J. Appl. Hort., 2(2):79-83, July-December, 2000 Effect of CaCl2 sprays, heat, and combined CaCl2-heat treatments on the quality of apples (Malus domestica Borkh.) R. Drisa, R. Niskanen a and El Assi N.b* a Department of Applied Biology, Horticulture, P.O. Box 27, FIN-00014 University of Helsinki, Finland b University of Jordan, College of Agriculture, Department of Horticulture and Plant Protection, P.O. Box 13367, Amman 11942, Jordan* Corresponding author. E-mail address: email@example.com Abstract ‘Lobo’ apple fruits were subjected to preharvest CaCl2 spraying treatment, pre-storage heat treatment, and combined CaCl2+heat treatment and were held at 2oC and 90-95% R.H. for six months. Respiration and ethylene production rates were monitored, soluble solids, juice pH, firmness, total dry matter and macronutrient (P, K, Ca, Mg, and N) contents were determined. Additionally, physiological disorders incidence and pathological disease occurrence were recorded. The respiration and ethylene production rates slightly decreased in heat-treated apples and increased in CaCl2-treated apples. CaCl2 treatment did not increase fruit firmness or Ca concentration. Combined CaCl2+heat treatment and heat treatment increased the pH. At the beginning of storage the firmness of heat and CaCl2+ heat-treated fruits was lower, but more than that of the control fruits at the end of storage period. After 6 months of storage, least disorder and disease symptoms were observed in the CaCl2+heat treatment. Key words: Apples, Malus domestica Borkh., calcium, macronutrients, ethylene, firmness, respiration, soluble solids Introduction The use of pre-, or post-harvest chemical treatments is becoming more restricted due to consumer preference for chemical-free food, and the increasing resistance of many microorganisms to commonly used chemicals. Calcium is associated with regulation of the ripening process and storage behaviour of apples (Marcelle, 1990). Many physiological storage disorders such as bitter pit and senescence breakdown are due to the low calcium concentration in apple flesh (Shear, 1974; Ferguson, 1984, Dris and Niskanen, 1998). Maintenance of relatively high calcium content in fruit tissues results in slower ripening and softening as well as reduced respiration and ethylene production rates (Bangerth et al., 1972; Faust and Shear, 1972; Lieberman and Wang, 1982). Pre-harvest calcium spraying treatments on apples have been reported to reduce the incidence of physiological storage disorders (Vang-Petersen, 1981; Raese et al., 1990; Peryea, 1991). Pre-storage heat treatment of apples has been shown to have beneficial effects on fruit quality during storage by delaying ripening and reducing ethylene production rate (Burg, 1962; Porritt and Lidster, 1978; Klein and Lurie, 1990). Limited research on the preharvest calcium spraying treatments has been done on apples grown in Finland (Dris and Niskanen, 1997; 1998), and pre-storage heat-treatment on apples has not been studied so far. Our objective was to study the effect of preharvest CaCl2 spray, pre-storage heat-treatments and combined CaCl2 and heat treatments on storage life of ‘Lobo’ apple fruits grown in the Åland Islands district. Materials and methods Twelve-year old ‘Lobo’ apple trees with A2 type rootstock grown commercially in two separate orchards in the district of the Åland Islands (56-61°N, 19-20°E) were used in this study. Two plots of 5 trees/ plot/orchard were chosen to conduct the present study. One plot/orchard was treated by foliar spraying with CaCl2 solution (Ca 1.3 g/l) six times during fruit development with a two-week interval, and the other plot was left untreated. CaCl2 sprays started in mid June (after fruit set) and ended early September (3 weeks before harvest). A total of 720 fruits were hand harvested on the third of October from the two orchards (designated ‘Lobo’1 and ‘Lobo’2), washed with water, air dried at ambient temperature, and transported on the same day to the laboratory. Fruits from untreated control (180 fruits) and CaCl2-treated plots (180 fruits) from each orchard were then divided further into 2 subgroups of 90 fruits each. One untreated and one CaCl2-treated subgroups were subjected to prestorage heat treatment in specific heat chambers at 38 oC for 4 days as described by Klein and Lurie (1990). Relative humidity was supplied by placing water trays on the floor of the heat chambers and maintained at 50% with the available equipment. Accordingly, the following four treatments were carried out on the fruits (90 in each treatment) from the two orchards: CaCl2-treated; heat-treated; CaCl2+ heat–treated; and untreated control-treatment. All fruits were stored for six months at 2oC and R.H. 90-95%. The assessment of quality characteristics and nutrient analysis were carried out at the beginning of the storage period and consequently after 1, 3 and 6 months storage, and the respiration (CO2 evolution) and ethylene production rates were monitored for 101 days. Measurements on fruits from the two orchards were taken separately, averaged, and then considered as replicates, except for the respiration and ethylene production rates determination. For monitoring respiration and ethylene production rates, two sound and uniformly sized apple fruits with two replicates for each treatment were weighed and placed in one-liter jars in the cold storage room at 2oC. Prior to taking gas samples, each jar was tightly closed with metal lid for 12 to 14 h, gas samples were drawn with a syringe through the rubber septum in the lid, then jars were uncovered. A 5 ml gas sample was injected into a gas 80 Journal of Applied Horticulture chromatograph (model HP 5890 Series II) equipped with a Porapak Q column for CO2 and a flame ionization detector for ethylene. Measurements were repeated 7 to 8 times during storage for ‘Lobo’ 1 and ‘Lobo’ 2 fruits from the first and second orchard, respectively. rates up to the 66th day of storage (Fig. 3 and 4). Similarly, CaCl2treated apple fruits showed slight increase in ethylene production rate up to the 66th day of storage (Fig. 4). Noticeably, a sharp increase in ethylene production rate was observed after 24 days in the CaCl2-treated apple fruits followed by a steady sharp Six apple fruits were randomly chosen for the textural test. The diameter (cm) of individual fruit was measured with sliding callipers, and firmness (kg) was determined by a penetrometer (The Instron Universal Testing Instruments, Instron TMM 100, Instron Ltd, England). The fruit was compressed for a distance of 1 cm (speed 10 cm min-1) with a 5.0 cm in diameter measuring device, and the force exerted was recorded. For measuring the total soluble solids (TSS) and pH, two apple fruits were cut transversally and pressed with a small juice extractor. The concentration of soluble solids was measured by a refractometer (model Abbe-3L Bausch & Lomb). The pH of the juice was measured using a pH-meter (Model PHM 83). The incidence of physiological storage disorders such as senescence breakdown, low temperature breakdown and water core etc., and diseases such as blue mould rot, brown rot etc. were evaluated. The affected fruits were calculated as a percentage of the total. Data was subjected to Analysis of Variance (ANOVA) with SAS General Linear Program Model procedure (SAS Institute, 1989). The mean separation was done by the Least Significant Difference (LSD) method at the 0.05 level. 350 µlC2H4(kg/hr) 300 250 200 150 100 50 Fig. 3 0 4 18 24 58 66 72 95 101 58 66 72 95 101 400 350 300 µlC2H4(kg/hr) For the nutrient analysis, seeds and stems of six apple fruits were removed with an apple borer (diameter 20 mm) after which the fruits were cut with a vegetable-slicing machine (Hobart VS 9A). The slices were frozen at -20°C, and then freeze-dried for 12-16 h and vacuum packed in laminate bags. The percentage of dry matter in fruit flesh was determined by weighing apple slices before and after drying. Fruit samples were pulverized and the concentration of dry matter was determined by drying the pulverized samples (2 g) at 105°C for 1.5 h. Nitrogen was determined by the Kjeldahl method (Tecator Digestion System 20, 1015 Digester and Kjeltec Auto 1030-analyzer, Tecator Ltd, Höganäs, Sweden). Fruit samples were ashed at 475°C and the ash was dissolved in 0.5 M HCl. Phosphorus in the ash extracts was determined by the ammonium vanadate method (Jackson, 1958), potassium by flame photometry, and calcium and magnesium by atomic absorption spectrophotometry (AAS). 250 200 Results and discussion Whole-fruit respiration, measured by CO2 production rate, fluctuated during storage between 0.5 to 7.0 mg-1 kg-1 hr -1 (Fig. 1 and 2). Even though the values showed slight to no significant differences among treatments, a slight decrease was noticed in the respiration rate of heat-treated apple fruits compared to the other treatments (Fig. 1). On the contrary, CaCl2-treated apple fruits showed a slight increase in their respiration rates (Fig. 1 and 2). The pronounced effect on apple fruits by the reduction of the respiration and ethylene production rate is in agreement with Klein and Lurie (1990) who observed that respiration rate of ‘Anna’ and ‘Granny Smith’ apple fruits declined after heat treatment and remained lower than that of the control fruits. Ethylene production rate followed a similar trend as described above, where the heattreated apple fruits from both orchards exhibited slightly lower 150 100 50 0 Fig. 4 4 Control 18 24 Days in storage CaCl 2 Heat CaCl2+Heat Leagends CO2 production rate (mg-1 kg-1 h-1)of Control, CaCl2-treated, Heattreated, and CaCl2 +Heat treated ‘Lobo’1 (Fig. 1) and ‘Lobo’2 (Fig. 2) apple fruit during storage at 2oC. C2H4 production rate (µl kg-1 h-1) of Control, CaCl2-treated, Heattreated, and CaCl2 +Heat treated ‘Lobo’1 (Fig. 3) and ‘Labo’2 (Fig. 4) apple fruit during storage at 2oC. CaCl2 sprays, heat, and combined CaCl2-heat treatments on quality of apples decline until the last measurement (Fig 4). Cultivar ‘Lobo’ showed a similar trend as ‘Anna’ apples (Klein and Lurie, 1990) in respect to ethylene production rate, which virtually ceased during heat treatment and after the treatment remained at a lower level than that in the control fruits. However, regardless of the type of the treatment, the rates of respiration and ethylene production were relatively low, which is in agreement with results obtained in fruit held at low temperature (Kader, 1992). Table 1. Effect of preharvest CaCl2, pre-storage heat, and CaCl2 + heat treatments on apple fruit texture (firmness/diameter- kg-1.cm-1) and dry matter (expressed as a percentage of fresh weight) during storage at 2oC for six months Parameter/1 Storage period (months) Treatment Texture Control CaCl2 Heat CaCl2+ Heat Dry Matter Control CaCl2 Heat CaCl2+ Heat 0 1 3 6 15.9 a2 14.3 ab 12.1 bcd 11.3 cd 13.7 abc 11.5 bcd 10.4 de 10.6 de 7.0 fg 6.3 gh 7.9 efg 9.9 def 3.7 h 3.7 h 11.6 bcd 9.2 defg 14.5 ab 15.5 a 15.6 a 14.5 ab 13.8 abc 13.2 abc 14.4 ab 14.5 ab 13.7 abc 11.8 c 13.5 abc 13.3 abc 12.9 bc 12.1 bc 13.7 abc 14.4 ab Table 2. Effect of preharvest CaCl2 sprays, pre-storage heat, and CaCl2 + heat treatments on apple fruit pH, TSS%, during storage at 2oC for six months* Parameter/1 Storage period (months) Treatment 0 1 3 6 pH Control 3.3 g2 3.2 g 3.8 de 4.1 bc CaCl2 3.4 fg 3.4 fg 3.7 ef 4.3 abc Heat 3.5 efg 3.4 fg 3.7 ef 4.5 a CaCl2+ Heat 3.7 ef 3.7 ef 4.0 cd 4.4 ab TSS% Control 10.4 ab 9.5 b 10.7 ab 10.8 ab CaCl2 9.8 ab 10.7 ab 10.4 ab 10.1 ab Heat 10.7 ab 10.7 ab 10.8 ab 10.3 ab CaCl2+ Heat 11.0 a 10.3 ab 10.9 ab 10.7 ab 1Each parameter is considered separately. 2Values for the corresponding parameter followed by the same letter (s) are not significantlydifferent at the 0.05 level by the LSD method. A steady decrease in apple weight was observed with time progression with no significant differences among the values obtained in all treatments (data not shown). The control and CaCl2-treated fruits exhibited considerable decrease in firmness values with time progression in storage (Table 1). Approximately 75% reduction was recorded in the firmness values for control and CaCl2-treated fruits. A decrease in firmness was also noticed in CaCl2+Heat-treated fruits, but it was gradual and minimal compared to the first two treatments (Table 1). This is in agreement with Davenport and Peryea (1990) and Peryea (1991) who observed that fruit firmness was not affected by calcium treatment. This could be due to the inadequate Ca distribution within the fruit tissues resulting in an insufficient 81 amount of Ca to exert an effect on firmness. However, our results contradict those obtained by Peryea (1991) where a slight increase in fruit firmness by frequent calcium treatments was observed. In the heat-treated fruits a pronounced decrease in firmness was observed up to the 3rd month, but a noticeable recovery in firmness values was observed in fruits after 6 months of storage (Table 1), which could be due to the inhibition of cell wall-degrading enzymes (Liu, 1978; Porritt and Lidster, 1978; Klein and Lurie, 1990). A steady decrease in the dry matter values obtained for all treatments with time progression in storage . However, significant difference between the initial and the final dry matter values was detected only in the CaCl2-treated fruits (Table 1). An increase in the pH values was noticed with time progression mainly after the 3rd and 6th months of storage with significant differences among the values obtained in all treatments (Table 2). As compared to the control, the combined CaCl2+ heat treatment increased the pH after 0 to 1 month storage and heat treatment after 6 months (Table 2). The percentage of the TSS showed fluctuations with no significant differences among the values (Table 2). Similar results of lower juice acidity and unaffected SSC were previously reported (Klein and Lurie, 1990). Table 3. Disorder incidence and disease occurrence in the control, preharvest CaCl2, pre-storage heat, and CaCl2 + heat-treated apple fruits during storage at 2 oC for six months Parameter/1 Storage period (months) Treatment 0 1 3 6 Disorders Control 0.01c2 0.0 c 2.5 c 52.5 ab CaCl2 0.0 c 0.0 c 5.0 c 60.0 a Heat 0.0 c 0.0 c 0.0 c 62.5 a CaCl2+ Heat 0.0 c 0.0 c 0.0 c 42.5 b Diseases Control 0.0 d 0.0 d 0.0 d 35.0 a CaCl2 0.0 d 0.0 d 0.0 d 7.5 ab Heat 0.0 d 0.0 d 15.0 bcd 35.0 a CaCl2+ Heat 0.0 d 0.0 d 10.0 cd 17.5 bc 1Each parameter is considered separately. 2Values for the corresponding parameter followed by the same letter (s) are not significantlydifferent at the 0.05 level by the LSD method. The combined CaCl2-heat treatment was effective in reducing the physiological disorders occurrence among all treatments (Table 3). Development of some physiological storage disorders have been reported to be reduced by heat treatment. Brooks and Harley (1934) effectively controlled soft scald in ‘Jonathan’ apples by heating at 35-40°C for 8 to 24 h. Heat treatment at 38°C for 4 to 6 days almost eliminated breakdown, core browning and decay of ‘Spartan’ apples and the incidence of physiological storage disorders in ‘Golden Delicious’ (Porritt and Lidster, 1978). In our study, neither heat treatment nor CaCl2 treatment was effective in decreasing physiological storage disorders incidence in apples. It is possible that such a result obtained in our experiments may be due to inadequate Ca distribution within the fruit tissue. Also early disease symptoms were detected after the 3rd month in the heat and CaCl2+heat treatments (Table 3). After 6 months of storage, the least disease symptoms were 82 Journal of Applied Horticulture observed in the CaCl2+ heat treatment (Table 3). However, CaCl2 treatment was relatively effective in reducing the disease occurrence (Table 3). There were only few significant differences in fruit nutrient concentrations between treatments and during storage period (Table 4 and 5). Heat treatment could be effective in prolonging the storability of apple fruits provided that high relative humidity (over 90%) was maintained during heat treatment. The influence of heat treatment might differ depending on apple cultivars (Mignani et al., 1994). Table 4. Effect of preharvest CaCl2, pre-storage heat, and CaCl2 + heat treatments on phosphorus and potassium (expressed as mg1. kg-1. fw-1) in apple fruits held at 2oC for six months. Parameter/1 Storage period (months) Treatment 0 1 3 6 Phosphorus Control 107.2 a2 122.3 a 121.2 a 109.6 a CaCl2 117.7 a 84.5 a 101.4 a 103.6 a Heat 111.4 a 109.0 a 99.1 a 119.1 a CaCl2+ Heat 111.6 a 105.6 a 106.9 a 119.6 a Potassium Control 1171.0d 1218.0 d 1723.0 a 1169.0 d CaCl2 1301.0 bcd 1039.0 d 1482.0 bcd 1083.0 d Heat 1255.0 cd 1253.0 cd 1708.0 abc 1179.0 d CaCl2+ Heat 1235.0 d 1956.0 a 1711.0 abc 1267.0 bcd Accordingly, our results indicate that preharvest CaCl2 treatment was not successful in achieving the expected results, neither was the heat treatment for such a long-term storage. Nevertheless, these treatments can be recommended only for a three-month storage period, but not for longer. The combined treatment of CaCl2+ heat resulted in maintaining firmness to some extent, decreasing the physiological and pathological disorders, provide evidence that it might be the best of the three treatment applied. Table 5. Effect of preharvest CaCl2, pre-storage heat, and CaCl2 + heat treatments on calcium and nitrogen contents (expressed as mg-1. kg-1.fw-1) in apple fruits held at 2oC for six months. Parameter/1 Storage period (months) Treatment 0 1 3 6 Calcium Control 55.3 bc2 47.4 c 68.4 bc 66.7 bc CaCl2 81.8 b 65.4 bc 68.5 bc 80.0 b Heat 80.0 b 118.2 a 65.1 bc 57.0 bc CaCl2+ Heat 54.8 bc 63.6 bc 84.2 b 64.9 bc Nitrogen Control 427 ab 458 ab 447 ab 432 ab CaCl2 540 a 418 ab 444 ab 541 a Heat 833 ab 448 ab 374 ab 462 ab CaCl2+ Heat 350 b 460 ab 456 ab 556 a 1 Each parameter is considered separately. 2 Values for the corresponding parameter followed by the same letter (s) are not significantly different at the 0.05 level by the LSD method. Calcium treatment has been reported to often increase Ca concentration in apples (Vang-Petersen, 1981; Raese, 1989; Raese et al., 1990). In our experiments, preharvest CaCl2 treatment did not increase the Ca level in apple flesh, rather decreased it. This could be explained by the results of Davenport and Peryea (1990), who considered that foliar application of Ca might fail to penetrate into the fruits and the small amount of Ca absorbed from foliar sprays could be analytically masked by background Ca levels in the whole fruit samples. In the study of Davenport and Peryea (1990), no effect of preharvest CaCl2 treatments was found on fruit mineral composition or other quality characteristics, and on the incidence of physiological storage disorders. In our experiments, CaCl2 treatment did not significantly affect the P, K, Ca, Mg, and N levels in apple fruits. However, in addition to the increased fruit N level in a previous experiment, also P and K were found to increase in Ca-treated fruits (Dris and Niskanen, 1997; Dris et al., 1999). Acknowledgments We express our gratitude to the Ministry of Agriculture and Forestry, the Government of the Åland Islands and Kemira Agro Ltd Horti for financial support. 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