Abstract
In this study, 130 uniform ‘Flame Seedless’ grape trees were selected for and subjected to the same cultural practices. The trees were sprayed three times, before flowering, during full bloom, and three weeks later with the following treatments: control (water only), 250, 500, and 750 ppm glycine; 50, 100, and 150 ppm folic acid (FA); 2%, 4%, and 6% leaf moringa aqueous extract (MLAE); and their combinations. High-performance liquid chromatography (HPLC) analysis of moringa leaf aqueous extract (MLAE) showed the presence of the phenolic compounds ellagic acid, vanillic acid, p-hydroxy benzoic acid, catechol, and gallic acid with values of 54.18, 18.79, 14, 12.32, and 12.12 mg/100 g, respectively. The obtained results showed that the foliar spraying of 250, 500, and 750 ppm glycine, 4% and 6% MLAE, and their combinations of glycine 500 ppm + FA 100 ppm + MLAE 4% and glycine 750 ppm + FA 150 ppm + MLAE 6% significantly increased the shoot length, shoot thickness, leaf chlorophyll content, yield, and fruit quality over the control. Glycine at 750 ppm was the best treatment followed by glycine at 500 ppm compared with the other applied treatments and the control in both experimental seasons.
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Application of Glycine, Folic Acid, and Moringa Extract as Bio-stimulants for Enhancing the Production of ‘Flame Seedless’ Grape Cultivar
Walid F. A. Mosa,a,* Mohamed Z. M. Salem,b,** Asma A. Al-Huqail,c,*** and Hayssam M. Ali c
In this study, 130 uniform ‘Flame Seedless’ grape trees were selected for and subjected to the same cultural practices. The trees were sprayed three times, before flowering, during full bloom, and three weeks later with the following treatments: control (water only), 250, 500, and 750 ppm glycine; 50, 100, and 150 ppm folic acid (FA); 2%, 4%, and 6% leaf moringa aqueous extract (MLAE); and their combinations. High-performance liquid chromatography (HPLC) analysis of moringa leaf aqueous extract (MLAE) showed the presence of the phenolic compounds ellagic acid, vanillic acid, p-hydroxy benzoic acid, catechol, and gallic acid with values of 54.18, 18.79, 14, 12.32, and 12.12 mg/100 g, respectively. The obtained results showed that the foliar spraying of 250, 500, and 750 ppm glycine, 4% and 6% MLAE, and their combinations of glycine 500 ppm + FA 100 ppm + MLAE 4% and glycine 750 ppm + FA 150 ppm + MLAE 6% significantly increased the shoot length, shoot thickness, leaf chlorophyll content, yield, and fruit quality over the control. Glycine at 750 ppm was the best treatment followed by glycine at 500 ppm compared with the other applied treatments and the control in both experimental seasons.
Keywords: Moringa extract; Glycine; Folic acid; Grape; Fruit quality
Contact information: a: Plant Production Department (Horticulture- Pomology), Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria 21531 Egypt; b: Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545 Egypt; c: Chair of Climate Change, Environmental Development and Vegetation Cover, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
Corresp. authors: *walidbreeder@yahoo.com; **zidan_forest@yahoo.com; ***aalhuqail@ksu.edu.sa
INTRODUCTION
Grape (Vitis vinifera L.) is a member of the Vitaceae family. In Egypt, the harvested area is 78853 ha, which produces 1759472 tons. It was stated by many authors that berries of grape have been utilized as table fruit, juice, and for the production of wine, and raisins, as well as in the industries of cosmetic from leaf, seed, and skin extracts (Iriti and Faoro 2006; Monagas et al. 2006). Moreover, grape skin, pulp, and seeds could be used to produce wine, terpenes, and norisoprenoids, as well as sugars, which could be converted to alcohol (Lund and Bohlmann 2006). It was reported by (Ruberto et al. 2007; Hogan et al. 2010; Drosou et al. 2015) that grapes are characterized by the high content of phenolic compounds, which are a valuable source of natural antioxidants. Grapes are a crucial exporter to potassium and manganese (Torres-Urrutia et al. 2011), rich in short-chain carbohydrates (Gibson and Shepherd 2012; Gibson 2017), and vitamin C (Moores 2013; Carr and Maggini 2017), which is important for immunology.
Glycine is an amino acid that has a crucial role in improving total chlorophyll and vegetative growth in grapes, as well as in increasing the availability of Fe, Zn, Mn, and Cu to the plants (Sekhon 2003; Liu et al. 2011; Ghasemi et al. 2013; Shan et al. 2016; Razavi et al. 2018). L-glycine works as a signal transduction molecule that contributes to making plant nutrients readily available (Teixeira et al. 2017). The plant height, leaf chlorophyll content, and fresh weight of the shoot and root of Coriandrum sativum were significantly improved by the application of glycine, especially at higher concentrations (Souri and Hatamian 2019). In addition, the application of glycine increased the total soluble solid and vitamin C contents compared with that in the controls. In comparison with the unfertilized plants, the leaf mineral contents of N, Ca, K, P, Fe, and Zn were significantly increased by the application of glycine (Jiang et al. 2020; Lo’ay and Doaa 2020).
Folic acid (FA) has a fundamental role in the metabolism of amino acids and in nucleic acids synthesis (Paucean et al. 2018). Moreover, FA was found to increase the productivity of Pisum sativum by affecting the leaf content of chlorophyll, as well as the yield, weight, and quality of the seed (Burguieres et al. 2007). Foliar spraying of sweet pepper plants with FA increased flowering, yield, and fruit quality (Al-Said and Kamal 2008). Additionally, exogenous FA showed a positive effect on the growth, yield, and quality of soybean and strawberry plants (Mansour 2014; Li et al. 2015). Spraying of FA at 0, 10, 20, and 30 mg/L on Luz de otono bean cultivar was investigated by Al-Maliky et al. (2019). The obtained results showed that spraying FA raised the height of plant, number of branches, leaf area, and shoot dry weight, as well as length, number and green weight of pods as compared to control. Moreover, 100 seeds weight, the production of green pods, and fresh seeds, the percentage of TSS, and dry matter, as well as protein and vitamin C and the concentration of 30 mg/L was the best concentration. Spraying FA at 150 µM/L under lack of water levels raised significantly plant growth, efficacy of water use, firmness of cell membrane, yield, total soluble solids, and protein content in snap beans (Phaseolus vulgaris L.) (Ibrahim et al. 2021).
Moringa oleifera contains around forty sex antioxidants such as ascorbate, carotenoids, phenols, and flavonoid (Iqbal and Bhanger 2006). Moringa leaf aqueous extract (MLAE) can be used as a bio-stimulant and contains macro- and micronutrients, amino acids, ascorbic acids, minerals, and growth-enhancing principles such as the hormone of the cytokinin type (Makkar et al. 2007). In addition, the growth hormone spray will cause the plants to be firmer and more resistant to pest and diseases. Moreover, M. oleifera is one of the best crop treatments with confirmed impacts on growth and yield, and it can be used by farmers as a source for nutrients instead of relying on inorganic fertilizers, which are costly and are associated with both land and soil degradation and environmental pollution (Phiri 2010). Additionally, M. oleifera is standardized to contain flavonoid, phenolic, and carotenoid, which can be used as antioxidants, one of them being quercetin (Alhakmani et al. 2013; Wang et al. 2017). Abbassy et al. (2020) reported that M. oleifera leaf extract is rich in prime metabolic compounds such as proteins, lipids, carbohydrates, minerals, vitamins, and amino acids. Moringa provides products that improve the growth and yield of various crops (Mohamed et al. 2020). Hollywood plum trees sprayed with 6% MLAE had an improved fruit set, fruit yield, fruit weight, firmness, color, total soluble solids, vitamin C, and the content of anthocyanin in the plum cultivar Hollywood, while they had reduced fruit drop compared to the use of 0%, 4%, or 5% MLAE (Shaaban et al. 2020).
Therefore, the objective of this study was to investigate the impacts of the foliar application of glycine (amino acid), folic acid, and MLAE as natural biostimulants to vegetative growth, yield, and fruit quality of the grape cv. ‘Flame Seedless’ to minimize the dependency on the chemical fertilization in grape orchards.
EXPERIMENTAL
Preparation of MLAE and HPLC Analysis of Phytochemical
Moringa oleifera Lam. leaves were obtained from Alexandria, Egypt and were shade-dried. The dried leaves were ground to powder using a small laboratory mill. Then, 100 g of the powdered leaves (200 g) were soaked in 2 L of distilled water for 24 h at room temperature (Mohamed et al. 2020). The mixture was filtered using Whatman No. 1 filter paper. The dissolved MLAE was concentrated by evaporation, then prepared at the concentrations 2%, 4%, and 6%. An Agilent 1260 Infinity 1260 II LC, System (HPLC Agilent, Santa Clara, CA, USA) was equipped with a Quaternary pump and a Zorbax Eclipse Plus C18 column (100 mm × 4.6 mm i.d.) (Agilent Technologies, Santa Clara, CA, USA) and operated at 30 °C; it was used to identify the phytochemical compounds in the MLAE. Separation conditions and standard phenolic compounds can be found in previously published works (Al-Huqail et al. 2019; Behiry et al. 2019; Salem et al. 2019; Ashmawy et al. 2020; Salem et al. 2020).
Experimental Location and Treatments
This experiment was carried out during two successive seasons, in 2018 and 2019, on 10-year-old ‘Flame Seedless’ grape trees (V. vinifera L.), planted with 3 m between rows and 2 m between trees in the same row, and grown in a calcareous soil under a drip irrigation system in a private orchard located at Nubaria, Beheira Governorate, Egypt. Physiochemical analysis of the experimental soil was performed (Table 1) (Sparks et al. 2020).
A total of 130 uniform trees were selected for this study, and all of them were subjected to the same cultural practices in the two seasons. They were sprayed with the experimental treatments three times in each season: before flowering, during full bloom, and three weeks later (Table 2). These applied treatments were arranged in a randomized complete block design where each treatment was composed of 10 replicates.
Measurement of Vegetative Parameters, Leaf Total Chlorophyll, Fruit Yield, Fruit Quality and Leaf Chemical Composition
Shoot length and thickness (cm) were measured at the end of each growing season. Total chlorophyll in the fresh leaves was determined with SPAD units as evaluated by a chlorophyll meter (SPAD-502; Konica Minolta, Osaka, Japan). The number of clusters per each vine and weight of each cluster was measured and recorded. The yield of vine (kg) was determined by the weight of clusters per vine multiplied by the number of clusters per vine. Yield in tons per hectare was estimated by multiplying yield of vine × number of vines per hectare.
For the leaf chemical composition, samples of 30 leaves were taken from the middle of the shoots and were randomly selected from each replicate after the harvest time in June to determine their content in terms of percentages of nitrogen (N), phosphorus (P), and potassium (K) (Arrobas et al. 2018). The leaf samples were washed first with tap water and then with distilled water and dried at 70 °C until a constant weight was obtained. Finally, the dried leaf samples were ground and acid digested using H2SO4 and H2O2 until the digested solution became clear. The digested solution was used for the determination of nitrogen using the micro-Kjeldahl method (Wang et al. 2016), phosphorus by the vanadomolybdate method (Weiwei et al. 2017), and potassium using a flame photometer (Banerjee and Prasad 2020).
Measuring of Fruit Quality in terms of Physical, and Chemical Characteristics
At the time of harvesting, 10 clusters from each vine/replicate were chosen randomly to determine their physical and chemical characteristics; cluster weight (g), size (cm3), length (cm), and width (cm). In addition, 100 berries were selected from all the chosen clusters of each vine to measure their weight (g), size (cm3), juice (%), berry weight (g), length (mm), and width (mm). Fruit firmness (lb/in2) using a Magness-Taylor pressure tester (mod. FT 02 (0-2 Lb., Via Reale, 63 – 48011 Alfonsine, Italy) with a 1/16-in plunger. Total soluble solids (TSS) were determined using a hand-held refractometer (ATAGO N-2E Brix 28-62 % made in Japan) and the results were expressed as percentages (%). The percentage of titratable acidity in the fruit juice of 100 berries was determined using the method described by Turner et al. (2011). Total sugars were determined calorimetrically using Nelson arsenate-molybdate colorimetric method (Nielsen 2010). Anthocyanin was determined at the stage of coloration (mg/100 g fresh weight peel) (Nangle et al. 2015).
Statistical Analysis
The obtained data were subjected to one-way analysis of variance (Ott and Longnecker 2015). A least significant difference at 0.05% was used to compare the means of the treatments and measured with CoHort Software (Pacific Grove, CA, USA).
RESULTS AND DISCUSSION
HPLC Analysis of Polyphenols of MLAE
Figure 1 shows the separation chromatograms of the identified chemical compounds in MLAE. Table 3 shows that ellagic acid (54.18), vanillic acid (18.79), p-hydroxy benzoic acid (14), catechol (12.32), and gallic acid (12.12) were the main identified phenolic compounds.
Fig. 1. HPLC chromatogram of phenolic compounds and caffeine identified in MLAE
Vegetative Parameters
Table 4 shows that the shoot length significantly increased as a consequence of the foliar spraying with glycine at 750 ppm, which gave the highest increment compared to the control in both seasons. In addition, it was enhanced by the foliar spraying of glycine at 250 and 500 ppm, MLAE at 4 and 6% and with the combinations of glycine 750 ppm + FA 150 ppm + 6% MLAE and glycine 500 ppm + FA 100 ppm + MLAE 4% in the two seasons as compared with the control. Compared with the control in both seasons, the shoot thickness was significantly enhanced by the applications of glycine at 250, 500, and 750 ppm, MLAE at 2%, 4%, and 6%, and FA at 50, 100, and 150 ppm. Additionally, it improved with the combinations of glycine 250 ppm + FA 50 ppm + 2% MLAE, glycine 500 ppm + FA 100 ppm + MLAE 4%, and glycine 750 ppm + FA 150 ppm + MLAE 6%. The total chlorophyll content in the leaves was greatly improved by the spraying with glycine at 250, 500, and 750 ppm, with MLAE at 2, 4, and 6%, and with FA at 100 and 150 ppm. In addition, it was raised with the combinations of glycine 250 ppm + FA 50 ppm + MLAE 2%, glycine 500 ppm + FA 100 ppm + 4% MLAE, and glycine 750 ppm + FA 150 ppm + 6% MLAE to a higher extent than the control in both experimental seasons. The best results were obtained by the foliar spraying of glycine at 750 ppm over the other applied treatments or the control in the two seasons.
Fruit Yield
Table 5 demonstrates that the foliar spraying with glycine at 250, 500, and 750 ppm, FA at 50, 100, and 150 ppm, and MLAE at 2, 4, and 6% significantly increased the clusters number, cluster weight, yield per vine (kg) and yield in ton per hectare comparing with the control in the two seasons. Moreover, they also were statistically raised by the foliar application of glycine 250 ppm + FA 50 ppm + MLAE 2%, glycine 500 ppm + FA 100 ppm + MLAE 4%, and glycine 750 ppm + FA 150 ppm + MLAE 6% combinations over the control. The highest increment and the best results were obtained by the foliar spraying of glycine, especially at 750 and 500 ppm, respectively, compared with the other applied treatments and the control in both experimental seasons.
Fruit Physical and Chemical Characteristics
Table 6 shows that the size, length, and width of the clusters had greatly increased by spraying glycine at 250, 500, and 750 ppm and by the combination of glycine 750 ppm + FA 150 ppm + MLAE 6% compared with the control and the other applied treatments in both seasons. The application of glycine at 750 ppm was superior and gave the best results in the two seasons rather than the other applied treatments.
Table 7 demonstrates that weight and size of 100 berries and berry length and width were greatly improved by foliar spraying with glycine at 250, 500, and 750 ppm, MLAE at 4 and 6%, the combinations of glycine 500 ppm + FA 100 ppm + MLAE 4% and glycine 750 ppm + FA 150 ppm + MLAE 6% compared with that of the control. The highest obvious effect was obtained by the spraying of glycine at 750 followed by 500 ppm comparing with the other applied treatments, in both experimental seasons.