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Vegetative growth, mineral change, and fruit quality of ‘Fuji’ tree as affected by foliar seawater application
Agricultural Water Management 126 (2013) 97–103
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Agricultural Water Management journal homepage: www.elsevier.com/locate/agwat
Vegetative growth, mineral change, and fruit quality of ‘Fuji’ tree as affected by foliar seawater application W.W. Zheng a,b,1 , I.J. Chun b,1 , S.B. Hong c , Y.X. Zang a,∗ a b c
College of Agriculture and Food Science, Zhejiang A & F University, Lin’an, Hangzhou 311300, China School of Bioresource Science, Andong National University, Andong 760-749, Republic of Korea Department of Biotechnology, University of Houston Clear Lake, Houston, TX 77058-1098, USA
a r t i c l e
i n f o
Article history: Received 5 March 2013 Accepted 5 May 2013 Available online 31 May 2013 Keywords: Foliar spray Seawater Apple Fruit quality Soluble solids Anthocyanin
a b s t r a c t Extensive researches have been conducted over the decades to investigate the effects of seawater on many crop plants either by irrigation or foliar spray in an attempt to enhance the yield and quality. The purpose of this study was to quantitatively determine the effects of different foliar seawater sprays on vegetative growth, fruit quality and yield of eight-year-old ‘Fuji’/M.9 apple trees. A field experiment was conducted using two different concentrations of seawater (100 or 50-fold dilution) starting from 130 DAFB (days after full bloom) at 5-day intervals between the treatments. Foliar seawater sprays to ‘Fuji’ tree led to increased levels of fruit soluble solids along with higher activities of sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (SS) (EC 2.4.1.13) and neutral invertase (EC 3.2.1.26). In addition, a significant increase in anthocyanin concentration was observed, especially when ‘Fuji’ trees were sprayed three times with 50-fold diluted seawater. Foliar seawater sprays also resulted in increases in Na+ concentration and K+ /Ca2+ ratio in fruit. In contrast to fruit, the levels of N, Na+ , K+ , Mg2+ in leaves remained unchanged regardless of different seawater treatments. Moreover, foliar seawater sprays did not affect the vegetative growth since leaf area, leaf fresh weight, shoot elongation, and chlorophyll content did not differ significantly from those of control plants. The results suggest that foliar seawater treatments could be useful for improving fruit quality without affecting the yield. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plants are frequently exposed to diverse abiotic stress conditions, such as high and low temperature, water deficit, UV light, oxidative stress, heavy metal toxicity and salinity. These factors greatly influence the growth and development of plants, as well as the crop productivity. In the course of evolution, plants have developed mechanisms to cope with and adapt to various adverse environmental stresses, most often by accumulating specific defensive secondary metabolites through signaling pathways (Ramakrishna and Ravishankar, 2011). So, moderate environmental stress conditions could be beneficial to improve the quality and to control the growth of certain crops. Salinity usually leads to a marked increase in soluble sugars and protein that may act as osmolytes for lowering the osmotic potential (Chen et al., 2009).
∗ Corresponding author at: College of Agriculture and Food Science, Zhejiang A & F University, 88 Huanchengbei Road, Lin’an, Hangzhou 311300, China. Tel.: +86 571 63742133. E-mail addresses: [email protected], [email protected] (Y.X. Zang). 1 These authors contributed equally to this work. 0378-3774/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.agwat.2013.05.010
Seawater has a salinity of nearly 3.5%, and the major components of the dissolved salts are sodium, magnesium, calcium and potassium; excessive accumulation is harmful to plant by causing osmotic injury and specific ion toxicity (Millero et al., 2008). Exogenous high salt application caused a reduction in pigment concentrations, as well as the uptake of K+ , Ca2+ , Mg2+ , and N in tissues (Cambrollé et al., 2011; Chakraborty et al., 2012). It was reported that high salinity of seawater reduces the ratio of Mg2+ / Na+ ; thus salinity may decrease photosynthesis by reducing concentration of Mg2+ , a major component of leaf chlorophyll. Several lines of evidence have suggested that seawater irrigation affects contents of specific metabolites in higher plants (Ferrante et al., 2011; Long et al., 2009; Ventura et al., 2011; Zhang et al., 2009). Moderate seawater treatment resulted in the marked accumulation of soluble sugar, protein, polyphenol, carotene, fatty acid (Long et al., 2008; Ventura et al., 2011). However, as the salinity was further increased, levels of electrolyte leakage and proline concentration rose, and this was typically accompanied by reduced levels of chlorophyll and carotenoid and more protein degradation (Ferrante et al., 2011; Long et al., 2009; Misra and Saxena, 2009). The high salinity stress resulted in reduced growth rate and biomass, shorter stature, smaller leaves, nutritional deficiency and mineral disorders (Ferrante et al., 2011; Long et al., 2009; Sekmen et al.,
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2012; Ventura et al., 2011; Zhang et al., 2009). The activity of superoxide dismutase was transiently stimulated and then decreased gradually under high salinity stress. Song and Ryou (2008) reported that as seawater spray frequency increased in grapevines, leaf browning and leaf drop occurred along with lower concentrations of soluble solids and anthocyanin in fruits. Thus, use of appropriate levels of salinity is important in agricultural practices, especially for the fruit quality improvement. Unlike grapevines, there was no leaf toxicity or symptoms of chlorosis and necrosis observed when olive trees were irrigated with up to 21.3% diluted seawater for five months (Vigo et al., 2005). The effects of saline water irrigation on growth and fruit quality have been investigated in other crop plants including melon and tomato (Botia et al., 2005; Maggio et al., 2004). To date a few scientific data about the effect of diluted seawater spray treatments on fruit quality of apple (Malus domestica Borkh.) have been reported. There is a lack of information on the proper timing and concentrations of seawater application in apple trees to improve fruit quality. In order to support agricultural extension service to fruit growers for the scientific management in seawater application, we investigated the effects of foliar seawater sprays on growth, fruit quality, and mineral nutrients of ‘Fuji’/M.9 apple over two consecutive years. 2. Materials and methods 2.1. Field condition, plant material and seawater dilution management This study was carried out on eight-year-old ‘Fuji’/M.9 cultivar apple trees for two consecutive seasons of 2010 and 2011. All the trees were planted with a distance of 3.8 m between rows and 1.8 m within the rows. Trees of 3 m high and 1.5 m wide were trained as a slender spindle, and those with similar growth vigor were selected as test materials. Soil properties were tested as pH 6.52, 13.2% organic matter, 0.75 g kg−1 available phosphorus, 0.46 g kg−1 available nitrogen, 0.48 g kg−1 available potassium. The trees were placed at yearly average temperature 11.8 ◦ C, sunshine hours 2698.4 h, average humidity 68%, precipitation 651.9 mm. The surface seawater was purified through a simple reverse osmosis system to eliminate dirt and microorganism and to reduce ion content. The desalted seawater (0.35 g kg−1 K+ , 0.31 g kg−1 Ca2+ , 0.94 g kg−1 Mg2+ , 0.89 g kg−1 Na+ ) was used for treatment. The experimental design consisted of nine treatments crossing two seawater concentrations. A standard randomized block design with six replications including one tree in replicate of each treatment was used. The trees to be tested were isolated in a distance with at least one untreated guard tree. All the trees were grown under the same environmental conditions with the same doses of irrigation, fertilization and phytosanitary treatments. All of the foliar seawater applications were performed by a hand sprayer (knapsack) at 20 L/six trees in the morning, starting from 130 DAFB (days after full bloom) at 5-day intervals between the sprays. In 2010, single, double and triple spray treatments with 100-fold diluted seawater were implemented, along with control treatment of water. In 2011, double and triple spray treatments with both 50- and 100-fold diluted seawater were implemented. The apple fruits were harvested at the time of optimum maturity which is at 175 DAFB. 2.2. Sampling and fruit characteristics Leaf area, fresh/dry weight, shoot elongation, and chlorophyll content were determined at 45 DAFFS (days after the first foliar spray application). Meanwhile fruits were selected based on the medium size, uniform shape and color scales in the morning.
To determine physical and chemical characteristics of fruit, each treated fruit sample was divided into two groups; one was used immediately and the other was stored in storage room maintaining 4 ◦ C and 80–85% relative humidity. Coloration was determined using a Color-Reader (KONICA MINOLTA SENSING, INC. Japan). Fruit firmness was measured by compression of individual apple fruits with a fruit texture analyzer (GUSS.ZA/GS-14). Soluble solid concentration and titratable acidity in apple juice were determined by a digital refractometer (PR-101, Cat. No. 3412, ATAGO, Japan) and a digital fruit acidity analyzer (Model: GMK-708, GVK, South Korea), respectively. Anthocyanin contents were determined as described by Rosso and Mercadante (2007). About 5 g of fresh fruit peel tissues from six randomly selected fruits in each treatment was pulverized and extracted with 1% (v/v) methanol. The homogenate was centrifuged at 19,000 × g for 15 min, and diluted with 1% HCl–methanol to a final volume of 500 ml. Absorbance of the diluent was measured at 530 nm. 2.3. Mineral analysis Leaves and fruits were washed in deionized water and dried at 80 ◦ C for 48 h, ground and stored in an oven at 60 ◦ C until analysis. Samples were then ashed in a muffle furnace at 600 ◦ C overnight, and dissolved in 0.1 N HCl. The N level in leaves was analyzed by Kjeldahl method (Page et al., 1982). The levels of Na+ , Ca2+ , Mg2+ , and K+ in fruits and leaves were analyzed by ICP-OES (Optima2000DV, Perkin Elmer Instruments Inc., CT, USA). All analyses were conducted in triplicate. 2.4. Enzyme activity assays Frozen mesocarp of pulp (0.5 g) was pulverized with pre-cooled mortar and pestle and mixed with 4 ml of extraction buffer containing 50 mM HEPES–NaOH (pH 7.5), 5 mM MgCl2 , 1 mM EDTA, 2.5 mM dithiothreitol (DTT), 0.2% of bovine serum albumin (BSA) and 0.05% Triton X-100. The mixture was centrifuged at 4 ◦ C for 10 min, and supernatant was desalted immediately through a Sephadex G-25 column pre-equilibrated with 50 mM Hepes–NaOH (pH 7.5), 5 mM MgCl2 , 2.5 mM DTT and 0.2% BSA. All procedures were carried out at 4 ◦ C or lower. Sucrose phosphate synthase (SPS) activity was assayed in a reaction mixture (0.3 ml) containing 7.5 mM UDP-glucose, 7.5 mM fructose-6-P, 10 mM glucose-6-P, 1.5 mM MgCl2 , 100 mM Tris–HCl (pH 7.5) and 50 l of the desalted extract at 30 ◦ C. The reaction was terminated by adding 75 l of 4 M NaOH after 20 min. The boiled enzyme was used as a control in each assay. Unreacted fructose-6P was removed by placing the reaction mixture in a boiling water bath for 10 min. After cooling, 0.25 ml of 0.1% (v/v) resorcinol in 95% (v/v) ethanol and 0.75 ml of 30% (w/v) HCl were added. The mixture was then incubated at 80 ◦ C for 8 min and allowed to cool, and its absorbance was determined at 540 nm. The procedure for sucrose synthase (SS) assay was identical to that of SPS except that the reaction mixtures contained 10 mM fructose instead of fructose-6-P. One unit of SPS and SS is defined as amount of the enzyme producing 1 mol sucrose per min. Acid and neutral invertase assays were performed at 30 ◦ C for 15 min in a total reaction volume of 300 l containing 50 l of desalted protein extract, 120 mM sucrose, and 100 mM citratephosphate (pH 5.0, for acid invertase), or 120 mM Hepes–NaOH buffer (pH 7.5, for neutral invertase). The reactions were terminated by boiling in a water bath for 5 min and the product (glucose) was assayed using dinitrosalicylic acid agent. Each assay had a boiled enzyme as control. One unit of acid and neutral invertase is defined as amount of the enzyme producing 1 mol glucose per min.
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Table 1 The effect of foliar seawater spray on vegetative growth of ‘Fuji’ trees in two consecutive years. Treatment
Leaf area (cm2 )
Leaf FW (g)
Leaf DW (g)
SPAD value
Shoot elongation (cm)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
28.1b 27.6bc 29.8a 27.2c
0.68a 0.69a 0.73a 0.64b
0.32a 0.30a 0.30a 0.30a
61.1a 61.6a 61.2a 60.7a
29.2a 32.5a 31.5a 30.7a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
27.3a 26.8a 23.4b 27.6a 27.7a
0.75a 0.71a 0.60b 0.76a 0.65b
0.29a 0.30a 0.24b 0.31a 0.26b
55.6a 57.7a 54.1a 60.8a 56.5a
32.1a 28.5a 28.9a 30.6a 34.5a
SW, seawater; FW, fresh weight; DW, dry weight. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
2.5. Statistical analysis Each sample was analyzed three times and each experiment was conducted in triplicate (n = 3). The results were expressed as means ± SE. Statistical comparisons were made by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (P = 0.05) in SAS (SAS, Inc., Cary, NC, USA) package program 9.1. 3. Results and discussion 3.1. Plant growth The response of plants to salt stress has been extensively studied using anatomical, physiological, ecological and molecular approaches (Jampeetong and Brix, 2009; Long et al., 2009; Naidoo et al., 2008). Salinity generally inhibits the development of leaf area, and decreases the leaf fresh/dry weight and chlorophyll content (Azuma et al., 2010; Kchaou et al., 2010; Pérez-Tornero et al., 2009; Suárez, 2011). In our study, however, no statistically significant differences were found for the two consecutive years, when diluted seawater was directly applied to ‘Fuji’/M.9 apple trees (Table 1). ‘Fuji’ trees growth, as indicated by leaf area and weight, was not affected, except for the high dose of triple-spray treatment with 50-fold diluted seawater. The discrepancy may be due to the difference in seawater concentration and the timing of pre-harvest foliar application; our foliar application was initiated at 130 days after full bloom when the fruit begins to enter ripening phase and vegetative growth nearly stops. The reduction in fresh leaf weight under the saline stress, which solely took place in triple-spray treatment with 50-fold diluted seawater, is known to be caused by lower water uptake and reduced water transport to the leaves (Botia et al., 2005; Chakraborty et al., 2012). A decrease in leaf weight is concomitant with the reduced photosynthesis (Azuma et al., 2010; Keutgen and Pawelzik, 2008; Suárez, 2011), and this decrease may be attributed to the reduction of CO2 diffusion to the chloroplast owing to stomatal closure triggered by high salinity stress and the subsequent changes in mesophyll structure (Omoto et al., 2012). However, this did not appear to happen in our treatment regime, because chlorophyll content remained unchanged even with triple-sprayed 50-fold diluted seawater as shown by chlorophyll content SPAD values (Table 1). 3.2. Yield and fruit size Previous researches showed that reduced marketable yield under moderate salinity is mainly due to the decreased fruit size, whereas under high salinity it is attributed to both smaller number
and size of fruit (Magán et al., 2008; Smith et al., 2010). In our study the size (weight) of fruits harvested from ‘Fuji’ trees applied with seawater was not significantly different from that of non-treated controls, except for that of double-spray of 50-fold diluted seawater treatment in 2011 (Table 2), which gave a lower yield mainly due to the reduction in fruit size rather than fruit number. No fruit drop was observed among all treatments with the approximate same number of fruit set after foliar seawater application. Thus, seawater used in this study was in such a moderate concentration that in most occasions both the yield and fruit formation did not appear to be affected by salt treatment.
3.3. Soluble sugars and related enzymes A foliar spray application with 50-fold dilution seawater gave slightly higher soluble solids concentration in fruits than 100-fold dilution seawater application (Table 2). Similar observations were noted in other fruits (Botia et al., 2005; Magán et al., 2008). Salinity may influence the allocation and distribution of photosynthates between the different plant organs. The higher soluble solid concentration is correlated with the higher accumulation of sucrose, glucose, and fructose in plants. Significant accumulations of reducing sugar, sucrose, glucose, and fructose were previously observed in other fruits under salinity stress (Hepaksoy, 2004; Rubio et al., 2009). Fructose is the dominant sugar in apple cultivars, followed by glucose, sucrose, and sorbitol (Wu et al., 2007). But under stress conditions sucrose was the highest, and fructose was the second highest content of sugar. In our study, sucrose phosphate synthase (SPS) activity increased in mesocarp fruit tissues after foliar application with diluted seawater (Table 3). Increased SPS activity was accompanied by the increased total soluble solid concentration (SSC) of sugar contents in the fruits of double and triple-spray of 50-fold diluted seawater treatments (Table 2). However, no correlation between SPS activity and SSC was observed in 100-fold diluted seawater treatments. Like SPS activity, similar observations were made in invertase and sucrose synthase (SS) (Table 3), and this is consistent with the previous result (Al-Hamdani and Barger, 2003). Plant having increased invertase and SS activities often accumulates higher amounts of glucose and fructose. In ‘Fuji’ apple, glucose and fructose contents may be increased as a result of foliar treatment of seawater, because of the increased activities of SS and neutral invertase in fruits (Table 3). Sucrose degradation to fructose and glucose is positively correlated with sucrose synthase (SS) and invertase activities. It is notable that acid invertase activity remained unchanged, whereas neutral invertase activity increased in response to foliar application of seawater. The results combined together suggest that increased concentrations of soluble solids in
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Table 2 The effect of foliar seawater spray on ‘Fuji’ fruit quality at harvest in two consecutive years. Treatment
Size (mm)
Weight (g)
Firmness (kg/0.5cm2 )
SSC (◦ Brix)
TA (%)
20.8a 20.9a 21.8a 21.7a
80.1a 83.6a 82.6a 83.1a
271.3a 270.1a 264.6a 267.2a
3.64a 3.49ab 3.46b 3.63a
14.11a 14.05a 14.24a 14.22a
0.24a 0.23a 0.25a 0.25a
18.1a 15.8c 17.1b 17.1b 16.8b
92.4a 87.0b 90.1ab 93.4a 91.2a
340.7a 284.6b 315.8ab 351.2a 330.7a
3.56b 3.64a 3.56b 3.41b 3.34bc
13.19ab 13.58a 13.54a 13.09b 13.05b
0.20b 0.21ab 0.22a 0.21b 0.21b
Hunter L
a
b
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
49.2a 50.3a 51.3a 50.8a
22.0a 20.6a 20.2a 21.8a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
48.2ab 46.6c 46.9bc 47.5abc 47.6abc
23.1b 26.0a 26.3a 23.9b 24.4b
SW, seawater; SSC, soluble solid concentration; TA, titratable acidity. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests. Table 3 The effect of foliar seawater spray on the activities of SPS, SS, acid invertase, and neutral invertase of ‘Fuji’ fruits in two consecutive years. Treatments
SPS (mol/min g FW)
SS (mol/min g FW)
Acid invertase (mol/min g FW)
Neutral invertase (mol/min g FW)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
8.71ab 7.81b 9.71ab 10.51a
9.34b 9.59b 11.18ab 12.37a
4.87a 5.03a 4.86a 5.17a
3.72b 4.60a 4.62a 4.95a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
6.92d 8.16bc 9.20a 7.29d 8.7ab
8.84d 9.48c 11.03a 9.37c 9.88b
3.78a 4.34a 4.54a 4.32a 4.08a
2.76c 2.96b 3.45a 3.04b 3.08b
SW, seawater; SPS, sucrose phosphate synthase; SS, sucrose synthase. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
fruit are accompanied by the increased activities of SPS, SS, and neutral invertase in 50-fold but not in 100-fold diluted seawater treatments. Edible quality of all fruits decreased gradually in the lapse of storage. A comparison among all treatments showed that seawater treatments did not adversely affect soluble sugar concentration, even after five-month storage (data not shown). 3.4. Color evaluation In 2010, ‘Fuji’ fruit lightness (L-value) and redness (a-value) were not affected by foliar seawater spray (Table 2), even if a high dose of triple spray with 50-fold diluted seawater was applied. On the other hand, in 2011 a more decrease in L-value and b-value, along with a more increase in a-value, was noted as compared to untreated control fruits, indicating the enhanced redness in fruit surface. Thus, foliar seawater spray improved marketable quality of ‘Fuji’ fruit. Among all treatments multiple applications of 50-fold diluted seawater remarkably increased fruit redness. The red coloration of apple is caused by water-soluble pigments anthocyanin that belongs to a class of flavonoid (Li et al., 2002). As shown in Fig. 1, ‘Fuji’ fruit accumulated higher anthocyanin content when treated with foliar seawater spray, indicating that redness expressed by a-value is positively correlated with anthocyanin level. Thus, seawater appears to enhance synthesis of fruit pigments in ‘Fuji’ skin. This assumption agrees well with the color intensities of fruits at the harvest time. Among all treatments, triple-spray of 50-fold diluted seawater yielded the highest anthocyanin content at the harvest (Fig. 1). Increasing levels of anthocyanin were also noted in salt-treated sugarcane (Wahid and
Ghazanfar, 2006). Accumulation of anthocyanin was assumed to allow plants to prevent stress-induced oxidative damage as well as to maintain osmotic balance (Sgherri et al., 2004). Elevated levels of soluble solids including fructose, glucose, and other hexoses in ‘Fuji’ fruits were observed in 2011 by foliar seawater spray (Table 2). Hexose is known to promote anthocyanin production through the shikimate, phenylpropanoid, and flavonoid pathways (Li et al., 2004). This could explain the increased anthocyanin accumulation after foliar seawater spray that triggers physiological changes (Fig. 1). Seawater treatments used in our study did not cause any negative effects on color evolution during storage (data not shown).
Fig. 1. The effect of foliar seawater spray on anthocyanins concentration of ‘Fuji’ fruit in 2011. Data are the mean ± SE of 10 fruits in triplicate.
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Table 4 The effect of foliar seawater spray on mineral concentration of ‘Fuji’ leaves in two consecutive years. Treatment
N (%)
Na (%)
K (%)
Mg (%)
S (%)
Ca (%)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
2.7a 2.7a 2.6a 2.6a
0.033a 0.038a 0.036a 0.036a
0.66a 0.68a 0.68a 0.66a
0.25a 0.23a 0.26a 0.26a
0.45a 0.39ab 0.35b 0.38ab
1.76a 1.64a 1.49a 1.42a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
2.2a 1.9a 2.3a 2.2a 2.1a
0.031a 0.033a 0.035a 0.039a 0.033a
0.63a 0.68a 0.71a 0.69a 0.62a
0.27a 0.27a 0.26a 0.26a 0.28a
0.25c 0.41b 0.57a 0.59a 0.64a
1.66ab 1.46b 1.44b 1.55b 1.65ab
SW, seawater. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
Table 5 The effect of foliar seawater spray on mineral concentration of ‘Fuji’ fruit in two consecutive years. Treatment
Na (%)
K (%)
Mg (%)
S (%)
Ca (%)
Mg/Ca
K/Ca
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
0.013b 0.016ab 0.016ab 0.018a
0.73a 0.71a 0.62ab 0.54b
0.019a 0.021a 0.019a 0.014b
0.12a 0.14a 0.13a 0.09a
0.023a 0.023a 0.021a 0.017b
0.83a 0.88a 0.90a 0.84a
29.39a 30.67a 31.85a 31.45a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
0.012d 0.015cd 0.017bc 0.019ab 0.021a
0.51a 0.53a 0.51a 0.60a 0.60a
0.022a 0.021a 0.022a 0.024a 0.021a
0.04b 0.06b 0.07b 0.07b 0.11a
0.027a 0.026a 0.027a 0.028a 0.024b
0.81a 0.81a 0.83a 0.87a 0.86a
18.92c 20.11b 18.93c 21.71b 24.99a
SW, seawater. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
3.5. Titratable acidity
3.7. Mineral analyses
Titratable acidity (TA) in ‘Fuji’ fruit does not appear to increase in response to foliar seawater spray except for the high dose of triple-spray treatment with 50-fold diluted seawater (Table 2). In line with this, many crops are known to respond to high salt stress by increasing fruit acidity and TA (Maggio et al., 2004; Botia et al., 2005). TA percentage of storage fruit decreased as storage period is prolonged regardless of different foliar seawater treatments. Nevertheless, no significant changes in TA were noted between the stored fruits of seawater treatments and non-treated controls (data not shown), indicating that seawater treatment regime employed in this study had no detrimental effects on ‘Fuji’ fruit quality during storage.
As shown in Table 4, Na+ accumulation in ‘Fuji’ leaves was not significantly different among all treatments in both seasons. Concentration of Na+ in leaf was found to be always under the toxic level of 0.2% regardless of salinity in diluted seawater treatments (Bernstein, 1975). Tolerant genotypes are assumed to have more capacity to either exclude or retain toxic ions than sensitive ones. The leaves of Fuji’/M.9 cultivar used in the present study accumulated around 0.03% of Na+ . Unlike the leaf tissue, foliar seawater application led to significantly higher accumulation of up to 0.02% of Na+ in ‘Fuji’ fruits, as compared to control fruits of all seasons (Table 5). The salt-stressed ‘Fuji’ leaves appear to accumulate less Ca2+ ranging from 1.42% to 1.76% than controls; the least accumulation was observed when the plant was treated at the highest 50-fold dilution with triple-spray frequency (Table 4). Fruit differs in calcium accumulation pattern from leaf in that the least accumulation occurred when the plant was treated at the highest triple-spray frequency of 100-fold dilution dose in both seasons (Table 5). This suggests that together with hexose carbons, a steady-state influx of salt ions into fruit may affect calcium deposition during fruit growth and ripening process. Calcium plays an important role in maintaining the structure and strength of cell wall and membrane that affect fruit firmness (Rubio et al., 2009). It is notable that fruit firmness was lowest when the plant was treated at the triple-spray frequency of 100-fold dilution (Table 2). Unlike Na+ and Ca+2 , the levels of K+ and Mg2+ in leaf and fruit remained relatively constant with respect to the different salinity treatments, except for the fruit of 100-fold diluted triple-spray in
3.6. Firmness The firmness of ‘Fuji’ fruits treated with double-spray of 100fold diluted seawater in 2010 and with triple-spray of 100-fold diluted seawater in 2011 was approximately 5–6% less than that of non-treated controls (Table 2). Statistical analysis showed that there is a tendency of decrease in fruit firmness by foliar treatments used in both years, except that fruit firmness was significantly increased by double-spray of 50-fold diluted seawater in 2011. From the data, salinity generally appears to reduce firmness. Similar observations of decreased pulp thickness and firmness were previously made in pepper fruit under salinity stress (Rubio et al., 2009).
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2010 (Tables 4 and 5). Similar observations were previously made in olive plant by Vigo et al. (2005). Overall, significant mineral deficiency did not appear to occur in leaf and fruit as a result of our foliar spray treatments. Salinity is known to increase K+ /Ca2+ and Mg2+ /Ca2+ ratio as a consequence of cation interactions in many plants (Kamel, 2002). K+ /Ca2+ ratio of ‘Fuji’ fruits remained constant in 2010 but appeared to increase in 2011 and the ratio of K+ /Ca2+ ranged from 18.9 to 31.9 (Table 5). Although the reason for this discrepancy is unknown, K+ /Ca2+ ratio of 2011 is consistent with the previous finding that variation of this ratio is between 19 and 46 in apple fruit (Dilmaghani et al., 2005). In contrast to K+ /Ca2+ , divalent cation ratio of Mg2+ /Ca2+ in apple fruit was stably maintained in both seasons. The multivariate analysis of apple fruits showed that K+ /Ca2+ ratio provides the best measure to distinguish between the fruits with and without physiological disorders, followed by Mg2+ /Ca2+ and N/Ca2+ ratio (Dilmaghani et al., 2005). Accordingly, our foliar spray treatments did not appear to bring about a significantly dysfunctional fruit physiology, as judged by the K+ /Ca2+ and Mg2+ /Ca2+ ratios. Fruit firmness is positively correlated with fruit Ca2+ concentration and is inversely correlated with K+ /Ca2+ ratio (Serrano et al., 2004). This finding may explain why firmness of ‘Fuji’ fruits of foliar seawater treatment was lower than untreated control at harvest. However, the loss of fruit firmness was not further accelerated during storage, since no significantly different strength of firmness was noted at different storage intervals for up to120 days (data not shown). 4. Conclusions The foliar seawater treatments used in the present study could be useful for increasing soluble solids and activities of SPS, SS, and neutral invertase in apple fruit, and this may in turn contribute to the enhanced quality of postharvest fruit. Also, anthocyanin concentration can be increased by foliar seawater, which will make fruits more attractive in the fresh-market. Although fruit firmness and some minerals such as Ca2+ were reduced, the beneficial effects on fruit quality without affecting the marketable yield outweigh the negative effects exerted by foliar seawater application. Further investigations are needed using different apple cultivars with respect to salt tolerance as well as yield and fruit quality. Acknowledgements This research was sponsored by the National Natural Science Foundation of China (Project No. 31201585, 31000916). We also gratefully acknowledge the financial support provided by Zhejiang A & F University (Project No. 2010FR089, 2009FR047). References Al-Hamdani, S.H., Barger, T.W., 2003. Influence of water stress on selected physiological response of three sorghum genotypes. Italian Journal of Agronomy 7, 15–22. Azuma, R., Ito, N., Nakayama, N., Suwa, R., Nguyen, N.T., Larrinaga-Mayoral, J.Á., Esaka, M., Fujiyama, H., Saneoka, H., 2010. Fruits are more sensitive to salinity than leaves and stems in pepper plants (Capsicum annuum L.). Scientia Horticulturae 125, 171–178. Bernstein, L., 1975. Effect of salinity on sodicity and on plant growth. Annual Review of Phytopathology 13, 295–312. Botia, P., Navarro, J.M., Cerda, A., Martinez, V., 2005. Yield and fruit quality of two melon cultivars irrigated with saline water at different stages of development. European Journal of Agronomy 23, 243–253. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Luque, T., Figueroa, M.E., 2011. Physiological responses to salinity in the yellow-horned poppy Glaucium flavum. Plant Physiology and Biochemistry 49, 186–194. Chakraborty, K., Sairam, R.K., Bhattacharya, R.C., 2012. Differential expression of salt overly sensitive pathway genes determines salinity stress tolerance in Brassica genotypes. Plant Physiology and Biochemistry 51, 90–101.
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Contents lists available at SciVerse ScienceDirect
Agricultural Water Management journal homepage: www.elsevier.com/locate/agwat
Vegetative growth, mineral change, and fruit quality of ‘Fuji’ tree as affected by foliar seawater application W.W. Zheng a,b,1 , I.J. Chun b,1 , S.B. Hong c , Y.X. Zang a,∗ a b c
College of Agriculture and Food Science, Zhejiang A & F University, Lin’an, Hangzhou 311300, China School of Bioresource Science, Andong National University, Andong 760-749, Republic of Korea Department of Biotechnology, University of Houston Clear Lake, Houston, TX 77058-1098, USA
a r t i c l e
i n f o
Article history: Received 5 March 2013 Accepted 5 May 2013 Available online 31 May 2013 Keywords: Foliar spray Seawater Apple Fruit quality Soluble solids Anthocyanin
a b s t r a c t Extensive researches have been conducted over the decades to investigate the effects of seawater on many crop plants either by irrigation or foliar spray in an attempt to enhance the yield and quality. The purpose of this study was to quantitatively determine the effects of different foliar seawater sprays on vegetative growth, fruit quality and yield of eight-year-old ‘Fuji’/M.9 apple trees. A field experiment was conducted using two different concentrations of seawater (100 or 50-fold dilution) starting from 130 DAFB (days after full bloom) at 5-day intervals between the treatments. Foliar seawater sprays to ‘Fuji’ tree led to increased levels of fruit soluble solids along with higher activities of sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (SS) (EC 2.4.1.13) and neutral invertase (EC 3.2.1.26). In addition, a significant increase in anthocyanin concentration was observed, especially when ‘Fuji’ trees were sprayed three times with 50-fold diluted seawater. Foliar seawater sprays also resulted in increases in Na+ concentration and K+ /Ca2+ ratio in fruit. In contrast to fruit, the levels of N, Na+ , K+ , Mg2+ in leaves remained unchanged regardless of different seawater treatments. Moreover, foliar seawater sprays did not affect the vegetative growth since leaf area, leaf fresh weight, shoot elongation, and chlorophyll content did not differ significantly from those of control plants. The results suggest that foliar seawater treatments could be useful for improving fruit quality without affecting the yield. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plants are frequently exposed to diverse abiotic stress conditions, such as high and low temperature, water deficit, UV light, oxidative stress, heavy metal toxicity and salinity. These factors greatly influence the growth and development of plants, as well as the crop productivity. In the course of evolution, plants have developed mechanisms to cope with and adapt to various adverse environmental stresses, most often by accumulating specific defensive secondary metabolites through signaling pathways (Ramakrishna and Ravishankar, 2011). So, moderate environmental stress conditions could be beneficial to improve the quality and to control the growth of certain crops. Salinity usually leads to a marked increase in soluble sugars and protein that may act as osmolytes for lowering the osmotic potential (Chen et al., 2009).
∗ Corresponding author at: College of Agriculture and Food Science, Zhejiang A & F University, 88 Huanchengbei Road, Lin’an, Hangzhou 311300, China. Tel.: +86 571 63742133. E-mail addresses: [email protected], [email protected] (Y.X. Zang). 1 These authors contributed equally to this work. 0378-3774/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.agwat.2013.05.010
Seawater has a salinity of nearly 3.5%, and the major components of the dissolved salts are sodium, magnesium, calcium and potassium; excessive accumulation is harmful to plant by causing osmotic injury and specific ion toxicity (Millero et al., 2008). Exogenous high salt application caused a reduction in pigment concentrations, as well as the uptake of K+ , Ca2+ , Mg2+ , and N in tissues (Cambrollé et al., 2011; Chakraborty et al., 2012). It was reported that high salinity of seawater reduces the ratio of Mg2+ / Na+ ; thus salinity may decrease photosynthesis by reducing concentration of Mg2+ , a major component of leaf chlorophyll. Several lines of evidence have suggested that seawater irrigation affects contents of specific metabolites in higher plants (Ferrante et al., 2011; Long et al., 2009; Ventura et al., 2011; Zhang et al., 2009). Moderate seawater treatment resulted in the marked accumulation of soluble sugar, protein, polyphenol, carotene, fatty acid (Long et al., 2008; Ventura et al., 2011). However, as the salinity was further increased, levels of electrolyte leakage and proline concentration rose, and this was typically accompanied by reduced levels of chlorophyll and carotenoid and more protein degradation (Ferrante et al., 2011; Long et al., 2009; Misra and Saxena, 2009). The high salinity stress resulted in reduced growth rate and biomass, shorter stature, smaller leaves, nutritional deficiency and mineral disorders (Ferrante et al., 2011; Long et al., 2009; Sekmen et al.,
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2012; Ventura et al., 2011; Zhang et al., 2009). The activity of superoxide dismutase was transiently stimulated and then decreased gradually under high salinity stress. Song and Ryou (2008) reported that as seawater spray frequency increased in grapevines, leaf browning and leaf drop occurred along with lower concentrations of soluble solids and anthocyanin in fruits. Thus, use of appropriate levels of salinity is important in agricultural practices, especially for the fruit quality improvement. Unlike grapevines, there was no leaf toxicity or symptoms of chlorosis and necrosis observed when olive trees were irrigated with up to 21.3% diluted seawater for five months (Vigo et al., 2005). The effects of saline water irrigation on growth and fruit quality have been investigated in other crop plants including melon and tomato (Botia et al., 2005; Maggio et al., 2004). To date a few scientific data about the effect of diluted seawater spray treatments on fruit quality of apple (Malus domestica Borkh.) have been reported. There is a lack of information on the proper timing and concentrations of seawater application in apple trees to improve fruit quality. In order to support agricultural extension service to fruit growers for the scientific management in seawater application, we investigated the effects of foliar seawater sprays on growth, fruit quality, and mineral nutrients of ‘Fuji’/M.9 apple over two consecutive years. 2. Materials and methods 2.1. Field condition, plant material and seawater dilution management This study was carried out on eight-year-old ‘Fuji’/M.9 cultivar apple trees for two consecutive seasons of 2010 and 2011. All the trees were planted with a distance of 3.8 m between rows and 1.8 m within the rows. Trees of 3 m high and 1.5 m wide were trained as a slender spindle, and those with similar growth vigor were selected as test materials. Soil properties were tested as pH 6.52, 13.2% organic matter, 0.75 g kg−1 available phosphorus, 0.46 g kg−1 available nitrogen, 0.48 g kg−1 available potassium. The trees were placed at yearly average temperature 11.8 ◦ C, sunshine hours 2698.4 h, average humidity 68%, precipitation 651.9 mm. The surface seawater was purified through a simple reverse osmosis system to eliminate dirt and microorganism and to reduce ion content. The desalted seawater (0.35 g kg−1 K+ , 0.31 g kg−1 Ca2+ , 0.94 g kg−1 Mg2+ , 0.89 g kg−1 Na+ ) was used for treatment. The experimental design consisted of nine treatments crossing two seawater concentrations. A standard randomized block design with six replications including one tree in replicate of each treatment was used. The trees to be tested were isolated in a distance with at least one untreated guard tree. All the trees were grown under the same environmental conditions with the same doses of irrigation, fertilization and phytosanitary treatments. All of the foliar seawater applications were performed by a hand sprayer (knapsack) at 20 L/six trees in the morning, starting from 130 DAFB (days after full bloom) at 5-day intervals between the sprays. In 2010, single, double and triple spray treatments with 100-fold diluted seawater were implemented, along with control treatment of water. In 2011, double and triple spray treatments with both 50- and 100-fold diluted seawater were implemented. The apple fruits were harvested at the time of optimum maturity which is at 175 DAFB. 2.2. Sampling and fruit characteristics Leaf area, fresh/dry weight, shoot elongation, and chlorophyll content were determined at 45 DAFFS (days after the first foliar spray application). Meanwhile fruits were selected based on the medium size, uniform shape and color scales in the morning.
To determine physical and chemical characteristics of fruit, each treated fruit sample was divided into two groups; one was used immediately and the other was stored in storage room maintaining 4 ◦ C and 80–85% relative humidity. Coloration was determined using a Color-Reader (KONICA MINOLTA SENSING, INC. Japan). Fruit firmness was measured by compression of individual apple fruits with a fruit texture analyzer (GUSS.ZA/GS-14). Soluble solid concentration and titratable acidity in apple juice were determined by a digital refractometer (PR-101, Cat. No. 3412, ATAGO, Japan) and a digital fruit acidity analyzer (Model: GMK-708, GVK, South Korea), respectively. Anthocyanin contents were determined as described by Rosso and Mercadante (2007). About 5 g of fresh fruit peel tissues from six randomly selected fruits in each treatment was pulverized and extracted with 1% (v/v) methanol. The homogenate was centrifuged at 19,000 × g for 15 min, and diluted with 1% HCl–methanol to a final volume of 500 ml. Absorbance of the diluent was measured at 530 nm. 2.3. Mineral analysis Leaves and fruits were washed in deionized water and dried at 80 ◦ C for 48 h, ground and stored in an oven at 60 ◦ C until analysis. Samples were then ashed in a muffle furnace at 600 ◦ C overnight, and dissolved in 0.1 N HCl. The N level in leaves was analyzed by Kjeldahl method (Page et al., 1982). The levels of Na+ , Ca2+ , Mg2+ , and K+ in fruits and leaves were analyzed by ICP-OES (Optima2000DV, Perkin Elmer Instruments Inc., CT, USA). All analyses were conducted in triplicate. 2.4. Enzyme activity assays Frozen mesocarp of pulp (0.5 g) was pulverized with pre-cooled mortar and pestle and mixed with 4 ml of extraction buffer containing 50 mM HEPES–NaOH (pH 7.5), 5 mM MgCl2 , 1 mM EDTA, 2.5 mM dithiothreitol (DTT), 0.2% of bovine serum albumin (BSA) and 0.05% Triton X-100. The mixture was centrifuged at 4 ◦ C for 10 min, and supernatant was desalted immediately through a Sephadex G-25 column pre-equilibrated with 50 mM Hepes–NaOH (pH 7.5), 5 mM MgCl2 , 2.5 mM DTT and 0.2% BSA. All procedures were carried out at 4 ◦ C or lower. Sucrose phosphate synthase (SPS) activity was assayed in a reaction mixture (0.3 ml) containing 7.5 mM UDP-glucose, 7.5 mM fructose-6-P, 10 mM glucose-6-P, 1.5 mM MgCl2 , 100 mM Tris–HCl (pH 7.5) and 50 l of the desalted extract at 30 ◦ C. The reaction was terminated by adding 75 l of 4 M NaOH after 20 min. The boiled enzyme was used as a control in each assay. Unreacted fructose-6P was removed by placing the reaction mixture in a boiling water bath for 10 min. After cooling, 0.25 ml of 0.1% (v/v) resorcinol in 95% (v/v) ethanol and 0.75 ml of 30% (w/v) HCl were added. The mixture was then incubated at 80 ◦ C for 8 min and allowed to cool, and its absorbance was determined at 540 nm. The procedure for sucrose synthase (SS) assay was identical to that of SPS except that the reaction mixtures contained 10 mM fructose instead of fructose-6-P. One unit of SPS and SS is defined as amount of the enzyme producing 1 mol sucrose per min. Acid and neutral invertase assays were performed at 30 ◦ C for 15 min in a total reaction volume of 300 l containing 50 l of desalted protein extract, 120 mM sucrose, and 100 mM citratephosphate (pH 5.0, for acid invertase), or 120 mM Hepes–NaOH buffer (pH 7.5, for neutral invertase). The reactions were terminated by boiling in a water bath for 5 min and the product (glucose) was assayed using dinitrosalicylic acid agent. Each assay had a boiled enzyme as control. One unit of acid and neutral invertase is defined as amount of the enzyme producing 1 mol glucose per min.
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Table 1 The effect of foliar seawater spray on vegetative growth of ‘Fuji’ trees in two consecutive years. Treatment
Leaf area (cm2 )
Leaf FW (g)
Leaf DW (g)
SPAD value
Shoot elongation (cm)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
28.1b 27.6bc 29.8a 27.2c
0.68a 0.69a 0.73a 0.64b
0.32a 0.30a 0.30a 0.30a
61.1a 61.6a 61.2a 60.7a
29.2a 32.5a 31.5a 30.7a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
27.3a 26.8a 23.4b 27.6a 27.7a
0.75a 0.71a 0.60b 0.76a 0.65b
0.29a 0.30a 0.24b 0.31a 0.26b
55.6a 57.7a 54.1a 60.8a 56.5a
32.1a 28.5a 28.9a 30.6a 34.5a
SW, seawater; FW, fresh weight; DW, dry weight. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
2.5. Statistical analysis Each sample was analyzed three times and each experiment was conducted in triplicate (n = 3). The results were expressed as means ± SE. Statistical comparisons were made by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (P = 0.05) in SAS (SAS, Inc., Cary, NC, USA) package program 9.1. 3. Results and discussion 3.1. Plant growth The response of plants to salt stress has been extensively studied using anatomical, physiological, ecological and molecular approaches (Jampeetong and Brix, 2009; Long et al., 2009; Naidoo et al., 2008). Salinity generally inhibits the development of leaf area, and decreases the leaf fresh/dry weight and chlorophyll content (Azuma et al., 2010; Kchaou et al., 2010; Pérez-Tornero et al., 2009; Suárez, 2011). In our study, however, no statistically significant differences were found for the two consecutive years, when diluted seawater was directly applied to ‘Fuji’/M.9 apple trees (Table 1). ‘Fuji’ trees growth, as indicated by leaf area and weight, was not affected, except for the high dose of triple-spray treatment with 50-fold diluted seawater. The discrepancy may be due to the difference in seawater concentration and the timing of pre-harvest foliar application; our foliar application was initiated at 130 days after full bloom when the fruit begins to enter ripening phase and vegetative growth nearly stops. The reduction in fresh leaf weight under the saline stress, which solely took place in triple-spray treatment with 50-fold diluted seawater, is known to be caused by lower water uptake and reduced water transport to the leaves (Botia et al., 2005; Chakraborty et al., 2012). A decrease in leaf weight is concomitant with the reduced photosynthesis (Azuma et al., 2010; Keutgen and Pawelzik, 2008; Suárez, 2011), and this decrease may be attributed to the reduction of CO2 diffusion to the chloroplast owing to stomatal closure triggered by high salinity stress and the subsequent changes in mesophyll structure (Omoto et al., 2012). However, this did not appear to happen in our treatment regime, because chlorophyll content remained unchanged even with triple-sprayed 50-fold diluted seawater as shown by chlorophyll content SPAD values (Table 1). 3.2. Yield and fruit size Previous researches showed that reduced marketable yield under moderate salinity is mainly due to the decreased fruit size, whereas under high salinity it is attributed to both smaller number
and size of fruit (Magán et al., 2008; Smith et al., 2010). In our study the size (weight) of fruits harvested from ‘Fuji’ trees applied with seawater was not significantly different from that of non-treated controls, except for that of double-spray of 50-fold diluted seawater treatment in 2011 (Table 2), which gave a lower yield mainly due to the reduction in fruit size rather than fruit number. No fruit drop was observed among all treatments with the approximate same number of fruit set after foliar seawater application. Thus, seawater used in this study was in such a moderate concentration that in most occasions both the yield and fruit formation did not appear to be affected by salt treatment.
3.3. Soluble sugars and related enzymes A foliar spray application with 50-fold dilution seawater gave slightly higher soluble solids concentration in fruits than 100-fold dilution seawater application (Table 2). Similar observations were noted in other fruits (Botia et al., 2005; Magán et al., 2008). Salinity may influence the allocation and distribution of photosynthates between the different plant organs. The higher soluble solid concentration is correlated with the higher accumulation of sucrose, glucose, and fructose in plants. Significant accumulations of reducing sugar, sucrose, glucose, and fructose were previously observed in other fruits under salinity stress (Hepaksoy, 2004; Rubio et al., 2009). Fructose is the dominant sugar in apple cultivars, followed by glucose, sucrose, and sorbitol (Wu et al., 2007). But under stress conditions sucrose was the highest, and fructose was the second highest content of sugar. In our study, sucrose phosphate synthase (SPS) activity increased in mesocarp fruit tissues after foliar application with diluted seawater (Table 3). Increased SPS activity was accompanied by the increased total soluble solid concentration (SSC) of sugar contents in the fruits of double and triple-spray of 50-fold diluted seawater treatments (Table 2). However, no correlation between SPS activity and SSC was observed in 100-fold diluted seawater treatments. Like SPS activity, similar observations were made in invertase and sucrose synthase (SS) (Table 3), and this is consistent with the previous result (Al-Hamdani and Barger, 2003). Plant having increased invertase and SS activities often accumulates higher amounts of glucose and fructose. In ‘Fuji’ apple, glucose and fructose contents may be increased as a result of foliar treatment of seawater, because of the increased activities of SS and neutral invertase in fruits (Table 3). Sucrose degradation to fructose and glucose is positively correlated with sucrose synthase (SS) and invertase activities. It is notable that acid invertase activity remained unchanged, whereas neutral invertase activity increased in response to foliar application of seawater. The results combined together suggest that increased concentrations of soluble solids in
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Table 2 The effect of foliar seawater spray on ‘Fuji’ fruit quality at harvest in two consecutive years. Treatment
Size (mm)
Weight (g)
Firmness (kg/0.5cm2 )
SSC (◦ Brix)
TA (%)
20.8a 20.9a 21.8a 21.7a
80.1a 83.6a 82.6a 83.1a
271.3a 270.1a 264.6a 267.2a
3.64a 3.49ab 3.46b 3.63a
14.11a 14.05a 14.24a 14.22a
0.24a 0.23a 0.25a 0.25a
18.1a 15.8c 17.1b 17.1b 16.8b
92.4a 87.0b 90.1ab 93.4a 91.2a
340.7a 284.6b 315.8ab 351.2a 330.7a
3.56b 3.64a 3.56b 3.41b 3.34bc
13.19ab 13.58a 13.54a 13.09b 13.05b
0.20b 0.21ab 0.22a 0.21b 0.21b
Hunter L
a
b
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
49.2a 50.3a 51.3a 50.8a
22.0a 20.6a 20.2a 21.8a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
48.2ab 46.6c 46.9bc 47.5abc 47.6abc
23.1b 26.0a 26.3a 23.9b 24.4b
SW, seawater; SSC, soluble solid concentration; TA, titratable acidity. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests. Table 3 The effect of foliar seawater spray on the activities of SPS, SS, acid invertase, and neutral invertase of ‘Fuji’ fruits in two consecutive years. Treatments
SPS (mol/min g FW)
SS (mol/min g FW)
Acid invertase (mol/min g FW)
Neutral invertase (mol/min g FW)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
8.71ab 7.81b 9.71ab 10.51a
9.34b 9.59b 11.18ab 12.37a
4.87a 5.03a 4.86a 5.17a
3.72b 4.60a 4.62a 4.95a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
6.92d 8.16bc 9.20a 7.29d 8.7ab
8.84d 9.48c 11.03a 9.37c 9.88b
3.78a 4.34a 4.54a 4.32a 4.08a
2.76c 2.96b 3.45a 3.04b 3.08b
SW, seawater; SPS, sucrose phosphate synthase; SS, sucrose synthase. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
fruit are accompanied by the increased activities of SPS, SS, and neutral invertase in 50-fold but not in 100-fold diluted seawater treatments. Edible quality of all fruits decreased gradually in the lapse of storage. A comparison among all treatments showed that seawater treatments did not adversely affect soluble sugar concentration, even after five-month storage (data not shown). 3.4. Color evaluation In 2010, ‘Fuji’ fruit lightness (L-value) and redness (a-value) were not affected by foliar seawater spray (Table 2), even if a high dose of triple spray with 50-fold diluted seawater was applied. On the other hand, in 2011 a more decrease in L-value and b-value, along with a more increase in a-value, was noted as compared to untreated control fruits, indicating the enhanced redness in fruit surface. Thus, foliar seawater spray improved marketable quality of ‘Fuji’ fruit. Among all treatments multiple applications of 50-fold diluted seawater remarkably increased fruit redness. The red coloration of apple is caused by water-soluble pigments anthocyanin that belongs to a class of flavonoid (Li et al., 2002). As shown in Fig. 1, ‘Fuji’ fruit accumulated higher anthocyanin content when treated with foliar seawater spray, indicating that redness expressed by a-value is positively correlated with anthocyanin level. Thus, seawater appears to enhance synthesis of fruit pigments in ‘Fuji’ skin. This assumption agrees well with the color intensities of fruits at the harvest time. Among all treatments, triple-spray of 50-fold diluted seawater yielded the highest anthocyanin content at the harvest (Fig. 1). Increasing levels of anthocyanin were also noted in salt-treated sugarcane (Wahid and
Ghazanfar, 2006). Accumulation of anthocyanin was assumed to allow plants to prevent stress-induced oxidative damage as well as to maintain osmotic balance (Sgherri et al., 2004). Elevated levels of soluble solids including fructose, glucose, and other hexoses in ‘Fuji’ fruits were observed in 2011 by foliar seawater spray (Table 2). Hexose is known to promote anthocyanin production through the shikimate, phenylpropanoid, and flavonoid pathways (Li et al., 2004). This could explain the increased anthocyanin accumulation after foliar seawater spray that triggers physiological changes (Fig. 1). Seawater treatments used in our study did not cause any negative effects on color evolution during storage (data not shown).
Fig. 1. The effect of foliar seawater spray on anthocyanins concentration of ‘Fuji’ fruit in 2011. Data are the mean ± SE of 10 fruits in triplicate.
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Table 4 The effect of foliar seawater spray on mineral concentration of ‘Fuji’ leaves in two consecutive years. Treatment
N (%)
Na (%)
K (%)
Mg (%)
S (%)
Ca (%)
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
2.7a 2.7a 2.6a 2.6a
0.033a 0.038a 0.036a 0.036a
0.66a 0.68a 0.68a 0.66a
0.25a 0.23a 0.26a 0.26a
0.45a 0.39ab 0.35b 0.38ab
1.76a 1.64a 1.49a 1.42a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
2.2a 1.9a 2.3a 2.2a 2.1a
0.031a 0.033a 0.035a 0.039a 0.033a
0.63a 0.68a 0.71a 0.69a 0.62a
0.27a 0.27a 0.26a 0.26a 0.28a
0.25c 0.41b 0.57a 0.59a 0.64a
1.66ab 1.46b 1.44b 1.55b 1.65ab
SW, seawater. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
Table 5 The effect of foliar seawater spray on mineral concentration of ‘Fuji’ fruit in two consecutive years. Treatment
Na (%)
K (%)
Mg (%)
S (%)
Ca (%)
Mg/Ca
K/Ca
2010 Control SW100 dilution for single spray SW100 dilution for double spray SW100 dilution for tri-spray
0.013b 0.016ab 0.016ab 0.018a
0.73a 0.71a 0.62ab 0.54b
0.019a 0.021a 0.019a 0.014b
0.12a 0.14a 0.13a 0.09a
0.023a 0.023a 0.021a 0.017b
0.83a 0.88a 0.90a 0.84a
29.39a 30.67a 31.85a 31.45a
2011 Control SW50 dilution for double spray SW50 dilution for tri-spray SW100 dilution for double spray SW100 dilution for tri-spray
0.012d 0.015cd 0.017bc 0.019ab 0.021a
0.51a 0.53a 0.51a 0.60a 0.60a
0.022a 0.021a 0.022a 0.024a 0.021a
0.04b 0.06b 0.07b 0.07b 0.11a
0.027a 0.026a 0.027a 0.028a 0.024b
0.81a 0.81a 0.83a 0.87a 0.86a
18.92c 20.11b 18.93c 21.71b 24.99a
SW, seawater. The means with the same letter within column (in the same year but not in the different year) are not significantly different at P = 0.05 according to Duncan’s multiple range tests.
3.5. Titratable acidity
3.7. Mineral analyses
Titratable acidity (TA) in ‘Fuji’ fruit does not appear to increase in response to foliar seawater spray except for the high dose of triple-spray treatment with 50-fold diluted seawater (Table 2). In line with this, many crops are known to respond to high salt stress by increasing fruit acidity and TA (Maggio et al., 2004; Botia et al., 2005). TA percentage of storage fruit decreased as storage period is prolonged regardless of different foliar seawater treatments. Nevertheless, no significant changes in TA were noted between the stored fruits of seawater treatments and non-treated controls (data not shown), indicating that seawater treatment regime employed in this study had no detrimental effects on ‘Fuji’ fruit quality during storage.
As shown in Table 4, Na+ accumulation in ‘Fuji’ leaves was not significantly different among all treatments in both seasons. Concentration of Na+ in leaf was found to be always under the toxic level of 0.2% regardless of salinity in diluted seawater treatments (Bernstein, 1975). Tolerant genotypes are assumed to have more capacity to either exclude or retain toxic ions than sensitive ones. The leaves of Fuji’/M.9 cultivar used in the present study accumulated around 0.03% of Na+ . Unlike the leaf tissue, foliar seawater application led to significantly higher accumulation of up to 0.02% of Na+ in ‘Fuji’ fruits, as compared to control fruits of all seasons (Table 5). The salt-stressed ‘Fuji’ leaves appear to accumulate less Ca2+ ranging from 1.42% to 1.76% than controls; the least accumulation was observed when the plant was treated at the highest 50-fold dilution with triple-spray frequency (Table 4). Fruit differs in calcium accumulation pattern from leaf in that the least accumulation occurred when the plant was treated at the highest triple-spray frequency of 100-fold dilution dose in both seasons (Table 5). This suggests that together with hexose carbons, a steady-state influx of salt ions into fruit may affect calcium deposition during fruit growth and ripening process. Calcium plays an important role in maintaining the structure and strength of cell wall and membrane that affect fruit firmness (Rubio et al., 2009). It is notable that fruit firmness was lowest when the plant was treated at the triple-spray frequency of 100-fold dilution (Table 2). Unlike Na+ and Ca+2 , the levels of K+ and Mg2+ in leaf and fruit remained relatively constant with respect to the different salinity treatments, except for the fruit of 100-fold diluted triple-spray in
3.6. Firmness The firmness of ‘Fuji’ fruits treated with double-spray of 100fold diluted seawater in 2010 and with triple-spray of 100-fold diluted seawater in 2011 was approximately 5–6% less than that of non-treated controls (Table 2). Statistical analysis showed that there is a tendency of decrease in fruit firmness by foliar treatments used in both years, except that fruit firmness was significantly increased by double-spray of 50-fold diluted seawater in 2011. From the data, salinity generally appears to reduce firmness. Similar observations of decreased pulp thickness and firmness were previously made in pepper fruit under salinity stress (Rubio et al., 2009).
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2010 (Tables 4 and 5). Similar observations were previously made in olive plant by Vigo et al. (2005). Overall, significant mineral deficiency did not appear to occur in leaf and fruit as a result of our foliar spray treatments. Salinity is known to increase K+ /Ca2+ and Mg2+ /Ca2+ ratio as a consequence of cation interactions in many plants (Kamel, 2002). K+ /Ca2+ ratio of ‘Fuji’ fruits remained constant in 2010 but appeared to increase in 2011 and the ratio of K+ /Ca2+ ranged from 18.9 to 31.9 (Table 5). Although the reason for this discrepancy is unknown, K+ /Ca2+ ratio of 2011 is consistent with the previous finding that variation of this ratio is between 19 and 46 in apple fruit (Dilmaghani et al., 2005). In contrast to K+ /Ca2+ , divalent cation ratio of Mg2+ /Ca2+ in apple fruit was stably maintained in both seasons. The multivariate analysis of apple fruits showed that K+ /Ca2+ ratio provides the best measure to distinguish between the fruits with and without physiological disorders, followed by Mg2+ /Ca2+ and N/Ca2+ ratio (Dilmaghani et al., 2005). Accordingly, our foliar spray treatments did not appear to bring about a significantly dysfunctional fruit physiology, as judged by the K+ /Ca2+ and Mg2+ /Ca2+ ratios. Fruit firmness is positively correlated with fruit Ca2+ concentration and is inversely correlated with K+ /Ca2+ ratio (Serrano et al., 2004). This finding may explain why firmness of ‘Fuji’ fruits of foliar seawater treatment was lower than untreated control at harvest. However, the loss of fruit firmness was not further accelerated during storage, since no significantly different strength of firmness was noted at different storage intervals for up to120 days (data not shown). 4. Conclusions The foliar seawater treatments used in the present study could be useful for increasing soluble solids and activities of SPS, SS, and neutral invertase in apple fruit, and this may in turn contribute to the enhanced quality of postharvest fruit. Also, anthocyanin concentration can be increased by foliar seawater, which will make fruits more attractive in the fresh-market. Although fruit firmness and some minerals such as Ca2+ were reduced, the beneficial effects on fruit quality without affecting the marketable yield outweigh the negative effects exerted by foliar seawater application. Further investigations are needed using different apple cultivars with respect to salt tolerance as well as yield and fruit quality. Acknowledgements This research was sponsored by the National Natural Science Foundation of China (Project No. 31201585, 31000916). We also gratefully acknowledge the financial support provided by Zhejiang A & F University (Project No. 2010FR089, 2009FR047). References Al-Hamdani, S.H., Barger, T.W., 2003. Influence of water stress on selected physiological response of three sorghum genotypes. Italian Journal of Agronomy 7, 15–22. Azuma, R., Ito, N., Nakayama, N., Suwa, R., Nguyen, N.T., Larrinaga-Mayoral, J.Á., Esaka, M., Fujiyama, H., Saneoka, H., 2010. Fruits are more sensitive to salinity than leaves and stems in pepper plants (Capsicum annuum L.). Scientia Horticulturae 125, 171–178. Bernstein, L., 1975. Effect of salinity on sodicity and on plant growth. Annual Review of Phytopathology 13, 295–312. Botia, P., Navarro, J.M., Cerda, A., Martinez, V., 2005. Yield and fruit quality of two melon cultivars irrigated with saline water at different stages of development. European Journal of Agronomy 23, 243–253. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Luque, T., Figueroa, M.E., 2011. Physiological responses to salinity in the yellow-horned poppy Glaucium flavum. Plant Physiology and Biochemistry 49, 186–194. Chakraborty, K., Sairam, R.K., Bhattacharya, R.C., 2012. Differential expression of salt overly sensitive pathway genes determines salinity stress tolerance in Brassica genotypes. Plant Physiology and Biochemistry 51, 90–101.
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