Effect of Foliar Application of Calcium on the Quality of Blueberry Fruits

Journal of Plant Nutrition, 31: 1299 1312, 2008 Copyright Taylor & Francis Group, LLC ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160802135076 Effect of Foliar Application of Calcium on the Quality of Blueberry Fruits Rosemarie Stückrath, Roberto Quevedo, Lucía de la Fuente, Astrid Hernández, and Viviana Sepúlveda Departamento de Ciencia y Tecnología de los Alimentos, Universidad de los Lagos, Osorno, Chile ABSTRACT Effect of foliar application of calcium (0.78 g, 4.68 g, and 7.8 g Ca 2+ )inpre-harvest, at three different growing conditions (tunnel, mesh, and ambient), on texture and pectin in blueberries (Vaccinium corymbosum)was studied. Calcium contents in leaves as well in fruits were different (P 0.05), affected by growing conditions and time. Differences (P 0.05) in calcium content were found in tunnel cultivar fruit, during the period to cell expansion toward harvest, at calcium foliar level of 5 ml per L (7.8 g). Fruit texture was significantly higher at the beginning of the cell expansion period in the tunnel cultivar fruit, and a linear correlation between calcium concentration and texture was established. Increment in low methoxyl pectin (LMP) was influenced by growing conditions, and was different (P 0.05) for tunnel cultivar fruit. A good correlation between LMP and calcium content was obtained with the high dose of calcium (5 ml per L). Keywords: Blueberries, calcium, low methoxyl pectin, texture INTRODUCTION Blueberries are grown in Chile, mainly for export, as a fresh-cooled or frozen product. Fruit texture, color, and size are important quality factors influencing market value and use in technological processes. Optimizing these characteristics will increase acceptability and decrease losses through transportation. An important characteristic of blueberry fruit is that it has a high percentage of low methoxyl pectins (LMP), representing approximately 60% of Received 01 May 2006; accepted 16 March 2007. Address correspondence to Rosemarie Stückrath, Departamento de Ciencia y Tecnología de los Alimentos, Universidad de los Lagos, Box 933, Fone 56-64-333346, Osorno, Chile. E-mail: rstuckra@ulagos.cl 1299

1300 R. Stückrath et al. total pectin substances in comparison to other berries, such as raspberry and cranberry (Stückrath et al., 1995, 1998, 2001). These substances are mainly distributed in the middle lamella of the cell wall, with which, according to estimations, at least 60% of the total calcium (Ca) in the plants is also associated (Poovaiah et al., 1988). The LMP, by means of their non-esterified carboxyl groups, interact with the calcium ions through chelator crosslinks, which influences fruit texture, and is directly involved in strengthening plant cell walls (Demarty et al., 1984). Calcium is the plant nutrient most frequently associated with fruit quality, in general, and firmness in particular (Sams, 1999). The mechanical property of the fruit primary wall is determined by a mixture of matrix (pectic and hemicellulosic) and fibrous (cellulose) polysaccharides. Those that confer on the wall two properties: its plasticity that enables it to expand as the cells enlarge during fruit development and its rigidity that confers strength and determines cell shape (Harker et al., 1997). The two chemical changes in cell wall composition, which have been reported for nearly every ripening fruit examined, are an increase in soluble pectin and a net loss of the noncellulosic neutral sugars galactose and arabinose. Modification of cell wall pectin involves two processes: solubilization and depolymerization. Enzymes that attack the neutral polymer side-chains could destabilize the pectin polysaccharide matrix and contribute to fruit softening (Harker et al., 1997). Calcium has a well-established role in strengthening the cell wall. The influence of calcium on diverse physiological and biochemical changes during fruit softening has been reviewed by Poovaiah et al. (1988). They have reported the benefit of direct calcium applications in reducing the incidence of physiological disorders. Postharvest treatments involving dipping or infiltrating with calcium are known to maintain firmness during storage of fruit (e.g., apple). Abbott et al. (1989) examined texture of apples infiltrated with calcium and they found a sigmoid relationship between concentration of calcium and increase in tissue strength. Conway and Sams (1987) found that Ca 2+ was more effective than magnesium (Mg 2+ )orstrontium (Sr 2+ )inincreasing firmness. Calcium is directly and integrally involved in texture changes and an adequate content in fruit at harvest is crucial in maintaining fruit quality. Pre-harvest calcium sprays also result in firmer fruit (Raese and Drake, 1993), which further confirms the involvement of calcium in maintaining texture. Sidiqui and Bangerth (1995) reported that pre-harvest calcium application in leaves might not always lead to firmer fruit at harvest, but may result in a better retention of firmness during storage. The purpose of this research is to determine the effect of calcium-salt application, in predetermined concentrations, during fruit growth, in which it could increase the endogenic calcium and result in firmer fruit with better texture.

Calcium on the Quality of Blueberry Fruits 1301 MATERIALS AND METHODS Sampling Blueberry cultivars (Vaccinium corymbosum), Elliot variety, from the period (1992 1993) belonging to Agrícola Giddings, Purranque (40 56 S, 73 07 W) Chile, were studied. Three growing conditions were created 1) Tunnel: a system covered with a non-pigmented polyethylene of low density under which microclimatic conditions are established, which shortens the growing period of the fruit; 2) Mesh: a system with 50% shade black cloths rachel ; 3) Ambient: a system without protection. Each system consisted of two rows of one hundred or one hundred and twenty plants each, one-meter between plants and 2.5 meters between rows, and divided into areas of five plants per row for a random sample. The foliar application started at the period of full flowering (day 1) and finished at the period of last harvest (day 134). Two fertilizers, based on calcium (120 g Ca 2+ L 1 ), were utilized. Fertilizer 1was applied once and fertilizer 2 was applied twice within a period of approximately twelve days, this period is designated a cycle. It was applied four cycles. Three different doses were utilized 0.5, 3.0, 5.0 ml of fertilizer per L of water, in foliar application. In this case, 13 L of water per one hundred plants, equivalent to 0.78 g, 4.68 g, and 7.8 g Ca 2+, respectively. In addition, these doses were reinforced using a mixture of fertilizer 1 + boron (B) solution. The boron would help the calcium translocation from the leaf to the fruit. The fertilization and sampling programs (leaves and fruit) in the nine procedures (three growing conditions, each with three different doses) are shown in Table 1. Calcium Determination in Leaves And Fruits The 7.1-Rev. 2000 method from the Comisión de Normalización y Acreditación de la Sociedad Chilena de la Ciencia del Suelo for vegetal tissues was used. Steps of the method were wash [water, hydrochloric acid (HCl) 0.1 N], dried (65 C 2 hours), milled (0.5 or 1 mm), calcination (500 C, HCl 1:1), and absorption and atomic emission spectrometry (flame air-acetylene, lantanne, 422.7 nm). The work was done in duplicate with a margin of error not higher than 5%. Pectic Substance Determination in Fresh Fruit Both the total pectic substances and those of the fractions (high methoxyl pectins, low methoxyl pectins and protopectins) were determined by the

1302 R. Stückrath et al. Table 1 Program of plant fertilisation of blueberry variety ELLIOT (1992 1993) Time (days) Application of fertiliser and period of sampling 1 Period of full flowering 4 Sampling: leaves 6 First cycle: fertiliser n o 1 18 First cycle: fertiliser n o 2 18 Sampling: leaves and fruits 27 First cycle: fertiliser n o 2 30 Sampling: leaves and fruit 34 Second cycle: fertiliser n o 1 38 Second cycle: fertiliser n o 2 50 Reinforcement: fertiliser n o 1 + Solution Boron 54 Sampling: leaves and fruits 57 Second cycle: fertiliser n o 2 65 Third cycle: fertiliser n o 1 65 Sampling: leaves and fruit 76 Third cycle: fertiliser n o 2 80 Sampling: leaves and fruits 82 Third cycle: fertiliser n o 2 94 Reinforcement: fertiliser n o 1 + Solution Boron 99 Sampling: leaves and fruits. 99 Harvest: tunnel cultivar 101 Fourth cycle: fertiliser n o 1 108 Harvest: Mesh and Ambient cultivars 117 Reinforcement: fertiliser n o 1 + Solution Boron 120 Sampling: leaves and fruits 131 Fourth cycle: fertiliser n o 2 133 Fourth cycle: fertiliser n o 2 134 Sampling: leaves and fruits methods used by Robertson (1979) and by Carbonell et al. (1989), which was modified by Stückrath et al. (1995, 1998) for raspberries, blueberries, and cranberries. The pectic substances are separated on the basis of their solubility in water, ammonium oxalate and cold alkali. Quantification of anhidrogalacturonic acid in each fraction was determined by the colorimetric method of Blumenkrantz and Asboe-Hansen (1973). This method utilizes m-hidroxydiphenyl, sensible reagent and specifies of uronic acids, with respect to the nonuronic carbohydrate presence. This method was modified by Kintner and Van Buren (1982), which recommended the use of a white sample to be able to discard interferences of other carbohydrates that are not uronics.

Calcium on the Quality of Blueberry Fruits 1303 Texture Determination Firmness index was carried out on whole fruit using a TA-XT2 Texture Analyzer, according to the following condition: cylindrical plunger (0.5 in. diameter); F was the compression force at 8.5 mm depth at a speed of 0.8 mm s 1. Ten determinations were used by treatment. Weight Determination Each sample consisted of 30 fruits. They were weighed individually on a scale with a sensitivity of 0.01 g. Reactives The reactives used were all of Analytical Grade from Aldrich Chemical Co. (Milwaukee. USA) and from Merck (Darmstadt, Germany) Statistical Analysis Analysis of variance was performed using the software StatGraphics Plus 4.0. (Statistical Graphics Corp.). Significant differences were determined at P 0.05. Tukey s multiple range tests were used in order to resolve differences among sample treatments. RESULTS AND DISCUSSION Foliar Calcium The concentration of foliar calcium increased steadily during the fertilization period and fruit development. The results show significant differences between the growing conditions (F = 111; df = 2; p = 0.0000) and over time (F = 252.7; df = 8; p = 0.0000). The foliar calcium was significantly lower in the tunnel cultivar (LS Mean: tunnel 0.33 g %; mesh 0.401; and ambient 0.413). In terms of time, the sampling period between 30 to 124 day were presents differences statistically significant (Table 2). On day 54, after two fertilization cycles and the first reinforcement (Calcium + Boron), significant differences were found between the calcium doses, the highest concentration of foliar calcium being found for the 5 ml per L dose (LS Mean 0.429 g % at different 0.375 and 0.365 g % for 3 and 5 ml per L) and for the growing conditions (LS Mean 0.465; 0.405; and 0.30 g % for ambient, mesh; and tunnel).

1304 R. Stückrath et al. Table 2 Multiple Range Tests: Tukey for Calcium (g %) in leaf; for Calcium (g %) in fruit and for weight (g) in fruit by time Time (day) Count LS Mean Leaf [Ca] Count LS Mean Fruit [Ca] LS Mean Fruit weight 4 18 0.2102 e 18 18 0.2415 e 9 0.1756 a 30 18 0.2809 d 9 0.1471 b 0.4133 d 54 18 0.3897 c 9 0.1273 bc 65 18 0.3959 c 9 0.1104 cd 0.6922 cb 80 18 0.4034 c 9 0.0928 d 0.8478 c 99 18 0.4458 b 9 0.0591 e 1.6900 a 120 18 0.5162 a 9 0.0482 e 1.7611 a 134 18 0.5486 a 9 0.0477 e 1.3911 b denotes a statistically significant difference. Fruit Calcium Calcium concentrations in fruit, determined throughout the development period, were significantly influenced by the growing method (F = 8.8; df = 2; p = 0.0003) and time (F = 229.7; df = 7; p = 0.0000). The highest concentrations were founded to the first period of the fertilization program (18 to 80 days), showing statistically significant differences (Table 2). Fruit shows an increase in calcium concentration at the beginning of growth, which is the cell division stage, characterized by a rapid increase in the calcium content of fruit. During the second stage, which is associated with the period of cell expansion, calcium absorption continues at a lower rate or ceases completely and concentration decreases through progressive dilution. This is exposed in the (Ca) vs. time graph (Figure 1) and is complemented by the fruit weight versus growing time graph (Figure 1) showing statistically significant differences (F = 207.62; df = 5; p = 0.0000). The highest weights were found in the 80 to 120 day period, this time being optimal for harvesting (Table 2). Serrano et al. (2002) in melon showed that the calcium content was significantly higher in control fruit than in those of low and removed calcium treatments. Therefore, calcium was probably accumulated in the fruit quite early in its development and before the calcium supply was totally interrupted during the two last weeks of fruit development. According to Bernadac et al. (1996) cited in Serrano et al. (2002), 80% of the calcium present in ripe melon fruits was already there 20 days after anthesis, and so calcium deficient applied to twenty day-old fruits had no significant effect on the calcium content at the time of harvest.

Calcium on the Quality of Blueberry Fruits 1305 0,25 0,2 A a A b 0,15 c % Ca 0,1 B d A B e A f f 0,05 0 0 20 40 60 80 100 120 140 Time (days) 2,5 a 2 a b Weight (g) 1,5 1 c c 0,5 d 0 0 20 40 60 80 100 120 140 160 Time (days) Tunnel 0.5 Tunnel 3 Tunnel 5 Mesh 0.5 Mesh 3 mesh 5 Ambient 0.5 Ambient 3 Ambient 5 Figure 1. Calcium concentration and weight in blueberry fruit during fruit development and ripening. Letters show a significant difference.

1306 R. Stückrath et al. After the fourth sampling and two foliar fertilization cycles, a statistically significant decrease in the calcium concentrations in fruit from the tunnel cultivar is observed (LS Mean 0.070 g % front a 0.103 and 0.105 for mesh and ambient). This one may be explained by the fact that the cell expansion stage of the fruit has begun. This growing method creates special environmental conditions, namely a microclimate that speeds up fruit growth. In respect of the dose of calcium applied, a significantly higher calcium concentration was obtained with the 5 ml per L dose in the fruits from the tunnel cultivar (LS Mean 0.083 a g%en contrast at 0.069 and 0.060 g %) during the cell expansion period (80 to 99 days). That is very important, since the first harvest took place on day 99. The high calcium concentration can be related to the low weight of the fruit, the highest correlation being found for this dose (Table 3). Thus, if one wish to modify the characteristics of calcium transport in order to increase calcium levels in the fruits, intervention should be make happen in the first stage of fruit development. Fruit Texture Important quality factors for blueberry fruits are texture and firmness. Firmness was measured as hardness, the peak force during the first compression cycle. Fruit firmness is an important economic trait in blueberry. Consumers perceive firm blueberries to be of higher quality. This perception can affect blueberry marketability and consumer demand (Ehlenfeldt et al. 2002). For influence of foliar calcium application, a study of fruit texture was effectuated throughout its development by measuring the force expressed in Newton (cm 2 ) 1 applied until the fruit broke apart. The statistical studies applied to the texture values throughout the fruit development period revealed differences with respect to time (F = 106.1; df = 5; p = 0.0000), but not with respect to calcium levels applied, nor to cultivation methods. On day 65 after fertilization started, all the fruits showed the highest texture values, the ones from the mesh cultivar being statistically higher [LS Mean 40 N (cm 2 ) 1 ] and those from the tunnel cultivar lower [LS Mean: 26,4 N (cm 2 ) 1 ]. Regarding the influence of Ca level in the tunnel cultivar, a higher, statistically significant, texture was obtained with the 5 ml per L dose [LS mean 30 N (cm 2 ) 1 ]. This time (day 65) coincides with the one of highest calcium absorption by the fruit from the mesh and ambient cultivars and with the beginning of cell expansion in the fruits from the tunnel cultivar. The best correlation values between calcium concentration in the fruit and texture were found with the 5 ml per L dose. During cell expansion, a linear correlation was found in tunnel fruits and fruit size, in general, was negatively correlated to firmness (Table 3).

Table 3 Regression coefficients between calcium content in blueberry fruits and the rest of the variables and between texture of fruits and the rest of the variables in the three types of growing conditions and with the three calcium doses Tunnel 0.5 Tunnel 3.0 Tunnel 5.0 Mesh 0.5 Mesh 3.0 Mesh 5.0 Ambient 0.5 Ambient 3.0 Ambient 5.0 %Calcium %Calcium %Calcium %Calcium %Calcium %Calcium %Calcium %Calcium %Calcium Texture 0.97 0.82 0.99 0.84 0.73 0.85 0.94 0.82 0.92 Weight 0.66 0.75 0.90 0.95 0.97 0.93 0.95 0.97 0.90 %PP 0.95 0.68 0.99 0.97 0.97 0.99 0.99 0.98 0.99 Texture Texture Texture Texture Texture Texture Texture Texture Texture % LMP 0.99 0.98 0.97 0.90 0.76 0.92 0.97 0.76 0.91 Weight 0.79 0.45 0.72 0.84 0.87 0.81 0.81 0.73 0.76 %PP 0.99 0.97 0.98 0.91 0.83 0.92 0.97 0.88 0.96 1307

1308 R. Stückrath et al. Environmental factors including light, temperature and moisture have pronounced effects on fruit texture. It is commonly accepted that the temperature during production affects growth and development. Temperature has a direct influence on metabolism and, thus, indirectly affects cellular structure and other components, which determine texture (Sams, 1999). Our results confirm the influence of temperature on fruit texture. The best texture was found in the fruit from mesh cultivar, in which a black mesh that lowers temperature protects plants. The lowest-quality texture was found in the fruit from the tunnel cultivar, where a microclimate is created. In general, firmness decreased as fruit matured. Peach (Kader et al., 1982), pear (Stow, 1988), and apple (Knee and Smith, 1989) fruits soften as they mature. In apple, there is a significant correlation between harvest firmness and firmness after storage (Knee and Smith, 1989; Cited in Sams, 1999). Bashir and Abu-Goukh (2003) studied fruit flesh firmness in two guava types and showed a progressive decline during ripening. The decline in firmness observed was about eight-fold from the hard mature green stage to the final soft ripe stage. Most of this decline occurred during the first ten days. Pectin Substances The fruit softening that occurs during development and ripening is primarily due to changes in the cell wall carbohydrate metabolism and to the action of cell wall hydrolases. Pectin-degrading enzymes have received most attention as potential causal agents in the softening of ripening fruit (Serrano et al., 2002). A study of the changes in pectic substances was carried out during fruit growth. Special attention was paid to low methoxyl pectins, because of the high percentage of this type of pectin in blueberries and because of the importance of their interaction with calcium ions as well as their effect on texture. The changes in low methoxyl pectins (LMP) and protopectins (PP) were statistically influenced by time (F = 111.53; df = 5; p = 0.0000) (F = 130.75; df = 5; p = 0.0000) and growing conditions (F = 10.59; df = 2; p = 0.0002) (F = 8.31; df = 2; p = 0.0009). At the beginning of the period there was a higher percentage of PP and a lower quantity of LMP (Figure 2) and high methoxyl pectins (HMP). However, eighty days after the beginning of fertilization, when fruit weight became significantly higher, PP began to decrease, and LMP to increase. The quantity of LMP was significantly in the fruits from the tunnel cultivar (Table 4). In general, the correlation between LMP and PP was linear and the best correlation between LMP and texture was found in the tunnel cultivar. The best correlation between LMP and calcium was obtained with the high dose of calcium in the tunnel cultivar and with the medium and high doses in the mesh cultivar (Table 3). The decrease in insoluble pectins is associated with the increase in soluble pectins. As ripening occurs, once pectins are released from their links

Calcium on the Quality of Blueberry Fruits 1309 100 80 a a % LMP 60 40 c A b 20 d d B 0 0 20 40 60 80 100 120 140 Time (days) Tunnel 0,5 Tunnel 3 Tunnel 5 Mesh 0,5 Mesh 3 Mesh 5 Ambient 0,5 Ambient 3 Ambient 5 Figure 2. Percentage of low methoxyl pectin (LMP) in blueberry fruit during fruit development and ripening. Letters show a significant difference. B with cellulose, they can desmethylate naturally, especially through the action of pectolitic enzymes. This allows them to participate in cross linking reactions through Ca ++, resulting in an increase in fruit firmness (Fennema, 1993; Wong, 1997). Table 4 Multiple Range Tests: Tukey for LMP (%) in fruit by growing conditions (cultivar) Cultivar Count LS Mean Day 80 LS Mean Day 120 LS Mean Day 134 Tunnel 3 41.97 a * 59.60 a 59.53 a Mesh 3 10.03 b 49.43 b 53.57 a Ambient 3 9.17 b 55.57 a 54.83 a denotes a statistically significant difference.

1310 R. Stückrath et al. Proctor and Peng (1989) determined changes in the pectin composition of Bluetta blueberries, there being a reduction in dilute alkali soluble pectin (PP) to 20%. Rising levels of chelator soluble pectin (CSP) suggest increasing demethylation, probably as a result of pectin methyl esterase (PME) activity, and subsequent binding of divalent cations. This is supported by a finding that the majority of cell wall bound calcium was inthe CSP fraction. The greater firmness of calcium-treated pears may result from calcium interacting directly with cell wall pectic substances, resulting in cell wall stiffening. Calcium may also act by reducing the activity of cell wall-degrading enzymes (Gerasopoulos and Richardson, 1999). Mango fruits (Evangelista et al., 2000) were sprayed pre-harvest with calcium chloride at 0, 2.5, and 5.0% to verify the influence of calcium on texture and activity of polygalacturonase, methylpectinesterase, and β-galactosidase. The fruits submitted to the treatment with calcium chloride at 5.0% presented firmer texture and less activity of the enzymes polygalacturonase and β-galactosidase. ACKNOWLEDGMENTS This study is part of Research Project 2002 in the Department of Research, Universidad de Los Lagos. We would like to express our gratitude to Mr. Julio Giddings and Mr. Roberto Giddings at Empresa Agrícola Giddings. REFERENCES Abbott, J. A., W. S. Conway, and C. E Sams. 1989. Post harvest calcium chloride infiltration affects textural attributes of apples. Journal of the American Society for Horticultural Science 114: 932 936. Bashir, H. A., and A.-B. A. Abu-Goukh. 2003. Compositional changes during guava ripening. Food Chemistry 80: 557 563. Bernadac, I., I. Jean-Baptiste, G. Bertoni, and P. Morard. 1996. Changes in calcium contents during melon (Cucumis melo L.) fruit development. Scientia Horticulturae 66: 181 189. Blumenkrantz N., and G. Asboe-Hansen. 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry 54: 484 489. Carbonell, E., E. Costell, and L.Durán. 1989. Evaluation of various methods for measurement of pectin content in jam. Journal - Association of Official Analytical Chemists 72 (4): 689 93. Conway, W. S., and C. E Sams. 1987. The effects of postharvest infiltration of calcium, magnesium, or strontium on decay, firmness, respiration, and ethylene production in apples. Journal of the American Society for Horticultural Science 112: 300 303.

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