*2.3. Magnesium Deficiency Index*

Magnesium deficiency (MD) results in interveinal yellowing or reddening on old leaves, beginning at the leaf edge and proceeding to the leaf veins' petiole-connected point. These symptoms progress to necrotic brown patches, and in severe MD, the leaves exhibit necrosis, dray, and premature fall. The Mg deficiency was inspected and scored on a scale from 0 (no injury) to 5 (very severe injury) [37].

#### *2.4. Leaf Pigments Content and Chlorophyll Fluorescence*

Total chlorophyll (Chls) and carotenoid (Car) content were determined spectrophotometrically [38] on the 7th leaf (ten leaves) from the shoot base.

Individual dark-leaf CF data were recorded. The data were acquired using a commercial fluorimeter (Mini-PAM, Walz, Effeltrich, Germany) and data gathering software (Win Control, Walz, Effeltrich, Germany). These data included F<sup>0</sup> (minimum fluorescence), Fm (light-saturated fluorescence), and the Fv/Fm ratio (the difference between maximum fluorescence and minimum fluorescence is Fv or variable fluorescence divided by maximum fluorescence). A fall in the Fv/Fm ratio below 0.75–0.78 suggests a decline in photosystem II photochemical transformation capability [39,40]. On the 7th leaf, CF parameters were determined.

#### *2.5. Leaf Area, Total Carbohydrate Content, Ion Leakage Percentage, and Malondialdehyde (MDA)*

On the 7th leaf, the Sokkia Planix 7 Digital Planimeter was used to quantify leaf area during four developmental stages. However, the vine canes' cumulative carbohydrate content was assessed according to [41]. The leaf petiole cell permeability was also tested. After three washes with deionized water, the rachis samples were put in 10 mL of 0.4 M mannitol at 24 ◦C for three hours. After measuring the EC of the aqueous phase (M1), the rachis samples were killed in a water bath at 100 ◦C for 20 min. This was followed by room-temperature cooling. Then, it was estimated as a percentage of the relative electrolyte loss from M1 rachis samples using the equation: ion leakage percent = (M1M2)/M1 × 100 [42,43]. However, MDA was a by-product of lipid peroxidation that accumulated during salinity stress. They used 2.5 g of leaf petiole samples for MDA extraction [44,45]. This was done by measuring 0–3 mM of TBARS (equal to 0–1 mM MDA) in 1,3,3-tetraethoxypropane (Sigma, St. Louis, MO, USA). During the assay's acid-heating halt, TEOP is stoichiometrically transformed to MDA.

#### *2.6. Leaf Minerals Content*

Leaf mineral content was measured on the 7th leaf from the base of the shoot during four vegetative growth stages. Nitrogen % [46], phosphorous [47], and potassium content [35] as well as the magnesium, calcium, chloride, and sodium content percentages were demined [48].

#### *2.7. Yield and Berry Properties*

At harvest, the number of clusters per vine, average cluster weight (Kg), and yield per vine (Kg) were determined. In addition, the pruned wood was weighted. The SSC % of berry juice was measured with a digital refractometer (PR32 ALA-GO Co., Tokyo, Japan) at lab temperature, and it was represented as a percentage. As for TA %, berry juice (20 mL) was used for titrating by NaOH (0.1N). The outcome was shown as a percentage. However, the SSC/TA-ratio was computed to judge bunch maturity [49,50].

#### *2.8. Statistical Analysis*

The experiment was designed as a randomized complete block in three-way ANOVA with three factors: seasons (2 levels), berry developmental phase (4 levels), and foliar magnesium forms (3 levels) with three replicates per treatment. The mean separations were run with Tukey's HSD test (*p* ≤ 0.05). Pearson's correlation matrix among the studied parameters and principal component analysis (PCA) were applied. Tukey's HSD test was run using the JMP Pro 16 software, with *p* < 0.05 taken as indicating a statistically significant difference (SAS Institute, Cary, NC, USA).

#### **3. Results**

#### *3.1. Magnesium Deficiency Index (MD-Index)*

Figure 1 depicts the magnesium deficiency index (MD-index), which is a function of berry developmental stages (BDSs) for all magnesium types. When seasons, BDSs, and magnesium application forms are examined, the MD-index demonstrates a significant influence of *p* < 0.05. Considering the different magnesium forms, it is obvious that the Mg-NPs treatment produced fewer symptoms of magnesium deficiency than the other magnesium forms. Observably, the effect of 'Mg-NPs' was that there was no evidence of deficient symptoms prior to the veraison stage (berry change color) and that it rose somewhat until the harvesting stage was completed. For vines treated with Mg-EDTA, MgSO4, and control treatments, deficit symptoms were observed prior to fruit set, increased significantly during veraison, and persisted until harvesting. However, during the vegetative growth stages, the 'Control' treatment exhibited the most deficiency symptoms. The severity of Mg was noticed on 'Control' vines that were unaffected by the Mg forms, but the control vines had more symptoms throughout the vegetative growth stages. On sandy soils, symptoms of a magnesium deficit appear on vines during the growth season, necessitating monthly spraying of vines to compensate for the shortfall and thereby avoiding deficient occurrence. Regardless of the magnesium supply to the vines, 750 g of magnesium sulfate per 600 L of irrigation water is employed to avoid magnesium shortages. It is distinguished by the yellowing of older leaves and a yellow tint between the veins of the leaves.

**Figure 1.** The influence of various magnesium fertilizer forms on 'Superior seedless' vines throughout four berry development phases (flowering, fruit set, veraison, and harvesting) under soil salinity conditions on magnesium shortage during the four stages. The values represent the mean affect levels in each application plus standard error (*n* = 3). Tukey's HSD test (*p* ≤ 0.05) used mean severance between blocks (capital letters) to detect significant differences between growing seasons and Mg applications (capital letters) to distinguish significant differences between Mg types.

#### *3.2. Photosynthetic Pigments: Chlorophyll (Chls) and Carotene (Car)*

Photosynthetic pigments as a function of BDSs for all foliar magnesium application forms are shown in Tables 2 and 3. Leaf pigments show a significant interaction at *p* ≤ 0.05 when the seasons, BDSs, and foliar magnesium treatments were considered. Generally, chlorophyll compounds (Chl A and Chl B) and carotenoid (Car) were raised gradually during BDSs until the harvest stage for all Mg treatments, whereas the untreated vines (control) treatment presented the lowest decreases in Chls and Car until the end of the experiment. Despite this, there is a significant variance between Mg treatment on pigment content that was observed during both growing seasons. The obvious outcomes are that the Mg-NPs presented the highest amount of Chl A and Chl B and Car compared to the other Mg treatments and control vines. They were marked with the highest amount at the harvest stage. Moreover, the Car exhibited the highest content at the harvest time stage compared to other foliar treatments. Regarding the Chl A:b ratio, the lowest rates at the harvesting

stage of the vegetative growth period decreased progressively until grape harvesting with all Mg treatments. Nevertheless, the Chl A:b ratio of Mg-NPs had more stable outcomes than those shown with other Mg treatments throughout the growing season.

#### *3.3. Parameters of Chlorophyll Fluorescence (CF) (Fv/Fm, Fm, and F0)*

A significant interaction between seasons and berry developmental stages was found as well as the influence of Mg treatments on Fm and F0 (*p* < 0.001). No significant variations in Fv/Fm ratio were observed for the interaction effect of seasons, berry developmental stages, and mg treatments, but significant differences in Fm and F0 were observed, whereas a significant difference (*p* < 0.01) was noted for the magnesium effect (*p* < 0.001). The Fv/Fm ratio of 'Superior seedless' vines was proposed as a function of BDSs; when seasons, BDSs, and foliar Mg form fertilization were considered, substantial results were obtained (Table 4). On average, untreated vines exhibit a higher decline in the Fv/Fm ratio than vines treated with other Mg compounds. It is drastically reduced until the harvest stage. Except for Mg-NPs treatment, the drop in the Fv/Fm ratio appears to be more gradual and progressive, including a trend toward a more inferior Fv/Fm ratio during vegetative growth stages.

Both Fm and F0 rates increased significantly in overall Mg treatments from the initial stage (flowering) to the veraison stage (Table 4), and this increase was significant for both Fm and F0. It was discovered that the effect of Mg treatments on Fm and F0 varied according to the Mg forms. Then, both are steady until the experiment's duration expires. In comparison to other treatments, the application of Mg-NPs resulted in the greatest Fm and F0 values. Thus, when the Fv/Fm ratio of the 'Superior Seedless' vine was changed, Mg-NPs enhanced CF parameters more than other Mg treatments. As a result, this sample fluorescence parameter can detect magnesium insufficiency.

#### *3.4. Leaf Area, Shoot Carbohydrate, Ion Leakage, and Malondialdehyde Content*

Table 5 presents the differences in leaf area, shoot carbohydrate, ion leakage, and malondialdehyde accumulation as a function of berry developmental stages. The interaction (*p* < 0.001) was significant between the berry developmental stages and the Mg foliar fertilization forms and season. The leaf area (cm<sup>2</sup> ) and shoot carbohydrate content (%) have significantly (*p* < 0.008) higher values when vines receive the Mg-NPs form than other forms. Whereas, when considering the ion leakage percent and MDA content, there were significantly (*p* < 0.0005) lower values throughout the berry developmental stages. This implies that there is variability based on Mg type for previous variables.

#### *3.5. Mineral Content in Leaves*

Tables 6 and 7 exhibit the significant variances (*p* > 0.001) between seasons, BDSs, and Mg application foliar form treatments in the 7th leaf from the base of the shoot N, P, K<sup>+</sup> , Ca++, Mg++, Na<sup>+</sup> , and Cl<sup>−</sup> content when all were considered as experimental factors. Na<sup>+</sup> and Cl− content significantly decreased with Mg-NPs application compared to other Mg forms. However, the rest of the mineral increased during the growth stages.

#### *3.6. Yield and Berry Quality Properties*

Table 8 presents the yield and berry quality properties. The quality variables were significantly affected by foliar fertilization at harvesting time by 5%. The yield was significantly affected more by using foliar Mg-NPs (9.13 kg vine−<sup>1</sup> ) compared to other forms and control treatments.

*Coatings* **2022**, *12*, 201

**Table 2.** The influence of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on leaf chlorophyll parameters pigment of 'Superior seedless' vines, which were used four times on various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).


were obtained at various stages of berry growth.

*Coatings* **2022**, *12*, 201

**Table 3.** The effect of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on leaf carotene pigment and the ratio of chlorophyll and carotenoid of 'Superior seedless' vines, which were used four times on various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).


**Table 4.** The impact of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on chlorophyll fluorescence parameters of 'Superior seedless' vines, which were used four times on various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).





**Table 5.** The impact of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on leaf area (cm2 ), shoot carbohydrate content percentage, ion leakage percentage, and malondialdehyde of 'Superior seedless' vines, which were used four times on various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).





**Table 6.** The effect of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on leaf mineral compositions of 'Superior seedless' vines, which were used four times on various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).


were obtained at various stages of berry growth.

*Coatings* **2022**, *12*, 201

**Table 7.** The influence of various magnesium fertilization types (MgSO4 , Mg-EDTA, and Mg-NPs) on the leaf mineral compositions of 'Superior seedless' vines was studied for four terms in various phases during berry growth (flowering, fruit set, version, and at harvest time) throughout two summers (2020 and 2021).


Control 0.440 ± 0.002 d 13.56 ± 0.233 c 5.91 ± 0.073 d 14.19 ± 0.134 d 3.46 ± 0.029 d 3.31 ± 0.008 d 15.95 ± 0.014 d 0.713 ± 0.001 a 22.35 ± 0.062 d 0.713 ± 0.001 a 22.35 ± 0.062 d MgSO4 0.502 ± 0.003 c 14.71 ± 0.020 b 7.39 ± 0.055 c 14.67 ± 0.023 c 3.85 ± 0.020 c 3.66 ± 0.005 c 16.71 ± 0.028 b 0.684 ± 0.002 c 24.41 ± 0.029 b 0.684 ± 0.002 c 24.41 ± 0.029 b Mg-EDTA 0.525 ± 0.002 b 15.51 ± 0.340 a 8.15 ± 0.210 b 15.94 ± 0.086 b 4.16 ± 0.029 b 4.17 ± 0.023 b 16.33 ± 0.014 c 0.699 ± 0.000 b 23.36 ± 0.020 c 0.699 ± 0.000 b 23.36 ± 0.020 c Mg-Nano 0.582 ± 0.002 a 15.98 ± 0.015 a 9.13 ± 0.038 a 16.85 ± 0.272 a 4.67 ± 0.021 a 4.56 ± 0.008 a 17.38 ± 0.038 a 0.661 ± 0.000 d 26.30 ± 0.066 a 0.661 ± 0.000 d 26.30 ± 0.066 a The main data of two seasons are analyzed using one-way (complete block randomized design) on 'Superior seedless' vines. Each value represents mean and ±SE (*n* = 4) replicates. The superscript letters differ (*p <* 0.05) and represent the significance between treatments using Tukey's HSD test at *p* ≤ 0.05. Data were collected at different berry developmental stages.

**Yield and Berry Properties Berry Juice Proprieties**

### *3.7. Multivariate Analysis of Leaf Parameters*

A PCA for physiological and biochemical variables data obtained from leaves was conducted from the tested different foliar magnesium fertilization forms (MgSO4, Mg-EDTA, and Mg-NPs) applied four times on different fruit developmental stages (flowering, fruit set, version, and at harvest time) throughout two growth seasons (2020 and 2021) of 'Superior Seedless' vines. The PCA separated the effect of magnesium forms under each seasonal stage. The PC1 explained 70.9% of the variability in the data, while PC2 explained 16.1% of the variability (Figure 2A). Figure 2B displays the negative correlation between MDindex with all the parameters except for EL%, MDA, Na+ %, and Cl− %. Chlorophyll a and b and total chlorophyll contents were negatively correlated with chlorophyll fluorescence variables (Fv/Fm; Fm, and F0). These four valuables (MD, MDA, Na+ %, and Cl− %) had a negative correlation with the other variables. Chl B showed negative correlation with Chl A:B. Chl A:B was positively correlated with Chls:Caro and Fv/Fm, whereas it had a negative correlation with the other valuables. Pearson's correlation matrix among the examined parameters shows the correlation and shows these results (Table 9).

**Figure 2.** Principal Component Analysis (PCA) representing seasons and magnesium application forms to 'Superior seedless' vine grown in sandy soil and salt conditions, plotted with the contribution of each parameter on the two PCA axes (**A**) and all the physiological and biochemical parameters measured in leaf during the growing season (**B**). Principal Component Analysis (PCA)-Variable correlation of 7th leaf.


84

*Coatings* **2022**, *12*, 201

#### **4. Discussion**

Magnesium is involved in a number of biochemical and physiological processes that influence plant growth and development [51]. As a result, the wounded bunches' earlystage leaves fall off throughout the growing season. However, under soil salinity conditions, a variety of mechanisms occur that result in Mg loss [52]. As a result, Mg insufficiency occurred on control vines earlier in the growth season than on vines treated with other Mg treatments. This can be seen in the slower transport of Mg through the soil profile, which results in more Mg adsorption [53]. In addition, changes in Ca and K content across Mg application rates suggest that Mg and two other cations interact throughout the season [54]. Foliar spraying is a common way for plants to adjust for nutritional deficiencies in the soil [55]. During the trial period, the efficiency of the nano-magnesium image revealed the fewest symptoms on the leaves. This result could be attributed to magnesium absorption being faster than the rest of the pictures, resulting in better photosynthetic efficiency [56]. These conclusions were reached because of the results shown in the graph. The presence of EDTA in chelated Mg form, on the other hand, has been shown to improve vine growth and biomass [57], and the sulfate part plays a critical role in the catalytic or electrochemical functions of the biomolecules in the cells [58].

Chlorophylls (Chls) are reputedly the most outstanding natural syntheses on the planet, as they are required for the photosynthesis process [59,60]. This method of vegetation occurs primarily based on gaining light rays by chlorophyll and especially chlorophyll A [61]. Photosynthesis is a very powerful method wherein it is supplied with 5 to 11 µmol CO<sup>2</sup> m−<sup>2</sup> s −1 . This process is involved in the biosynthesis of essential organic molecules required for plant growth and development [62]. The photosynthesizing cells need a large amount of assimilatory pigment that reaches up to 5% of typical dry matter [63,64]. Most plant species have photosynthetic pigment content in their leaves (chlorophyll and carotene), which plays a fundamental function in the physiological overall performance of plants [65,66]. Mg participates in a variety of biochemical and physiological processes that contribute to vine growth. It is a critical component of the chlorophyll molecule, affecting both its structure and function [67]. Foliar magnesium fertilization compensates for deficits in the vines' growth stages. Additionally, it reflected the quantity and activity of photosynthetic pigments [54]. Mg is a mineral activator constituent of the chlorophyll molecule, which is responsible for photosynthetic regulation [68]. As a result, as compared to other Mg forms, the usage of Mg-NPs increased the chlorophyll components and carotene content [69]. Our findings corroborated those published in Tables 1 and 2. This comparison most likely reflects Mg-NPs' superior mobility and absorption capacity when compared to other forms [70].

Chlorophylls are critical functional and structural cofactors for all photosynthetic pigment proteins involved in oxygenic and anoxic photosynthesis, and so magnesium fertilization throughout the growing season contributes to photosynthesis's efficacy. The pigments' distinctiveness is owing to the porphyritic chromophore's extensive electron system, which chelates the Mg2+ ion in the center [71]. The results in Table 3 can be clarified by the variation in the Fv/Fm ratios of the various forms of foliar magnesium fertilization applied at various growth stages. In comparison to other forms, Mg-NPs dramatically boosted nucleic acid and carbohydrate enzymes [68]. However, the onset of magnesium deficit during the growing season may result in a reduction in chlorophyll and carotene levels [72]. Our findings established that Mg-NPs boosted photosynthetic pigment in comparison to other Mg forms, and our findings corroborated those of [56].

Since magnesium is required for carbohydrate accumulation in plants, its absence has an effect on the overall biomass production and distribution among plant sections [73,74]. This shows that three major factors could influence Mg effects. These are the magnesium forms, mobility, and absorption capacity of magnesium [75]. Our data indicated that the Mg-NPs increased the leaf area and carbohydrate content of the shoots during the growing season, owing to the higher photosynthesis performance. We observed reduced values for ion leakage and MDA quantity when vines were treated with Mg-NPs compared to

other types. One may argue that increasing magnesium absorption in nano form [69] resulted in a reduction in the size of the cell wall, which was most likely because of its role in ion transport across the membrane and involvement in membrane-center ATPase activity [76,77]. This conclusion was consistent with previous research on citrus [78], banana [19], and coffee [79]. On the current experiment, we discovered a similar pattern of carbohydrate accumulation in vines stressed with evident leaf symptoms in the presence of a magnesium deficiency.

Normally, in plants, an element's uptake and distribution are controlled by both its supply conditions and interactions with other elements [80]. Mg, K, and Ca have been considered to exhibit opposing interactions as cation ions. Mg absorption was restricted when K or Ca concentrations increased and vice versa [81,82]. However, under salinity stress, the application of Mg-NPs increases the content of macro and micro-nutrients (Tables 5 and 6). This may be explained by the inaction between Ca++ and K<sup>+</sup> and Mg++ , which increased the abortion of both cations by using Mg-NPs more than other forms [52]. The achieved outcomes regarding the effect of foliar Mg forms on leaf mineral content proved that the magnesium nano form has a pronounced effect on micronutrient status. The results agree with the findings of [56]. In addition, the foliar magnesium fertilizer improved the leaf mineral content of the mentioned fruit crop species.

This could be explained by the fact that the Mg-NPs enhanced photosynthesis during the growth stages [54]. As a result, the carbohydrate content of the product increased [7]. Our findings established that Mg-NPs raised carbohydrate content more than other forms (Table 4) and wood-trimmed weight more than other forms (Table 8). However, Mg-NPs had a considerably greater effect on berry quality features than other treatments, as measured by SSC percent (17.50%), TA percent (0.805%), and SSC:TA ratio (21.63%) (Table 7). The lowest SSC:TA ratio observed with Mg-NPs application might be read as indicating that bunches collected from vines treated with other Mg forms had a significantly longer shelf life. Additionally, magnesium has a role in protein synthesis as a bridge element that aids in ribosome assembly [83]. Additionally, it catalyzes about 300 enzymes, including phosphoenolpyruvate carboxylase, glutathione synthase, phosphatases, kinases, RNA polymerases, and ATPases [74].

A negative connection was detected between Chl B and Chl A:B. Chls:Caro and Fv/Fm were positively linked with Chl A:B but negatively with the other assets. Our observations were acknowledged by both parties [19].

#### **5. Conclusions**

The outcomes of this research recommend that the Fv/Fm ratio during the growth season of 'Superior Seedless' vines may be a good tool to assess magnesium fertilization effects before visible deficiency symptoms appear. Mg-NPs are more effective at improving 'Superior Seedless' vine growth than the other magnesium forms. Moreover, a comparison validated that the application of different forms of Mg foliar fertilization for 'Superior Seedless' vines does affect the yield and berry quality at harvest time as a final determination of the impact of Mg foliar fertilization. Overall, Mg-NPs are the most effective form for application to 'Superior Seedless' vines when compared to other Mg forms under saline soil. It enhanced biochemical and bunched quality variables.

**Author Contributions:** Conceptualization, S.M.A.-Q.; Data curation, S.F.A.E.-E; Formal analysis, H.M.A.; Funding acquisition, N.A.A.-H.; Investigation, Z.A.A.; Methodology, S.F.A.E.-E.; Project administration, S.M.A.-Q.; Resources, Z.A.A.; Software, L.A.A., N.A.A.-H. and H.M.A.; Supervision, L.A.A. and M.A.A.; Writing—original draft, M.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Relevant data applicable to this research are within the paper.

**Acknowledgments:** The author Lo'ay, A.A. extend their thanks, appreciation, and gratitude to Sally F. Abo EL-Ezz for their constructive cooperation during the research stages. This research is presented as a tribute to the soul of our dead colleague, Sally F. Abo EL-Ezz.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

