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Preharvest calcium chloride sprays affect ripening of Eksotika II’papaya fruits during cold storage
Scientia Horticulturae 171 (2014) 6–13
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Preharvest calcium chloride sprays affect ripening of Eksotika II’papaya fruits during cold storage Babak Madani a,∗ , Mahmud Tengku Muda Mohamed a,∗∗ , Christopher B. Watkins b , Jugah Kadir c , Yahya Awang a , Taha Roodbar Shojaei d a
Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Horticulture, College of Agriculture and Life Science, Cornell University, Ithaca, NY 14853, USA c Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia d Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b
a r t i c l e
i n f o
Article history: Received 25 December 2013 Received in revised form 15 March 2014 Accepted 20 March 2014 Available online 12 April 2014 Keywords: Papaya Preharvest spray Calcium chloride Physicochemical
a b s t r a c t The effects of preharvest application of calcium chloride on ripening, activity patterns of pectin modifying enzymes and overall quality of papaya (Carica papaya L. cv. ‘Eksotika II’) fruits have been investigated. Foliar sprays of 0, 0.5%, 1%, 1.5% and 2% (w/v) calcium chloride were applied six times during the growing season. After harvest, fruits were stored at 12 ◦ C for up to three weeks. Higher calcium concentrations in the sprays coincided with increasing calcium concentrations in peel and pulp tissues, higher firmness and titratable acidity, and reduced respiration rate, ethylene production and soluble solids concentrations, compared with those of the untreated control fruits. The overall quality of fruits treated with calcium was greater than the control fruits. Also, fruits sprayed with calcium had decreased activities of polygalacturonase (PG) and pectin methyl esterase (PME) during storage. Microscopic results confirmed that the middle lamallae of cell walls was more intact in calcium chloride-treated fruits. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Papaya is an important export product for Malaysia (Ali et al., 2014) and new cultivar development is ongoing. The world trade of fruit is increasing, but marketing is limited due to improper handling, transport and postharvest diseases, that cause low quality and storage life. ‘Eksotika II’, released by Malaysian Agricultural Research and Development Institute, and has gained popularity in the local and export markets due to high yield and good quality. However, fruits soften rapidly and are susceptible to disease (Shukor and Shokri, 1997; Madani et al., 2013). One of the most economically significant postharvest pathogens of papaya is anthracnose that is caused by Colletotrichum gloeosporioides Penz (Gamagae et al., 2003). Postharvest diseases are mainly controlled by using synthetic chemical fungicides, but consumer concern about pesticide residues on foods, as well as pathogen resistance
∗ Corresponding author at: Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 123338642. ∗∗ Corresponding author at: Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 389474823. E-mail addresses: [email protected], [email protected] (B. Madani), [email protected] (M.T.M. Mohamed). http://dx.doi.org/10.1016/j.scienta.2014.03.032 0304-4238/© 2014 Elsevier B.V. All rights reserved.
to many currently registered pesticides, has increased. Alternative methods for decay control has become more important, including improvement of fruit resistance to pathogens before harvest. It is known that one approach is the use of calcium salts. Preharvest application of calcium salts can control or reduce certain physiological disorders, reduce the incidence of fungal pathogens, maintain fruit firmness and reduce mechanical damage in many fruit types (Ferguson and Watkins, 1989; Cheour et al., 1991; Oms-Oliu et al., 2010; Ciccarese et al., 2013). Most research on calcium sprays has been centred on temperate fruits such as apple, and relatively less is known for tropical and subtropical fruits. Available studies, however, show positive responses. For example, in pineapple, calcium sprayed fruits had higher calcium concentrations, and decreased black heart symptoms (Hewajulige et al., 2006). Calcium sprayed guava and pomegranate fruits had higher ascorbic acid concentrations (Ramezanian et al., 2009; Goutam et al., 2010), lower respiration rates, better appearance and marketability in mango (Singh et al., 1993). In papaya, Ramakrishna et al. (2001) reported that preharvest application of calcium chloride and calcium nitrate reduced weight loss, enhanced firmness and increased titratable acidity during storage at ambient temperatures. Softening of climacteric fruits is mainly related with changes in pectin fraction in cell wall, and large increase in the pectin
B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
solubilization correlated with softening (Lohani et al., 2004). In papaya, softening is associated with higher activities of polygalacturonase and pectin methyl esterase (Ali et al., 2004), which are synthesized de novo during ripening (Manganaris et al., 2005). Calcium is thought to maintain cell wall structure by interacting with pectin in the cell wall to form calcium pectate complexes, which support bonding between components of the cell wall and prevent the action of enzymes associated with cell wall disassembly. While preharvest calcium treatment of peaches reduced activities of PG and PME (Manganaris et al., 2005), little is known about the effects of calcium on these enzymes in tropical fruits such as papaya. The objective of the current study was to investigate the effects of a range of calcium concentrations applied before harvest on postharvest quality and activities of associated cell wall degrading enzymes activities of papaya fruits during cold storage.
2. Materials and methods 2.1. Plant materials Papaya trees (Carica papaya L. cv. Eksotika II) growing at the Agro-Tech Unit, University Agriculture Park (TPU), Universiti Putra Malaysia, Serdang, Selangor (3◦ 00 21.34 N, 101◦ 42 15.06 E, 37 m elevation) were used in both 2012 and 2013. Forty and twenty four eight month old trees, approximately 2.2 m tall, were used for experiments in 2012 and 2013, respectively. The experiments were conducted in a randomized complete block design with four blocks including two plants in each 3 m × 3 m block. Commercial fertilization (12:12:17:2 N:P:K:Mg) were utilized monthly around the canopy periphery for all treatments (Basir, 2005). Irrigation was carried out with overhead sprinklers at approximately 4 day intervals.
2.2. Treatments Calcium chloride (CaCl2 ·2H2 O (99% CC, SYSTERM® , Malaysia) at concentrations of (0, 0.5, 1.0, 1.5 and 2.0% (w/v)) were sprayed to the fruits and leaves (approximately 1.5 L per tree (with a Knapsack Sprayer). Sprays were applied every two weeks for six times from 21 days after flower anthesis until 10 days before harvest. In 2013, 0, 1.5% and 2% calcium was sprayed on trees till 2 days before fruit harvest. In all other respects the 2013 trial was performed using the same procedures as in the previous season. In both years, 64 fruits in each treatment with uniform size (500–550 g) and shape were harvested at ripening index 2 (green with trace of yellow), washed with water and allowed to air dry before randomly divided into five different lots for 2012 and three lots for 2013. Fruit samples for each treatment were taken at harvest and during storage. Fruits were packed in commercial boxes (EXOTIC STAR® , Kajang, Selangor, Malaysia) and stored at 12 ± 2 ◦ C and 85–90% relative humidity for up to three weeks.
2.3. Determination of calcium concentration in fruit Approximately 20 g of peel and pulp samples were taken from two fruits from each of four blocks per treatment. The samples were dried at 70 ◦ C in an air-circulating oven. Dried peel and pulp (0.25 g) were digested in 5 mL 98% H2 SO4 on hot plate at 285 ◦ C, and 2 mL 50% H2 O2 added to the mixture. The solutions were made up to 100 mL with distilled water. Calcium concentration was measured using an atomic absorption spectrophotometer (Perkin–Elmer, Model AAS 3110, Palo Alto, California, USA), and results were expressed as percentages.
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2.4. Respiration rate and ethylene production Respiration rate and ethylene production were measured using method described by Saltveit (1982). For each treatment eight fruits were used to measure ethylene production and respiration rate. The weight and volume of each single fruit was measured and kept in 1.9 L airtight container for 2 h in storage (12 ± 2 ◦ C). One mL of accumulated gases in the sealed container was injected into the injector port of gas chromatograph (Clarus-500, Perkin–Elmer, Shelton, CT, USA) to determine CO2 and C2 H4 concentrations. Instruments were adjusted by certified standards. The amount of C2 H4 and CO2 was expressed by L kg−1 h−1 and mL kg−1 h−1 , respectively. 2.5. Fruit quality factors Titratable acidity (TA) and soluble solids concentration (SSC) of 10 g pulp tissues were measured by the methods of Ali et al. (2011). TA was determined by homogenizing 10 g pulp tissues using a kitchen blender (MX-799S, Panasonic, Shah Alam, Malaysia) with 40 mL of distilled water, and filtering through cotton wool. Then, 1.5 mL of filtrate with two drops of phenolphthalein (0.1%) as indicator, was titrated with 0.1 N NaOH to an endpoint pink (pH 8.1). Results are expressed as percentage of citric acid per 100 g fresh weight. The SSC of the pulp was determined by a hand refractometer (N3000E, Atago Co., Tokyo, Japan) from the filtrate prepared from TA determination. The reading was corrected for the dilution resulting from blending (factor of five). Results are expressed % SSC. Two fruits per block per week arranged in randomized complete block design were used for TA and SSC measurement. Flesh firmness was measured by Instron Universal Testing Machine (Model 5540, USA) and results expressed in Newtons (N). Peel colour of fruits was measured by Minolta (CR-300, Minolta Corp, Osaka, Japan) colourimeter and expressed as L* , C* and h◦ . Two fruits per block in each week arranged in randomized complete block design were used for firmness and colour measurements. 2.6. Microscopy Cubes of flesh (1.5 mm3 ) from mid-region of each fruit were fixed in 4% glutaraldehyde at 4 ◦ C for 24 h. Samples were washed with 0.1 mol L−1 sodium cacodylate buffer (pH 7.6) for three times, and post fixed in 1% (w/v) osmium tetraoxide for 2 h. The fixed samples were then rinsed again in 0.1 mol L−1 sodium cacodylate buffer (pH 7.6), and dehydrated in graded series of acetone at (35–100%). Subsequently, dehydrated samples were embedded in beam capsules and polymerized at 60 ◦ C for 2 days. Ultra–slim sections (60–90 nm) of samples were cut and mounted onto copper grids. Sections were stained with lead citrate and saturated uranyl acetate (Reynolds, 1963) and investigated using transmission electron micrscope (TEM) (Hitachi, H-7100, Tokyo, Japan). 2.7. Extraction and assay of cell wall associated enzyme activities The procedure for extraction of polygalacturonase and pectin methyl esterase activity was performed using methods described by Lazan et al. (1989) and Ali et al. (2004) with some modification. Ten grams of pulp tissues were homogenized in a blender for 2 min with 20 mL of cold 0.1 mol L−1 sodium citrate, pH 4.6 buffer (containing 1 mol L−1 NaCl, 13 mmol L−1 EDTA, 10 mmol L−1 -mercaptoethanol, and 1% (w/v) polyvinylpyrrolidone). Afterwards, the mixture was incubated at 4 ◦ C for 60 min with stirring. The supernatant was subsequently recovered by centrifugation at 15,000 × g for 30 min at 4 ◦ C in a refrigerated centrifuge (Scan speed 1730R, Scala Scientific, Netherlands). The clear supernatant (crude enzyme extract) was used to determine enzyme activity.
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Polygalacturonase (PG) activity was assayed by the method described by Pathak and Sanwal (1998) and Lohani et al. (2004) with slight modification. The reaction mixture contained 0.4 mL sodium acetate (200 mmol L−1 , pH 4.5) buffer, 0.1 mL NaCl (200 mmol L−1 ), 0.4 mL polygalacturonic acid (PGA 1% aqueous solution adjusted to pH 4.5) and 0.1 mL of enzyme extract in a total volume of 1.0 mL. The control comprised the same components but with the enzyme extract boiled for 5 min. The reaction was initiated by the addition of the PGA substrate. The mixture was incubated at 37 ◦ C for 1 h and 0.1 mL 3, 5-dinitrosalicylate (DNS) reagent added. The reaction mixture was heated in boiling water for 5 min. When the mixture reached to room temperature (25 ± 2 ◦ C) the absorbance was measured at 450 nm using a spectrophotometer (WPA, Biochrom, Cambridge, England). The formation of reducing groups was estimated against d-galacturonic acid as a standard. One unit of enzyme activity is defined as the amount of enzyme required to liberate 1 nM of galactronic acid per min per g of original fresh weight of flesh. Pectin methyl esterase (PME) activity was measured according to the method described by Hagerman and Austin (1986) and Lohani et al. (2004). The reaction mixture was prepared in a 3 mL glass cuvette and was composed of 1 mL pectin solution (0.01% aqueous solution adjusted to pH 7.5 using 0.1 mol L−1 NaOH, 0.2 mL NaCl (0.15 mol L−1 ), 0.1 mL bromothymol blue solution (0.01%), 0.2 mL sterilized water and 0.1 mL crude enzyme extract. After adding the enzyme prepared, the cuvette was shaken gently. The absorbance of the reaction mixture was measured immediately at 620 nm using a spectrophotometer (WPA, Biochrom, Cambridge, England). The absorbance was again measured after 3 min. The difference in absorbance between 0 and 3 min was the measure of PME activity. Calculation of the activity was carried out against a standard curve of galacturonic acid (Hagerman and Austin, 1986). One unit is defined as the amount of the enzyme required to release 1 mol of methyl ester per min per g of original fresh weight of flesh. For enzyme activities, two fruits per block in each week arranged in a randomized complete block design with two factors (weeks in storage and calcium concentrations), were used. 2.8. Determination of disease severity and overall quality Disease severity was measured after three weeks in storage as surface of fruit with anthracnose disease and expressed by percentage. Six fruits per blocks arranged in randomized complete block design were used for disease severity measurement. To investigate overall quality, a panel of six untrained judges were selected. Overall quality was performed utilizing the Hedonic scale by allotting the number from 0 to 5, where 0 for very poor and 5 for excellent and measured after three weeks. 2.9. Statistical analysis Data were subjected to Analysis of Variance (ANOVA) using the Statistical Analysis System (SAS) version 8.2 (SAS Institute Inc., Cary, NC, USA). The means were compared by the Duncan’s Multiple Range Test (DMRT) at significance level of 0.05.
3. Results and discussion Preharvest calcium sprays have been shown to delay ripening and senescence processes and inhibit the development of physiological and pathological disorders (Ferguson and Watkins, 1989; Cheour et al., 1991; Oms-Oliu et al., 2010; Ciccarese et al., 2013), but most research has been focused on temperate crops. Here we
show that these sprays can have dramatic effects on a tropical fruit, the papaya. 3.1. Calcium concentration in fruit In this study, calcium was sprayed six times throughout the growing season. In 2012, preharvest calcium increased calcium concentration in peel (77–129%) and pulp (30%) of fruits sprayed with 1.5% and 2% calcium relative to that in control fruits at harvest (Table 1). In 2013, differences in calcium were detected for 1.5% and 2% calcium for calcium concentration in peel and pulp compared with control after week 1. No differences were detected between 1.5% and control in peel and pulp tissues at the first storage sampling (Table 1). Because the results for 1.5% and 2% calcium sprays were similar, we did not use higher amounts of calcium in 2013. Also, because no symptoms of leaf and fruit damage from calcium applications were detected at any calcium concentration, we used the lower and more economical rate. Interestingly, Qiu et al. (1995) found that calcium sprays did not increase calcium concentrations in papaya flesh. Environmental factors, presence of stomata or other irregular surfaces such as cracks, and genetically differences in the cultivars can affect calcium absorption in fruits (Saure, 2005). 3.2. Ethylene production and respiration rate Differences in ethylene production between fruits treated with 1.5% and 2% calcium compared with those of the control were detected at harvest in 2012 (Table 1). However, no differences were found among preharvest calcium concentrations after a week of storage. After three weeks differences were detected, though not between the control and 0.5% calcium treatments. In 2013, differences were found between 1.5% and 2% calcium compared with control treatments at each week of storage (Table 1). The respiration rate of fruits was lower only in 1.5% and 2% calcium treatments relative to control treatments at harvest time and after one week of storage in 2012 (Table 1). However, at the second and third weeks of storage there were differences between 1%, 1.5% and 2% calcium treated fruit compared with untreated fruit. In 2013, differences found between 1.5% and 2% calcium relative to control for all weeks with the exception of week two when no differences between the 1.5% calcium treatment and the control were detected (Table 1). Papaya is characterized by rapid softening once ripening has been initiated, reflecting behaviour of a typical climateric fruit (Paull and Duarte, 2011). Calcium is hypothesized to delay ripening by reducing disintegration of tissues and maintaining membrane integrity (Sams and Conway, 1984; Torre et al., 1999). It has also been proposed that calcium delays ethylene production by preventing solubilization of calcium binding sites in cell walls which activate the ethylene generation system located in cell wall plasma membrane complex (Agusti et al., 2004). 3.3. Firmness, cell wall integrity, and activity of cell wall enzymes Flesh firmness is the most important parameter in papaya fruits during storage and marketing, because flesh softening is associated with senescence and increased susceptibility of fruits to injury during handling. Fruits sprayed with 1%, 1.5% and 2% calcium were firmer than those of the untreated fruits at harvest and at weeks two and three in 2012 (Table 1). All fruits softened markedly between weeks two and three, but all calcium treatments above 0.5% resulted in firmer fruits than the untreated controls. In 2013, differences were found between 1.5% and 2% calcium treatments relative to the control treatment with the exception of week two when the 1.5% calcium treatment did not show any differences with control (Table 1). Softening of fruits is associated with dissolution
B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
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Table 1 Calcium concentration, ethylene production, respiration rate, and pulp firmness of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride in storage period in 2012 and 2013. Quality parameters
Calcium chloride concentration (%)
Storage period (Week)
0
1
2
3
2012
Calcium in peel (%)
0 0.5 1 1.5 2
1.14dz 1.40 cd 1.72bc 2.02b 2.61a
1.43c 1.49bc 1.50bc 1.76ab 1.98a
1.22c 1.30bc 1.37bc 1.58ab 1.82a
1.13c 1.41c 1.52bc 1.85b 2.39a
Calcium in pulp (%)
0 0.5 1 1.5 2
0.55b 0.58ab 0.61ab 0.71a 0.72a
0.51b 0.58ab 0.67a 0.69a 0.69a
0.51b 0.61a 0.65a 0.68a 0.69a
0.61b 0.78b 1.18a 1.19a 1.12a
0 0.5 1 1.5 2
0.8a 0.8ab 0.7ab 0.6b 0.6b
3.5a 3.4a 3.3a 3.0a 3.0a
7.8a 7.5a 5.5b 5.4b 5.3b
11.1a 11.0a 5.4b 3.5c 3.4c
0 0.5 1 1.5 2
15.9a 15.1ab 14.4ab 14.1b 11.9c
17.3a 17.2a 16.5a 14.2b 13.9b
20.1a 19.4ab 17.7bc 17.1c 17.0c
23.1a 21.8a 17.7b 17.1b 17.0b
0 0.5 1 1.5 2
34.0c 38.0c 46.1b 53.1a 54.1a
25.1b 27.8ab 30.2ab 32.1ab 33.8a
16.7c 19.2bc 23.3ab 25.3a 26.9a
3.8c 5.7bc 7.6ab 8.5a 8.4a
−1
Ethylene (L kg
−1
h
−1
Respiration (mL kg
)
−1
h
)
Firmness (N)
2013 Calcium in peel (%)
0 1.5 2
1.66c 2.41b 2.96a
1.25b 1.79ab 2.20a
1.15b 1.57a 1.86a
1.03c 1.82b 2.34a
Calcium in pulp (%)
0 1.5 2
0.51b 0.66a 0.67a
0.57b 0.65ab 0.68a
0.60b 0.80a 0.86a
0.62b 1.08a 1.07a
Ethylene(L kg−1 h−1 )
0 1.5 2
5.4a 4.3b 3.9b
6.3a 5.0b 4.6b
9.5a 5.7b 5.2b
12.4a 6.1b 5.9b
Respiration (mL kg−1 h−1 )
0 1.5 2
16.4a 12.1b 11.6b
19.4a 13.9b 12.3b
22.7a 18.5ab 17.7b
26.4a 18.8b 17.7b
Firmness (N)
0 1.5 2
36.2b 52.0a 56.0a
26.6b 37.3a 35.3a
16.9b 21.3ab 24.1a
3.2b 8.6a 8.9a
z
Small letters in columns show the mean comparison among concentrations of calcium chloride. Means with the same letter are not significantly different at P = 0.05.
of the middle lamallae with turnover in the composition, structure and linkages between polysaccharides (Vicente et al., 2007). The effects of calcium are presumably mediated by reduced ethylene production and respiration rate (Recasens et al., 2004). However, maintenance of firmness is also a result of direct interaction of calcium with the cell walls, where it binds to pectins and forms bridges between pectic acids. Calcium also stabilizes pectin–protein complexes in the middle lamallae thereby acting as an intermolecular binding factor (Dey and Brinson, 1984), and may thicken the middle lammelae due to increased calcium pectate deposition (Gupta et al., 1984). TEM images confirmed that the middle lamallae of calciumtreated fruits was maintained more intact than that of untreated fruits. The middle lamallae in untreated fruits had completely disappeared after three weeks in storage compared with that in fruits on the day of harvest (Fig. 1A and B), while in both 1.5% and 2% calcium, middle lamallae had been preserved after three weeks of storage (Fig. 1C and D).
Disassembly of the cell wall and middle lamellae of papaya is associated with increased activities of PG and PME (Ali et al., 2004). In the current study, PG activity decreased in fruits from the 1.5 and 2% calcium treatments at weeks two and three compared with the control treatment (Table 3). PME activity decreased from week one to three in the 1.5 and 2% calcium-treated fruits compared with control. PG activity increased progressively in all calcium treated fruits in second and third week of storage compared with at harvest and the first week of storage, while PME increased significantly in all concentrations of calcium in all weeks of storage relative to harvest (Table 3). Fruits with higher calcium concentrations have higher level of calcium pectate that maintains tissue resistant to degradation by PG (Agusti et al., 2004). Calcium ions can form salt-bridge cross-links with carboxyl groups of the pectin, thereby making the cell wall less accessible to the enzymes responsible for softening. PG only hydrolyses homogalacturonan regions when uronic acid residues have been previously de-methylated by PME (De Assis et al., 2001; Chuni et al., 2010). Since pectins are
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B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
Table 2 Colour changes, soluble solids concentrations, and titratable acidity of ‘Ekzotika II’ papaya fruit in relation to pre-harvest sprays of calcium chloride in storage period in 2012 and 2013. Quality parameters
Calcium chloride concentration (%)
Storage period (Weeks)
0
1
2
3
2012
*
0 0.5 1 1.5 2
59.9az 58.7a 54.2b 53.0b 52.1b
61.3a 58.8a 53.4b 51.1b 51.1b
63.2a 62.2a 60.0ab 56.7bc 55.7c
65.2a 64.2ab 63.3b 62.8b 62.8b
Chroma (C )
0 0.5 1 1.5 2
46.8a 45.8a 42.3b 40.6b 40.1b
47.4a 44.4ab 43.6ab 41.9b 41.7b
48.3a 48.2a 47.6a 43.0b 41.6b
51.2a 49.9a 49.5ab 48.0bc 46.9c
Hue angle (h )
0 0.5 1 1.5 2
158.1a 159.4a 160.3a 161.5a 163.3a
142.8b 143.5b 144.1b 146.5ab 149.6a
Titratable acidity (%)
0 0.5 1 1.5 2
0.29b 0.30b 0.31ab 0.31ab 0.35a
0.25c 0.26c 0.27bc 0.31ab 0.33a
0.17c 0.18bc 0.21abc 0.23ab 0.24a
Soluble solid concentration (%)
0 0.5 1 1.5 2
6.8a 6.4b 6.1c 6.1c 5.5d
8.3a 8.1ab 7.1b 6.1c 5.6d
9.4a 9.2a 7.8b 7.5b 7.4b
11.6a 11.5a 9.5b 7.5c 7.4c
Lightness (L* )
0 1.5 2
59.7a 53.7b 53.6b
60.1a 55.7ab 54.2b
65.9a 55.3b 53.0b
67.4a 62.6b 62.0b
Chroma (C* )
0 1.5 2
47.2a 41.8b 41.2b
48.4a 43.8b 42.8b
50.6a 43.8b 43.3b
52.1a 47.1b 46.7b
Hue angle (h◦ )
0 1.5 2
154.5a 160.2a 160.4a
146.0a 150.1a 152.2a
110.7b 131.3a 141.1a
102.6b 145.8a 140.2a
Titratable acidity (%)
0 1.5 2
0.28b 0.39a 0.43a
0.20b 0.33a 0.34a
0.26a 0.29a 0.30a
Soluble solid concentration (%)
0 1.5 2
6.6a 6.1b 5.4c
7.3a 6.3ab 6.1b
7.9a 6.1b 6.1b
Lightness (L )
*
◦
115.1b 120.0ab 120.1ab 122.5a 124.2a
101.8c 128.2b 147.5a 148.0a 145.0a 0.11c 0.14bc 0.15b 0.20a 0.20a
2013
0.23b 0.31a 0.33a 10.7a 4.3b 4.2b
z Small letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different according to the Multiple Range Test at P = 0.05.
Table 3 Polygalacturonase (PG) and pectin methyl esterase (PME) activity of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride during storage in 2013. Enzymes
Calcium chloride concentration (%)
Storage period (Week)
0
1
2
3
PG activity (U g−1 fw−1 )
0 1.5 2
0.95by Az 0.89bA 0.69cA
1.1bA 0.91bA 0.86cA
2.05aA 1.58aB 1.27bB
2.39aA 1.75aB 1.55aC
PME activity (U g−1 fw−1 )
0 1.5 2
0.14dA 0.13dAB 0.09cB
0.22cA 0.20cB 0.19bC
0.38bA 0.30bB 0.23bC
0.47aA 0.42aB 0.35aC
y
Small letters in rows show the mean comparison among weeks in storage. Means with the same letter are not significantly different at (p = 0.05) at DMRT. Capital letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different at (p = 0.05) at DMRT. z
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Fig. 1. Transmission-electron microscopy images of untreated pulp (A = at harvest, B = 3 weeks after harvest) and treated calcium (C = 1.5% calcium chloride, D = 2% calcium chloride after 3 weeks). Note thickened middle lamallae (ML) in C and D relative to the thin ML in A and no ML in B (arrows) Bar = 1 m.
synthesized and deposited on the cell wall are largely esterified (Staehelin and Moore, 1995; Chuni et al., 2010) the negative, charges generated by PME are necessary for Ca to bind onto the cell wall and delay cell wall disassembly. 3.4. Quality factors Fruit luminosity or brightness is greater in ripened fruit; loss of chlorophyll induces yellow and red colours due to the increase in carotenoids and other pigments (Yahia and Ornelas-Paz, 2009). The lightness value of papaya increased during ripening, from 59.9 at harvest to 65.2 after three weeks in storage in 2012 in control and from 59.7 to 67.4 in control in 2013 (Table 2). Also, increased calcium concentrations decreased values in both years. In 2012, at all weeks except for week two, there was a significant decrease in 1%, 1.5% and 2% calcium treatments relative to the control treatment. In 2013, at all weeks except for the second week there were significant differences between 1.5% and 2% calcium treatments compared with the control (Table 2). Chroma values indicate the degree of saturation or colour intensity. Chroma values increased from 46.8 at harvest to 51.2 after three weeks of storage in control fruits in 2012 and from 47.2 to 52.1 in 2013 (Table 2). Fruits treated with higher concentrations of calcium showed smaller increases in the chroma indicating that colour intensity of sprayed fruits was lower compared to the control. In both years, at all weeks except at harvest, decreases between 1.5% and 2% calcium treatments relative to control treatments were observed. However, in 2012 at harvest significant differences were
observed between 1%, 1.5% and 2% calcium treatments relative to the control treatment. The hue angle was reduced from 158.1 to 101.8 in 2012 and from 154.5 to 102.6 in 2013 for control (Table 2). Increased calcium concentrations resulted in higher hue angles after three weeks of storage but were not affected at harvest. The SSC of untreated fruits reached to 11.6% and 10.7% in 2012 and 2013, respectively. In 2012, lower SSC was observed at all weeks for 1%, 1.5% and 2% calcium treated fruit compared with control fruits. However, at harvest, the SSC in fruits from all treatments differed relative to control. In 2013, significant differences were detected between 1.5% and 2% calcium with the control fruits after week 1 (Table 2). Fruits from the higher calcium treatments resulted in higher TA compared to those of the control in 2012 (Table 2). In 2013, significant increases were found between 1.5% and 2% calcium compared with control at all weeks except for second week when no differences were found among treatments (Table 2). Collectively, calcium sprays delayed fruit ripening as assessed by colour changes, SSC and TA, and consistent with effects found for other fruits such as mango (Suntharalingam, 1996). Peel colour changes from green to orange-yellow during ripening in papaya fruit result from loss of chlorophyll and synthesis of carotenoids (Rohani, 1994), and is affected by ethylene action (Njoroje et al., 1998). Calcium treatments decrease TSS, probably due to slowing down respiration and as a result slower change from carbohydrates to sugars (Rohani et al., 1997). Similarly, citric acid, the primary organic acid in papaya, decreases during ripening (Rohani, 1994),
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Table 4 Disease severity and overall quality of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride after three weeks in storage in 2012 and 2013. Disease severity (%)
Overall quality (Scale)
2012 0 0.5 1 1.5 2
25.4az 14.6b 10.2bc 3.4cd 0.0d
2.5b 2.6b 3.1a 3.1a 3.2a
2013 0 1.5 2
20.4a 3.0b 0.0b
2.2b 3.4a 3.4a
Calcium chloride concentration (%)
z Small letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different at P = 0.05.
and presumably higher acidity in calcium treated fruits was related to due to delayed ripening and lower respiration rates (Goutam et al., 2010). 3.5. Disease severity and overall quality The lowest disease severity occurred in fruits from the 1.5% and 2% treatments (Table 4). Overall quality was higher with increasing calcium concentrations (Table 4). The 1%, 1.5% and 2.0% calcium treatments resulted in the maximum score in 2012, while maximum scores were found in the 1.5% and 2% calcium treatments in 2013 (Table 4). Lower disease severity in calcium treated fruit might be due to the fact that calcium delays ripening (Ferguson, 1984). Calcium decreases disease directly by reducing spore germination and indirectly by maintaining cell wall integrity (Ciccarese et al., 2013). Calcium deficiencies are associated with increased membrane permebility and ion leakage, and increased activity of enzymes such as PG and PME that accelerate ripening and softening and increases fungal attack. At the market level, only products that are acceptable by consumers are appropriate. Therefore, it is critical to estimate the effects of calcium treatments on overall quality of fruits and vegetables (Tzortzakis et al., 2007). Calcium treatments maintain overall quality of fruit by maintaining firmness and decreasing fungal spots. In conclusion, calcium application in the field appears to be a useful tool to decrease disease severity and maintain firmness of papaya fruits. Further studies are required to explore the effects of preharvest applications of calcium on quality and storage life of papaya in cultivars and different growing conditions. References Agusti, M., Juan, M., Martinez-Fuentes, A., Mesejo, C., Almela, V., 2004. Calcium nitrate delays climacteric of persimmon fruit. Ann. Appl. Biol. 144, 65–69. Ali, A., Mahmud, T.M.M., Sijam, K., Siddiqui, Y., 2011. Effect of chitosan coatings on the physico-chemical characteristics of Eksotika II papaya (Carica papaya L) fruit during cold storage. Food Chem. 124, 620–626. Ali, A., Ong, M.K., Forney, C.F., 2014. Effect of ozone pre-conditioning on quality and antioxidant capacity of papaya fruit during ambient storage. Food Chem. 142, 19–26. Ali, Z.M., Chin, L.H., Lazan, H., 2004. A comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruits. Plant Sci. 167, 317–327. Basir, T.G., 2005. Land and Agricultural Policy. Malaysian Palm Oil Board, Malaysia. Cheour, F., Willemot, C., Arul, J., Makhlouf, J., Desjardins, Y., 1991. Postharvest response of two strawberry cultivars to foliar application of CaCl2 . HortScience 26, 1186–1188. Chuni, S.H., Awang, Y., Mohamed, M.T.M., 2010. Cell wall enzymes activities and quality of calcium treated fresh-cut red flesh dragon fruit (Hylocereus polyrhizus). Int. J. Agric. Biol. 12, 713–718. Ciccarese, A., Stellacci, M., Gentilesco, G., Rubino, P., 2013. Effectiveness of pre- and post-veraison calcium applications to control decay and
maintain table grape fruit quality during storage. Postharvest Biol. Technol. 75, 135–141. De Assis, S.A., Lima, D.C., De Faria Oliveira, O.M.M., 2001. Activity of pectin methylesterase, pectin content and vitamin C in acerola fruit at various stages of fruit development. Food Chem. 74, 133–137. Dey, P.M., Brinson, K., 1984. Plant cell walls. Adv. Carbohydr. Chem. Biochem. 42, 265–382. Ferguson, I.B., 1984. Calcium in plant senescence and fruit ripening. Plant Cell. Environ. 7, 477–489. Ferguson, I.B., Watkins, C.B., 1989. Bitter pit in apple fruit. Hortic. Rev. 11, 289–355. Gamagae, S.U., Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C., 2003. Use of sodium bicarbonate and Candida oleophila to control anthracnose in papaya during storage. Crop Prot. 22, 775–779. Goutam, M., Dhaliwal, H.S., Mahajan, B.V., 2010. Effect of preharvest calcium sprays on post-harvest life of winter guava (Psidium guajava L). J. Food Sci. Technol. 47, 501–506. Gupta, O.P., Singh, B.P., Singh, S.P., Chauhan, K.S., 1984. Effect of calcium compounds as preharvest spray on the shelf-life of peach cv. ‘Sharbati’ Punjab Hort. J. 24, 105–110. Hagerman, A.E., Austin, P.J., 1986. Continuous spectrophotometric assay for plant pectin methyl-esterase. J. Agric. Food Chem. 34, 440–444. Hewajulige, I.G.N., Wijeratnam, S.W., Wijesundera, R.L.C., 2006. Preharvest application of calcium to control black heart disorder in Mauritius pineapples during low-temperature storage. J. Sci. Food Agric. 86, 420– 424. Lazan, H., Ali, Z.M., Liang, K.S., Yee, K.L., 1989. Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment. Physiol. Plant. 77, 93–98. Lohani, S., Trivedi, P.K., Nath, P., 2004. Changes in activities of cell wall hydrolases during ethylene-induced ripening in banana: effect of 1-MCP ABA and IAA. Postharvest Biol. Technol. 31, 119–126. Madani, B., Mahmud, T.M.M., Awang, Y., Kadir, J., Patil, V.D., 2013. Effects of calcium treatment applied around the root zone on nutrient concentration and morphological traits of papaya seedlings (Carica papaya L. cv. Eksotika II). Austal. J. Crop Sci. 5, 568–572. Manganaris, G.A., Vasilakakis, M., Mignani, I., Diamantidis, G., Tzavella-Klonari, K., 2005. The effect of preharvest calcium sprays on quality attributes physicchemical aspects of cell wall components and susceptibility to brown rot of peach fruit. Sci. Hortic. 107, 43–50. Njoroje, C.K., Kerbel, E.L., Briskin, D.P., 1998. Effect of calcium and calmodulin antagonists on ethylene biosynthesis in tomato fruits. J. Sci. Food Agric. 76, 209–214. Oms-Oliu, G., Rojas-Graü, M.A., González, L.A., Varela, P., Soliva-Fortuny, R., Hernando, M.I.H., Munuera, I.P., Fiszman, S., Martín-Belloso, O., 2010. Recent approaches using chemical treatments to preserve quality of fresh-cut fruit: a review. Postharvest Biol. Technol. 57, 139–148. Pathak, N., Sanwal, G.G., 1998. Multiple forms of polygalacturonase from banana fruits. Phytochemistry 48, 249–255. Paull, R.E., Duarte, O., 2011. Tropical Fruits. CABI Publisher, Wallingford, pp. 400. Qiu, Y., Nishina, M.S., Paull, R.E., 1995. Papaya fruit growth, calcium uptake, and fruit ripening. J. Am. Soc. Hortic. Sci. 120, 246–253. Ramakrishna, M., Haribabu, K., Reddy, Y.N., Purushotham, K., 2001. Effect of preharvest application of calcium on physicochemical changes during ripening and storage of papaya. Indian J. Hortic. 58, 228–231. Ramezanian, A., Rahemi, M., Vazifehshenas, M.R., 2009. Effects of foliar application of calcium chloride and urea on quantitative and qualitative characteristics of pomegranate fruits. Sci. Hortic. 121, 171–175. Recasens, I., Benavides, A., Puy, J., Casero, T., 2004. Preharvest calcium treatments in relation to the respiration rate and ethylene production of ‘Golden Smoothee’ apples. J. Sci. Food Agric. 84, 765–771. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212. Rohani, M., 1994. Papaya Fruit Development, Postharvest Physiology, Handling and Marketing in ASEAN. ASEAN Food Handling Bureau, Malaysia, pp. 144. Rohani, M.Y., Zaipun, M.Z., Norhayati, M., 1997. Effect of modified atmosphere on the storage life and quality of Eksotika papaya. J. Trop. Agric. Food Sci. 25, 103–113. Saltveit, M.E., 1982. Procedure for extracting and analyzing internal gas samples from plant tissue by gas chromatography. HortScience 17, 878–881. Sams, C.E., Conway, W.S., 1984. Effect of calcium infiltration on ethylene production, respiration rate, soluble polyuronide content, and quality of ‘Golden Delicious’ apple fruit. J. Am. Soc. Hortic. Sci. 109, 53–57. Saure, M.C., 2005. Calcium translocation to fleshy fruit: its mechanism and endogenous control. Sci. Hortic. 105, 65–89. Shukor, A.R.A., Shokri, A.O., 1997. Respiratory activity and compositional changes in Eksotika papaya fruit following storage in low oxygen atmosphere. J. Trop. Agric. Food Sci. 25, 85–93. Singh, B.P., Tandon, D.K., Kalra, S.K., 1993. Change in post-harvest quality of mangoes affected by preharvest application of calcium salts. Sci. Hortic. 54, 211– 219. Staehelin, L.A., Moore, I., 1995. The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 261–268.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Preharvest calcium chloride sprays affect ripening of Eksotika II’papaya fruits during cold storage Babak Madani a,∗ , Mahmud Tengku Muda Mohamed a,∗∗ , Christopher B. Watkins b , Jugah Kadir c , Yahya Awang a , Taha Roodbar Shojaei d a
Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Horticulture, College of Agriculture and Life Science, Cornell University, Ithaca, NY 14853, USA c Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia d Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b
a r t i c l e
i n f o
Article history: Received 25 December 2013 Received in revised form 15 March 2014 Accepted 20 March 2014 Available online 12 April 2014 Keywords: Papaya Preharvest spray Calcium chloride Physicochemical
a b s t r a c t The effects of preharvest application of calcium chloride on ripening, activity patterns of pectin modifying enzymes and overall quality of papaya (Carica papaya L. cv. ‘Eksotika II’) fruits have been investigated. Foliar sprays of 0, 0.5%, 1%, 1.5% and 2% (w/v) calcium chloride were applied six times during the growing season. After harvest, fruits were stored at 12 ◦ C for up to three weeks. Higher calcium concentrations in the sprays coincided with increasing calcium concentrations in peel and pulp tissues, higher firmness and titratable acidity, and reduced respiration rate, ethylene production and soluble solids concentrations, compared with those of the untreated control fruits. The overall quality of fruits treated with calcium was greater than the control fruits. Also, fruits sprayed with calcium had decreased activities of polygalacturonase (PG) and pectin methyl esterase (PME) during storage. Microscopic results confirmed that the middle lamallae of cell walls was more intact in calcium chloride-treated fruits. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Papaya is an important export product for Malaysia (Ali et al., 2014) and new cultivar development is ongoing. The world trade of fruit is increasing, but marketing is limited due to improper handling, transport and postharvest diseases, that cause low quality and storage life. ‘Eksotika II’, released by Malaysian Agricultural Research and Development Institute, and has gained popularity in the local and export markets due to high yield and good quality. However, fruits soften rapidly and are susceptible to disease (Shukor and Shokri, 1997; Madani et al., 2013). One of the most economically significant postharvest pathogens of papaya is anthracnose that is caused by Colletotrichum gloeosporioides Penz (Gamagae et al., 2003). Postharvest diseases are mainly controlled by using synthetic chemical fungicides, but consumer concern about pesticide residues on foods, as well as pathogen resistance
∗ Corresponding author at: Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 123338642. ∗∗ Corresponding author at: Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 389474823. E-mail addresses: [email protected], [email protected] (B. Madani), [email protected] (M.T.M. Mohamed). http://dx.doi.org/10.1016/j.scienta.2014.03.032 0304-4238/© 2014 Elsevier B.V. All rights reserved.
to many currently registered pesticides, has increased. Alternative methods for decay control has become more important, including improvement of fruit resistance to pathogens before harvest. It is known that one approach is the use of calcium salts. Preharvest application of calcium salts can control or reduce certain physiological disorders, reduce the incidence of fungal pathogens, maintain fruit firmness and reduce mechanical damage in many fruit types (Ferguson and Watkins, 1989; Cheour et al., 1991; Oms-Oliu et al., 2010; Ciccarese et al., 2013). Most research on calcium sprays has been centred on temperate fruits such as apple, and relatively less is known for tropical and subtropical fruits. Available studies, however, show positive responses. For example, in pineapple, calcium sprayed fruits had higher calcium concentrations, and decreased black heart symptoms (Hewajulige et al., 2006). Calcium sprayed guava and pomegranate fruits had higher ascorbic acid concentrations (Ramezanian et al., 2009; Goutam et al., 2010), lower respiration rates, better appearance and marketability in mango (Singh et al., 1993). In papaya, Ramakrishna et al. (2001) reported that preharvest application of calcium chloride and calcium nitrate reduced weight loss, enhanced firmness and increased titratable acidity during storage at ambient temperatures. Softening of climacteric fruits is mainly related with changes in pectin fraction in cell wall, and large increase in the pectin
B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
solubilization correlated with softening (Lohani et al., 2004). In papaya, softening is associated with higher activities of polygalacturonase and pectin methyl esterase (Ali et al., 2004), which are synthesized de novo during ripening (Manganaris et al., 2005). Calcium is thought to maintain cell wall structure by interacting with pectin in the cell wall to form calcium pectate complexes, which support bonding between components of the cell wall and prevent the action of enzymes associated with cell wall disassembly. While preharvest calcium treatment of peaches reduced activities of PG and PME (Manganaris et al., 2005), little is known about the effects of calcium on these enzymes in tropical fruits such as papaya. The objective of the current study was to investigate the effects of a range of calcium concentrations applied before harvest on postharvest quality and activities of associated cell wall degrading enzymes activities of papaya fruits during cold storage.
2. Materials and methods 2.1. Plant materials Papaya trees (Carica papaya L. cv. Eksotika II) growing at the Agro-Tech Unit, University Agriculture Park (TPU), Universiti Putra Malaysia, Serdang, Selangor (3◦ 00 21.34 N, 101◦ 42 15.06 E, 37 m elevation) were used in both 2012 and 2013. Forty and twenty four eight month old trees, approximately 2.2 m tall, were used for experiments in 2012 and 2013, respectively. The experiments were conducted in a randomized complete block design with four blocks including two plants in each 3 m × 3 m block. Commercial fertilization (12:12:17:2 N:P:K:Mg) were utilized monthly around the canopy periphery for all treatments (Basir, 2005). Irrigation was carried out with overhead sprinklers at approximately 4 day intervals.
2.2. Treatments Calcium chloride (CaCl2 ·2H2 O (99% CC, SYSTERM® , Malaysia) at concentrations of (0, 0.5, 1.0, 1.5 and 2.0% (w/v)) were sprayed to the fruits and leaves (approximately 1.5 L per tree (with a Knapsack Sprayer). Sprays were applied every two weeks for six times from 21 days after flower anthesis until 10 days before harvest. In 2013, 0, 1.5% and 2% calcium was sprayed on trees till 2 days before fruit harvest. In all other respects the 2013 trial was performed using the same procedures as in the previous season. In both years, 64 fruits in each treatment with uniform size (500–550 g) and shape were harvested at ripening index 2 (green with trace of yellow), washed with water and allowed to air dry before randomly divided into five different lots for 2012 and three lots for 2013. Fruit samples for each treatment were taken at harvest and during storage. Fruits were packed in commercial boxes (EXOTIC STAR® , Kajang, Selangor, Malaysia) and stored at 12 ± 2 ◦ C and 85–90% relative humidity for up to three weeks.
2.3. Determination of calcium concentration in fruit Approximately 20 g of peel and pulp samples were taken from two fruits from each of four blocks per treatment. The samples were dried at 70 ◦ C in an air-circulating oven. Dried peel and pulp (0.25 g) were digested in 5 mL 98% H2 SO4 on hot plate at 285 ◦ C, and 2 mL 50% H2 O2 added to the mixture. The solutions were made up to 100 mL with distilled water. Calcium concentration was measured using an atomic absorption spectrophotometer (Perkin–Elmer, Model AAS 3110, Palo Alto, California, USA), and results were expressed as percentages.
7
2.4. Respiration rate and ethylene production Respiration rate and ethylene production were measured using method described by Saltveit (1982). For each treatment eight fruits were used to measure ethylene production and respiration rate. The weight and volume of each single fruit was measured and kept in 1.9 L airtight container for 2 h in storage (12 ± 2 ◦ C). One mL of accumulated gases in the sealed container was injected into the injector port of gas chromatograph (Clarus-500, Perkin–Elmer, Shelton, CT, USA) to determine CO2 and C2 H4 concentrations. Instruments were adjusted by certified standards. The amount of C2 H4 and CO2 was expressed by L kg−1 h−1 and mL kg−1 h−1 , respectively. 2.5. Fruit quality factors Titratable acidity (TA) and soluble solids concentration (SSC) of 10 g pulp tissues were measured by the methods of Ali et al. (2011). TA was determined by homogenizing 10 g pulp tissues using a kitchen blender (MX-799S, Panasonic, Shah Alam, Malaysia) with 40 mL of distilled water, and filtering through cotton wool. Then, 1.5 mL of filtrate with two drops of phenolphthalein (0.1%) as indicator, was titrated with 0.1 N NaOH to an endpoint pink (pH 8.1). Results are expressed as percentage of citric acid per 100 g fresh weight. The SSC of the pulp was determined by a hand refractometer (N3000E, Atago Co., Tokyo, Japan) from the filtrate prepared from TA determination. The reading was corrected for the dilution resulting from blending (factor of five). Results are expressed % SSC. Two fruits per block per week arranged in randomized complete block design were used for TA and SSC measurement. Flesh firmness was measured by Instron Universal Testing Machine (Model 5540, USA) and results expressed in Newtons (N). Peel colour of fruits was measured by Minolta (CR-300, Minolta Corp, Osaka, Japan) colourimeter and expressed as L* , C* and h◦ . Two fruits per block in each week arranged in randomized complete block design were used for firmness and colour measurements. 2.6. Microscopy Cubes of flesh (1.5 mm3 ) from mid-region of each fruit were fixed in 4% glutaraldehyde at 4 ◦ C for 24 h. Samples were washed with 0.1 mol L−1 sodium cacodylate buffer (pH 7.6) for three times, and post fixed in 1% (w/v) osmium tetraoxide for 2 h. The fixed samples were then rinsed again in 0.1 mol L−1 sodium cacodylate buffer (pH 7.6), and dehydrated in graded series of acetone at (35–100%). Subsequently, dehydrated samples were embedded in beam capsules and polymerized at 60 ◦ C for 2 days. Ultra–slim sections (60–90 nm) of samples were cut and mounted onto copper grids. Sections were stained with lead citrate and saturated uranyl acetate (Reynolds, 1963) and investigated using transmission electron micrscope (TEM) (Hitachi, H-7100, Tokyo, Japan). 2.7. Extraction and assay of cell wall associated enzyme activities The procedure for extraction of polygalacturonase and pectin methyl esterase activity was performed using methods described by Lazan et al. (1989) and Ali et al. (2004) with some modification. Ten grams of pulp tissues were homogenized in a blender for 2 min with 20 mL of cold 0.1 mol L−1 sodium citrate, pH 4.6 buffer (containing 1 mol L−1 NaCl, 13 mmol L−1 EDTA, 10 mmol L−1 -mercaptoethanol, and 1% (w/v) polyvinylpyrrolidone). Afterwards, the mixture was incubated at 4 ◦ C for 60 min with stirring. The supernatant was subsequently recovered by centrifugation at 15,000 × g for 30 min at 4 ◦ C in a refrigerated centrifuge (Scan speed 1730R, Scala Scientific, Netherlands). The clear supernatant (crude enzyme extract) was used to determine enzyme activity.
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Polygalacturonase (PG) activity was assayed by the method described by Pathak and Sanwal (1998) and Lohani et al. (2004) with slight modification. The reaction mixture contained 0.4 mL sodium acetate (200 mmol L−1 , pH 4.5) buffer, 0.1 mL NaCl (200 mmol L−1 ), 0.4 mL polygalacturonic acid (PGA 1% aqueous solution adjusted to pH 4.5) and 0.1 mL of enzyme extract in a total volume of 1.0 mL. The control comprised the same components but with the enzyme extract boiled for 5 min. The reaction was initiated by the addition of the PGA substrate. The mixture was incubated at 37 ◦ C for 1 h and 0.1 mL 3, 5-dinitrosalicylate (DNS) reagent added. The reaction mixture was heated in boiling water for 5 min. When the mixture reached to room temperature (25 ± 2 ◦ C) the absorbance was measured at 450 nm using a spectrophotometer (WPA, Biochrom, Cambridge, England). The formation of reducing groups was estimated against d-galacturonic acid as a standard. One unit of enzyme activity is defined as the amount of enzyme required to liberate 1 nM of galactronic acid per min per g of original fresh weight of flesh. Pectin methyl esterase (PME) activity was measured according to the method described by Hagerman and Austin (1986) and Lohani et al. (2004). The reaction mixture was prepared in a 3 mL glass cuvette and was composed of 1 mL pectin solution (0.01% aqueous solution adjusted to pH 7.5 using 0.1 mol L−1 NaOH, 0.2 mL NaCl (0.15 mol L−1 ), 0.1 mL bromothymol blue solution (0.01%), 0.2 mL sterilized water and 0.1 mL crude enzyme extract. After adding the enzyme prepared, the cuvette was shaken gently. The absorbance of the reaction mixture was measured immediately at 620 nm using a spectrophotometer (WPA, Biochrom, Cambridge, England). The absorbance was again measured after 3 min. The difference in absorbance between 0 and 3 min was the measure of PME activity. Calculation of the activity was carried out against a standard curve of galacturonic acid (Hagerman and Austin, 1986). One unit is defined as the amount of the enzyme required to release 1 mol of methyl ester per min per g of original fresh weight of flesh. For enzyme activities, two fruits per block in each week arranged in a randomized complete block design with two factors (weeks in storage and calcium concentrations), were used. 2.8. Determination of disease severity and overall quality Disease severity was measured after three weeks in storage as surface of fruit with anthracnose disease and expressed by percentage. Six fruits per blocks arranged in randomized complete block design were used for disease severity measurement. To investigate overall quality, a panel of six untrained judges were selected. Overall quality was performed utilizing the Hedonic scale by allotting the number from 0 to 5, where 0 for very poor and 5 for excellent and measured after three weeks. 2.9. Statistical analysis Data were subjected to Analysis of Variance (ANOVA) using the Statistical Analysis System (SAS) version 8.2 (SAS Institute Inc., Cary, NC, USA). The means were compared by the Duncan’s Multiple Range Test (DMRT) at significance level of 0.05.
3. Results and discussion Preharvest calcium sprays have been shown to delay ripening and senescence processes and inhibit the development of physiological and pathological disorders (Ferguson and Watkins, 1989; Cheour et al., 1991; Oms-Oliu et al., 2010; Ciccarese et al., 2013), but most research has been focused on temperate crops. Here we
show that these sprays can have dramatic effects on a tropical fruit, the papaya. 3.1. Calcium concentration in fruit In this study, calcium was sprayed six times throughout the growing season. In 2012, preharvest calcium increased calcium concentration in peel (77–129%) and pulp (30%) of fruits sprayed with 1.5% and 2% calcium relative to that in control fruits at harvest (Table 1). In 2013, differences in calcium were detected for 1.5% and 2% calcium for calcium concentration in peel and pulp compared with control after week 1. No differences were detected between 1.5% and control in peel and pulp tissues at the first storage sampling (Table 1). Because the results for 1.5% and 2% calcium sprays were similar, we did not use higher amounts of calcium in 2013. Also, because no symptoms of leaf and fruit damage from calcium applications were detected at any calcium concentration, we used the lower and more economical rate. Interestingly, Qiu et al. (1995) found that calcium sprays did not increase calcium concentrations in papaya flesh. Environmental factors, presence of stomata or other irregular surfaces such as cracks, and genetically differences in the cultivars can affect calcium absorption in fruits (Saure, 2005). 3.2. Ethylene production and respiration rate Differences in ethylene production between fruits treated with 1.5% and 2% calcium compared with those of the control were detected at harvest in 2012 (Table 1). However, no differences were found among preharvest calcium concentrations after a week of storage. After three weeks differences were detected, though not between the control and 0.5% calcium treatments. In 2013, differences were found between 1.5% and 2% calcium compared with control treatments at each week of storage (Table 1). The respiration rate of fruits was lower only in 1.5% and 2% calcium treatments relative to control treatments at harvest time and after one week of storage in 2012 (Table 1). However, at the second and third weeks of storage there were differences between 1%, 1.5% and 2% calcium treated fruit compared with untreated fruit. In 2013, differences found between 1.5% and 2% calcium relative to control for all weeks with the exception of week two when no differences between the 1.5% calcium treatment and the control were detected (Table 1). Papaya is characterized by rapid softening once ripening has been initiated, reflecting behaviour of a typical climateric fruit (Paull and Duarte, 2011). Calcium is hypothesized to delay ripening by reducing disintegration of tissues and maintaining membrane integrity (Sams and Conway, 1984; Torre et al., 1999). It has also been proposed that calcium delays ethylene production by preventing solubilization of calcium binding sites in cell walls which activate the ethylene generation system located in cell wall plasma membrane complex (Agusti et al., 2004). 3.3. Firmness, cell wall integrity, and activity of cell wall enzymes Flesh firmness is the most important parameter in papaya fruits during storage and marketing, because flesh softening is associated with senescence and increased susceptibility of fruits to injury during handling. Fruits sprayed with 1%, 1.5% and 2% calcium were firmer than those of the untreated fruits at harvest and at weeks two and three in 2012 (Table 1). All fruits softened markedly between weeks two and three, but all calcium treatments above 0.5% resulted in firmer fruits than the untreated controls. In 2013, differences were found between 1.5% and 2% calcium treatments relative to the control treatment with the exception of week two when the 1.5% calcium treatment did not show any differences with control (Table 1). Softening of fruits is associated with dissolution
B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
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Table 1 Calcium concentration, ethylene production, respiration rate, and pulp firmness of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride in storage period in 2012 and 2013. Quality parameters
Calcium chloride concentration (%)
Storage period (Week)
0
1
2
3
2012
Calcium in peel (%)
0 0.5 1 1.5 2
1.14dz 1.40 cd 1.72bc 2.02b 2.61a
1.43c 1.49bc 1.50bc 1.76ab 1.98a
1.22c 1.30bc 1.37bc 1.58ab 1.82a
1.13c 1.41c 1.52bc 1.85b 2.39a
Calcium in pulp (%)
0 0.5 1 1.5 2
0.55b 0.58ab 0.61ab 0.71a 0.72a
0.51b 0.58ab 0.67a 0.69a 0.69a
0.51b 0.61a 0.65a 0.68a 0.69a
0.61b 0.78b 1.18a 1.19a 1.12a
0 0.5 1 1.5 2
0.8a 0.8ab 0.7ab 0.6b 0.6b
3.5a 3.4a 3.3a 3.0a 3.0a
7.8a 7.5a 5.5b 5.4b 5.3b
11.1a 11.0a 5.4b 3.5c 3.4c
0 0.5 1 1.5 2
15.9a 15.1ab 14.4ab 14.1b 11.9c
17.3a 17.2a 16.5a 14.2b 13.9b
20.1a 19.4ab 17.7bc 17.1c 17.0c
23.1a 21.8a 17.7b 17.1b 17.0b
0 0.5 1 1.5 2
34.0c 38.0c 46.1b 53.1a 54.1a
25.1b 27.8ab 30.2ab 32.1ab 33.8a
16.7c 19.2bc 23.3ab 25.3a 26.9a
3.8c 5.7bc 7.6ab 8.5a 8.4a
−1
Ethylene (L kg
−1
h
−1
Respiration (mL kg
)
−1
h
)
Firmness (N)
2013 Calcium in peel (%)
0 1.5 2
1.66c 2.41b 2.96a
1.25b 1.79ab 2.20a
1.15b 1.57a 1.86a
1.03c 1.82b 2.34a
Calcium in pulp (%)
0 1.5 2
0.51b 0.66a 0.67a
0.57b 0.65ab 0.68a
0.60b 0.80a 0.86a
0.62b 1.08a 1.07a
Ethylene(L kg−1 h−1 )
0 1.5 2
5.4a 4.3b 3.9b
6.3a 5.0b 4.6b
9.5a 5.7b 5.2b
12.4a 6.1b 5.9b
Respiration (mL kg−1 h−1 )
0 1.5 2
16.4a 12.1b 11.6b
19.4a 13.9b 12.3b
22.7a 18.5ab 17.7b
26.4a 18.8b 17.7b
Firmness (N)
0 1.5 2
36.2b 52.0a 56.0a
26.6b 37.3a 35.3a
16.9b 21.3ab 24.1a
3.2b 8.6a 8.9a
z
Small letters in columns show the mean comparison among concentrations of calcium chloride. Means with the same letter are not significantly different at P = 0.05.
of the middle lamallae with turnover in the composition, structure and linkages between polysaccharides (Vicente et al., 2007). The effects of calcium are presumably mediated by reduced ethylene production and respiration rate (Recasens et al., 2004). However, maintenance of firmness is also a result of direct interaction of calcium with the cell walls, where it binds to pectins and forms bridges between pectic acids. Calcium also stabilizes pectin–protein complexes in the middle lamallae thereby acting as an intermolecular binding factor (Dey and Brinson, 1984), and may thicken the middle lammelae due to increased calcium pectate deposition (Gupta et al., 1984). TEM images confirmed that the middle lamallae of calciumtreated fruits was maintained more intact than that of untreated fruits. The middle lamallae in untreated fruits had completely disappeared after three weeks in storage compared with that in fruits on the day of harvest (Fig. 1A and B), while in both 1.5% and 2% calcium, middle lamallae had been preserved after three weeks of storage (Fig. 1C and D).
Disassembly of the cell wall and middle lamellae of papaya is associated with increased activities of PG and PME (Ali et al., 2004). In the current study, PG activity decreased in fruits from the 1.5 and 2% calcium treatments at weeks two and three compared with the control treatment (Table 3). PME activity decreased from week one to three in the 1.5 and 2% calcium-treated fruits compared with control. PG activity increased progressively in all calcium treated fruits in second and third week of storage compared with at harvest and the first week of storage, while PME increased significantly in all concentrations of calcium in all weeks of storage relative to harvest (Table 3). Fruits with higher calcium concentrations have higher level of calcium pectate that maintains tissue resistant to degradation by PG (Agusti et al., 2004). Calcium ions can form salt-bridge cross-links with carboxyl groups of the pectin, thereby making the cell wall less accessible to the enzymes responsible for softening. PG only hydrolyses homogalacturonan regions when uronic acid residues have been previously de-methylated by PME (De Assis et al., 2001; Chuni et al., 2010). Since pectins are
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B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
Table 2 Colour changes, soluble solids concentrations, and titratable acidity of ‘Ekzotika II’ papaya fruit in relation to pre-harvest sprays of calcium chloride in storage period in 2012 and 2013. Quality parameters
Calcium chloride concentration (%)
Storage period (Weeks)
0
1
2
3
2012
*
0 0.5 1 1.5 2
59.9az 58.7a 54.2b 53.0b 52.1b
61.3a 58.8a 53.4b 51.1b 51.1b
63.2a 62.2a 60.0ab 56.7bc 55.7c
65.2a 64.2ab 63.3b 62.8b 62.8b
Chroma (C )
0 0.5 1 1.5 2
46.8a 45.8a 42.3b 40.6b 40.1b
47.4a 44.4ab 43.6ab 41.9b 41.7b
48.3a 48.2a 47.6a 43.0b 41.6b
51.2a 49.9a 49.5ab 48.0bc 46.9c
Hue angle (h )
0 0.5 1 1.5 2
158.1a 159.4a 160.3a 161.5a 163.3a
142.8b 143.5b 144.1b 146.5ab 149.6a
Titratable acidity (%)
0 0.5 1 1.5 2
0.29b 0.30b 0.31ab 0.31ab 0.35a
0.25c 0.26c 0.27bc 0.31ab 0.33a
0.17c 0.18bc 0.21abc 0.23ab 0.24a
Soluble solid concentration (%)
0 0.5 1 1.5 2
6.8a 6.4b 6.1c 6.1c 5.5d
8.3a 8.1ab 7.1b 6.1c 5.6d
9.4a 9.2a 7.8b 7.5b 7.4b
11.6a 11.5a 9.5b 7.5c 7.4c
Lightness (L* )
0 1.5 2
59.7a 53.7b 53.6b
60.1a 55.7ab 54.2b
65.9a 55.3b 53.0b
67.4a 62.6b 62.0b
Chroma (C* )
0 1.5 2
47.2a 41.8b 41.2b
48.4a 43.8b 42.8b
50.6a 43.8b 43.3b
52.1a 47.1b 46.7b
Hue angle (h◦ )
0 1.5 2
154.5a 160.2a 160.4a
146.0a 150.1a 152.2a
110.7b 131.3a 141.1a
102.6b 145.8a 140.2a
Titratable acidity (%)
0 1.5 2
0.28b 0.39a 0.43a
0.20b 0.33a 0.34a
0.26a 0.29a 0.30a
Soluble solid concentration (%)
0 1.5 2
6.6a 6.1b 5.4c
7.3a 6.3ab 6.1b
7.9a 6.1b 6.1b
Lightness (L )
*
◦
115.1b 120.0ab 120.1ab 122.5a 124.2a
101.8c 128.2b 147.5a 148.0a 145.0a 0.11c 0.14bc 0.15b 0.20a 0.20a
2013
0.23b 0.31a 0.33a 10.7a 4.3b 4.2b
z Small letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different according to the Multiple Range Test at P = 0.05.
Table 3 Polygalacturonase (PG) and pectin methyl esterase (PME) activity of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride during storage in 2013. Enzymes
Calcium chloride concentration (%)
Storage period (Week)
0
1
2
3
PG activity (U g−1 fw−1 )
0 1.5 2
0.95by Az 0.89bA 0.69cA
1.1bA 0.91bA 0.86cA
2.05aA 1.58aB 1.27bB
2.39aA 1.75aB 1.55aC
PME activity (U g−1 fw−1 )
0 1.5 2
0.14dA 0.13dAB 0.09cB
0.22cA 0.20cB 0.19bC
0.38bA 0.30bB 0.23bC
0.47aA 0.42aB 0.35aC
y
Small letters in rows show the mean comparison among weeks in storage. Means with the same letter are not significantly different at (p = 0.05) at DMRT. Capital letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different at (p = 0.05) at DMRT. z
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Fig. 1. Transmission-electron microscopy images of untreated pulp (A = at harvest, B = 3 weeks after harvest) and treated calcium (C = 1.5% calcium chloride, D = 2% calcium chloride after 3 weeks). Note thickened middle lamallae (ML) in C and D relative to the thin ML in A and no ML in B (arrows) Bar = 1 m.
synthesized and deposited on the cell wall are largely esterified (Staehelin and Moore, 1995; Chuni et al., 2010) the negative, charges generated by PME are necessary for Ca to bind onto the cell wall and delay cell wall disassembly. 3.4. Quality factors Fruit luminosity or brightness is greater in ripened fruit; loss of chlorophyll induces yellow and red colours due to the increase in carotenoids and other pigments (Yahia and Ornelas-Paz, 2009). The lightness value of papaya increased during ripening, from 59.9 at harvest to 65.2 after three weeks in storage in 2012 in control and from 59.7 to 67.4 in control in 2013 (Table 2). Also, increased calcium concentrations decreased values in both years. In 2012, at all weeks except for week two, there was a significant decrease in 1%, 1.5% and 2% calcium treatments relative to the control treatment. In 2013, at all weeks except for the second week there were significant differences between 1.5% and 2% calcium treatments compared with the control (Table 2). Chroma values indicate the degree of saturation or colour intensity. Chroma values increased from 46.8 at harvest to 51.2 after three weeks of storage in control fruits in 2012 and from 47.2 to 52.1 in 2013 (Table 2). Fruits treated with higher concentrations of calcium showed smaller increases in the chroma indicating that colour intensity of sprayed fruits was lower compared to the control. In both years, at all weeks except at harvest, decreases between 1.5% and 2% calcium treatments relative to control treatments were observed. However, in 2012 at harvest significant differences were
observed between 1%, 1.5% and 2% calcium treatments relative to the control treatment. The hue angle was reduced from 158.1 to 101.8 in 2012 and from 154.5 to 102.6 in 2013 for control (Table 2). Increased calcium concentrations resulted in higher hue angles after three weeks of storage but were not affected at harvest. The SSC of untreated fruits reached to 11.6% and 10.7% in 2012 and 2013, respectively. In 2012, lower SSC was observed at all weeks for 1%, 1.5% and 2% calcium treated fruit compared with control fruits. However, at harvest, the SSC in fruits from all treatments differed relative to control. In 2013, significant differences were detected between 1.5% and 2% calcium with the control fruits after week 1 (Table 2). Fruits from the higher calcium treatments resulted in higher TA compared to those of the control in 2012 (Table 2). In 2013, significant increases were found between 1.5% and 2% calcium compared with control at all weeks except for second week when no differences were found among treatments (Table 2). Collectively, calcium sprays delayed fruit ripening as assessed by colour changes, SSC and TA, and consistent with effects found for other fruits such as mango (Suntharalingam, 1996). Peel colour changes from green to orange-yellow during ripening in papaya fruit result from loss of chlorophyll and synthesis of carotenoids (Rohani, 1994), and is affected by ethylene action (Njoroje et al., 1998). Calcium treatments decrease TSS, probably due to slowing down respiration and as a result slower change from carbohydrates to sugars (Rohani et al., 1997). Similarly, citric acid, the primary organic acid in papaya, decreases during ripening (Rohani, 1994),
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B. Madani et al. / Scientia Horticulturae 171 (2014) 6–13
Table 4 Disease severity and overall quality of ‘Ekzotika II’ papaya fruit in relation to preharvest sprays of calcium chloride after three weeks in storage in 2012 and 2013. Disease severity (%)
Overall quality (Scale)
2012 0 0.5 1 1.5 2
25.4az 14.6b 10.2bc 3.4cd 0.0d
2.5b 2.6b 3.1a 3.1a 3.2a
2013 0 1.5 2
20.4a 3.0b 0.0b
2.2b 3.4a 3.4a
Calcium chloride concentration (%)
z Small letters in columns show the mean comparison among calcium chloride concentrations. Means with the same letter are not significantly different at P = 0.05.
and presumably higher acidity in calcium treated fruits was related to due to delayed ripening and lower respiration rates (Goutam et al., 2010). 3.5. Disease severity and overall quality The lowest disease severity occurred in fruits from the 1.5% and 2% treatments (Table 4). Overall quality was higher with increasing calcium concentrations (Table 4). The 1%, 1.5% and 2.0% calcium treatments resulted in the maximum score in 2012, while maximum scores were found in the 1.5% and 2% calcium treatments in 2013 (Table 4). Lower disease severity in calcium treated fruit might be due to the fact that calcium delays ripening (Ferguson, 1984). Calcium decreases disease directly by reducing spore germination and indirectly by maintaining cell wall integrity (Ciccarese et al., 2013). Calcium deficiencies are associated with increased membrane permebility and ion leakage, and increased activity of enzymes such as PG and PME that accelerate ripening and softening and increases fungal attack. At the market level, only products that are acceptable by consumers are appropriate. Therefore, it is critical to estimate the effects of calcium treatments on overall quality of fruits and vegetables (Tzortzakis et al., 2007). Calcium treatments maintain overall quality of fruit by maintaining firmness and decreasing fungal spots. In conclusion, calcium application in the field appears to be a useful tool to decrease disease severity and maintain firmness of papaya fruits. Further studies are required to explore the effects of preharvest applications of calcium on quality and storage life of papaya in cultivars and different growing conditions. References Agusti, M., Juan, M., Martinez-Fuentes, A., Mesejo, C., Almela, V., 2004. Calcium nitrate delays climacteric of persimmon fruit. Ann. Appl. Biol. 144, 65–69. Ali, A., Mahmud, T.M.M., Sijam, K., Siddiqui, Y., 2011. Effect of chitosan coatings on the physico-chemical characteristics of Eksotika II papaya (Carica papaya L) fruit during cold storage. Food Chem. 124, 620–626. Ali, A., Ong, M.K., Forney, C.F., 2014. Effect of ozone pre-conditioning on quality and antioxidant capacity of papaya fruit during ambient storage. Food Chem. 142, 19–26. Ali, Z.M., Chin, L.H., Lazan, H., 2004. A comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruits. Plant Sci. 167, 317–327. Basir, T.G., 2005. Land and Agricultural Policy. Malaysian Palm Oil Board, Malaysia. Cheour, F., Willemot, C., Arul, J., Makhlouf, J., Desjardins, Y., 1991. Postharvest response of two strawberry cultivars to foliar application of CaCl2 . HortScience 26, 1186–1188. Chuni, S.H., Awang, Y., Mohamed, M.T.M., 2010. Cell wall enzymes activities and quality of calcium treated fresh-cut red flesh dragon fruit (Hylocereus polyrhizus). Int. J. Agric. Biol. 12, 713–718. Ciccarese, A., Stellacci, M., Gentilesco, G., Rubino, P., 2013. Effectiveness of pre- and post-veraison calcium applications to control decay and
maintain table grape fruit quality during storage. Postharvest Biol. Technol. 75, 135–141. De Assis, S.A., Lima, D.C., De Faria Oliveira, O.M.M., 2001. Activity of pectin methylesterase, pectin content and vitamin C in acerola fruit at various stages of fruit development. Food Chem. 74, 133–137. Dey, P.M., Brinson, K., 1984. Plant cell walls. Adv. Carbohydr. Chem. Biochem. 42, 265–382. Ferguson, I.B., 1984. Calcium in plant senescence and fruit ripening. Plant Cell. Environ. 7, 477–489. Ferguson, I.B., Watkins, C.B., 1989. Bitter pit in apple fruit. Hortic. Rev. 11, 289–355. Gamagae, S.U., Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C., 2003. Use of sodium bicarbonate and Candida oleophila to control anthracnose in papaya during storage. Crop Prot. 22, 775–779. Goutam, M., Dhaliwal, H.S., Mahajan, B.V., 2010. Effect of preharvest calcium sprays on post-harvest life of winter guava (Psidium guajava L). J. Food Sci. Technol. 47, 501–506. 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Respiratory activity and compositional changes in Eksotika papaya fruit following storage in low oxygen atmosphere. J. Trop. Agric. Food Sci. 25, 85–93. Singh, B.P., Tandon, D.K., Kalra, S.K., 1993. Change in post-harvest quality of mangoes affected by preharvest application of calcium salts. Sci. Hortic. 54, 211– 219. Staehelin, L.A., Moore, I., 1995. The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 261–268.
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