1. 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 in other provinces, including Khyber Pakhtunkhwa and Gilgit-Baltistan [
4]. In recent years, the Pothwar region (Northern Punjab) has gained momentum in terms of grapevine cultivation. However, the yield of grapes in Pakistan is just four tons per hectare, which is very low when compared with other high-yielding countries including Brazil, which produces more than 25 tons per hectare. This minor level of production as compared to developed countries is due to poor management practices, a poor nutritional profile, the unavailability of improved crop materials and the lack of research work in the region, affecting the production of grapes.
The Pothwar region has extensive land for crop production, with the limitations of low rainfall and less fertile lands. The area is vulnerable to drought, with average rainfall ranging from 30 to 200 mm and with temperatures up to 47 °C. As a result, salts amass over the soil surface, subsequently resulting in soil salinity [
5]. Hot summers in the region result in a high rate of evapotranspiration, leading to higher electrical conductivity, i.e., >4 mScm
−1 [
6]. Sodicity (the deposition of surplus Na
+ as a result of salinity) in soil has several adverse effects on its physiochemical properties, including disruption of the structure, hydraulic properties and nutrient availability of soil [
7]. On the other hand, soil fertility is reduced due to continuous cropping without adequate fertilization [
8]. Likewise, the loss of nutrients due to soil erosion causes a decline in soil productivity [
9]; erosion may affect soil properties including its organic matter, tilth, and water holding capacity, as well as the structure and texture of the soil [
10,
11]. Soils of the Pothwar region are mostly calcareous in nature (having large concentrations of calcium carbonate) with more than 7.5 soil pH. The reason behind this calcareous soil is that these properties are inherited from their parent soils, i.e., calcareous alluvial and loess [
12]. Increases in soil erosion elevate calcareousness, which raises pH [
13]. With each increase in unit pH, nutrient availability (especially micronutrients) reduces manyfold [
14]. Micronutrients (Cu, Fe, Mn, Ni and Zn) are strongly bound with soil particles at high pH and, as a result, they are unavailable to the plants [
15]. Soil erosion also has a negative impact on soil organic matter content. Most of the organic matter loss from fields is associated with eroded sediments [
16].
Due to these limitations, the soils of the Pothwar region are deficient in micronutrients, including iron, zinc and boron [
11,
12]. Therefore, grapevines need proper nutrition management to fulfill their needs. As basic mineral nutrients, micronutrients (Fe, Zn, B, etc.) are also measured as essential nutrients for grapevine growth, metabolism, fruit development and quality because, as a co-factor, they activate many metabolic enzymes [
17]. Micronutrients, especially Fe, Zn and B, are essential elements utilized by plants for healthy growth. Each of these elements has a significant role in plant growth [
18,
19]. Poor soil conditions bind these nutrients, ultimately affecting their availability and uptake. Under deficient conditions, the exogenous supply of these substances through an integrated approach can effectively regulate the viability of plants. These supplements compensate for the reduced supply of nutrients from the soil throughout different stages of development [
20].
To overcome these requirements, at present, foliar application of these nutrients is gaining traction because of their multiple benefits, including their immediate effects and the low amount of fertilizer required in the solution [
21]. Foliar fertilizers are more effective under high nutrient demands, especially when the soil supply and root uptake may be inadequate [
22]. Therefore, in the current study, we planned to investigate the role of foliar sprays of micronutrients on the growth, development, yield and quality of the table grape cv. ‘Flame seedless’.
2. Materials and Methods
This experiment was conducted in a private vineyard at ‘Muradi Janjeel’ Tehsil ‘Gujjar Khan’, ‘Rawalpindi’ District (33°14′ N, 73°08′ E), Pakistan. It is semi-arid during the winter season and sub-humid during the summer season. Sixty to 70% of the rainfall takes place during the rainy season viz. mid-July to mid-September. The physical and chemical properties of the soil are shown in
Table 1. This trial was carried out at the facility of the 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.
2.1. 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:
2.2. 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:
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:
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:
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:
2.3. 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.
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 H2SO4 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.
2.4. 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.
2.5. 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].
4. 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 (
Table 2 and
Table 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.