Foliar application of calcium chloride and borax affects the fruit skin strength and cracking incidence in litchi (Litchi chinensis Sonn.) cultivars

The influence of foliar application of calcium chloride and borax calcium on fruit skin strength and cracking incidence in litchi (Litchi chinensis Sonn.) fruit was investigated at 25 days interval and also the evaluation of fruit skin calcium and boron contents, skin strength, ion leakage from skin discs and fruit cracking in four litchi cultivars. Significant variations were observed in different litchi cultivars and CaCl2 and borax treatments. The means revealed that cultivar Gola had the highest fruit skin calcium content (7.67 mg/100 g DW), ion leakage from skin discs (31.11%) and fruit cracking (21.91%) but the least skin strength (2.44 kg cm -2 ). Skin boron content was the highest in cultivars Gola (0.203 mg/100 g DW). Cultivar Bedana had the highest fruit skin strength (3.39 kg cm -2 ) and the least mean ion leakage (18.86%) and fruit cracking (13.26%). Foliar application of CaCl2 and borax significantly increased the mean fruit skin calcium content (4.79 mg/100 g DW), boron content (0.109 mg/100 g DW), and skin strength (2.43 kg cm -2 ) from the least in the control to the highest 8.88 mg/100 g DW , 0.247 mg/100 g DW and 3.01 kg cm -2 with CaCl2 3 + boron 1.5% treatment respectively, while ion leakage (35.17%) and fruit cracking (25.40%) in the control decreased to 16.17 and 11.14% respectively with CaCl2 3% + boron 1.5% treatment. The Ca content of the litchi fruit skin in the control vs. Rest, control vs. CaCl2 and control vs. CaCl2 + borax increased from 4.79 to 7.36 mg/100 g DW, 4.85 and 8.20 mg/100 g DW respectively. The boron content of the fruit skin also increased in the control vs. Rest (0.11 vs. 0.21 mg/100 g DW), control vs. CaCl2 (0.11 vs. 0.14 mg/100 g DW), control vs. CaCl2 + Borax (0.11 vs. 0.23 mg/100 g DW) and CaCl2 vs. CaCl2 + Borax (0.14 vs. 0.23 mg/100 g DW) treatments. The mean ion leakage decreased from 35.17 in the control to 24.10% in control vs. Rest, 30.26% in control vs. CaCl2 and 22.05% in control vs. CaCl2+Borax. Similarly, the ion leakage decline from 30.06 to 22.05 in CaCl2 vs. CaCl2 + Borax means.


INTRODUCTION
The litchi (Litchi chinensis Sonn.) is a popular fruit of the family Sapindaceae (Huang et al., 2005).The fruits are high in sugar and contain several vitamins and minerals (Robert et al., 2000).It can be processed into juice, wine, pickles, preserves, ice cream and yoghurt (Huang et al., 2004).In Pakistan it is being grown in the Sind, Punjab *Corresponding author.E-Mail: abdurrabaup@gmail.com.Tel: 092-91-9216541, 092-300-9055870.Fax: 092-91-9216520.and parts of KP province on a reported total area of 572 ha with a production average of 9250 tons (Shah, 2003).The litchi is in high demand due to its unique taste, health benefits and cuisine value.It therefore, captures good price in the markets.Much of the litchi fruit is used in fresh form but a small percentage is processed (Rajwana et al., 2010).The litchi is a non-climacteric fruit, which is characterized by low rates of respiration and ethylene production (Chen et al., 1986).The edible portion of the fruit is well protected against water loss, insects and pests by a rough skin.Yet, the litchi fruits have a short post-harvest life (Kaiser, 1994).The pericarp of litchi fruit rapidly turns brown and brittle resulting in skin cracking, which exposes the aril to pathogens (Kaiser, 1994).While, significant variations are reported in fruit's resistance to cracking in different litchi cultivars (Huang et al., 2005), yet fruit cracking is a major physiological disorder in litchi (Huang, 2005).The cracking of litchi pericarp is generally initiated at the early stage of fruit development (Huang et al, 2005).Research on fruit cracking in litchi, reveals that low relative humidity, high temperature, hot dry winds, drought or excessive irrigation are the major environmental factors that promote fruit cracking (Li et al., 2001).Evidence suggests that calcium is involved in cracking resistance in litchi fruit because trees with lower cracking incidence have higher calcium levels, while, a low exchangeable calcium in plants results in high cracking incidence (Li et al., 2001).Similarly, the cracked fruits have significantly lower concentrations of calcium in fruit as compared to the unaffected fruit of the same cultivars (Li et al., 2001).Furthermore, cultivars characterized by resistance to cracking have higher calcium concentration in the pericarp than the cracking-susceptible cultivars (Huang et al., 2005).The calcium movement within the plant is generally from the soil to the leaves and with very little from the leaves to the fruit (Kadir, 2005).Therefore, foliar application of calcium on the fruit is essential to decrease the calcium related physiological disorders (Fallahi et al., 1997).
Boron is another important micronutrient involved in cell wall development, cell division, phloem development, and movement of sugars, metabolism of nitrogen and phosphorus and absorption of salts (Dale and Krystyna, 1998).Experiment with litchi fruit indicates that borax at 0.4% not only decreased the fruit cracking but total soluble solids (TSS) and total sugar contents of the fruits were also higher (Ruby et al., 2001).Boron deficiency causes a decrease in the amount of Ca associated with pectin constituents of the cell wall suggesting that boron may be important in Ca metabolism in the cell wall (Yamaguchi et al., 1986).When different litchi cultivars were treated with borax, it was found that 0.8% boron resulted in significant decline in fruit cracking (Kumar et al., 2001).
The present study was initiated to explore the possibility of using foliar application of calcium chloride and borax to enhance fruit quality especially by decreasing fruit cracking.

Experiment location
The study on the influence of foliar applications of calcium chloride and borax on fruit skin strength and cracking incidence in litchi fruit in four litchi cultivars that is, China, Gola, Surahi and Bedana was conducted in the agroclimatic conditions of Haripur, Hazara region of KP Province, Pakistan during the years 2007 and 2008.

Experiment planning
The experiment was planned in randomized complete block design (RCBD) two factors factorial arrangement having litchi cultivars as factor A in main block while different calcium chloride and Borax concentrations in subplots.Each treatment comprised three trees.Data was recorded on fruit skin calcium and boron contents, skin strength, ion leakage from skin discs and fruit cracking.

Preparation of chemicals and application
All nutrient solutions were prepared from AnalaR Grade chemicals.Since calcium chloride dehydrate was being used, therefore, the molecular weight of 2H2O was subtracted from the total molecular weight of CaCl2.2H2O(147 g) to the weight of CaCl2 only (111 g).Calcium chloride (1, 2 and 3%) and Borax (0.5, 1 and 1.5%) solutions were made.The calcium chloride solutions were applied either alone or in combination with 0.5, 1 and 1.5% borax.A power sprayer was used to carry out the foliar sprays.After each treatment, the pump was washed thoroughly.A teaspoon of a commercial washing powder was added as wetting agent.Plain water was sprayed on the plants in the controlled treatment.All the foliar sprays were carried out early in the morning starting immediately after fruit set with 21 days interval till harvest.All other agronomic practices were kept uniform throughout the experiment.For recording the physical and chemical characters, the same procedures were adopted as described in experiment-1 while for recording the ion leakage from the skin disc, the procedure described in experiment 2 was adopted.

Calcium determination in fruit skin
Fruit skin calcium content (mg/100 g DW) was determined by the technique suggested by Isaac and Kerber (1971).The fruits were harvested and thoroughly washed for 10 min with tap water and then with distilled water.The fruit skins from the samples were peeled and once again washed with distilled water and allowed to oven dry at 70°C until the achievement of a constant weight.The dried samples were ground to powder and sealed in Petri dishes for chemical analysis.The calcium percentage in the fruit skin was determined by the atomic absorption flame spectrophotometer (GBC AA, 932).The spectrophotometer was calibrated with a standard solution of 5 µgml -1 as per recommendations of the manufacturer.

Boron determination in fruit skin
For fruit skin Boron content (mg/100 g DW ) determination, the skins of the selected fruits were washed thoroughly first with tap water and then with distilled water.Ten grams (10 g) of the sample was taken and dried for 12 h at 75°C in oven and then ashed for 3 h at 525°C.Ashes were extracted with 10 ml of 2M HNO3 and were heated on a hot plate.Filtered contents after dissolution were diluted to a final volume of 50 ml.This solution was used for the determination of boron content in the fruit skin by the azomethine -H method using atomic absorption flame spectrophotometer (GBC AA 932) as described by (Harp, 1997).Boric acid standard solution (1000 mgl -1 ) was used for standardization.

Ion leakage
Ion leakage was recorded by incising 1 cm 2 skin discs made of the skin taken from the equatorial regions of the selected fruits and putting them into 45 ml copolymer polypropylene vials having 20 ml of double distilled water.The base line conductivity of the double distilled water was recorded with the help of HI 9813 Portable conductivity meter (Hanna Instruments, Inc) in µScm -1 .Each vial containing double distilled de-ionized water was then added 3 discs and the vials were then fixed in a stand and vibrated for 30 min on a rotary shaker.On completion of 30 min, the conductivity of the water, representing ion leakage from cell walls was recorded (Saltveit, 2005).The vials were then given three freeze -thaw cycles to determine the total conductivity as described earlier (Saltveit, 2005).The ion leakage from the cell was approximated by the formula: (conductivity of water with discs after 30 min) -(conductivity of water) % Ion leakage = × 100 Total conductivity after 3 freeze thaw cycles

Fruit skin strength determination
Fruit skin strength was determined using hand held penetrometer as described by data pertaining to fruit firmness (Effigi, 11 mm Prob).The fruit were punctured at the equatorial area and the resistance to puncturing pressure was taken as skin strength of litchi fruit (Pocharski et al., 2000).

Fruit cracking
Fruit skin cracking percentage was recorded by visually observing and counting the number of total and cracked fruits on the tagged branches and converting the differential into percentage.Fruits with even the slightest of cracks were counted as cracked fruits.

Data analysis
The data was analyzed using M. Stat-C Software and means were separated by LSD α = at 0.05.

Calcium content of litchi fruit skin (mg/ 100 g DW)
The mean calcium content of litchi fruit skin varied significantly with the highest (7.67 mg/ 100 g DW) in cultivar Gola followed by cultivar China and Surahi with 7.59 and 7.32 mg Ca / 100 g DW respectively while the least calcium content (4.375 mg/100 g DW) was recorded in cultivar Bedana (Table 1).Treatment with CaCl 2 and borax also resulted in significant increase in Ca content of the litchi fruit skin.The minimum mean calcium content in the control (4.79 Ca /100 g DW) increased significantly to 4.88 with 3% CaCl 2 application.The calcium content of the litchi fruit skin increased further with CaCl 2 + borax treatments to the maximum of 8.88 mg Ca / 100 g dry weight with application of CaCl 2 3 + 1.5% Borax treatment (Table 1).The cultivar × treatment interaction revealed significant differences among cultivars and treatments in calcium content of litchi fruit skin.The minimum calcium content (4.21 mg/100 g DW) in cultivar Bedana increased to the maximum of 7.38 mg/100 g DW CaCl 2 3 + borax 1.5% treatment.In the control treatment, Ihsan-ul-Haq and Rab 2447 cultivar China had the highest calcium content (5.25 mg/100 g DW) but it was the highest (9.75 mg/100 g DW) in cultivar Gola with CaCl 2 3 + Borax 1.5% treatment, the differences with cultivar China (9.63) with the same treatment was, however, non significant (Figure 1).1).
Calcium is an important nutrient involved in the structure of cell walls and cell membranes (Peter, 2005).The calcium concentration in healthy plant tissues is about 0.1 to 1% of the dry matter (White and Broadley, 2003).Thus, a regular calcium supply is needed to ensure vigorous growth, avoid physiological disorders and enhance tolerance to abiotic stresses (White and Broadley, 2003;Del-Amor and Marcelis, 2006).Litchi cultivars may exhibit significant variation in mobilization of calcium to cell in the skin tissue (Huang et al., 2004).It was reported earlier that preharvest application of calcium chloride increase the calcium content of the fruit (Cronje et al., 2009).The relative small increase in calcium content in the skin tissue with only CaCl 2 application can be attributed to low absorption and incorporation of calcium in the skin cell wall (Huang et al., 2005).The control vs. CaCl 2 + borax resulted in 41.54% increase in calcium content of litchi fruit skin as compared to 40.85% in CaCl 2 vs. CaCl 2 + borax treatments (Table 1) indicating that boron might promote calcium incorporation into the cell wall (Wojvik et al., 1999).The control vs. CaCl 2 + Borax resulted in 41.54% increase in calcium content of litchi fruit skin as compared to 40.85% in CaCl 2 vs. CaCl 2 + Borax treatments (Table 1) indicating that boron might help in the incorporation of calcium in the cell wall (Wojvik et al., 1999;Khalifa et al., 2009).

Boron content of litchi fruit skin (mg/ 100 g DW)
Significant differences among litchi cultivars were observed in mean boron content of fruit skin with the highest boron content (0.203 mg/ 100 g DW) in cultivar Gola followed by cultivar China and Bedana with 0.202 and 0.198 mg/ 100 g DW respectively.Cultivar Surahi had the lowest boron content with 0.196mg / 100 g DW (Table 1).The boron content in litchi fruit skin was not affected by calcium chloride alone but increased significantly with CaCl 2 + borax treatments.The minimum boron content in litchi fruit skin in the control (0.109 mg/100 g DW) increased but non significantly to 0.146 mg/100 g DW with increasing CaCl 2 concentration to 3%.Calcium chloride 1% + borax increments (0.5 to 1.5%) resulted in significant increase in boron content of the skin (0.213 to 0.218 mg/100 g DW) but the difference among borax doses being non significant.Increased CaCl 2 concentration to 2% resulted in further increase in boron content of litchi fruit skin which continued to increase with increasing borax increments from 0.5 to 1.0 and 1.5%.At CaCl 2 3%, the maximum boron content in litchi fruit skin was observed with CaCl 2 3 + borax 1.5% (Table 1).The planned paired means comparison of control vs. Rest revealed significant increased in boron content from 0.11 to 0.21 mg/100 g DW and from 0.11 in control to 0.14 in control vs. CaCl 2 treatment means.The control vs. CaCl 2 + borax comparison revealed further increase in boron content from 0.11 to 0.23 mg/100 g DW.The boron content of the litchi fruit skin was also significantly higher (0.23 mg/100 g DW) with CaCl 2 + borax as compared to 0.14 mg/100 g DW, recorded with CaCl 2 alone (Table 1).Boron is an important micronutrient involved in cell wall development, cell division, phloem development, and the movement of sugars and metabolism of nitrogen, phosphorus (Patile et al., 2008).Application of boron increases the fruit set and fruit yield (Dale and Krystyna, 1998) and improve fruit quality (Ruby et al., 2001) while its deficiency has been associated with fruit discoloration, cracking, or rotting tubers and roots (Dale and Krystyna, 1998).Application of boron has been shown to decrease litchi fruit cracking, a disorder associated with calcium levels (Kumar et al., 2001;Ruby et al., 2001), yet the increased levels of boron in fruit skin could be significant in strengthening the cell walls (Loomis and Durst, 1992).The application of B increased Ca uptake by the fruits, thus, decrease Ca deficiency related disorders (Wojvik et al., 1999) such as fruit cracking (Kumar et al., 2001), probably by enhancing Ca uptake by the fruits or its metabolism in cell wall (Wojvik et al., 1999) but increase in boron content was more with CaCl 2 + borax rather than CaCl 2 alone.

Skin strength (kg cm -2 )
The mean fruit skin strength in cultivar Bedana (3.39 kg cm -2 ) was significantly higher than the rest of the cultivars, while it was the least in cultivar Gola (2.44 kg cm -2 ).The skin strength increased significantly from 2.43 kg cm -2 in control to 2.46 kg cm -2 with the application of 3% calcium chloride but then further to 2.79 kg cm -2 and 2.8 kg cm -2 when 1.0 and 2% calcium chloride was added with 1% borax and finally reached the maximum of 3.02 kg cm -2 with CaCl 2 3 + 0.5 or 1.0% borax (Table 1).The interaction of cultivars and calcium chloride + borax treatments was also significant.The skin strength was the highest in cultivar Bedana in the control, which increased 3.58 kg cm -2 with CaCl 2 2 + borax 0.5% but thereafter remained non significant.By contrast, the skin strength in the other cultivars continued to increase with increasing calcium chloride or borax concentrations (Figure 2).The control vs. Rest paired means also showed significantly higher skin strength (2.43 vs. 2.78 kg cm -2 ).Similarly, increased skin strength was observed in control vs. CaCl 2 + borax (2.43 vs. 2.89 kg cm -2 ) and CaCl 2 vs. CaCl 2 + borax (2.46 vs. 2.89 kg cm -2 ), while control vs. CaCl 2 means were not significant (Table 1).The tensile characteristic of fruit skin is important in cracking resistance (Hershko et al., 1994).The skin strength in litchi fruit is an important and desirable characteristic (Christensen, 1994) because an imbalance in cell turgidity (Peter, 2005) and extensibility (Pilling and Hofte, 2003) could lead to fruit cracking (Christensen, 1994).The greater strength in cultivar Bedana may be due its high calcium content (Leyla and Husnu, 2004).

Ion leakage (%)
The highest mean ion leakage percentage from fruit skin discs was in cultivars Gola (31.11%), followed by 28.24 and 21.60% in cultivars Surahi and China respectively, while the lowest ion leakage (18.86%) was observed in cultivar Bedana (Table 1).1).The cultivar × treatment interaction revealed the maximum ion leakage (35.17%) in cultivar Gola which declined to the minimum of 16.17 with CaCl 2 3 + Borax 1.5% treatment.However, cultivar Bedana had the lowest ion leakage at any treatment as compared to other cultivars.The ion leakage in Bedana in the control (18.86%) decreased to the minimum of 13.72% with CaCl 2 3 + borax 1.5% treatment (Figure 3).
The planned paired means comparison revealed significant decrease in ion leakage in control vs. Rest (35.17 to 24.10), control vs. CaCl 2 (35.17 to 33.24).Borax application decreased the ion leakage more than control or CaCl 2 treatments means as evident from control vs. CaCl 2 + borax (33.24 vs. 22.05%) and CaCl 2 vs. CaCl 2 + borax (30.06 vs. 22.05) (Table 1).The electrolyte leakage is an indicator of integrity of cell membrane and cell wall (Saltveit. 2005) and has been shown to correlate with pericarp browning in litchi fruit (Song et al., 2006).Since the CaCl 2 and borax treated fruit had high calcium contents, the decreased electrolyte leakage with calcium application may be due to increased cell wall integrity and stability (Patile et al., 2008).

Fruit cracking (%)
Different litchi cultivars varied significantly in tendency of fruit cracking, with the highest fruit cracking percentage observed in cultivars Gola (21.91%), followed by 19.13 and 15.12% in cultivars Surahi and China respectively, while the lowest fruit cracking (13.26) was recorded in cultivar Bedana (Table 1).
The CaCl 2 and borax treatments also significantly decreased the fruit cracking.The highest fruit cracking in control (25.40%) declined to 19.15% with 3% CaCl 2 application and declined further to the minimum 11.34% with CaCl 2 + 1.5% borax treatment.The cultivar × treatment interaction revealed the maximum fruit cracking (43.23%) in cultivar Gola which declined to the minimum of 17.87 with CaCl 2 3 + borax 1.5% treatment (Figure 4).The incidence of fruit cracking was significantly decreased by calcium and boron treatments as well as the interaction of cultivars x treatment (Table 1).1).It is interesting to observe that the influence of CaCl 2 alone had much less impact on decreasing fruit cracking than in combination with borax.The relative less influence of CaCl 2 may be due to poor absorption of calcium by litchi fruit and its incorporation in the skin cell wall (Huang et al., 2005).The application of Borax at 0.8% has been show to decrease fruit cracking (Kumar et al., 2001).
The fruit cracking in litchi fruit is a serious postharvest problem (Li et al., 2001) that leads to poor visual quality due to browning in exposed aril (Huang et al., 1985).The fruit cracking also enhances desiccation (Underhill and Simons, 1993).It is reported that differences in the thickness of cuticle and spongy layers in different cultivars could contribute to susceptibility of a cultivar to cracking (Huang et al., 2004).Since the litchi cultivars also show significant variation in calcium accumulation in cell walls of the fruits and skin, it is likely to observe variation in cracking susceptibility (Leyla and Husnu, 2004).Since calcium is required for the stability and extensibility of the cell wall, it has been wide studied and considerable evidence suggest that calcium is involved in cracking resistance in litchi (Huang et al., 2005).For example, the pericarp of cracked fruit has significantly lower concentrations of calcium than those of the unaffected fruit within the same cultivars (Lin, 2001), the lower cracking incidence in plant having higher calcium content or trees in calcium rich soil (Li et al., 2001), litchi fruit with high calcium content have greater resistance to fruit cracking (Huang et al., 2004;Leyla and Husnu, 2004).Similarly, treatment with 0.5 and 1% calcium nitrate and 0.4 and 0.8% borax have been shown to decrease fruit cracking (Kumar et al., 2001).Thus, it can be attributed to a synergism as application of borax may help in calcium metabolism in cell wall (Wojvik et al., 1999).

Conclusions
The litchi cultivars varied significantly in the skin calcium and born contents, skin strength, ion leakage and fruit cracking.Cultivar Bedana had the highest skin strength, least ion leakage and least fruit cracking, despite relatively lower calcium and boron content in fruit skin.
The foliar application of CaCl 2 and borax increased the calcium and boron content of the litchi fruit skin with a concomitant increase in skin strength and decrease in ion leakage and fruit cracking percentage.

Figure 2 .
Figure 2. Effect of calcium chloride and Borax applications on skin strength (Kg/cm2) in litchi cultivars.The error bars represent the least significant difference at = 0.05.

Figure 3 .
Figure 3. Influence of CaCl2 and Borax application on ion leakage (%) of litchi fruit.

Figure 4 .
Figure 4. Influence of CaCl2 and Borax application on fruit cracking incidence in litchi cultivars.

Table 1 .
Influence of calcium and boron application on specific gravity, reducing and non reducing sugars (%), calcium and boron content (mg/100 g DW) of skin in Litchi fruit.
Means followed by similar letters in a column are non significantly different from each other at α 0.05.NS, Non significant.