Foliar-Supplied Molybdenum Improves Phyto-Nutritional Composition of Leaves and Fruits of Loquat ( Eriobotrya japonica Lindl.)

: This study was conducted to analyze the effect of molybdenum (Mo) on the level of macronutrients (Mg, K, Ca), micronutrients (Na, Zn, B, Mo, Mn, Fe, Co, Ni, Cu) and toxic elements (Cd, Sn, As, Cr, Hg, Pb) in leaves and fruit tissues (peel, pulp & seed) of loquat using inductively coupled plasma-mass spectrometry (ICP-MS). Fruits were obtained from a loquat orchard located in Fujian (Yun Xiao/Zhangzhou). Loquat leaves showed 3.99, 23.90, 57.68 and 9.79-fold increase in the concentrations of Mg, K, Ca and B as affected by exogenously applied Mo, while a signiﬁcant ( p ≤ 0.05) reduction was observed in Na, Mo, Mn, Co, Ni, Cu, Sn, As, Cr, Hg, and Pb contents. In fruit peel, Mo induced 1.05, 1.56, 2.26, 1.15, 1.39, 1.30 and 1.89-fold increase in Mg, K, Na, Zn, B, Co and Ni, respectively, and Mo, Mn, Fe, Cu, Cr, and Pb contents were reduced. Fruit pulp showed elevated contents of Na, B, Co, Ni, and Sn by 25.65, 26.57, 70.09, 12.33 and 0.99%, respectively, and decreased concentrations of Mg, K, Ca, Zn, Mo, Mn, Fe, Cu, Cd, and Cr. Similarly, in loquat seeds, under the inﬂuence of Mo application, levels of K, Na, Zn, Fe, and Cr were uplifted by 14.75, 41.54, 12.71, 536.47 and 656.27%, respectively, while Mg, Ca, Mn, Co, Cu, Sn, and Hg were reduced signiﬁcantly ( p ≤ 0.05). Overall, mineral concentrations detected in the leaves and fruit tissues of loquat were greatly inﬂuenced by the application of Mo. Although the concentration of Sn in fruit pulp was increased by the foliar application of Mo, the fruits were under safe limits for human consumption.


Introduction
Fruits and vegetables are low in energy content but have high nutritive value [1,2]. They are a rich source of vitamins, fibers, and minerals which are much essential for the human body [3]. 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 [4]. To avoid certain cardiovascular diseases, it is recommended to consume 440 g fresh fruits and vegetables per day [5]. Heavy metals can influence the function of the nervous system, and cause mental illness by affecting the blood circulatory system [6]. Hence, it is very important to investigate the mineral contents of fruits and vegetables [7].
Loquat (Eriobotrya japonica Lindl.) is an evergreen fruit tree originated from China. It belongs to the family Rosaceae, subfamily Maloideae. It is a rich source of vitamin A, vitamin B6, potassium, magnesium, and dietary fiber [8]. It is a very beautiful orangecolored fruit with a mild sweet and sour taste [9]. 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 [10]. China is the leading producer and exporter of loquat and grows it on more than 100,000 hectares. The annual production of loquat in China reaches up to 380,000 tons [11]. More than 30 species of loquat are being grown in temperate and subtropical regions of Asia [12].
Fertilizer use is one of the main factors influencing the mineral contents of the plants [13][14][15]. Foliar application of fertilizers is 10-20% more influential than soil application [16][17][18]. Molybdenum is an essential nutrient for plant growth and is required in trace quantity. It is an integral component of nitrate reductase and nitrogenase, and vital for the nitrates' assimilation in the soil. Apart from its role in nitrate reductase, its function in higher plants is not well known [19]. Therefore, the role of Mo is closely associated with nitrogen metabolism, and its deficiency leads to N deficiency in plants [20]. Foliar fertilization of Mo can effectually increase internal Mo and improve the performance of molybdo-enzymes [21].
Since not much is studied about the nutritional effects of foliar-applied Mo on leaves and fruits of loquat, a field experiment was conducted at Yun Xiao, Fujian, to investigate the nutritional response of loquat to exogenous application of Mo. The contents of macronutrients (Mg, K, Ca), micronutrients (Na, Zn, B, Mo, Mn, Fe, Co, Ni, Cu), and heavy metals (Cd, Sn, As, Cr, Hg, Pb) in different tissues of loquat (leaf blade, fruit peel, pulp, and seed) were analyzed at different maturity stages through Inductively coupled plasma-mass spectrometry (ICP-MS).

Plant Material
Field experiment was carried out in a loquat orchard located at Yun Xiao, Fujian, China (23 • 57 13.5 N 117 • 20 36.0 E). Twenty-four young loquat trees (Cv. Jiefang zhong) were selected and tagged for periodic monitoring after foliar application of ammonium molybdate (CAS No. 12054-85-2, ≥99% purity, Sigma Aldrich, Milwaukee, USA). Loquat trees were 4-5.5 m tall with canopy diameters of 4-5 m. The plantation distance among trees was 6 × 6 m 2 , approximately. Loquat trees were systematically pruned and thinned, and fertilized with NPK (15:15:15) at concentration of 5 kg per plant per season for three growing seasons. The experiment contained two treatments including the control (water spray) and 0.05% ammonium molybdate. The foliar application was carried out twice at immature green fruiting stage with a 3-week interval. Each treatment consisted of four replicates, and each replicate had three trees.

Sample Collection and Preparation
Samples of the leaves and fruits were taken from the south-exposed tree canopy, at about 3-5 m height, at three different fruit maturity stages (i.e., immature green (IMG) (at the time of foliar spray), 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 sub-samples around the trunk were taken to make a composite soil sample for each tree [7]. Samples were transported to the laboratory (Institute of Subtropical Fruits, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou), oven-dried at 40 • C, and crushed to pass through a 2-mm sieve, then ground to <60 µm for element determinations [7,22]. The soil's structure was sandy loam with pH and EC of 6.2 and 0.396 dS m −1 , respectively. The pH and EC were determined by using digital pH meter (Hanna, HI-98,107, Mauritius) and EC meter (HI-98,304, Hanna Instruments Inc., Mauritius), respectively. The nutritional status of the soil was determined using ICP-MS before the foliar spray of Mo (Table 1). The values in Table 1 show the mean ± standard error of four replicates.

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., Mg, K, Ca, Na, Zn, B, Mo, Mn, Fe, Co, Ni, Cu, Cd, Sn, As, Cr, 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 m·mhos) was prepared locally. In this experiment, the working parameters of the inductively coupled plasma mass spectrometer were optimized before the test [7].

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, Düsseldorf, 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 [7]. 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
Treatment allocation to loquat trees was done according to randomized complete block design (RCBD) arrangement. Collected data were subjected to analysis of variance (ANOVA), and Fisher's LSD technique was used to compare means (p ≤ 0.05) 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
The foliar application of 0.05% ammonium molybdate increased the concentrations of Mg, K, and Ca in loquat leaves by 248%, 672%, and 632% at mature green stage, while 299%, 2290%, and 5668% at full ripe stage as compared to control. The results showed gradual increase in macronutrients with the foliar application of 0.05% ammonium molybdate from mature green fruiting stage to full ripe stage, except in case of Mg, when Mg contents were higher at mature green stage than full ripe stage ( Table 2).

Micronutrients
Overall concentration of Na was reduced at full ripe stage as compared to immature and mature green stage. At mature green stage, the Na level increased by 4.43% under the influence of Mo application as compared to control and recorded maximum among all observations. While at full ripe stage, Mo application reduced the Na concentration by 18% as compared to control. Zinc contents remained unchanged with the fruit maturity but significantly elevated at mature green stage in untreated plants. Plants treated with Mo exhibited a 25% decrease in Zn concentration at mature green stage, as compared to control, while remained unchanged at full ripe stage. The maximum level of B (33.9 mg·Kg −1 ) was observed in loquat leaves treated with 0.05% ammonium molybdate, at full ripe stage, followed by same treatment at mature green stage. The foliar application of Mo caused gradual increase in B concentration, while untreated plants showed constant level of B in leaves with fruit maturity. The plants receiving Mo application exhibited 9.8 and 7.7-fold decrease in Mo concentration at mature green and full ripe stage, respectively. Whereas untreated plants showed significantly increased Mo level with fruit maturity. Manganese concentration in the leaves of untreated plants reduced with fruit maturity (38.8%), whereas the plants treated with 0.05% ammonium molybdate showed further reduction in Mn level (Table 3). Untreated plants showed significantly increased Fe level at mature green stage (15.4 g·Kg −1 ) followed by full ripe stage (12.86 g·Kg −1 ) as compared to immature green stage (11.39 g·Kg −1 ). While, foliar application of Mo severely hampered the Fe concentration (106 and 247-fold reduction at mature green and full ripe stage, respectively, as compared to control). Similarly, Co concentration was also reduced with Mo application in comparison with control (12.64 and 14.42-fold reduction at mature green and full ripe stage, respectively). Whereas, untreated plants exhibited gradual decrease in Co level with fruit maturity. Nickle concentration was also reduced with fruit maturity, in untreated plants. But under the influence of Mo, maximum Ni (9.7 mg·Kg −1 ) was recorded at mature green stage, while minimum (1.0 mg·Kg −1 ) at full ripe stage. In case of Cu, untreated plants exhibited steady enhancement with fruit maturity from immature green to full ripe. While Mo treated plants showed maximum Cu level (13.8 mg·Kg −1 ) at mature green stage, while lowest at full ripe stage (1.8 mg·Kg −1 ) ( Table 3).

Heavy Metals
Cadmium concentration in the leaves of loquat increased with increase in fruit maturity. Maximum Cd contents (197.3 µg·Kg −1 ) were observed in the plants received foliar application of 0.05% ammonium molybdate, which was 2.5-fold higher than control. In contrast with Cd, Sn concentration decreased with increased fruit maturity, as well as with Mo application. Maximum Sn (7745.3 µg·Kg −1 ) was recorded in untreated plants at mature green stage, while minimum concentration (89.1 µg·Kg −1 ) was observed in Mo treated plants at same maturity stage. Foliar application of Mo significantly reduced As concentration in loquat leaves. At full ripe stage, untreated plants showed 42.3 mg·Kg −1 As, while in comparison, plants treated with Mo exhibited only 9.2 µg·Kg −1 As, which was 4700 times less than control, approximately. Similarly, Maximum Cr concentration (4.4 mg·Kg −1 ) was observed in the leaves of untreated loquat plants at mature green stage, while minimum (504.6 µg·Kg −1 ) found in Mo treated plants at same maturity stage. Although Hg concentration increased with increase in fruit maturity, Mo application significantly reduced (96 and 95.7% at mature green and full ripe stage, respectively) Hg contents at mature green and full ripe stage. In contrast with Hg, Pb concentration decreased with increase in fruit maturity. Loquat plants treated with Mo exhibited 545 and 235-fold reduction at mature green and full ripe stage, respectively (Table 4).

Macronutrients
Magnesium concentration in fruit peel of loquat significantly reduced with increase in maturity stage. The foliar application of 0.05% ammonium molybdate caused increase in Mg concentrations by 12.81% and 5.76% at mature green and full ripe stage, respectively. The results showed gradual increase in K contents with increase in maturity stage. The foliar application of 0.05% ammonium molybdate boosted up the increasing trend of K by 56.5% at full ripe stage. While, Ca concentration increased with increase in fruit maturity, regardless of treatment applied. At full ripe stage, Ca concentration was 3.5-fold higher than that at mature green stage (Table 5).

Micronutrients
The concentration of Na increased at full ripe stage as compared to mature green stage. The foliar application of 0.05% ammonium molybdate boosted up the increasing trend of Na by 126.8% at full ripe stage. Similarly, a gradual increase in Zn contents was observed with increase in maturity stage. At full ripe stage, the Zn level increased by 14.5% under the influence of Mo application as compared to control and recorded maximum among all observations. Similar trend was observed in case of B concentration in fruit peel of loquat. Leaf B elevated by 39.12% at full ripe stage in Mo treated plants. Untreated plants showed 208% increase in Mo concentration with increase in maturity (from mature green to full ripe stage). While Mo treated plants exhibited 30% decrease in leaf Mo concentration at full ripe stage. The plants receiving Mo application exhibited 1.08 and 1.32-fold decrease in Mn concentration at mature green and full ripe stage, respectively. Whereas untreated plants showed significantly increased Mn level with fruit maturity. Iron concentration in the leaves of untreated plants reduced with fruit maturity (56.6%). Whereas the plants treated with 0.05% ammonium molybdate showed further reduction (1.3-fold) in Mn level, at full ripe stage. Cobalt concentration increased with increase in fruit maturity, as further increase with the foliar application of Mo in comparison with control (1.4 and 1.3-fold increase at mature green and full ripe stage, respectively). Nickle concentration in fruit peel of untreated loquat plants also increased with increase in fruit maturity. Under the influence of Mo, maximum Ni (1.3 mg·Kg −1 ) was recorded at full ripe stage followed by 0.9 mg·Kg −1 at mature green stage. Regardless of treatments applied, plants exhibited significant reduction in Cu concentration with increasing fruit maturity. The foliar application of Mo enhanced Cu contents by 12.26% at mature green stage, while reduced by 3.87% at full ripe stage (Table 6). Table 6. Micronutrient concentrations in fruit peel of loquat as affected by the foliar application of molybdenum.

Fruiting Stage
Treatments

Heavy Metals
Cadmium concentration in fruit peel of loquat increased with increase in fruit maturity. Maximum Cd contents (190.5 µg·Kg −1 ) were observed in the plants received foliar application of 0.05% ammonium molybdate, which was 1.03-fold higher than control. Similarly, Sn concentration also increased with increase in fruit maturity, as well as with Mo application. Maximum Sn (1156.6 µg·Kg −1 ) was recorded in Mo treated plants at full ripe stage, while minimum concentration (160.14 µg·Kg  (Table 7).

Macronutrients
The foliar application of 0.05% ammonium molybdate enhanced the Mg concentrations at mature green stage by 8.6%, and reduced at full ripe stage by 10.6%. Overall Mg level was reduced with increase in fruit maturity. Like in case of Mg, Mo treated plants exhibited increased K concentration at mature green stage by 71.3%, and decrease at full ripe stage by 45.2%. The trend of K level in untreated plants was reciprocal to that of Mg level i.e., K level was enhanced with increase in fruit maturity. Similar to Mg contents, Ca contents reduced with increase in fruit maturity. Fruit pulp Ca was reduced up to 90% at full ripe stage as compared to control. The foliar application of Mo further reduced Ca level in fruit pulp of loquat by 27.8 and 33.5% at mature green and full ripe stage, respectively (Table 8).

Micronutrients
Overall concentration of Na remained unchanged at full ripe stage as compared to immature green stage, and reduced with respect to mature green stage. At mature green stage, the Na level increased (2.64-fold) as compared to immature green stage and recorded maximum among all observations. At same maturity stage, the foliar application of Mo reduced the Na concentration to same level as immature green stage had. While at full ripe stage, Mo application enhanced the Na concentration by 25.65% as compared to control. Zinc contents were reduced with the fruit maturity. Minimum Zn contents (21.58 mg. Kg −1 ) were observed at mature green stage in Mo treated plants. Plants treated with Mo exhibited 17% decrease in Zn concentration at full ripe stage, as compared to control. The maximum level of B (36.3 mg·Kg −1 ) was observed in fruit pulp of loquat treated with 0.05% ammonium molybdate, at mature green stage, followed by same treatment at full ripe stage. The untreated plants showed slight but significant increase in B contents in fruit pulp with fruit maturity. The foliar application of Mo caused further increase in B concentration. The plants receiving Mo application exhibited 1.37 and 1.01-fold decrease in Mo concentration at mature green and full ripe stage, respectively. The untreated plants also showed significant decrease in Mo level with fruit maturity. Manganese concentration in the fruit pulp of untreated plants reduced with fruit maturity (up to 82%). Whereas, the plants treated with 0.05% ammonium molybdate showed slight but significant increase in Mn level at mature green stage, and further reduction at full ripe stage as compared to control (Table 9). Untreated plants showed slight but significant increase in Fe level at full ripe stage (76.1 mg·Kg −1 ) as compared to immature green stage (68.9 mg·Kg −1 ). While, foliar application of Mo significantly hampered the Fe concentration (1.21 and 1.38-fold reduction at mature green and full ripe stage, respectively, as compared to control). Cobalt concentration was observed increasing with increased fruit maturity. Molybdenum application induced 1.4 and 1.7-fold increase in Co level of loquat fruit pulp at mature green and full ripe stage, respectively. The maximum Ni concentration (10.6 mg·Kg −1 ) was observed in fruit pulp of untreated plants at mature green stage. The foliar application of Mo induced 3.5-fold reduction in Ni level at mature green stage. While at full ripe stage, Ni contents were elevated by 12.33% as compared to control. In case of Cu, the maximum level (8.5 mg·Kg −1 ) was observed in fruit pulp of untreated plants at mature green stage. The foliar application of Mo induced 2 and 1.4-fold reduction in Cu level at mature green and full ripe stage, respectively (Table 9).

Heavy Metals
Cadmium concentration in fruit pulp of loquat decreased with increase in fruit maturity. Maximum Cd contents (298.1 µg·Kg −1 ) were observed in the untreated plants at immature green stage, which was 4.3-fold higher than those at full ripe stage. Foliar application of Mo induced 1.8 and 1.86-fold reduction in Cd level at mature green and full ripe stage. In contrast with Cd, Sn concentration increased with increased fruit maturity, as well as with Mo application. Maximum Sn (101.8 µg·Kg −1 ) was recorded in Mo treated plants at full ripe stage, while minimum concentration (58.4 µg·Kg −1 ) was observed in untreated plants at mature green stage. Foliar application of Mo significantly reduced As concentration in fruit pulp of loquat at mature green stage, while observed unchanged at full ripe stage as compared to control. At mature green stage, untreated plants showed 21.7 µg·Kg −1 As, while in comparison, plants treated with Mo exhibited only 0.5 µg·Kg −1 As, which was 47 times less than control, approximately. Similarly, Maximum Cr concentration (1.5 mg·Kg −1 ) was observed in fruit pulp of untreated loquat plants at immature green stage, while minimum (535.1 µg·Kg −1 ) found in Mo treated plants at full ripe stage. Mercury concentration remained unchanged throughout fruit maturity. Molybdenum application significantly enhanced Hg contents in fruit pulp of loquat at mature green stage, while recorded unchanged at full ripe stage as compared to control. Regardless of treatment applied, Pb concentration decreased with increase in fruit maturity (Table 10).

Macronutrients
The Mg and Ca concentration in untreated loquat seeds increased by 91 and 46.4% at mature green stage, and decreased by 52 and 90%, respectively at full ripe stage as compared to immature green stage. While seeds of plants receiving foliar application of Mo exhibited 1.4 and 1.5-fold reduction in Mg and Ca contents at full ripe stage, respectively. The K concentration showed reciprocal trend to the Mg and Ca concentrations. Magnesium concentration in untreated loquat seeds decreased by 48.74% at mature green stage, and increased by 35.27% at full ripe stage as compared to immature green stage. This fluctuation indicated an extraordinary reduction in K level (10,956.3 mg·Kg −1 ) at mature green stage. While seeds of plants receiving foliar application of Mo exhibited 1.03 and 1.14-fold increment in K contents at mature green and full ripe stage, respectively (Table 11).

Micronutrients
Sodium contents in seeds were observed to be reduced with increase in fruit maturity of loquat. While, Mo application significantly improved the Na percentage in loquat seeds at mature green (195%) and full ripe stage (41.5%) as compared to control. Zinc concentration in untreated loquat seeds increased by 62.2% at mature green stage, and decreased by 70% at full ripe stage as compared to immature green stage. This fluctuation indicated an extraordinary enhancement in Zn level (37 mg·Kg −1 ) at mature green stage. While seeds of plants receiving foliar application of Mo exhibited 1.38 and 1.12-fold enhancement in Zn contents at mature green and full ripe stage, respectively. Regardless of treatment applied, B contents remained unchanged with the fruit maturity, but significantly supressed at mature green stage in untreated plants. The maximum level of Mo (4.7 mg·Kg −1 ) was observed in loquat seeds treated with 0.05% ammonium molybdate, at mature green stage. All other observations exhibited non-significant difference among each other. Manganese concentration in untreated loquat seeds increased by 668.4% at mature green stage, and decreased by 93.4% at full ripe stage as compared to immature green stage. This fluctuation indicated an extraordinary enhancement in Mn level (402.2 mg·Kg −1 ) at mature green stage. While seeds of plants receiving foliar application of Mo exhibited 2.02 and 1.85-fold reduction in Mn contents at mature green and full ripe stage, respectively (Table 12). Untreated plants showed significantly increased Fe level at full ripe stage (205.4 mg·Kg −1 ) followed by mature green stage (66.3 mg·Kg −1 ) as compared to immature green stage (45.9 mg·Kg −1 ). While, foliar application of Mo significantly enhanced the Fe concentration (6.36-fold) at full ripe stage as compared to control. Cobalt concentration in untreated loquat seeds increased by 176.4% at mature green stage, and decreased by 18.35% at full ripe stage as compared to immature green stage. This fluctuation indicated an extraordinary enhancement in Co level (0.6 mg·Kg −1 ) at mature green stage. While seeds of plants receiving foliar application of Mo exhibited 3.06 and 1.75-fold reduction in Co contents at mature green and full ripe stage, respectively. The Ni concentration showed reciprocal trend to the Co concentration. Nickel concentration in untreated loquat seeds decreased by 71.82% at mature green stage, and increased by 196.83% at full ripe stage as compared to immature green stage. This fluctuation indicated an extraordinary reduction in Ni level (0.4 mg·Kg −1 ) at mature green stage. While seeds of plants receiving foliar application of Mo exhibited 1.9-fold reduction in Ni contents at mature green stage. In case of Cu, untreated plants exhibited steady enhancement with fruit maturity from immature green to full ripe. While Mo treated plants showed maximum Cu level (2.5 mg·Kg −1 ) at full ripe stage, while lowest at mature green stage (1.7 mg·Kg −1 ) (Table 12).

Heavy Metals
Cadmium concentration in the seeds of loquat decreased with increase in fruit maturity. Maximum Cd contents (268.2 µg·Kg −1 ) were observed in the plants received foliar application of 0.05% ammonium molybdate, which was non-significantly different than control. In contrast with Cd, Sn concentration increased with increase in fruit maturity, while decreased with Mo application. Maximum Sn (484.2 µg·Kg −1 ) was recorded in untreated plants at full ripe stage, while minimum concentration (80.24 µg·Kg −1 ) was observed in untreated plants at immature green stage. Foliar application of Mo significantly reduced As concentration in loquat seeds at mature green stage. At full ripe stage, untreated plants showed 4.4 µg·Kg −1 As, while in comparison, plants treated with Mo exhibited 9.09 µg·Kg −1 As. In contrast, Maximum Cr concentration (80.7 mg·Kg −1 ) was observed in the seeds of Mo treated loquat plants at full ripe stage, while minimum (1.3 mg·Kg −1 ) found in untreated plants at immature green stage. Molybdenum application significantly increased Hg contents by 78.2% at mature green stage, while reduced by 68.8% at full ripe stage. Similarly, loquat plants treated with Mo exhibited 1.65-fold enhanced Pb at mature green as compared to control. While, there was no significant change in Pb contents observed by Mo application at full ripe stage (Table 13).

Principle Component Analysis
To delineate the effect of multiple variables, Principal Component Analysis (PCA) among Mo treatment and nutritional concentrations in leaves and fruits of loquat was determined ( Figure 1). Factor F1, covering 39.81% variability in data (eigenvalue 7.167), showed highest squared cosine values of Mg, K, Ca, Fe, Cu, Sn, As, Hg and Pb in leaves with both treatments (Control and 0.05% ammonium molybdate) at immature green and full ripe stage, as well as in seeds at immature green stage, suggesting positive relation of aforementioned response and treatment variables. Second factor, covering 24.06% variability in data (eigenvalue 4.331), showed highest squared cosine values of Zn, Mo, Mn and Cd in fruit peel with both treatments at mature green stage, in fruit pulp both treatments at immature green stage and Mo treatment at mature green and full ripe stage, in seed both treatments at mature green stage, and control treatment at full ripe stage. Third factor of PCA, covering 12.59% variability in data (eigenvalue 2.268), showed highest cosine values of B, Ni and Cu in leaves with foliar application of Mo at mature green stage, in fruit peel both treatments at full ripe stage, in pulp control treatment at mature green and full ripe stage. Factor 4, covering 9.44% variability in data (eigenvalue 1.7), showed highest cosine values of Na and Cr in loquat seeds with foliar application of Mo at full ripe stage. Thus, PCA delineated the effect of treatment variables on macro-nutrients, micro-nutrients and heavy metals in leaves and fruit tissues of loquat.

Discussion
There were differences in the concentrations of nutrients and heavy metals between leaves and fruit tissues of loquat observed on three different maturity stages i.e., immature green, mature green, and full ripe stage (Table 1-13). The nutrient concentrations in plant tissues are known to fluctuate throughout the growing season due to environmental

Discussion
There were differences in the concentrations of nutrients and heavy metals between leaves and fruit tissues of loquat observed on three different maturity stages i.e., immature green, mature green, and full ripe stage (Tables 1-13). The nutrient concentrations in plant tissues are known to fluctuate throughout the growing season due to environmental factors (i.e., soil water, temperature) between periods of adequate soil supply of micronutrients and periods of insufficient soil supply of the applied micronutrient [23,24]. Therefore, in present study, the fluctuation in concentrations of nutrients was observed throughout maturity of loquat. Current study suggests that Mg, K, and Ca content increased by the foliar application of 0.05% ammonium molybdate in leaves of loquat. In plants, K, Na, Ca, and Mg not only maintain the balance of potential energy, but are also components or key supporting elements of important functional enzymes [25]. The obtained results regarding the effect of foliar applied Mo on leaf Mg, K, and Ca content are in line with earlier reported studies. It has been reported that Mo application improved leaf Mg, K, and Ca content of sunflower by 12%, 3%, and 6%, respectively [26]. Foliar application of 1.5 cm·L −1 Mo increased the concentrations of Mg, K, and Ca by 6.6%, 5%, and 9.8% in the leaves of 'Le-Conte' pear. Same treatment, when applied along with 3 g·L −1 Fe, enhanced the Mg, K, and Ca level by 18.3%, 12.3%, and 30.3% in the leaves of 'Le-Conte' pear [27].
Molybdenum is an essential element for plant growth and plays important role in different metabolic processes. Moreover, it has vital role in the function of nitrate and nitrite reductase [21]. After the uptake of nitrates by roots, these nitrates are readily moveable in plants and their accumulation occurs in vacuoles. However, nitrates reduction to ammonium (NH 4 + ) is necessary to support the synthesis of organic compounds and proteins in plants [28]. The nitrates reduction to NH 4 + is supported by different enzymes in two steps. In first step, the transformation of NO 3 − to NO 3 − is occurred by nitrate reductase in cytoplasm. In second step, the conversion of NO 2 − to NO 4 + is occurred in proplastids or chloroplasts by nitrite reductase [29]. The reduction of NO 3 − to NO 2 − depends upon several factors including plant species, plant carbohydrates, activity of nitrate and nitrite reductase, and environmental conditions i.e., light, pH, CO 2, and NO 3 − [30]. The foliar application of Mo is highly associated with N accumulation in plant leaves. The absence of Mo promotes nitrates accumulation and indicates less N assimilation by the plants [28]. Exogenously applied Mo improved Mo concentration in plant tissues of 'Le-Conte' pear [27], grapes (Cv. Merlot) [31], sunflower [26], peanut [32] and lettuce [28]. Our findings are in line with aforementioned studies, suggesting that foliar application of 0.05% ammonium molybdate increased the concentration of Mo in seeds of loquat. The reduction of Mo in leaves can be associated with the availability NH 4 + along with Mo. It has been reported earlier than the availability of NH 4 + lowers the activity of nitrate reductase causing Mo deficiency in plants [33]. A similar observation was made by Phillips [34] who reported lower molybdenum concentrations in leaf petioles from previously treated grape vines than untreated controls. Furthermore, the data presented by Williams et al. [35] over a three-year period also showed a successive decline in leaf molybdenum concentration.
Foliar application of 0.05% ammonium molybdate severely hampered the Fe concentration in leaves, fruit peel, and pulp of loquat. While, in 'Le-Conte' pear, Fe concentration was significantly increased with Mo application [27]. Similarly, Zn concentration was observed increased in fruit peel and seed under the influence of foliar-applied Mo. Our findings are in corroboration with Abd-El-Latif et al. [27] who also reported a remarkable increase in Zn concentration of pear leaves under the influence of 1.5 cm.L −1 Mo. Abd-El-Latif et al. [27] also reported an increase in Mn concentration as the results of Mo application on 'Le-Conte' pear, which was in contrast with our findings. Similarly, synergism between Mn and Mo was found in canola [36] 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 [37]. Loquat leaves are rich in phenolics and triterpenes [38]. Concerning human health, it is very important to investigate the effect of foliar-supplied nutrients on phytonutritional composition of loquat leaves as the uptake of elements by plants can be associated with the availability of other elements. The quadrupole inductively coupled plasma mass spectrometry (ICP-MS) is the most suitable method for the determination of trace elements in fruits and vegetables and is prevailed as the most appropriate practice for clinical quantification [39][40][41][42]. The element detection through ICP-MS has become a predominant methodology, because of its rapidity, determination limits, and the sample quantity needed for analysis [43].
Schroeder and Balassa [44] were the first to identify that fertilizers were implicated in raising heavy metal concentrations in food crops. Studies of heavy metals uptake by plants have often revealed their accumulation at a level, toxic to human health. More than 70% of cadmium intake by humans is sourced from fruits and vegetables [45]. According to the findings of various scientists, cadmium is a mobile element in the soil whereas it is immobile in plants, that is why it is found abundantly in plant roots [46][47][48]. The results indicate a significant decrease in cadmium concentration in loquat fruits from mature green to fruit ripening stage. The reduction of cadmium concentration over time in leaves of holm oak and fruit pulp of olives has also been reported earlier [22]. Although Cd is an immobile element in plant body, its reduction under the influence of Mo has also been reported in several studies [49,50].
Loquat fruit is one of those fruit crops which are highly sensitive to quality of irrigation water [51]. 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 [52]. There are many studies reported on high-level toxicity of arsenic in fruits, vegetables, and cereal crops irrigated on arsenic-contaminated water [53]. Our findings of the arsenic concentration in different plant tissues of loquat indicate that the toxicity of arsenic reduced with the progression from mature green stage to full ripe stage. The results are in corroboration with Madejon et al. [22] who reported reduction in the concentration of As with the passage of time in leaves and fruits of olive and holm oak. Overall, results suggested that Mo improved 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 concentration and availability of phytonutrients in loquat leaves and fruit tissues under the influence of the foliar application of molybdenum. Exogenous application of molybdenum proved to be successful for improving mineral nutrition of loquat, as evidenced by increased macro and micro-nutrients and decreased heavy metals in leaves and fruit tissues. There is need to investigate Mo-modulated molecular mechanisms regulating minerals uptake and accumulation in leaves and fruit tissues.