Exogenous Application of Mg, Zn and B Inﬂuences Phyto-Nutritional Composition of Leaves and Fruits of Loquat ( Eriobotrya japonica Lindl.)

: This study was conducted to analyze the effect of magnesium (Mg), zinc (Zn) and boron (B) on the level of macronutrients (Na, Mg, K, Ca), micronutrients (Zn, B, Mo, Mn, Fe, Co, Ni, Cu), and heavy metals (Cd, As, Hg, Pb) in loquat leaves and fruit tissues (peel, pulp, and seed) using inductively coupled plasma-mass spectrometry (ICP-MS). Fruits were obtained from a loquat orchard located in Fujian (Yun Xiao/Zhangzhou). The results revealed that the foliar application of Mg increased the concentrations of Mg, K, Ca, Zn, B, Mn, Ni, and Cu in leaves; Mg, Ca, Zn, B, Mo, and Mn in fruit pulp; and Na, K, B, Mo, Co, Ni, and Cu in seeds. Zinc increased Mg, K, Ca, Zn, B, Ni, and Cu in leaves; Fe, Co and Ni in fruit peel; K, B, Mn, Fe, and Co in fruit pulp; and Na and K in seeds. Similarly, B application increased the concentrations of Na, Mg, K, Ca, and B in leaves; Ca and Ni in fruit peel; Na, Mg, Ca, Zn, B, Mn, Fe, and Co in fruit pulp; and Na, K, Ca, Zn, B, Mo, Mn, Co, and Ni in loquat seeds. Overall, mineral concentrations detected in the leaves and fruit tissues of loquats were greatly inﬂuenced by the application of Mg, Zn and B. Although heavy metal concentrations in fruit pulp were increased by the foliar application of B, the fruits were under safe limits for human consumption.


Introduction
The loquat (Eriobotrya japonica Lindl.) is an evergreen fruit tree originating from the People's Republic of China. It belongs to the family Rosaceae, subfamily Maloideae. It is a rich source of vitamin A, vitamin B6, potassium, magnesium, and dietary fiber [1]. It is a very beautiful orange-colored fruit with a mild sweet and sour taste [2]. It is most widely grown in Japan, Korea, India, Pakistan, and the south-central region of China. It is also grown as an ornamental shrub in California [3]. China is the leading producer and exporter of loquats, and grows it on more than 100,000 hectares. The annual production of loquats in China reaches up to 380,000 tons [4]. More than 30 species of loquat are being grown in temperate and subtropical regions of Asia [5].
Fruits and vegetables are low in energy content but have high nutritive value. They are a rich source of vitamins, fibers, and minerals, which are very essential for the human body [6]. The sufficient intake of fruits and vegetables can remove saturated fats and sugars from the body and enhance the consumption of healthy nutrients and dietary fiber [7]. To avoid certain cardiovascular diseases, a consumption of 440 g fresh fruits and vegetables per day is recommended [8]. Heavy metals (e.g., Hg, As, Cd, and Pb) can influence the functioning of the nervous system and cause mental illness by affecting the blood circulatory system [9]. Hence, it is very important to investigate the mineral contents of fruits and vegetables.
Fertilizer use is one of the main factors influencing the mineral contents of plants [10][11][12]. Foliar application of fertilizers is 10-20% more influential than soil application [13]. Magnesium helps the plants by strengthening cell walls, and it improves the uptake of certain nutrients (e.g., N, P, and S) [14]. Magnesium regulates the balance of cations and anions in cells and plays a significant role with K in maintaining cell turgor pressure [15,16]. There is 75% of leaf Mg involved in protein synthesis, and 15-20% has a significant role in chlorophyll pigmentation [17] and acts as a co-factor of a number of enzymes in photosynthetic metabolism [18][19][20]. Zinc is one of the essential micronutrients, and is crucial for mineral nutrition in plants. Zinc is involved in essential physiological functions in plant metabolism, influencing the growth and productivity of plants [21]. Boron nutrition affects nutrient status in plants by modulating the uptake and translocation of other nutrients [22]. For example, B plays a positive role in uptake of N, P, K, Cu, Zn, and Fe, whereas it negatively interacts with Ca, Mg, and Mn [23][24][25][26].
The quadrupole inductively coupled plasma mass spectrometer (ICP-MS) is the most suitable method for determining trace elements in fruits and vegetables, and has prevailed as the most appropriate practice for clinical quantification [27][28][29][30]. Element detection through ICP-MS has become a predominant methodology because of its rapidity, determination limits, and the sample quantity needed for analysis [31]. In the present experiment, the contents of macronutrients (Na, Mg, K, Ca), micronutrients (Zn, B, Mo, Mn, Fe, Co, Ni, Cu), and heavy metals (Cd, As, Hg, Pb) were analyzed in different loquat tissues (leaf blade, fruit peel, pulp, and seed) affected by the foliar application of Mg, Zn, and B via inductively coupled plasma-mass spectrometry (ICP-MS).

Plant and Soil Sampling
Twenty young loquat trees, growing in the subtropical area of Fujian province (Yun Xiao) (23 • 57 13.5" N 117 • 20 36.0" E), were selected and marked for periodic monitoring after foliar application of Mg, Zn, and B. Loquat trees were 4-5.5 m tall with canopy diameters of 4-5 m. The plantation distance among trees was 6 × 6 m, approximately. Loquat trees were systematically pruned and thinned, and fertilized with NPK (15:15:15) at concentrations of 5 kg per plant per season for three growing seasons. The experiment contained five treatments including the control (water spray) (T 0 ), 2% MgSO 4 ·7H 2 O (T 1 ), 0.1% ZnSO 4 ·7H 2 O (T 2 ), 0.2% Na 2 B 4 O 7 ·10H 2 O (T 3 ), and T 1 +T 2 +T 3 (1:1:1) (T 4 ). The foliar application was carried out twice at the full bloom stage with a 3 week interval. Each tree was considered as one replicate of each treatment (four replicates per treatment).
Samples of the leaves and fruits were taken from the south-exposed tree canopy, at about 3-5 m height, at two different fruit maturity stages (i.e., mature green (MG) (40 days after foliar spray) and fully ripe (FR) (70 days after foliar spray). Samples were washed (for 10 s approximately) with a solution of phosphate-free detergent, then with a 0.1 N HCl solution, and finally with distilled water. Leaves, fruit peel, pulp, and seeds were dried at 70 • C, ground, and passed through a 500-µm stainless-steel sieve.
Soil samples were taken from the root zone of each tree, about 2 m from the trunk, and at two depths (0-25 and 25-40 cm) using a spiral auger with a 2.5 cm diameter. Three subsamples around the trunk were taken to make a composite soil sample for each tree. Samples were transported to the laboratory (Institute of Subtropical Fruits, Fujian Agriculture and Forestry University), oven-dried at 40 • C, and crushed to pass through a 2-mm sieve, then ground to <60 µm for element determinations [32]. The soil's structural type was sandy loam with electric conductivity and pH of 0.396 dS m −1 and 7.6, respectively. The electric conductivity and pH were recorded with an EC meter (HI-98,304, Hanna Instruments Inc., George Washington, RI, USA) and digital pH meter (Hanna, HI-98,107, Mauritius), respectively. Table 1 shows the level of mineral nutrients and heavy metals in loquat soil and plant tissues determined through ICP-MS before foliar application of Mg, Zn, and B. The values in Table 1 show the mean of four replicates. The optimum ranges of mineral nutrients in loquat leaves are given after the findings of Quiñones et al. [33] and Reig et al. [34].

Instrumentation and Reagents
The inductively coupled plasma mass spectrometer (Agilent7700X, Agilent, Santa Clara, CA, USA) used in this study was combined with a high-efficiency sample introduction desolvating system equipped with a quartz cyclonic spray chamber. Before each analysis, the instrument was tuned for daily performance using Agilent 7700X Sensitivity Detection Limit Solution, Agilent, USA. This is an aqueous multi-element standard solution of Li, Y, Co, Ce, and Tl for consistent sensitivity ( 7 Li,59 Co, 89 Y, 140 Ce, 205 Tl) and minimum doubly charged and oxide species levels ( 140 Ce). The concentration of elements in the solution was 10 µg L −1 . The internal standard solution (Agilent, USA) had a concentration of 0.01 µg ml −1 for each element ( 226 Re, 115 In, 73 Ge, 45 Sc).
Standard solution (1000 µg L −1 ) for each element (i.e., Na, Mg, K, Ca, Zn, B, Mo, Mn, Fe, Co, Ni, Cu, Cd, As, Hg, and Pb) was purchased from the National Standard Material Research Centre. Nitric acid (HNO 3 ) used in the experiment was analytically pure, obtained from CNW Technologies GmbH, Germany. The deionized water (1.83 × 10 10 mmhos) was prepared locally. In this experiment, the working parameters of the inductively coupled plasma mass spectrometer were optimized before the test, and the parameter settings are shown in Table 2.

Sample Preparation
After cleaning, glassware and a Pure Teflon (PTFE) digestion tank were soaked in 20% nitric acid (1 + 4, V + V) for more than 12 h, then rinsed with deionized water three times before use. After lyophilizing, 0.5 g of freeze-dried loquat tissue was added into a poly-tetra-fluoroethylene digestion tank. Then, 5 mL of concentrated nitric acid (analytical grade, CNW Technologies GmbH, Dusseldorf, Germany) was added. The digestion tank was gently shaken to completely immerse the sample, and microwave digestion (Mars5, HY−20−164, CEM, Matthews, NC, USA) occurred under the set conditions (Table 3). After microwave digestion, the solution was cooled naturally. The digestion solution was transferred to a 25-mL volumetric flask. The blank sample (control) was prepared simultaneously.

Determination
A single standard series solution was prepared. The mixed standard series solution and sample solution were measured by ICP−MS, and the standard curve method was used for quantification. The mixed internal standard was added to correct for matrix interference and instrument signal drift.

Statistical Data Analysis
Collected data were analyzed for analysis of variance (ANOVA) and Fisher's least significance difference (LSD) method for the pair-wise comparison of mean values at a 5% significance level using the analytical software package 'Statistix 8.1'. Principal component analysis (PCA) and correlation coefficient values were determined with the Pearson (n) method using XLSTAT Ver. 2019.

Macronutrients
Sodium concentration in loquat leaves was reduced after foliar applications of T 1 , T 2 , and T 4 , while it increased with the application of T 3 . Leaves of plants treated with T 4 showed the minimum level of sodium (Na) at the mature green stage (56.27 mg kg −1 ), whereas at the fully ripe stage the minimum Na (74.16 mg kg −1 ) was recorded in plants treated with T 2 . The maximum Na concentration occurred in the plants treated with T 3 at both maturity stages. Regarding Mg, the plants treated with T 1 , T 2 , T 3 and T 4 showed increased concentrations of Mg as compared to the control, with the maximum Mg was observed in leaves of plants treated with T 1 (MG−2694.88; FR−2745.70 mg kg −1 ) followed by T 3 , T 2 , and T 4 at both maturity stages (MG and FR). Similarly, with the applications of T 1 , T 2 , T 3 , and T 4 , the K level in loquat leaves increased. The plants that received T 2 showed a maximum K level (15,569.89 mg kg −1 ) at the mature green stage, whereas at the fully ripe stage the maximum K level (23,508.60 mg kg −1 ) was depicted in the plants treated with T 3 .
The loquat leaves that received foliar applications of T 1 , T 2 , T 3 , and T 4 had enhanced Ca concentrations as compared to the leaves of the untreated plants (T 0 ). Foliar application of T 1 was proven better as compared to the other treatments in terms of increasing the level of Ca (16,944.45 mg kg −1 ) at the mature green stage, while at the fully ripe stage the plants treated with T 4 showed their maximum Ca concentration (9950.87 mg kg −1 ) (Figure 1). stage the maximum K level (23,508.60 mg kg −1 ) was depicted in the plants treated with T3.
The loquat leaves that received foliar applications of T1, T2, T3, and T4 had enhanced Ca concentrations as compared to the leaves of the untreated plants (T0). Foliar application of T1 was proven better as compared to the other treatments in terms of increasing the level of Ca (16,944.45 mg kg −1 ) at the mature green stage, while at the fully ripe stage the plants treated with T4 showed their maximum Ca concentration (9950.87 mg kg −1 ) ( Figure  1).  Concentrations of Ni and Cu in leaves of a loquat were observed to increase with the applications of T 1 , T 2 , T 3 , and T 4 at the mature green stage, whereas at the fully ripe stage the concentrations of Ni increased with applications of T 1 and T 2 , and decreased with applications of T 3 and T 4 . The concentrations of Cu recorded at the fully ripe stage decreased with the foliar applications of T 1 , T 3 , and T 4 , but not with T 2 (Figure 1).

Heavy Metals
There were significant increases in Cd concentrations in loquat leaf blades with applications of T 1 , T 2 , T 3 , and T 4 at the fully ripe stage, while at the mature green stage there were variations among treatments (increasing with T 1 and T 3 , decreasing with T 2 and T 4 ). The maximum As level (MG−58,776.82; FR−42,313 µg kg −1 ) was recorded in the leaves of the untreated plants (T 0 ) at both maturity stages, while with the applications of T 1 , T 2 , T 3 , and T 4 the As level reduced significantly. Regarding Hg and Pb, concentrations reduced with the applications of T 1 , T 2 , T 3 , and T 4 as compared to T 0 . The minimum concentrations of Hg and Pb were recorded in plants treated with T 3 and T 2 , respectively, at the mature green stage (1.42 and 390.49 µg kg −1 , respectively), while at the fully ripe stage, their concentrations in all treated plants remained non-significantly different ( Figure 1).

Macronutrients
The Na concentrations in the loquat fruit's peel were reduced by the foliar applications of T 1 and T 3 , and increased with the applications of T 2 and T 4 at the mature green stage. The plants treated with T 2 showed their maximum level of Na at the mature green stage (187.66 mg kg −1 ), whereas, at the fully ripe stage maximum Na (193.44 mg kg −1 ) was recorded in the plants treated with T 3 . Regarding Mg, the plants treated with T 4 exhibited increased concentrations of Mg (1705.86 mg kg −1 ) as compared to the control (T 0 ) at the mature green stage, whereas at the fully ripe stage Mg was reduced by the applications of T 1 , T 3 , and T 4 . Similarly, with the application of T 4 , K level in the loquat fruit's peel was observed to increase at both maturity stages (MG−12,171.99; FR−26,687.45 mg kg −1 ). The loquat fruit's peels that received foliar applications of T 1 and T 3 showed enhancement in Ca concentration as compared to the untreated plants (T 0 ) at the mature green stage. The foliar application of T 3 proved better than to other treatments, showing the maximum level of Ca (15,437.72 mg kg −1 ) at the mature green stage, while the Ca level was reduced at the fully ripe stage in plants treated with T 1 , T 2 , and T 3 ( Figure 2).

Micronutrients
The plants that received foliar applications showed increased levels of Zn as compared to the untreated plants (T 0 ) (21.08 mg kg −1 ), except when treated with T 1 (33.32 mg kg −1 ) and T 2 (16.97 mg kg −1 ) at the mature green stage, and T 4 (25.36 mg kg −1 ) at the fully ripe stage. The foliar applications of T 1 , T 2 , T 3 , and T 4 on the loquat fruit's peel showed that the B concentration was increased in the plants that received a foliar application of T 4 as compared to untreated plants (T 0 ) at the fully ripe stage, while at the mature green stage the B level was reduced by applications of T 1 , T 2 , and T 4 . In contrast with B, the Mo level decreased with the applications of T 1 (107.09 µg kg −1 ) and T 3 (76.09 µg kg −1 ), as compared to T 0 (129.56 µg kg −1 ) at the mature green stage. Similarly, the plants that received foliar applications showed a decreased level of Mo at the fully ripe stage.
The plants that received the foliar applications showed a non-significant level of Mn as compared to the untreated plants (T 0 ) (48.80 mg kg −1 ), except when treated with T 1 (407.34 mg kg −1 ) and T 2 (64.82 mg kg −1 ) at the mature green stage, and T 2 (68.34 mg kg −1 ) at the fully ripe stage. Similarly, the plants treated with T 2 showed a maximum concentration of iron at the mature green stage, whereas at the fully ripe stage the plants that received foliar applications showed a decrease in Fe level as compared to the control. The plants

Heavy Metals
There was a significant increase in Cd concentrations in the loquat fruit's peel with applications of T 1 and T 3 at the mature green stage, while at the fully ripe stage the plants that received foliar applications showed a decrease in Cd level as compared to the control. The maximum As level (MG−123.01; FR−15.52 µg kg −1 ) was recorded in the fruit peels of the plants treated with T 3 at both maturity stages. Regarding Hg, toxicity was significantly increased with applications of T 1 and T 3 at the mature green stage as compared to the control. Similarly, at the fully ripe stage, concentrations of Hg were reduced with foliar applications except in the plants treated with T 4 (13.21 µg kg −1 ). Lead concentrations decreased with foliar applications of T 1 , T 2 , T 3, and T 4 at both stages. There was a significant decrease in Pb toxicity resulting from changes in the maturity stage ( Figure 2).

Macronutrients
The Na concentrations in the loquat fruits' pulp were reduced with foliar applications of T 1 , T 2 , T 3 , and T 4 at the mature green stage, while at the fully ripe stage they decreased except in the plants treated with T 3 . The plants treated with T 1 showed a minimum level of Na at the mature green stage (75.57 mg kg −1 ), whereas, at the fully ripe stage, minimum Na (60.65 mg kg −1 ) was recorded in plants treated with T 2 (Figure 3). Regarding Mg, the plants treated with T 1 , T 2 , T 3 , and T 4 showed variations in concentrations of Mg, where the maximum Mg was observed in the plants treated with T 2 (2594.59 mg kg −1 ) followed by T 4 , T 3 , and T 1 at the mature green stage, and T 3 (2543.08 mg kg −1 ) followed by T 1 , T 2 , and T 4 at the fully ripe stage. Similarly, through the application of T 1 , T 2 , T 3 , and T 4 , the K level in the loquat fruits' pulp was uplifted at the mature green stage. The plants that received T 1 showed their maximum K level (13,135.76 mg kg −1 ) at the mature green stage, whereas the maximum K level (12,788.59 mg kg −1 ) was depicted at the fully ripe stage by the plants treated with T 2 . The plants that received foliar applications of T 1 , T 2 , T 3 , and T 4 showed enhancement in Ca concentration as compared to the pulp of the untreated fruits (T 0 ). The foliar application of T 1 was proven to be better than other treatments, showing its maximum level of Ca (MG−10,214.13; FR−20,459.14 mg kg −1 ) at both maturity stages ( Figure 3).

Heavy Metals
There were significant increases in Cd concentrations in the loquat fruit pulp following applications of T1 and T2, and decreases with applications of T3 and T4, at both maturity

Micronutrients
The plants that received foliar applications of T 2 and T 4 showed decreased levels of Zn concentration as compared to the untreated plants (T 0 ) at the mature green stage, while at the fully ripe stage there were variations among treatments (increased by T 1 and T 3 and decreased by T 2 and T 4 ). B concentrations increased in the fruit pulp of the plants that received foliar applications of T 1 , T 2 , T 3 , and T 4 as compared to the untreated plants (T 0 ) at the mature green stage. The maximum level of B (40.62 mg kg −1 ) was found in the plants treated with T 1 followed by T 2 , T 3 , and T 4 at the mature green stage, whereas at the fully ripe stage, the maximum value of B concentration was observed in plants treated with T 2 . Similarly, Mo level was also increased by application of T 1 and T 3 at the mature green stage, and T 1 and T 2 at the fully ripe stage. The maximum concentration of Mo was seen in the plants treated with T 1 at the mature green stage and T 2 at the fully ripe stage (Figure 3).

Heavy Metals
There were significant increases in Cd concentrations in the loquat fruit pulp following applications of T 1 and T 2 , and decreases with applications of T 3 and T 4 , at both maturity stages. The maximum As level (21.69 µg kg −1 ) was recorded in the leaves of the untreated plants (T 0 ) at the mature green stage, whereas at the fully ripe stage the maximum As was observed in the pulp of the fruits treated with T 3 . Regarding Hg, toxicity increased with applications of T 1 , T 2 , T 3 , and T 4 as compared to the control (T 0 ). The maximum concentration was recorded in plants treated with T 2 at the mature green stage (3.21 µg kg −1 ), while at the fully ripe stage the maximum concentration of Hg was recorded in the plants treated with T 3 . Pb concentration was reduced with the foliar application of T 2 at both maturity stages ( Figure 3).

Macronutrients
Na concentrations were reduced with foliar applications of T 1 and T 3 at the mature green stage, while they increased with foliar applications of T 2 and T 4 . Na concentrations were universally reduced at the fully ripe stage, except in the plants treated with T 3 . The plants treated with T 3 showed their minimum level of Na at the mature green stage (47.44 mg kg −1 ), whereas, at fully ripe stage, the minimum Na (73.63 mg kg −1 ) was recorded in the untreated plants (T 0 ) (Figure 4). Regarding Mg, the plants treated with T 1 , T 2 , T 3 , and T 4 showed variations in their concentrations of Mg, with the maximum Mg observed in plants treated with T 4 (2959.40 mg kg −1 ) followed by the control (T 0 ) and T 2 at the mature green stage, and T 3 (1241.59 mg kg −1 ) followed by T 0 , T 1 , T 2 , and T 4 at the fully ripe stage. With the application of T 3 , K levels in loquat seeds were slightly uplifted at the mature green stage, but decreased with the application of other treatments as compared to the control. Plants that received T 1 showed their maximum K level (22,396.23 mg kg −1 ) at the fully ripe stage. Loquat seeds indicated enhanced Ca concentrations in the plants that received foliar applications of T 2 and T 4 as compared to the untreated plants (T 0 ).
The foliar application of T 4 proved better, however, showing its maximum level of Ca (22,752.88 mg kg −1 ) at the mature green stage, while T 1 showed its maximum level at the fully ripe stage (Figure 4).
T4 showed variations in their concentrations of Mg, with the maximum Mg observed in plants treated with T4 (2959.40 mg kg −1 ) followed by the control (T0) and T2 at the mature green stage, and T3 (1241.59 mg kg −1 ) followed by T0, T1, T2, and T4 at the fully ripe stage. With the application of T3, K levels in loquat seeds were slightly uplifted at the mature green stage, but decreased with the application of other treatments as compared to the control. Plants that received T1 showed their maximum K level (22,396.23 mg kg −1 ) at the fully ripe stage. Loquat seeds indicated enhanced Ca concentrations in the plants that received foliar applications of T2 and T4 as compared to the untreated plants (T0). The foliar application of T4 proved better, however, showing its maximum level of Ca (22,752.88 mg kg −1 ) at the mature green stage, while T1 showed its maximum level at the fully ripe stage (Figure 4).

Micronutrients
The plants that received foliar applications of T1, T2, T3, and T4 showed decreased levels of Zn as compared to the untreated plants (T0) at the mature green stage, while at the fully ripe stage there were variations among treatments (increased by T1 and T3 and decreased by T2 and T4). B concentrations increased in the seeds of the plants that received foliar applications of T1 and T3 as compared to the untreated plants (T0) at both maturity

Micronutrients
The plants that received foliar applications of T 1 , T 2 , T 3 , and T 4 showed decreased levels of Zn as compared to the untreated plants (T 0 ) at the mature green stage, while at the fully ripe stage there were variations among treatments (increased by T 1 and T 3 and decreased by T 2 and T 4 ). B concentrations increased in the seeds of the plants that received foliar applications of T 1 and T 3 as compared to the untreated plants (T 0 ) at both maturity stages. The maximum level of B (19.58 mg kg −1 ) was found in the plants treated with T 1 followed by T 3 , T 2 , and T 4 at the mature green stage, whereas at the fully ripe stage, the maximum value of B was observed in the plants treated with T 3 . Similarly, the Mo level also increased with applications of T 1 and T 3 at both maturity stages. The maximum concentration of Mo was seen in the plants treated with T 3 at both maturity stages (MG−152.42; FR−235.82 µg kg −1 ). The maximum concentration of Mn was observed in the untreated plants (T 0 ) (402.29 mg kg −1 ) at the mature green stage, while at the fully ripe stage the maximum Mn (102.43 mg kg −1 ) was observed in the plants treated with T 1 . Iron concentrations decreased with the foliar applications except in the plants treated with T 2 at the mature green stage. The concentrations also decreased with a change in maturity stage. The plants that received foliar applications of T 1 and T 3 showed increases in Co concentration as compared to the untreated plants (T 0 ) at the fully ripe stage, while the maximum level of Co (979.14 µg kg −1 ) was shown at the mature green stage by the seeds of those plants that were treated with T 1 . Concentrations of Ni and Cu increased with applications of T 1 and T 4 in loquat seeds at the mature green stage, whereas Ni concentrations increased with applications of T 1 and T 3 at the fully ripe stage and decreased when T 2 and T 4 were applied. Copper concentrations decreased with foliar applications at the fully ripe stage except when T 1 and T 2 were applied (Figure 4).

Heavy Metals
There was a significant increase in the Cd concentration in loquat seeds with an application of T 4 , and a decrease with T 1 and T 3 at the mature green stage. While at the fully ripe stage, the plants that received foliar applications of T 1 and T 3 showed increased Cd as compared to the untreated plants (T 0 ). The maximum As (91.11 µg kg −1 ) was recorded in the seeds of the untreated plants (T 0 ) at the mature green stage, whereas the maximum As was observed in the seeds of the plants at the fully ripe stage treated with T 3 (87.24 µg kg −1 ). Regarding Hg, toxicity decreased with applications of T 1 and T 3 up to 1/2-fold and 1/3-fold, respectively, at the mature green stage. Similarly, at the fully ripe stage, the concentrations of Hg were reduced with the foliar applications except in the plants treated with T 3 (3.14 µg kg −1 ). Lead concentrations decreased with foliar applications of T 1 , T 2, T 3 , and T 4 at both maturity stages. Toxicity was also reduced with a change in maturity stage from mature green to fully ripe except in the plants treated with T 1 (Figure 4).

Principle Component Analysis
Principal component analysis (PCA) was applied to Mg, Zn, and B treatments (T 0 , T 1 , T 2 , T 3 and T 4 ) and phyto-nutritional element concentrations in loquat leaves and fruit peel, pulp, and seed ( Figure 5). The macro-and micro-nutrients of leaves in the 2nd and 3rd quadrants demonstrated an inverse relationship with T 0 and positive relationships with T 1 , T 2 , T 3 , and T 4 . This suggests that the application of Mg, Zn, and B increased the concentrations of macro-and micro-nutrients except for those near to T 0 (Fe, Mo, and Co). A cluster of heavy metals (As, Hg, and Pb) located in the 1st quadrant was near to T 0 , indicating an association with the untreated plants. In the peel, phyto-nutrients in the 1st quadrant were found increased in the plants treated with T 0 . These elements included Mo, Fe, Cu, and Pb at the mature green stage and Ca, Zn, Mn, Mo, Cu, and Cd at the fully ripe stage, whereas the elements in the 4th quadrant had a direct relationship with T 2 and T 4 . In the case of fruit pulp, T 0 was in the 2nd quadrant, while T 1 and T 3 were in 1st, and T 2 and T 4 were in 3rd quadrant, indicating a significant difference between the treated and untreated plants. In loquat seeds, concentrations of Pb, Mn, Zn, Mg, and Hg increased in untreated plants at the green mature stage (2nd quadrant). The Ni, Co, and Cu concentrations were greatly influenced by the application of T 1 at both maturity stages (1st quadrant). Similarly, the concentrations of Na, K, Ca, Zn, B, Mo, Cd, and Hg were positively influenced by T 3 treatments. Thus, PCA delineated the effects of Mg, Zn, and B treatments on macro-nutrients, micro-nutrients, and heavy metals in the leaves and fruit tissues of loquats. untreated plants at the green mature stage (2nd quadrant). The Ni, Co, and Cu concentrations were greatly influenced by the application of T1 at both maturity stages (1st quadrant). Similarly, the concentrations of Na, K, Ca, Zn, B, Mo, Cd, and Hg were positively influenced by T3 treatments. Thus, PCA delineated the effects of Mg, Zn, and B treatments on macro-nutrients, micro-nutrients, and heavy metals in the leaves and fruit tissues of loquats.

Discussion
There were differences in the concentrations of nutrients and heavy metals between the leaves and fruit tissues of loquats observed at two different maturity stages (i.e., mature green and fully ripe; Figures 1-4). Our findings suggest that Na content in loquat leaves and fruit tissues were suppressed with an application of 2% magnesium sulphate at both maturity stages, while it was increased with a foliar application of 0.2% borax, whereas Mg, K, and Ca contents were increased with an application of 2% magnesium sulphate in leaves of loquat. The obtained results regarding the effects of magnesium sulphate on Mg, K, and Ca contents in loquat leaves are in line with the findings of Ahmed and Morsy [35] on Canino apricots; Mostafa et al. [36] on bananas; Fawzi et al. [37] on Le Conte pears and Hanafy Ahmed et al. [38] on Washington navel oranges. It has been reported that magnesium fertilizer improved leaf Mg content of the aforementioned fruit species. Furthermore, an adequate level of magnesium is required for photosynthesis and chlorophyll formation, which produces carbohydrates in leaves and leads to good yields and high fruit quality [39]. The foliar application of magnesium sulphate at 0.5-3.0% and

Discussion
There were differences in the concentrations of nutrients and heavy metals between the leaves and fruit tissues of loquats observed at two different maturity stages (i.e., mature green and fully ripe; Figures 1-4). Our findings suggest that Na content in loquat leaves and fruit tissues were suppressed with an application of 2% magnesium sulphate at both maturity stages, while it was increased with a foliar application of 0.2% borax, whereas Mg, K, and Ca contents were increased with an application of 2% magnesium sulphate in leaves of loquat. The obtained results regarding the effects of magnesium sulphate on Mg, K, and Ca contents in loquat leaves are in line with the findings of Ahmed and Morsy [35] on Canino apricots; Mostafa et al. [36] on bananas; Fawzi et al. [37] on Le Conte pears and Hanafy Ahmed et al. [38] on Washington navel oranges. It has been reported that magnesium fertilizer improved leaf Mg content of the aforementioned fruit species. Furthermore, an adequate level of magnesium is required for photosynthesis and chlorophyll formation, which produces carbohydrates in leaves and leads to good yields and high fruit quality [39]. The foliar application of magnesium sulphate at 0.5-3.0% and boric acid at 0.20-1.0% has previously significantly raised percentages of K and Mg in the leaves of Canino apricot compared to a control [40]. Another study revealed that the application of magnesium−potassium based fertilizer decreased Mg and Ca level in leaves, and increased it in Fuji apple fruits [41], although no significant effect was observed of Mg application on the leaf Mg content of olive trees cv. Picual [42].
Loquat leaves have high medicinal value and have been used as folk medicines for thousands of years. Cough, inflammation, chronic bronchitis, cancer, and diabetes are treated with the extract of loquat leaves in Chinese folk medicine [43]. Loquat leaves are rich in phenolics and triterpenes [44]. Concerning human health, it is very important to investigate the effect of foliar-supplied nutrients on the phytonutritional compositions of loquat leaves, as the uptake of elements by plants can be associated with the availability of other elements. A foliar application of 0.1% zinc sulphate decreased the concentrations of Na in leaf blades and fruit pulps, but increased its amount in seeds and fruit peels at the green mature stage (Figures 1, 2 and 4). While at the fully ripe stage, concentrations of Na were found to be decreased in loquat leaves and fruit tissues but not their seeds (Figure 4). The decreased level of Na in loquat plants treated with zinc sulphate have been might be due to less uptake of Na by roots [45]. The concentrations of Mg, K, and Ca in loquat leaves increased as the result of the foliar application of 0.1% zinc sulphate (Figure 1), while Mg was found decreased in the seeds (Figure 4) and K and Ca were also decreased in the peel (Figure 2). Our findings are in line with the findings of Aboyeji et al. [46], who stated that the application of zinc at a concentration of 8 kg ha −1 can increase the K concentration, but that increasing the Zn concentration can lead to a decreased level of Ca in groundnut seeds. The increase in K and Mg could be due to the stimulating effect of Zn X K on wheat [47] and Zn X Mg on tung trees [48]. The reduction in Ca content of loquat seeds could be attributable to the antagonistic effect of Zn, which makes Ca unavailable to plants. Kalyanasundaram and Mehta [49] reported a reduced availability of Ca under high-Zn applications.
In a study, seven rootstocks of citrus were compared under a limited supply of boron, and it was found that the concentration of boron decreased in the leaves from 83.6% to 72.7% in all seedlings. Decreases in Ca, K, Mg, and Zn content and increases in Fe and Mn concentrations were observed in the leaf tissues of scion [50]. Our results also suggest that the loquat plants treated with 0.2% borax showed increased concentrations of Na, Mg, K, Ca, and B, with decreased concentrations of Mo, Mn, Fe, Co, Cu, As, Hg, and Pb in leaves ( Figure 1). In accordance with our results (Figure 4), application of 900 mL B ha −1 significantly increased concentrations of Mg, K, and Ca in groundnut seeds [46].
The Zn concentration in the leaves significantly increased with the application of 0.1% zinc sulphate as compared to other treatments observed at the fully ripe stage (Figure 1). The foliar application of zinc sulphate significantly increased concentrations of zinc in the leaf tissues of mango, rice, and coffee, respectively [51][52][53]. The increase in Zn concentration was found at the fully ripe stage rather than the mature green stage, indicating a slow mobility of Zn in leaves ( Figure 1). It has previously been reported that Zn has limited mobility in leaves, moving <25 mm from the application point after 24 h [54]. Our results suggest that the overall concentrations of Zn, Fe, and Cu in leaves and fruits were reduced with a change in maturity (from the mature green to the fully ripe stage). These results are supported by the findings of Madejon et al. [32], who reported that the concentrations of Zn, Fe, and Cu in the leaves, fruits, and seeds of oak and olive significantly reduce over time.
Boron is an essential element for plant growth that plays an important role in different metabolic processes (i.e., cell wall development, respiration, lignification, transport of sugar, phenolic metabolism, IAA metabolism, etc.) [55]. Moreover, it has a vital significance with respect to pollen tube development, the production of flower adhesive material, and pollen viability [56]. Boron concentration in the loquat leaves decreased with the application of borax as compared to the untreated plants ( Figure 1). In contrast, the leaves of sweet orange treated with boric acid, zinc borate, and calcium borate showed increased concentrations of boron [57]. Boron concentration in the leaves of coconut palm increased in a dose-dependent manner when boron was applied (0-24 g/plant) [58]. The concentration of boron in the fruit tissues of loquat increased with the application of borax (Figures 2-4). Similarly, the boron concentration in grains of wheat [59], rice [60], and sunflower tops [61] also increased with the application of boron in a dose-dependent manner. The concentration of Ni in the leaves and fruit pulp of loquat reduced with a change in maturity (Figures 1 and 3). These results are supported by the findings of Madejon et al. [32], who reported that the concentrations of Ni in the leaves and fruit pulp of oak significantly reduced over time.
In order to maintain nutrient homeostasis, plants must regulate nutrient uptake and respond to changes in the soil as well as within the plant. Thus, plant species utilize various strategies for the mobilization and uptake of nutrients as well as chelation, the transport between the various cells and organs of the plant, and storage to achieve whole-plant nutrient homeostasis [62,63]. Following penetration of the cuticle, the uptake of solutes into the cell interior depends primarily on the electrochemical concentration gradient from outside into the cell, but also on the plasma membrane permeability coefficient of the molecule and the degree of cell-mediated active uptake. There are various fates of foliarapplied fertilizer nutrients including retention in the cuticle in solution in the lipoidal layer; penetration of the cuticle followed by movement into the aqueous phase of the apoplast and diffusion into the inner leaf structure; penetration of the cuticle followed by movement with the transpiration water into the mesophyll and symplast; and translocation out of the leaf via the petiole [64].
Schroeder and Balassa [65] were the first to identify that fertilizers are implicated in raising heavy metal concentrations in food crops, and since then researchers have been urged to investigate the impact of impurities in fertilizers on the crop uptake of potentially heavy metals. Studies of heavy metal uptake by plants have often revealed their accumulations at a level that is toxic to human health. More than 70% of cadmium intake by humans is sourced from fruits and vegetables [66]. According to the findings of various scientists, cadmium is a mobile element in the soil, whereas it is immobile in plants, which is why it is found abundantly in plant roots [67][68][69]. The results indicate a significant decrease in cadmium concentration in leaves ( Figure 1) and fruits (Figures 2-4) from the mature green to the ripening stage. A reduction in cadmium concentration over time in the leaves of holm oak and the fruit pulp of olives has also been reported [32]. The maximum concentration of cadmium was observed in plants treated with magnesium sulphate as compared to other treatments (Figures 1-3). This might be due to a reduced translocation of cadmium in plant tissues resulting from the application of Mg [70].
Loquat is one of those fruit crops that is highly sensitive to the quality of irrigation water [71]. Arsenic (As) is a heavy metal that is present in the groundwater of many areas of the world and can contaminate fruits through irrigation water [72]. Many studies have reported on the high level of toxicity in fruits, vegetables, and cereal crops irrigated with arsenic-contaminated water [73]. Our findings show that the toxicity of arsenic concentrations in different loquat plant tissues was reduced with the progression from the mature green stage to the fully ripe stage. These results are in agreement with Madejon et al. [32], who reported a reduction in concentration of As with the passage of time in the leaves and fruits of olives and holm oak. Overall, results suggested that Mg, Zn, and B improved the nutritional composition of loquat leaves and fruit tissues and alleviated the toxicity of heavy metals.

Conclusions
Concerning human health, it is very important to investigate the effect of foliarsupplied nutrients on heavy metal accumulation in fruits, as the uptake of elements by plants can be associated with the availability of other elements. Therefore, this study was conducted to confirm the concentrations and availabilities of different phytonutrients in loquat leaves and fruit tissues under the influence of the foliar applications of Mg, Zn, and B. Exogenous application of Mg, Zn, and B proved to be successful for improving mineral nutrition in loquat, as evidenced from the increased macro-and micro-nutrients, and the decreased heavy metals in leaves and fruit tissues. There is need to further investigate Mg-, Zn-, and B-modulated molecular mechanisms regulating mineral uptake and accumulation in leaves and fruit tissues.