of tomato fruit in relation to ripening and senescence. J. Am. Soc. Hort. Sci. 111, 201 – 204. Bergevin, M., L’Heureux, G.P., Willemot, C., 1993. Tomato fruit chilling tolerance in relation to internal atmosphere after return to ambient temperature. HortScience 28, 138 – 140. Brown, E.D., Yada, R.Y., Stanley, D.W., 1989. Chilling injury and modified atmosphere storage of mature green tomatoes. Can. Inst. Food Sci. Technol. 22, 339 – 344. Cabrera, R.M., Saltveit, M.E.J., 1990. Physiological response to chilling temperatures of intermittently warmed cucumber fruit. J. Am. Soc. Hort. Sci. 115, 256 – 261. Cheng, T.S., Shewfelt, R.L., 1988. Effect of chilling exposure of tomatoes during subsequent ripening. J. Food Sci. 53, 1160 – 1162.
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de Vries, H.S.M., Wasono, M.A.J., Harren, F.J.M., Woltering, E.J., van der Valk, H.C.P.M., Reuss, J., 1996. Ethylene and CO2 emission rates and pathways in harvested fruits investigated, in situ, by laser photothermal deflection and photoacoustic techniques. Postharvest Biol. Technol. 8, 1 – 10. Ferna´ndez-Trujillo, J.P., Cano, A., Arte´s, F., 1998. Physiological changes in peaches related to chilling injury and ripening. Postharvest Biol. Technol. 13, 109–119. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., Fischer, R.L., 1992. Polygalacturonase and tomato fruit ripening. Hortic. Rev. 13, 67 – 103. Hobson, G.E., 1981. The short-term storage of tomato fruit. J. Hort. Sci. 56, 363 – 368. Lurie, S., Sabehat, A., 1997. Prestorage temperature manipulations to reduce chilling injury in tomatoes. Postharvest Biol. Technol. 11, 57 –62. Lyons, J.M., Breidenbach, R.W., 1990. Relation of chilling stress to respiration. In: Wang, C.Y. (Ed.), Chilling Injury of Horticultural Crops, 14. CRC Press, Boca Raton, Florida, pp. 223 – 234. Marangoni, A.G., Jackman, R.L., Stanley, D.W., 1995. Chilling-associated softening of tomato fruit is related to increased pectinmethylesterase activity. J. Food Sci. 60, 1277 – 1281. Marangoni, A.G., Palma, T., Stanley, D.W., 1996. Membrane effects in postharvest physiology. Postharvest Biol. Technol. 7, 193 – 217.
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Mutschler, M.A., 1984. Ripening and storage characteristics of the ‘Alcobaca’ ripening mutant in tomato. J. Am. Soc. Hort. Sci. 109, 504 – 507. Palma, T., Marangoni, A.G., Stanley, D.W., 1995. Environmental stresses affect tomato microsomal membrane function differently than natural ripening and senescence. Postharvest Biol. Technol. 6, 257 – 273. Saltveit, M.E. Jr., 1991. Prior temperature exposure affects subsequent chilling sensitivity. Physiol. Plant. 82, 529 – 536. Sankat, C.K., Mohammed, A., 1993. Controlled cyclic cooling and warming of the Julie mango. Proc. 6th Int. CA Research Conf., NRAES-71. Cornell University, Ithaca, NY, pp. 402 – 417. Wang, C.Y., Adams, D.O., 1982. Chilling-induced ethylene production in cucumbers (Cucumis sati6us L.). Plant Physiol. 69, 424 – 427. Watkins, C.B., Haki, J.M., Frenkel, C., 1988. Activities of polygalacturonase, a-D-mannosidase, and a-D- and b-Dgalactosidases in ripening tomato. HortScience 23, 192 – 194. Watkins, C.B., Picton, S., Grierson, D., 1990. Stimulation and inhibition of expression of ripening-related mRNAs in tomatoes as influenced by chilling temperatures. J. Plant Physiol. 136, 318 – 323. Watkins, C.B., Bramlage, W.J., Cregoe, B.A., 1995. Superficial scald of Granny Smith apples is expressed as a typical chilling injury. J. Am. Soc. Hort. Sci. 120, 88 – 94.
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Physiological responses of tomato fruit to cyclic intermittent temperature regimes Francisco Arte´s *, Francisco Garcı´a, Jose´ Marquina, Antonio Cano 1, J. Pablo Ferna´ndez-Trujillo 2 Posthar6est and Refrigeration Laboratory, Food Science and Technology Department, CEBAS-CSIC, PO Box 4195, E-30080, Murcia, Spain Received 18 July 1997; accepted 5 August 1998
Abstract Long life tomatoes (Lycopersicon esculentum Mill. cultivar ‘Durinta’) at breaker stage, treated with 0.5 g l − 1 iprodione or washed in water, were stored at 9, 12 or 20°C for up to 28 days. Fruit stored at 9°C were warmed to 20°C for 1 day every week, whereas fruit stored at 12°C were cooled to 2°C for 1 day every week. Fungicide treatment reduced decay and pitting on fruit stored more than 3 weeks. At 9°C, a slight synergistic effect on fruit pitting was observed from fungicide alone or fungicide plus intermittent warming. Compared with fruit stored at a constant 9°C, intermittently warmed tomatoes had better surface colour and flavour, were slightly less firm with less severe pitting, and were in better condition both at the end of the storage period and after a 3-day shelf-life. In fruit held at constant 20°C, 9°C or intermittently warmed, increased pectolytic enzyme activity accompanied a fall in respiration rate and ethylene production. In fruit held at a continuous 9°C, polygalacturonase activity was reduced slightly during the third week of storage. Intermittently cooled fruit showed enhanced taste and appearance compared with fruit held at a constant 12°C, but had more decay and pitting after the second cooling treatment. After cooling, slight reductions in L*a*b* Colour Space lightness were detected. During the shelf-life period, tomatoes previously stored continuously for 2 or 3 weeks at 9 or 12°C produced more ethylene and had higher respiration rates. By the third week, ethylene production was severely reduced by cooling at 2°C, but a possible relationship between pitting and non-ripening-dependent ethylene production was indicated. The respiration rate was not affected by the disorder incidence. We conclude that intermittent warming is more beneficial than intermittent cooling because of pitting development at 2°C in intermittently cooled fruit. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Ethylene; Iprodione; Colour; Pectolytic enzymes; Pitting; Respiration
* Corresponding author. E-mail: [email protected] 1 Present address: Department of Plant Biology (Plant Physiology), University of Murcia, E-30100, Murcia, Spain. 2 Present address: Department of Fruit and Vegetable Science, Cornell University, Ithaca, NY 14853, USA. 0925-5214/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0925-5214(98)00055-6
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1. Introduction Tomato fruit are susceptible to chilling injury (CI) at storage temperatures below 12°C. Typical symptoms of CI in tomato include surface pitting, uneven ripening or failure to ripen, increased fungal development, and a less acceptable flavour profile (Hobson, 1981; Cheng and Shewfelt, 1988). Ripening development in conventional cultivars of tomato is characterised by chlorophyll degradation and lycopene synthesis, an increase in respiration rate (RR) and ethylene production (EP), and increased endopolygalacturonase (endoPG) activity associated with softening (Giovannoni et al., 1992). Physiological changes during ripening that are induced by CI stress include a decreased rate of red colour development and a delayed rise in endoPG activity (Palma et al., 1995), high pectinmethylesterase (PME) activity with a previous loss of pericarp or whole fruit firmness (Marangoni et al., 1995), transient stimulation of EP, and modifications in RR (Cheng and Shewfelt, 1988). CI therefore limits quality and storage life in tomato. Chilling injury of tomatoes has been reduced by intermittent warming (IW) treatments, effectiveness being dependent on cultivar and time-temperature regimes (Hobson, 1981; Arte´s and Escriche, 1994). Arte´s and Escriche (1994) found that storage of fruit at 9°C with cycles of IW of 1 day at 20°C every 6 days reduced chilling injury in two cultivars, although the same cycles in fruit stored at 6°C did not. We have developed a technique in which cycles of cooling are applied to chilling-sensitive fruit between temperatures that would normally induce CI after a previous non-chilling temperature (intermittent cooling or IC). The advantages of IC include a delay in senescence (Arte´s et al., 1993; Sankat and Mohammed, 1993), but sometimes increased fruit susceptibility to CI and decay associated with water condensation also occurs (Sankat and Mohammed, 1993). Other treatments using temperature manipulations, such as gradual cooling prior to or during storage, have reduced CI in tomatoes (Marangoni et al., 1996; Lurie and Sabehat, 1997).
The aim of the present work was to study the effectiveness of cycles of IW or original cycles of IC, with or without a fungicide pretreatment to reduce disorders and particularly surface pitting, on the storage and physiological behaviour of a long life tomato cultivar.
2. Materials and methods
2.1. Plant material and experimental design The vine tomato (Lycopersicon esculentum Mill.) cultivar Durinta (W-424 F1 line from Western Seed, Las Palmas de Gran Canaria, Spain) is the product of two long life tomato parents (red fruits, long shelf life (3 weeks) gene independent from softness, without the ripening inhibitor (rin) characters; van Vliet, 1996, Western Seed, personal communication). The tomatoes were grown in a greenhouse in Mazarro´n (Murcia), on the Mediterranean southeast coast of Spain. Plants were grown in a mixture of sand and gravel with the following characteristics: pH5 8.5; electrical conductivity 7.85 dS m − 1; 63.5 mmol Na + ; 2.22 mmol K + ; 1.34 mmol Ca2 + ; 4.94 mmol Mg2 + ; 66.4 mmol Cl − ; 1.12 mmol CO23 − ; 4.82 mmol SO24 − . Localised fertigation was employed using 1.5 l water day − 1. Electrical water conductivity at 3 dS m − 1 was increased to 1.5 dS m − 1 by the application of 1N-1P-3K soluble fertiliser as needed. Fruit were harvested at stage 2 (breaker) according to the USDA tomato colour chart, transported 45 km by ventilated car on the day of harvest to the laboratory at Murcia, and sorted for uniform size and freedom from defects and blemishes. Calyces were carefully removed to avoid possible CO2 accumulation in the internal atmosphere of the chilled fruit after transfer to 20°C (Hobson, 1981; Bergevin et al., 1993). Mean fruit weights (9 S.E.) at harvest (n= 24) were 1179 4.7 g, equatorial diameter 63.191 mm and longitudinal diameter 50.29 0.7. Quality parameters at harvest (n= 6 replicates of four fruit each) were: pH 4.19 0.1 (Crison pH-meter); total soluble solids= 4.379 0.04° Brix (Atago
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N1 hand refractometer, Tokyo, Japan); titratable acidity= 92.693.3 mmol H + l − 1 (titration with 0.1 N NaOH up to pH 8.1; 5 ml samples). Fruit were then washed with water and randomised into seven lots of 24 fruits. Two lots were used for flesh firmness evaluation and hedonic tests, one for colour development measurements and three for quality evaluation. For measurement of pectolytic enzyme activities, three additional replicates of 24 fruits each were used in the three treatments. Half of the fruit lots were then dipped in an aqueous solution of iprodione [3-(3,5-dichlorophenyl)-N-(1-methylethyl)2-4-dioxo-1-imidazolidinecarboxamide (Rovral Aquaflo), Rhoˆne-Poulenc Agro, Madrid, Spain] at 0.5 g l − 1 active ingredient, at 15°C for 2 min. The fruit were air-dried at 15°C for about 30 min and any remaining liquid removed by blotting. Treatments were: Continuous storage at 20, 12 or 9°C for 28 days; storage at 9°C with three cycles of IW of 1 day at 20°C every 6 days (IW); storage at 12°C with three cycles of IC of 1 day at 2°C every 6 days (IC). For all treatments, an additional shelf-life period of 3 days at 20°C was used. Following the method previously described by Arte´s and Escriche (1994) and Arte´s et al. (1993), plastic boxes with moulded plastic trays containing the fruit were placed inside 360-l gas-tight chambers, with an air flow of 360 l h − 1, located in cold rooms at 9, 12 or 20°C and 95% RH. Warming or cooling was applied by removing the corresponding boxes from the cold room to another room at 20 or 2°C (95% RH) respectively. After 1 day the fruit were transferred back to continuous storage.
2.2. Temperature recording Temperatures were recorded automatically at 2-min intervals on three fruit used only for this purpose with a datalogger (Multitracker system, Datapaq Inc., Cambridge, UK) connected to six thermocouples equipped with needles inserted under the peel or in the central part of the columella.
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2.3. Quality losses Losses due to dehydration, wilting, pitting, decay, microcracking (also known as russeting) and senescence were evaluated weekly, after removal from cold storage and on the same fruit after 3 days of shelf-life at 20°C and 75–80% RH. Microcracking was observed as single, lignified radial, or in some cases lateral, fractures in the cuticle or skin (0.5–2 mm width, usually 10–30 mm length) without any necrotic tissues. All fruit were visually examined and disorders recorded using four classes as previously reported (Ferna´ndez-Trujillo et al., 1998). In the very slight to slight disorder class, less than 5% of the tomato skin was affected, and pitted areas were less than 3 mm diameter. Only moderate and severe levels were considered in total losses, when disorders covered between 5 and 15% of the surface (areas of 3–10 mm diameter in the case of pitting). Fungi were identified according to conventional methodology (Arte´s and Escriche, 1994). Losses from storage disorders, decay and dehydration were recorded as percentages on a fresh weight basis. Each fruit sample was inspected weekly for presence of physiological disorders and a disorder index calculated according to Ferna´ndez-Trujillo et al. (1998). The index represents the degree of the disorder on a 0–100 scale, while total losses represent the severity of injuries.
2.4. Colour, flesh firmness and hedonic tests Tomato colour and flesh firmness were measured according to Arte´s and Escriche (1994) and Ferna´ndez-Trujillo et al. (1998). The Minolta CR300 tristimulus colorimeter was calibrated with a white reference plate, C illuminant and 2° observer of standard C.I.E. L*a*b* Colour Space coordinates. The hue angle [tan − 1(b* · a* − 1)] was calculated. Hedonic tests (flavour, taste, visual quality and overall quality) were carried out (n= 6 fruit per treatment) by a panel of six trained people on fruit after 4 weeks of storage and additional subsequent ripening. Evaluation was on a 0 to 5 point scale: 0, extremely poor; 1, poor; 2, fair
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(fairly fresh, limit of edibility); 3, good (fresh, limit of marketability); 4, very good; 5, excellent. An average score plus standard error (n = 6 judges) was calculated for each treatment.
2.5. Respiration rate and ethylene production CO2 and C2H4 were measured with a Hewlett Packard 5730A gas chromatograph fitted with TCD and FID detectors. Measurements were made on individual fruit in hermetically sealed jars over given times according to Ferna´ndez-Trujillo et al. (1998), using five fruits for each treatment, except in continuous 2°C storage or in intermediate transfer to ripening after 2 or 3 weeks of continuous storage at 9 or 12°C when four and three fruit were used, respectively.
2.6. Pectolytic enzyme acti6ity Pectolytic enzymes were extracted by homogenizing outer pericarp tissue combined from three tomato fruit per replicate, in an aqueous solution containing 0.5 M NaCl and 1% polyvinylpyrrolidone (Arte´s et al., 1996). Homogenates were filtered through four layers of cheesecloth and centrifuged at 10000×g for 10 min at 4°C. Enzyme assays for PG (EC 3.2.1.15) and PME (EC 3.1.1.11) were conducted according to Arte´s et al. (1996). PG activity was expressed as mmol of reducing groups (ml extract) − 1 h − 1. PME was expressed as mequiv. H + (ml extract) − 1 h − 1.
2.7. Statistical analysis Data for flesh firmness and hedonic tests of iprodione-treated fruit were subjected to fully factorial multivariate analysis of variance (M)ANOVA. Colour analysis data obtained on the 5th, 12th, 19th, 26th and 29th day after harvest were used for statistical analysis of repeated measurements on each fruit (Ato and Lo´pez, 1994). Percent loss data, except weight loss and indices, were transformed to arcsin for statistical analysis. A similar (M)ANOVA design of the repeated measurements was also performed for losses and indices using data from the first or the
second week to the end of storage. Data from variables related to quality losses after shelf-life at 20°C were not included in the inspection time factor. In the experimental design used for losses, iprodione and storage regime were the ‘betweensubject’ factors and inspection time was the ‘within-subject’ factor. Mean comparisons were performed using LSDs except for pitting losses and indices, when the Bonferroni test (Ato and Lo´pez, 1994) was used. 3. Results
3.1. Fruit temperatures In the IC treatment, the time to acquire a constant temperature regime (differences lower than 0.2°C between air and fruit temperatures, or between peel and columella) was 6.67 h (from 12 to 2°C) or 8.77 h (from 2 to 12°C). In the IW treatment, these times were 7.70 h (from 9 to 20°C) or 6.73 h (from 20 to 9°C). In both treatments, with warming or cooling, differences between air and columella tissue were less than 1°C within 4.5 h.
3.2. Flesh firmness, colour and hedonic sensory tests Fruit ripened after harvest softened from 61 to 16 N after 28 days at 20°C. Firmness of fruit from 12°C and IC regimes averaged 26 N (data not shown). Firmness of fruit at 9°C (38 N) was greater than with IW or other treatments after storage (P 50.01), but not after 3 days of shelflife at 20°C. Fruit kept at 20°C became increasingly red as indicated by decreasing hue angles (Fig. 1A). Hue angles indicated that fruit kept at 12°C ripened more slowly and were not affected by IC. Fruit kept at 9°C also ripened more slowly, but IW fruit tended to ripen faster after the first warming. However, by the time of the third IW, no difference between IW fruit held at 9 and 12°C treatments could be detected. After 4 weeks of continuous storage at 9°C, fruit did not ripen as rapidly as those from any other treatment, even after a shelf-life period.
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Fig. 1. Changes in hue angle and lightness (L*) and during storage and after 3 days of shelf-life at 20°C. Bars are 2 ×S.E. (n = 24). Treatments were: continuous storage at 9, 12 and 20°C; IW, intermittent warming of 1 day at 20°C every 6 days of storage at 9°C; IC, intermittent cooling of 1 day at 2°C every 6 days of storage at 12°C. Dashed lines indicate shelf-life. Arrows indicate transfer times of fruit to intermittent warming or cooling periods (bold solid line).
A slight reduction in L* was detected during the cooling periods in IC fruit. This effect was not related to the induction of pitting in IC because 9°C untreated fruit exhibited similar onset of pitting but not a decrease in L* values
(Fig. 1BFig. 2AFig. 3A). Fruit colour was blotchy orange to red after the first cooling, but to a lesser extent thereafter, because the IC effect on red colour was reversible at 12°C (Fig. 1B).
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Fig. 2. Mean losses, on a percentage fresh weight basis, during storage of ‘Durinta’ tomatoes washed with water (A) or additionally treated with 0.5 g l − 1 of iprodione (B). Thin or bold arrows indicate transfer times of fruit to intermittent warming or cooling (IW and IC, respectively), or shelf-life at 20°C respectively. Bars are 2 ×S.E. (n = 3). For treatment details, see Fig. 1.
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Fig. 3. Physiological disorder indices (0 to 100 scale) during storage of ‘Durinta’ tomatoes washed with water (A) or additionally treated with 0.5 g l − 1 of iprodione (B). Thin or bold arrows indicate transfer times of fruit to intermittent warming or cooling, or shelf-life at 20°C, respectively. Bars are 2 × S.E. (n=3). See Fig. 1 for treatment details.
The shelf-life of fruit kept at 20°C was 2 weeks (taste of 2.9 9 0.3 and overall quality 3.8 9 0.2 U). After 4 weeks of storage, no significant differences
in visual and overall quality of fruit among continuous and cyclical treatments were observed, except reduced flavour in fruit kept continuously
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at 9°C compared with IW fruit (2.3 9 0.4 and 3.5 90.6 U, respectively, P B0.05). After subsequent ripening, fruit from continuous and cyclical treatments were similar, although the best overall quality was attained by IC fruit, particularly due to taste and visual quality (3.890.4 and 4.49 0.2 U, respectively).
3.3. Quality losses 3.3.1. Weight losses and decay After the shelf-life period, iprodione was effective in reducing weight losses only in fruit from 9°C and IW treatments (iprodione ×treatment interaction was significant, P 5 0.001). This fact was probably associated with a reduction in CI in both treatments, because 9°C and IW treatments showed the highest weight losses when untreated (three-way interaction significant at P 50.001; Fig. 2). Untreated fruit developed fungal decay after 3 weeks (Fig. 2). After a shelf-life period, IW fruit were the least susceptible to decay, but at 9°C fruit developed Alternaria spp. on pitted areas. Increased losses after shelf-life occurred in IC fruit (pitting with or without Alternaria or Cladosporium spp. growth) and in fruit at 12°C (shrivelling and senescence). Minimum decay occurred in the IW treatment even when fruit were not treated with iprodione (Fig. 2). Compared with continuous storage at 12°C, IC did not exacerbate the decay that appeared after 3 weeks of storage (Fig. 2). Iprodione reduced decay and total losses, especially at 12°C (Fig. 2). Iprodione was also effective in reducing and delaying Alternaria spp. growth on senescent 20°C fruit (1.4% in treated fruit after 4 weeks at 20°C; 10.9% in untreated fruit after the 3rd week at 20°C). With IW, only 0.7% of fruit ripened unevenly during storage and 1.4% after shelf-life. After 28 days at 20°C, high levels of senescence were detected (see below). 3.3.2. Pitting Slight pitting occurred in iprodione-treated fruits stored for 28 days at 9 or 12°C but its incidence increased in both treated and untreated
IW fruits, and in 9°C and IC untreated fruit (Fig. 2). Slight symptoms of surface pits appeared after 1 week of storage in all treatments except in continuous 12°C storage (Fig. 3). However, pitted skin was rapidly colonised by fungi in 9°C fruit, probably masking the real incidence of pitting. After the shelf-life period, Alternaria spp. colonised most of the pitted areas (except 0.7% (w/w) in IW and 1.4% in 9°C but untreated, and 1.4% of IC treated). IC fruit showed pitting after the second cooling, with fungi colonising pitted areas after the third cooling. Slight pitting developed after the first exposure to IW, the location of pitting mainly corresponding to the fruit zone that was last to ripen. Pitting losses and CI index only slightly increased after the second warming period, which induced partial ripening (Fig. 1). In untreated fruit after 4 weeks of storage plus shelf-life at 20°C, about 20% fruit suffered pitting without significant differences among 9°C, IW and IC treatments, while 12°C fruit was slightly affected (6%). However, in fungicide-treated fruit subjected to these treatments, pitting was detected (10, 8 and 15%, respectively), 12°C fruit being again slightly affected (4%). Iprodione reduced fruits affected by moderate to severe pitting (by 5%) and also had a synergistic effect when combined with 9°C or IW storage (an additional 5%). However, the increased pitting indicated by the pitting index was only reduced by storage at 12°C with or without iprodione pretreatment (Fig. 3).
3.3.3. Senescence, annular breakdown and microcracking After the second week of storage, senescence symptoms appeared particularly in 20°C fruit (2, 50, 65 and 85% losses in the 2nd, 3rd and 4th week, and subsequent ripening inspections). Other treatments were practically free from advanced senescence at the end of subsequent ripening (9°C, IW treated, 12°C untreated) or slightly affected (2.8% in 12°C treated fruit and 1.4% in other treatments). A form of wilting was identified, manifested as dehydration around the calyx zone associated with browning of the tissues affected. This disor-
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der, which to the best of our knowledge has not been previously reported, was called ‘annular breakdown’. Its occurrence may be related to the removal of sepals prior to storage. Symptoms of this disorder resembled the ‘stem end rind breakdown’ in citrus fruit. After subsequent ripening, less than 1% moderate annular breakdown appeared in both IW and IC untreated fruit or in 12°C treated fruit. Annular breakdown in untreated fruit was more frequent than in treated fruit but only slight severity (Fig. 3). Occasional increases in this disorder occurred after 3 weeks of storage. Microcracking symptoms were observed mainly in fruit held continuously at 9°C without fungicide treatment (Fig. 3). In treated fruit, annular breakdown and microcracking indices after shelf-life were lower than 3.8 and 2.5, respectively in cold treatments and 5.6 and 2.8 in control fruit (Fig. 3). At 9°C, iprodione reduced microcracking after the second week of storage (fungicide × storage time× storage regime interaction significant at P B0.01), but at 20°C exacerbated the increase of annular breakdown during storage (P B 0.001) and to a lesser extent microcracking (Fig. 2).
3.4. Respiration and ethylene production In ‘Durinta’ tomatoes, RR and EP decreases progressively, without a clear respiratory climacteric onset or EP peak during the shelf-life period (Figs. 4 and 5). During storage at 2°C, EP declined but RR remained constant (Figs. 4 and 5). During the shelf-life of this fruit, EP increased continuously, indicating a possible response to low temperatures and failure to ripen (data not shown). Even after the 4th day of shelf-life, RR and EP in these fruits remained higher than in other treatments (Figs. 4 and 5). In fruit kept at 9 or 12°C, transfer to 20°C after 2 or 3 weeks of storage resulted in increased RR and EP compared with that in fruit kept continuously at 20°C (Figs. 4 and 5). In the fourth week of storage, a peak of EP was slightly delayed in fruit from the treatments compared to that in fruit at 20°C. In IW fruit, EP was stimulated to similar degrees during successive warmings periods, rates ranging from 3 to 4.5 ml C2H4 kg − 1 h − 1 (Fig. 5).
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A slight EP stimulation after return to 9°C after the third warming period could be related to a slight increase in pitting index in this case (Fig. 3 section B, Fig. 5). EP was transiently stimulated after the second and third cooling periods when fruit returned to 12°C storage (Fig. 5). This stimulation was in parallel with the first pitting symptoms (Fig. 2BFig. 3B). In the third cooling period, EP reached the lower levels attained at continuous 2°C (0.23 9 0.03 ml C2H4 kg − 1 h − 1), and more than three times lower than in the previous cooling periods (Fig. 5).
3.5. Pectolytic enzyme acti6ities Activities of pectolytic enzymes increased rapidly during ripening at 20°C, but were delayed in fruit from 9°C or IW treatments (Fig. 6). PME activities were similar. Slight uneven ripening in fruit from the 9°C treatment between the second and the third weeks of storage (Fig. 1) was associated with slightly lower PG activity compared with that in IW fruit (Fig. 6). PG or PME activities in fruit from 9°C or IW treatments did not reach those found in fruit kept continuously at 20°C (Fig. 6).
4. Discussion IW reduced pitting and decay the most, and resulted in good fruit firmness and colour, and in the lowest total losses after storage and shelf-life, without detrimental sensory effects. Overall fruit quality in the IW treatment was better than that reported for a conventional cultivar (Arte´s and Escriche, 1994). Pitting incidence and severity developed to a similar extent after the second warming period (more advanced fruit ripening stage). Variation in chilling sensitivity during tomato ripening has been reported as biphasic, with a decline at the onset of ripening followed by a senescence-related increase (Autio and Bramlage, 1986). IC resulted in increased pitting. Also, water condensation that occurs at 12°C on the fruit subjected to IC probably accelerated senescence
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Fig. 4. Respiration rate during 26 days of storage and additional shelf-life at 20°C of ‘Durinta’ tomatoes. See Fig. 1 for treatment details (a continuous storage at 2°C is included). Vertical arrows represent transfer to intermittent warming (bold dashed line) or cooling periods (bold solid line). All fruits were treated with 0.5 g l − 1 of iprodione. Horizontal arrows ( ) represent transfer to ripening at 20°C (dashed lines). Bars are S.E. (n= 5 except at 2°C or in intermediate transfer to ripening when n =4 and n =3, respectively).
and/or skin deterioration resulting in stimulated fungal attacks, as shown in mangoes (Sankat and Mohammed, 1993). Clearly IC treatment conditions require adjustment to reduce pitting and
water condensation, while retaining the positive effect on taste. The differences in ripening-dependent storage time for the application of 1 day of IC or IW without stimulating pitting is evident,
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Fig. 5. Ethylene production during 26 days of storage and additional shelf-life at 20°C of ‘Durinta’ tomatoes. See Fig. 1 for treatment details (a continuous storage at 2°C is included). Vertical arrows represent transfer to intermittent warming (bold dashed line) or cooling periods (bold solid line). All fruits were treated with 0.5 g l − 1 of iprodione. Horizontal arrows ( ) represent transfer to ripening at 20°C (dashed lines). Bars are S.E. (n= 5 except at 2°C or in intermediate transfer to ripening when n = 4 and n= 3, respectively).
perhaps related to differences in membrane repair after cold storage (Marangoni et al., 1996). The relatively small effect on ripening at low temperatures could be related to low sensitivity to CI of this cultivar (Brown et al., 1989). The effect
of cooling at 2°C on lycopene biosynthesis was reversible but the onset of pitting was not. Reversibility of chilling sensitivity, evaluated as the ability to develop red colour (Saltveit, 1991), was strongly dependent on the length of the warming and cooling periods.
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Beneficial effects of IW in comparison with continuous 9°C storage on development of Alternaria spp. and Cladosporium spp. were remarkable, confirming previous studies on Alternaria spp. (Arte´s et al., 1987; Arte´s and Escriche, 1994). Iprodione was effective in reducing pitting and weight losses particularly during storage at 9°C alone or combined with IW. A single or synergistic iprodione effect does not appear to have been previously reported. Colour and flesh firmness differences in ‘Durinta’ fruit do not appear to be directly associated with differences in EP, RR or pectolytic enzyme activities. The decrease in RR and EP during ripening is typical behaviour similar to that found in ‘Alcobaca’ (alc) tomato fruits with 95% red colour developed, although the PG activity is not as low for the hybrids (Mutschler, 1984; Watkins et al., 1988). The delayed EP peaks found in all
Fig. 6. Enzyme activities assayed in fruit washed with water during continuous storage at 20°C (), 9°C ( –), or IW () (intermittent warming of 1 day at 20°C every 6 days at 9°C). (A) PME activity: PMEu is defined as mequiv. H + released (ml of extract) − 1 h − 1. (B) PG activity: PGu is defined as mmol of reducing groups (ml of extract) − 1 h − 1. Arrows indicate intermittent warming (dashed lines). Bars are S.E. (n= 3).
the treatments during the fourth week of storage (Fig. 5) was rather a sign of senescence (microcracking, annular breakdown, shrivelling) appearing at this time than a delayed response to the climacteric. Typically, in chilling sensitive plants, RR increases initially, but as chilling proceeds it decreases. When the burst of RR does not return to those found at nonchilling temperatures, CI symptoms usually become apparent (Lyons and Breidenbach, 1990). As reported in other cultivars (Arte´s et al., 1987; Cheng and Shewfelt, 1988), the onset of RR in ‘Durinta’ occurred as a late response to CI (Fig. 4). The stimulation of EP during shelf-life after continuous 2°C storage, or during subsequent ripening for 2 or 3 weeks at continuous 9 or 12°C fruit compared with 20°C fruit, agrees with results from Cheng and Shewfelt (1988). These treatments resulted in different ripening stages during storage (Fig. 1), but treatment differences on EP were evident only during shelf-life when pitting developed (Fig. 5). Increased EP following chilling has also been shown in cucumbers to be the result of an increased capacity of the tissue to synthesize ACC and/or a change in compartmentalisation of the reactants (Wang and Adams, 1982; Cabrera and Saltveit, 1990). However, this chilling-induced EP stimulation did not occur in tomato with different degrees of inhibition of lycopene synthesis (Watkins et al., 1990). This suggests that chilling-induced EP shown in our experiment (Fig. 5) was mostly a response to pitting or eventually other biochemical processes in spite of uneven ripening. Other evidence for the relationship between EP and skin disorders is found in the different rates of EP during normal ripening and the general trend for EP to decrease to a constant level (Fig. 5). The stem scar is the main channel for carbon dioxide and ethylene release (Bergevin et al., 1993; de Vries et al., 1996). ‘Durinta’ tomatoes were only affected in the skin adjacent to this zone by storage (Fig. 3B, annular breakdown disorder). Therefore, a possible explanation of a high upsurge or increase in EP is the onset of skin disorders as a consequence of chilling or senescence, since uneven ripening was in general unimportant.
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Slight chilling stress during continuous storage at 12°C was indicated by quite similar and even higher RR and lower EP than 20°C fruit, as previously described (Brown et al., 1989). When IC was applied, an increase in pitting was accompanied by an impairment of EP after the second cooling period (Figs. 2, 3 and 5). This also occurred in the continuous 2°C treatment (Fig. 5). The effect of chilling on EP could be explained by the stimulation of ACC oxidase activity after short exposures to chilling (the case of shelf-life after 9 or 12°C continuous storage) (Watkins et al., 1990) and the loss of ACC oxidase activity during longer exposures (Cabrera and Saltveit, 1990). In tomato, transcripts for the gene encoding the oxidase increased during cold storage (Watkins et al., 1990). The relationship between pitting and EP after cooling or warming periods may be associated with the alleviation of CI due to ethylene-stimulated metabolism in some IW cycles, associated with proposed effects of IW in conditioning fruit tissues against cell damage (Watkins et al., 1995). Fruit from both IC and continuous 12°C, and to a lesser extent from IW and continuous 9°C, were at similar ripening stages during storage (Fig. 1). This ensured that comparisons of physiological effects were not confounded by differences in ripening stages. The slight stimulation of EP by warming only partly agrees with previous results in a conventional cultivar of tomato (Arte´s et al., 1987), in peaches (Ferna´ndez-Trujillo et al., 1998) and in apples (Watkins et al., 1995), probably due to different ripening behaviour and CI symptoms in these latter fruit. PG activity increased during ripening and was lower in fruit kept at 9°C, but patterns of change were not directly related to EP (e.g. see from the first to the second week of storage, Figs. 5 and 6). Temperatures lower than 9°C (e.g. 4°C, see Palma et al., 1995) or different IW conditions than the regime assayed are required for a higher inhibition of this activity. For commercial purposes, IW or IC regimes involve moving produce from one room to another, requiring the availability of specific rooms and methods to control condensation on the fruits. For IW, energy costs are compensated for
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by reduction of pitting, Alternaria spp. infection and chemical usage.
Acknowledgements Financial support was provided by the Spanish CICYT (ALI-95/530 Project). J.P. Ferna´ndezTrujillo is indebted to CSIC for a PhD grant. A. Cano is indebted to Caja de Ahorros del Mediterra´neo for a grant. Thanks are due to J.A. Martı´nez for technical help, Dura´n S.A.T. for supplying tomatoes and other facilities, and Dr C.B. Watkins for a critical review of the manuscript.
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