Quality Responses of Table Grapes ‘Flame Seedless’ as Effected by Foliarly Applied Micronutrients

: Micronutrient (iron, zinc and boron) deﬁciencies are a basic and prominent factor affecting grape quality and yield in the Pothwar region. To overcome these deﬁciencies, different levels of micronutrients were applied foliarly on grapevines at ﬁve different berry developmental stages during two consecutive growing seasons (2018 and 2019). The data suggested that foliar treatment of micronutrients signiﬁcantly increased the yield, number of bunches per vine, bunch weight, yield per vines, bunch length, berry number per cluster, berry diameter, berry weight and cluster compactness. The biochemical quality attributes of berries, including sugars (reducing, non-reducing as well as total sugars), ascorbic acid content, pH and TSS values, were at their highest levels in grapevines supplemented with Fe, Zn and B treatment at 200 ppm, respectively, i.e., the highest concentrations used. Biochemical leaf values, including chlorophyll a and b and leaf micronutrient content (Fe, Zn and B), were also highest in grapevines that were sprayed with Fe, Zn and B at 200 ppm. Overall, the results revealed that the performance of grapevine cv. ‘Flame Seedless’ growing in agroclimatic conditions of the Pothwar region was improved as a result of the foliar application of Fe, Zn and B at 200 ppm. The results also suggested that a further increase in the concentration of each nutrient might be helpful to obtain berries of improved quantity and quality. Thirty-nine disease-free, 4-year-old, and uniform size grape vines ( Vitis vinifera ) of ‘Flame Seedless’ were selected for this study. Vines of ‘Flame seedless’ grapes were planted in east–west direction on the Y-trellis system, with 8 feet vine to vine and 10 feet row-to-row distance. Standard cultural practices were applied to all experimental vines during the year 2018. Micronutrient treatments, i.e., Fe-EDTA (50, 100, 150 and 200 ppm), ZnSO 4 (50, 100, 150 and 200 ppm) and Boric acid (50, 100, 150 and 200 ppm), were sprayed at ﬁve different developmental stages viz. (i) before sprouting, (ii) during sprouting, (iii) after 10 days of sprouting, (iv) during blooming, and (v) after 10 days of blooming, i.e., a total number of 5 sprays. Tween-20 was used as surfactant for the sprays. The nutrient solution was sprayed thoroughly on the leaves and branches; hence, 1.5 L of spray per tree were used during the 1st two times, while 3 L per tree were used for the rest of sprays. The plant was sprayed thoroughly.


Introduction
Grape (Vitis vinifera L.) belongs to the family Vitaceae and is native to the riverbanks of Asia, North Ameria and Europe. It is considered the highest ranked fruit in the world because of its manifold benefits [1] and has gained significant importance due to its high nutritional value, taste, multiple uses and the superior returns obtained by farmers [2]. It is one of the dominant commercial fruit crops of temperate to tropical regions and covers more land than any other fruit, representing more than half of total fruit production of the world [3].
In Pakistan, the production of grapes is gaining attention, where mostly European grapes are being produced because of their harmony with the local climate [4]. Approximately 70% of the country's grapes are produced in Baluchistan, with minor amounts Department of Horticulture, PMAS-Arid Agriculture University Rawalpindi, Pakistan. Thirty-nine disease-free, 4-year-old, and uniform size grape vines (Vitis vinifera) of 'Flame Seedless' were selected for this study. Vines of 'Flame seedless' grapes were planted in east-west direction on the Y-trellis system, with 8 feet vine to vine and 10 feet row-to-row distance. Standard cultural practices were applied to all experimental vines during the year 2018. Micronutrient treatments, i.e., Fe-EDTA (50, 100, 150 and 200 ppm), ZnSO 4 (50, 100, 150 and 200 ppm) and Boric acid (50, 100, 150 and 200 ppm), were sprayed at five different developmental stages viz. (i) before sprouting, (ii) during sprouting, (iii) after 10 days of sprouting, (iv) during blooming, and (v) after 10 days of blooming, i.e., a total number of 5 sprays. Tween-20 was used as surfactant for the sprays. The nutrient solution was sprayed thoroughly on the leaves and branches; hence, 1.5 L of spray per tree were used during the 1st two times, while 3 L per tree were used for the rest of sprays. The plant was sprayed thoroughly. Grape bunches were taken from all possible locations on grapevines so that the samples represent correct yield and quality attributes.

Morphological Analysis
Different physical parameters were calculated manually, including number of clusters per vine, average bunch weight (g), average bunch length (cm), average berry number per cluster, average berry diameter (mm), and average berry weight (g). Average yield per vine and cluster compactness were calculated by using the following formulas: (1)

Biochemical Quality Analysis
A sample of 50 berries was selected from bunches of each replicate and juice was extracted. Chemical attributes such as soluble solids contents (SSC), pH, titratable acidity (TA), ascorbic acid (Vitamin C), reducing sugars, total sugars and non-reducing sugars were determined from the juice. Soluble solid contents were determined [23] using a handheld refractometer (Model: SG-103) at room temperature, while pH was measured with a digital pH meter at 18 ± 2 • C. To determine titratable acidity, extracted juice (10 mL) was mixed with 40 mL distilled water and 4-5 drops of phenolphthalein were added in the juice. A 10 mL aliquot was placed in a titration flask and titrated against 0.1 normal (N) NaOH until a permanent light pink color appeared. After titration, titratable acidity was calculated by the given formula: NaOH used × N of NaOH × Equivalent weight of Tartaric acid Volume of juice used for titration (ml) × 100 Ascorbic acid contents were calculated [24]. Five grams of pulp of grapes from 30 berries were ground, using a mortar and pestle, with 5 mL 1.0% HCL and the mixture was centrifuged for 10 min at 10,000 rpm. Absorbance of the extracted supernatant was noted at a wavelength of 243 nm by a spectrophotometer (sp3000 plus model, Optima Japan). Sugar content (total, reducing and non-reducing sugars) in the fruits was determined [25]. A 10 mL (juice) sample was taken in a 250 mL volumetric flask, to which 100 mL distilled water, 25 mL lead acetate solution (430/1000 mL) and 10 mL of (20%) potassium oxalate were added. In a conical flask, 10 mL of Fehling's (5 mL of both Fehling A and B) solution was taken. Sample aliquots were placed in a burette and left to run dropwise into the conical flask containing Fehling's solution. During titration, slow boiling continued until the appearance of a brick red color. Two to three drops of methyl blue were added and titration continued until a brick red color appeared again. The reading of the sample aliquot used was noted and the percent of reducing sugar was calculated as below: Reducing sugar (%) = 6.25 (X/Y) where X = mL of standard sugar solution reading used against 10 mL Fehling's solution. Y = mL of sample aliquot used against 10 mL Fehling's solution.
For total sugars, 25 mL of aliquot already prepared for reducing sugars was taken in a 100 mL flask in which 20 mL distilled water and 5 mL of concentrated hydrochloric acid were added to convert non-reducing sugars to reducing sugars. To complete the conversion process, the solution was kept at ordinary temperature for a day. It was then neutralized with about 1 N NaOH solution using 2-3 drops of phenolphthalein as an indicator, and again, neutralized with HCL and made up to the volume of 100 mL with distilled water. The prepared solution was taken in a burette and titrated against 10 mL Fehling's solution to the brick red color end point, using methylene blue as an indicator; the same procedure was followed for calculation of the reducing sugars. Total sugar was calculated by the given formula: Total sugar (%) = 25 × (X/Z) where X = mL of standard sugar solution reading used against 10 mL Fehling's solution. Y = mL of sample aliquot used against 10 mL of Fehling's solution. Non-reducing sugars were calculated with the formula given below:

Biochemical Leaf Analysis
Leaf chlorophyll a and b contents were determined [26]. Chlorophyll contents in leaves were determined by extracting an accurately weighted fresh plant leaf sample of 0.5 g in 15 mL acetone. The homogenized sample was centrifuged at 10,000 rpm for 15 min. The supernatant was separated, and 0.5 mL was mixed with 4.5 mL acetone. The solution mixture was analyzed with a spectrophotometer at wavelengths 663 nm and 645 nm (using a spectrophotometer, model: SP-3000 plus, Optima, Japan). The following formulas were used to calculate the chlorophyll a and b content in the leaves.
Chlorophyll a (C a) = 11.75 A663 -2.350 A645 Chlorophyll b (C b) = 18.61 A645 -3.960 A663tag2 Micronutrient content in the leaves was calculated through the dry ashing method [27]. One gram of powdered sample from each treated grapevine was placed in porcelain crucibles and transferred into a muffle furnace. The furnace was then gradually ignited up to 550 • C and heating was continued for a further 5-6 h after reaching the required temperature. After the designated time, the muffle furnace was switched off and crucibles with white ash were cooled. Cooled ash was dissolved in 5 mL of 2 N HCl and thoroughly mixed with a plastic rod. The total volume of this solution was made up to 50 mL using distilled water. The mixture was allowed to stand for 30 min. After filtering the mixture, the obtained aliquot was analyzed for concentrations of zinc and iron through atomic absorption spectrophotometry (SHIMADZU AA-6300). The obtained results were expressed in ppm.
To find out the concentration of boron in the leaf sample, absorbance was determined using a spectrophotometer at wavelength 420 nm with little modification of dry-ashing. One gram of powdered leaf sample was heated in the muffle furnace up to 550 • C for 6 h to ensure the formation of white ash. Crucibles with ash were taken out and cooled ash was mixed with 5 drops of distilled water as well as 10 mL of 0.36 N H 2 SO 4 solution. The solution was continuously stirred for a few intervals for 1 h. The mixture was filtered through Whatman No. 1 filter paper and the obtained aliquot was visualized under a spectrophotometer.

Organoleptic Evaluation of Grapes
Organoleptic evaluation for texture, flavor and overall acceptability of the samples was performed by using the Hedonic scale [28].
A panel of five judges was selected on the basis of their consistency and reliability of judgment. This method involved presenting the judges with fruit samples, to assess organoleptic factors. Judges were also allowed to retaste a sample, if required. Judges were advised to score each sample by allotting numbers according to the following scale: 1 = Extremely disliked, 2 = Disliked very much, 3 = Moderately disliked, 4 = Slightly disliked, 5 = Neither liked nor disliked, 6 = Slightly liked, 7 = Moderately liked, 8 = Liked very much and 9 = Liked extremely.

Statistical Analysis
A randomized complete block design was used in the experiment, while a least significant difference test (LSD) at the 5% level of significance was used to compare the means obtained for the treatments used in the experiment [29]. . During both study years, controlled grapevines produced lighter bunches, with the lowest bunch weights being 628 g and 632 g, respectively. Almost all the parameters discussed above have the lowest results when grapevines received only foliar water treatment (control); however, with the increase in individual foliar treatment of micronutrients Fe, Zn and B at 50, 100, 150 and 200 ppm, these parameters increased gradually until significantly higher values were observed when grapevines were treated with 200 ppm of nutrients (Table 2). Similarly, bunch compactness, berry weight, and yield were significantly higher at higher concentrations of Fe, Zn and B. Grapevines sprayed with micronutrients exhibited compact bunches; the highest bunch compactness values recorded in 2018 were 7.43 and 7.41 at 200 ppm Fe and B, respectively, while comparable results were obtained in 2019 in 200 ppm Fe, Zn and B sprayed grapevines. Similar findings for berry weight were observed. The highest berry weight was observed in the highest doses (200 ppm) of Fe, Zn and B, respectively. During 2019, maximum berry weight was recorded in berries of vines treated with 200 Fe, Zn and B foliar application. Likewise, yield attribute varied significantly among all foliar treatments. Yield per vine increased significantly by application of micronutrients, but substantially higher yields were recorded at terminal concentrations of Fe, Zn and B (200 ppm) compared to small concentrations of micronutrients and control treatments during both years, i.e., 2018 and 2019 (Table 3).

Biochemical Fruit Quality Analysis
Application of foliar micronutrients, viz. Fe, Zn and B, significantly improved almost all fruit quality attributes as compared to the control (Tables 3 and 4  Values are mean ± standard error, means within columns with the same letters are statistically insignificant (p ≤ 0.05).  TA percentage of juice shows that the highest acidity (1.41% as well as 1.35% during 2018 and 2019, respectively) was observed in grapevines that received no supplemental micronutrients, whereas application of micronutrients significantly reduced acidity percentage, which was observed in 200 ppm of Fe-, Zn-and B-supplemented vines (Table 3). Table 4 shows that all quality parameters including pH, ascorbic acid and sugars (reducing, non-reducing and total sugar) varied significantly with increase in concentration of foliar nutrients. pH indicates the amount of acid present in the juice, which determines their quality and flavor. More acidic berries (with lower pH) were observed in controlled grapevines, while lower doses of nutrients show little change in acidity as compared to the control but are still statistically insignificant with respect to control. However, application of Fe, Zn and B at 150 and 200 ppm shows a significantly higher level of pH as compared to the control.
Ascorbic acid (AA) concentration in the berries is given in Table 4. Control and 50 ppm foliar treatment of individual nutrient (Fe, Zn and B) concentrations resulted in the lowest values. In contrast, treatment of Fe, Zn and B significantly increased AA concentration with respect to control until the highest amount of AA (6.05/6.11%, 5.95/5.77% and 5.87/5.95% during the two study years 2018/2019, respectively) was observed in 200 ppm foliar treatment of Fe, Zn and B, respectively.
Sugar contents (%) in the berries of different grapevines are given in Table 4. It is obvious from the data that the concentration of micronutrients has a direct relationship with sugar (%), i.e., higher in the higher doses and vice versa. Non-reducing sugars in the berries shows that controlled grapevines have the lowest amount of non-reducing sugars (1.14/1.

Biochemical Leaf Analysis
Results of the biochemical leaf analysis are given in Table 5  Foliar application of Zn similarly has no significant effect on Fe and B nutrient concentration in the leaves, whereas Zn mineral nutrient concentration significantly increased only in response to the increase in Zn foliar application with respect to the control. The highest amount of Zn in the leaves ( (Table 5).

Organoleptic Evaluation
Organoleptic evaluation of fruits of grapevines as a result of foliar treatment of different micronutrients Fe, Zn and B is given in Table 6. Flavor, texture, taste and acceptability of fruits were improved statistically, with respect to the control, when foliar treatments with different micronutrients were applied.  Values are mean ± standard error, means within columns with the same letters are statistically insignificant (p ≤ 0.05).

Discussion
All yield-related attributes significantly increased as a result of foliar application of micronutrients and the highest values were almost always observed in the highest doses of nutrients applied. Increases in bunch number per vine, bunch length, berry number per bunch, berry diameter, and bunch weight might be affected by the induction of flowers into fruits as a result of foliar treatment of nutrients, leading to increased grapevine yield (Tables 2 and 3). Such upturn in function of iron in fruit through different enzyme reactions and chlorophyll amalgamation might have increased photosynthesis. Improved berry diameter could be ascribed to increased chlorophyll content in the leaf, which is associated with a high production of photosynthate in a plant [30]. Zinc (Zn) increases vegetative growth (stem diameter) by synthesizing tryptophan and regulates growth and production of grapevines [31]. Our results can be correlated with previous findings [32][33][34], where foliar application of Fe, Zn and Br significantly improved for bunches per vine, bunch weight and the quality of grapevines. Increases in bunch weight of grapevines sprayed with B and Zn could be attributed to the increase in berry set and higher number of berries per bunch along with improved cell size [35]. Increases in yield attributes as a result of foliar application of B might be attributed to its synthetic role in different hormones and other metabolic reactions. Previously, foliar application of B increased fruit yield in naval orange [36] and comparative observations regarding B application were also reported in grapevines [31,37], whereas Fe regulated functions directly influencing fruit setting, fruit retention percentage, bunch number per vine, bunch length, berry number per bunch, berry diameter, bunch weight, bunch compactness, berry weight and yield per vine [38,39].
Fruit quality can also be assessed by parameters such as SSC, titratable acidity, firmness, size and color [40]. The gradual increase in TSS as a result of foliar application of B (Table 3) shows their direct relation with TSS, which might be due to B involvement in photosynthesis. Our findings are in agreement with previous experiments on grapevines [41,42], where foliar treatment of B significantly increased TSS level in grape berries. Zn being an essential micronutrient helps in the activation of enzymes (fructose-1 and 6-bis phosphatase) that play an important role in biochemical reactions accumulating sugars in the fruits [43,44]. The present results are supported by previous studies where foliar application of B, Fe, and Zn increased TSS of 'Perlette' grapes, mango, and strawberry fruit, respectively [31,45,46]. The inverse relationship of TA with foliar spray of Fe (Table 3) might be due to an increase in the metabolic rate that increases the conversion of organic acids into lower carbohydrates in the berry solution, resulting in a reduction in the acidity, whereas Zn helps in the translocation of carbohydrates from leaves to fruits that increases the berry quality and quantity with an improved amount of sugar content and reduced titratable acidity by their conversion into sugars. Similar results were reported [46,47] where application of B and other micronutrients (Zn, Fe) resulted in decreased TA in grape and strawberry. Meanwhile, ascorbic acid contents of grape berries were increased with increasing concentrations of micronutrients (Fe, Zn, B) and the maximum value was recorded in berries sprayed with 200 ppm B.
Ascorbic acid is an important dietary ingredient that works as a strong antioxidant as well as helping in the electron transport chain and it regulates enzymatic activities by performing their roles as co-enzymes. In the current study, foliar application of micronutrients (Fe, Zn, B) increased the ascorbic acid contents of grape berries during both growing years (Table 4). B as a micronutrient participates in a variety of biochemical processes and has been reported to increase ascorbic acid contents as it is a fundamental part of important cell structures [48]. Increases in ascorbic acid contents in the berries as a result of supplemental micronutrient treatments could be related to the regulation of various essential metabolic activities and similar findings have been reported in previous studies [49,50]. Our results showed that reducing, non-reducing and total sugar contents of grape berries were increased with increasing concentration of micronutrients (Table 4). Such an increase in sugar contents of grape berries could be related to the increased chlorophyll contents and the higher photosynthesis rate achieved through supplemental sprays of Fe, B and Zn [51]; similar findings were reported in apple and grapevines [52][53][54]. Higher sugar contents as a result of foliar sprays of micronutrients have also been reported in strawberry, pomegranate, and grapes [55][56][57].
Grapevines sprayed with supplemental sprays of Fe, Zn, and B exhibited significantly higher values of respective nutrients in the leaves, while each nutrient remained ineffective in increasing the contents of other nutrients in the leaves significantly (Table 5). Though insignificant, higher doses of each nutrient increased the concentration of other nutrients, which could be ascribed to the synergistic effect of these nutrients on each other [58][59][60][61].
The results of the present study depicted that grapevines sprayed with micronutrients exhibited higher chlorophyll contents in leaves (Table 5). This increase in chlorophyll content could be owed to the higher nutrient level of Mg, Fe, K and Ca as a result of the application of supplemental micronutrients. Previously, the application of micronutrients increased chlorophyll a and chlorophyll b contents of leaves of peas as well as peace lily [62,63]. Previously, foliar as well as soil application of Fe-EDTA increased chlorophyll a concentration in the foliage of wheat [64], whereas Zn has been proven to be helpful in improving photosynthetic efficiency in plants [65] by stabilizing the activity of carbonic anhydrase, which has a role in the accumulation of chloroplast and chlorophyll synthesis [66]. Likewise, foliar application of B increases the expression of auxin biosynthesis gene BnNIT1 [67], which is related to chlorophyll synthesis [68]. Similar observations were recorded where B application increased chlorophyll contents in olive, cashew, cucumber, and pepper leaves [69][70][71][72]. Sensory attributes of flame seedless grapes, including taste, texture, aroma, and acceptability, were improved with increasing concentrations of foliar sprays of micronutrients (Table 6). Zn is believed to be associated with auxin synthesis in plants and plays a vital role in enzymatic reactions that describe the final quality of the fruit. Zn helps in enzymatic reactions that lead to the transformation of carbohydrates, formation of cellulose and change in sugars [73]. Furthermore, Dutta and Dhua [73] observed an improvement in mango sensory attributes by application of micronutrients. Our results are comparable with the findings of Bhoyar and Ramdevputra [74], where the application of Zn, B and Fe increased the overall sensory quality of guava fruit. Similar findings were published [75], observing an improvement in sensory attributes of different pomegranate cultivars by the application of micronutrients.

Conclusions
The experiment was conducted to improve the quality of "Flame Seedless" table grapes by the foliar application of micronutrients, i.e., Fe, Zn and B, under the arid conditions of Pakistan. The results obtained from the present study proved that foliar application of micronutrients (Fe, Zn, and B) was the most effective treatment in improving the physical and chemical parameters of grape berries. To improve the yield and quality of "Flame Seedless" table grapes under the conditions in Pothwar, micronutrients (Fe, Zn and B) can be applied as foliar spray to the vineyards in order to enhance their efficiency and avoid losses. To our knowledge, this is the first report of micronutrient foliar sprays and fruit quality of grapes under the arid conditions of Pakistan. Thus, keeping in view the trends obtained from the data, further study is recommended with higher doses and combinations of the abovementioned micronutrients.