Performance of Mango Trees under the Spraying of Some Biostimulants

: Ensuring the sustainability of horticultural production is crucial to meeting consumer demand, and the most effective approach involves enhancing the efﬁcient utilization of resources to support the production of wholesome foods. Plant biostimulants are substances deﬁned by their ability, when applied to plants or the rhizosphere, to enhance root development, resulting in heightened vigor. They facilitate the more effective uptake and translocation of nutrients and water throughout the growing season. Additionally, these biostimulants contribute to improved plant health and quality by supplying essential nutrients that stimulate overall growth and metabolic processes. Therefore, this study was performed to examine the impact of some biostimulants on the possibility of improving vegetative growth, yield, fruit quality, and the leaf mineral content of mango cv. Keitt. Mango trees were sprayed with Putrescine at 25, 50, and 100 mg/L; Brassinosteroids at 0.5, 1, and 2 mg/L; and Chitosan at 500, 1000, and 1500 mg/L four times starting from mid-April, with three-week intervals during the seasons of 2021 and 2022. The results showed that the foliar application of these three biostimulants improved the growth, yield, and fruit quality of mango trees as opposed to untreated trees during two study seasons. The results also showed that the foliar application of 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put gave the highest increments for inducing the number, length, and thickness of shoots, leaf area, and leaf chlorophyll over the other applied treatments. In addition, it also heightened the fruit set, fruit yield, and the fruit’s physical and chemical characteristics in both experimental seasons.


Introduction
Mango (Mangifera indica L.) belongs to the Anacardaceae family and is characterized as an evergreen tree and has significant importance as one of the most fundamental fruits in tropical and subtropical regions.Mango trees have a substantial annual nutrient demand as their optimal growth and productivity require a well-balanced supply of both micronutrients and macronutrients, with a focus on high accessibility [1].It is one of the most popular and well-liked fruit harvests in the world, accounting for the third largest crop in Egypt after citrus and grapes [2].It is renowned as the "King of fruits" [3].A distinct fruit reveals the high quality and abundance of nutrients it provides.A mango may meet up to 40% of daily dietary fiber requirements [4] and is particularly popular due to its versatility, increased nutritional content, variety, delicious taste, and exceptional flavor.Raw and ripe mangoes are used to manufacture a range of cuisines, including dried mango pulp, pickles, jam, chutney, and other condiments [5].
Mango trees cv.Keitt are medium-sized, strong, and straight, and the fruit is normally greenish-yellow but sometimes pink or red in color.It has an oval shape with a circular base, measuring 13-15 cm in length, 9-11 cm in width, 8.5-10 cm in thickness, and their weights range from 510 to 2000 g.The skin is dense and rough, the flesh is steady and succulent, with low lemon-yellow fiber, and it also has a sweet and nice taste with an excellent flavor.The seed of this cultivar is monoembryonic, which is covered by a woody stone [6].
Mango trees are highly susceptible to adverse environmental conditions like drought, salinity, and extreme temperatures, which can negatively affect their growth and development.Additionally, these factors also lead to a consistent reduction in flower retention, fruit-setting percentages, and photosynthetic rates while they increase respiration and transpiration rates, which consequently reduce the annual production by as much as 50-70% over time [7].
Polyamines are low-molecular-weight aliphatic nitrogenous bases with significant bioactivity, and they are crucial for plant growth, development, and the way plants respond to different environmental stresses [8].In addition, Putrescine (Put) serves as a significant polyamine with vital functions in regulating plant growth, differentiation, and their ability to respond to various environmental stresses [9].Notably, when Picual olive trees were sprayed with Put at 15 and 30 ppm, it significantly improved the fruit yield, as well as the physical and chemical features of the fruit [10].Applying Put through spraying on 'Zaghloul' dates has been found to increase both yield and fruit quality.This is reflected in the improved levels of amino acids, sugars, and essential minerals such as nitrogen, phosphorus, and potassium.Additionally, there is an augmentation of phytohormones like GA 3 , auxins, and cytokinins, along with increased concentrations of photosynthetic pigments [11].Furthermore, the application of Put has the capacity to enhance cell division [12] and promote flower formation [13].Applying Put at a concentration of 150 ppm through spraying on papaya cv.Arka Surya resulted in positive effects.The treatment enhanced pulp thickness, fruit firmness, pulp weight, the pulp-peel ratio, seed number, and fresh and dry weights of seeds.Additionally, it increased the levels of ascorbic acid, soluble solids, the TSS-acid ratio, total, reducing, and non-reducing sugars, and carotenoids content, and extended the fruits' shelf life.Conversely, it led to a reduction in fruit acidity compared to untreated trees [14].
Brassinosteroids (BRs) represent the sixth crucial category of phytohormones in plants, playing an essential role in normal growth and various developmental processes [15].These steroidal compounds play a crucial role not only in plant growth but also in mitigating the undesirable effects of stressful stresses; these include water, temperature, and highsalinity stresses [16].Additionally, BRs are identified as natural, non-toxic, environmentally friendly phytohormones.This makes them well-suited for agricultural and horticultural applications to ameliorate growth, productivity, and fruit quality [17].Brs are crucial in diverse developmental processes, encompassing cell division, stem elongation, vascular tissue formation, the regulation of photosynthesis, activation of enzymes, the control of senescence, photomorphogenesis, reproductive development, leaf aging, and responses to external stressors [18].Spraying foliage with BRs has been observed to increase levels of total soluble solids and ascorbic acid in fruits, along with prolonging the shelf life of the fruits [19].Further, the implementation of 0.4 mg L −1 BRs effectively raised the content of grapefruits from total anthocyanin in contrast to non-treated trees [20].Additionally, it has been reported that BRs contribute to an increase in soluble sugars, pigments, and potassium content in both the leaves and roots of loquat [21].
Chitosan (Chit) is an eco-friendly and cost-effective natural plant growth agent because it can improve plant growth and its tolerance to environmental stresses, raising fruit trees' productivity and quality [22].Chit also functions as a biostimulant and is derived from amino polysaccharides found in insect cuticles, fungal cell walls, or the shells of seafood [23].It exhibits a range of functions, serving as an anti-transpirant and promoter of both plant growth and beneficial microorganisms [24], enhancing photosynthesis via enlarging chloroplasts and stimulating the biosynthesis of photosynthetic pigments like chlorophyll a, b, and carotenoids [25].When Chit is applied, it boosts the levels of indole-3-acetic acid as a compound that stimulates cell division.Additionally, it triggers the activation of the PIN1 gene, which plays a role in governing the movement of auxin [26].
Therefore, the aim of this study was to assess how the foliar application of Put, Brs, and Chit influences the vegetative growth, yield, physical and chemical characteristics of fruit, and the nutritional content of leaves in mango cv.Keitt.

Experimental Site Description and Its Design
This study was performed to investigate the influence of the foliar spraying of Putrescine (Put) at 25, 50, and 100; Brs at 0.5, 1, and 2 mg/L; and Chit at 500, 1000, and 1500 mg/L compared to the control (sprayed with water only).The experiment was performed on mango cv.Keitt, which was at the age of ten years in the Nubaria region, which is located at a latitude of 30.6667 and longitude of 30.0667,El Beheira governorate, Egypt, under drip irrigation during the seasons of 2021 and 2022.The applied treatments were arranged in a randomized block design (RCBD) in six replicates for a total of sixty trees that were the same size, growth, and vigor.The physical and chemical properties of the experimental soil are shown in Table 1, and climate data from the experimental region are included in Table 2.The trees were sprayed four times starting in mid-April, with three-week intervals during the seasons of 2021 and 2022.The effects of the aforesaid treatments were evaluated by analyzing their influence on the following parameters:

Vegetative Growth Parameters
In October, and after picking the fruits for the seasons of 2021 and 2022, the shoot length and diameter were measured in cm, and the shoot number was accounted.Chlorophyll content in the leaves was measured using a chlorophyll apparatus (SPAD-502, Hangzhou Mindfull Technology Co., Ltd., A-3-405, Hangzhou, China), registering 10 readings from each replicate (tree).The trunk girth was measured in cm.Leaf area (cm 2 ) was measured in the 1st week of October by taking twenty leaves using Equation (1) [27].
where LA = the leaf area in cm 2 ), L = the maximum length of the leaf (cm), and W = the maximum width of the leaf (cm).

Fruit Set Percentages, Fruit Drop Percentages and Fruit Yield
Fruit set and fruit drop percentages were calculated according to Equations ( 2) and ( 3), respectively, according to El-Hady et al. [28] Fruit set% = Fruit yield was assessed by calculating the yield for each replicate/tree in kilograms and then converting it to tons per hectare.This conversion was achieved by multiplying the average tree yield by the total number of trees in a hectare.

Fruit Quality 2.4.1. Fruit Physical Characteristics
During the harvesting period in October 2021 and 2022, ten fruits were randomly chosen from each replicate for analysis.These selected fruits were assessed for various characteristics, including their weight, size, pulp weight, peel weight, and seed weight.To measure the dimensions of the fruit, such as their length and thickness, a digital Vernier caliper from Cangxian Sanxing Hose Clamp Co., Ltd.(Cangzhou, China) was utilized.Furthermore, fruit firmness was determined using a Magness and Taylor pressure tester equipped with a 7/18-inch plunger [29].

Fruit Chemical Characteristics
Total soluble solids (TSS %) were measured using a handheld refractometer.To determine the fruit's acidity, a calorimetric method relying on the estimation of citric acid was employed.This involved taking five milliliters of fruit juice and titrating it with a known normality of 0.1 N NaOH, using phenolphthalein as an indicator, following the procedure described in [30].The vitamin C content (mg/100 mL juice) was assessed using 3% oxalic acid and 2,6-dichlorophenol indophenols, as described by Nielsen [31].Total and reducing sugars were estimated via calorimetric methods involving phenol and sulfuric acid, utilizing 5 g of fresh pulp, following the method described by Nielsen [32].Non-reducing sugars are the differences between total sugars and reducing sugars.

Mineral Content in Mango Leaves and Fruits
At the end of the growing season in October 2021 and 2022, a total of twenty leaf and fruit samples were collected for mineral content analysis, specifically for nitrogen, potassium, and phosphorus, as described by Arrobas et al. [33].The collected leaves were initially cleaned with tap water and then laundered with distilled water.Subsequently, they were dried in an oven at 70 • C until they reached a consistent weight and were then crushed.To analyze the samples, they underwent a digestion process using a mixture of H 2 SO 4 and H 2 O 2 .By using micro-Kjeldahl method procedure, nitrogen content was assessed [34], while vanado-molybdate method was utilized to estimate phosphorus content Wieczorek et al. [35].Both the nitrogen and phosphorus measurements were conducted using a spectrophotometer set at a wavelength of 405 nm.Furthermore, the potassium content was assessed using a flame photometer from SKZ International Co., Ltd., located in Jinan Shandong, China [36].

Statistical Analysis
Data from the present study were subjected to analysis of variance (ANOVA) utilizing a Randomized Complete Block Design (RCBD).To evaluate the distinctions among the means of treatment, Duncan's test was conducted at a significance level of 0.05.Subsequently, a further comparison of means was executed using the least significant difference approach at a 5% probability [37], using CoHort Software version 6.311 (Pacific Grove, CA, USA).

Results
The foliar application of Put, Brs, and Chit had an advantageous effect on the vegetable growth parameters; the number, length, and thickness of shoots contrasted with untreated trees (Table 3).The most remarkable increments in shoot number, shoot length, and shoot thickness were obtained via the spraying of Chit at 1500 mg/L, followed by Brs at 2 mg/L in contrast with unsprayed trees.It was noticed that the beneficial influence of Put, Brs, and Chit increased gradually by raising the doses used in the seasons of 2021 and 2022.The differences between the effect of the leafy implementation of 1000 mg/L Chit, 2 mg/L Brs, and 100 mg/L Put on the shoot number were so slight as to be significant.The results in Table 4 indicate that the exogenous implementation of mango trees using Put, Brs, and Chit was a helpful way to improve the leaf chlorophyll content, leaf area, and truck girth over control trees during the seasons of 2021 and 2022.Additionally, the application of Chit at 1500 mg/L was the best treatment, which achieved the most remarkable increments in the vegetative measured characteristics.The results demonstrate that the application of Chit was higher in its effect than the spraying of Brs, which was higher than Put in both trial seasons.Moreover, it was also noted that the spraying of 1000 mg/L Chit, 2 mg/L Brs, and 100 mg/L Put markedly raised the leaf chlorophyll content, leaf area, and trunk girth compared to untreated trees.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.
Table 5 indicates that the spraying of Chit at 1500 mg/L and 2 mg/L Brs remarkably raised the fruit set percentages and fruit yields in kg or tons in contrast to untreated trees.The influence of the highest concentrations was greater than the lowest ones for the two seasons.Besides, the spraying of 100 mg/L of Put also improved the fruit set percentages, comparing 25 or 50 mg/L between the two seasons.On the contrary, the same applied treatments reduced the fruit drop percentages in contrast with non-sprayed trees.Additionally, the fruit yields in kg per tree and in tons per hectare were also remarkably impacted by the spraying of 1000 mg/L of Chit, 1 mg/L of Brs, and 50 mg/L of Put rather than unsprayed trees, but the differences between these effects were so slight as to not be significant in the two seasons.Fruit weight, size, length, and diameter were greatly improved by the foliar implication of applied biostimulants: Put, Brs, and Chit in contrast to untreated trees in the seasons of 2021 and 2022 (Table 6).In addition, these physical characteristics were markedly improved by the application of 1500 Chit in contrast with the other sprayed treatments, where it was the superior treatment in the two seasons.Moreover, the same mentioned fruit's physical characteristics were also improved by the use of 2 mg/L of Brs and 100 mg/L of Put in the two seasons.The fruit length and diameter were markedly ameliorated by the application of 1000 mg/L of Chit and 1 mg/L of Brs as opposed to untreated trees, but the differences between their influence were not significant.
Spraying mango trees cv.Keitt with Put, Brs, and Chit improved fruit firmness, as well as the weights of the peel, seed, and pulp against untreated trees in 2021 and 2022 (Table 7).In total, 1500 mg/L of Chit was the superior treatment, followed by the spraying of 2 mg/L of Brs and 100 mg/L of Put, which improved fruit firmness and the weights of peel, seeds, and pulp rather than the other sprayed treatments for the two seasons.Additionally, the same physical fruit characteristics were considerably improved by the leafy spraying of 1000 mg/L of Chit and 1 mg/L of Brs over untreated trees.When Put, Brs, and Chit were applied through foliar spraying, there was a considerable increase in the fruit content of carotene and total soluble solids, as well as a notable improvement in the TSS-acidity ratio during the seasons of 2021 and 2022 (Table 8).Conversely, these treatments led to a reduction in fruit acidity during the same period.The results also showed that the highest values of TSS % were obtained via the leafy spraying of 1500 mg/L of Chit and 2 mg/L of Brs in both seasons.In the second season, the extern spraying of 1000 mg/L of Chit, 1 mg/L of Brs, and 100 mg/L of Put markedly raised the fruit content from TSS percentages.The highest values for the fruit content from carotene were obtained via the application of 1500 mg/L of Chit, while this treatment gave the least significant percentage for fruit acidity in the two seasons.Concerning the TSS-acidity ratio, it was remarkably increased by the implementation of 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put in the two seasons.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.
The findings exhibited in Table 9 denoted that when Put, Brs, and Chit were applied, there was a notable increase in the fruit content of various components such as VC, total sugars, reduced sugars, and non-reduced sugars contrasted to trees that were not treated across the study seasons.Moreover, among the different concentrations tested, the application of 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put was more effective at enhancing these fruit components than the lower concentrations of 1000 or 500 mg/L of Chit, 0.5 or 1 mg/L of Brs, and 50 or 25 mg/L of Put 200 ppm, respectively.Additionally, there were no notable differences observed between the effects of spraying 1500 mg/L of Chit and 2 mg/L of Brs in terms of the fruit content from VC and reduced and non-reduced sugar percentages in the two study seasons.
The application of Put, Brs, and Chit on the mango trees of the Keitt cultivar clearly resulted in an increase in the mineral content of leaves from nitrogen, phosphorus, and potassium in comparison to trees that were not treated (as shown in Table 10).Notably, during the seasons of 2021 and 2022, the most significant increases in the leaf mineral content from nitrogen, phosphorus, and potassium percentages were observed when 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put were applied.Interestingly, no noteworthy differences were noticed between the impact of the extern spraying with 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put on the leaf mineral content for these nutrients in both experimental seasons.

Discussion
The results demonstrate the significant positive impact of enhancing the vegetative growth, yield, physical and chemical attributes of fruit, and nutrient contents from nitrogen, potassium, and phosphorus through the foliar application of Put, Brs, and Chit as opposed to unsprayed trees.
These findings were previously explained by several authors who have shown that the exogenous application of Put increased photosynthetic rates by mitigating the effects caused by environmental stress and maintaining the composition of chloroplasts and photosynthetic pigments [38].Applying Put at 15 or 30 ppm on olive trees markedly ameliorated shoot length, the leaf number and area in contrast to non-treated trees [39].Furthermore, when Put was applied at concentrations of 0.1 mM and 1.0 mM to date palm cv.Kabkab, it led to an obvious increment in fruit yield; meanwhile, it clearly minimized the fruit drop percentage as opposed to untreated trees [40].Furthermore, the application of Put at 0.45 mM on mango trees greatly raised the fruit set percentage, fruit yield, and quality, such as the fruit peel chlorophyll content, pulp quality, and the content of TSS %, reducing sugars, and the TSS-TA ratio.Additionally, Put positively impacted amino acids, free phenolic compounds, as well as fruit's phosphorous, potassium, and nitrogen content, while it decreased the fruit drop percentage [41].The exogenous application of 1 mM of Put on date palm trees cv.Zaghloul at the stages of cell division and cell elongation significantly improved the fruit content from dry matter, ash, soluble solids, and a reduction in non-reducing and total soluble sugars, as well as carbohydrate, protein, amino acid, N, P, K, Ca, Mg, Zn, Fe, and Mn content.In addition, the levels of phytohormones such as indole-3-acetic acid, cytokinins, GA 3 , and SA were also greatly improved, but the applied treatment, on the other hand, minimized the fruit content from the percentages of moisture, total acidity, total phenols, and tannins [42].
BRs can enhance photosynthetic efficiency, particularly under stress conditions, contributing significantly to increased plant growth and playing a crucial role in plant adaptation to stress [43].BRs had a positive impact on apricot when treated at doses of 0.5, 1, and 2 mg/L.Shoot length, leaf area, and total chlorophyll were ameliorated, as well as fruit set, productivity, fruit weight, size, length, diameter, hardness, total soluble solids %, and vitamin C content.Furthermore, the use of BRs resulted in higher percentages of total, reduced, and non-reduced sugars.In addition, the leaf mineral content improved, including both macro and micronutrients, while fruit acidity decreased in comparison to unsprayed trees [44].The external implication of Brs at 0.25, 0.5, and 0.75 mg/L on sweet cherries led to a notable rise in the fruit's anthocyanin and ascorbic acid.Furthermore, it improved the firmness, weight, diameter, and length of fruits and ultimately resulted in a higher fruit yield [45].In addition, the exogenous application of Brs on walnut trees at 1, 10, and 50 mg/L led to a remarkable increment in the number of flowers and fruits, pollen tube development, fruit set percentages, and fruit weights as opposed to untreated trees [46].Similarly, Tepkaew et al. [47] documented that the foliar application of BRs on mango trees, at 0.01 or 0.1 M during the large bud swell stage markedly increased the length of the panicle, the flower number, pollen viability, their germination, and development rates, and thus raised the fruit set percentage, while the percentage of malformed inflorescences decreased.
Chit has been observed to positively influence the development and productivity of various crops, and its stimulatory effect on plant growth is probably attributed to its capacity to enhance water availability and nutrient uptake [48].Due to its amino groups, Chit can fulfill the nutritional requirements of plants and improve soil texture when combined with fertilizers [49].The influence of Chit is likely linked to its ability to enhance the release of nitrogen, which can be utilized to synthesize components for the photosynthetic system [50].Furthermore, Khalil and Eldin [51] found that applying Chit externally at concentrations of 5 and 10 g•dm −3 on grapevines led to enhanced plant growth, root weight, overall plant production, and increased tolerance to water-deficit conditions.The researchers highlighted that chitosan acts as an anti-transparent agent, reducing water loss through plant leaves.This characteristic can result in heightened leaf water potential and relative leaf water content, thereby improving a plant's capacity to withstand drought stress.Chit serves multiple functions, acting as a water efficiency enhancer, a carrier for nutrients, and a remover of heavy metals [52].Additionally, Muley et al. [53] noted that Chit, containing amino groups, can stimulate plant growth and productivity.The foliar application of Chit has been shown to increase stomatal conductance and water use efficiency by reducing the transpiration rate [54].Furthermore, the treatment of tea with Chit led to improvements in the chlorophyll content and soluble sugar levels [55].

Conclusions
The results of the experiment indicate that the foliar applications of Put, Brs, and Chit are effective, environmentally friendly, and safe biostimulants.These applications prove to be a valuable strategy for enhancing the growth attributes, yield, fruit quality, and leaf mineral content from nitrogen, phosphorus, and potassium.Significantly, the most substantial improvements in the measured factors were seen when using 1500 mg/L of Chit, 2 mg/L of Brs, and 100 mg/L of Put compared to other treatments.In terms of some measured parameters such as the fruit set percentage, fruit yields, and fruit content from TSS percentages and the TSS-acidity ratio, VC, and total, reduced, and non-reduced sugars %, it is clear from the results that the differences were not significant between the extern spraying of 1500 mg/L and 2 mg/L of Brs during the two seasons.

Table 1 .
Physical and chemical composition of experimental soil.

Table 2 .
Climate data of the experimental region.

Table 3 .
The spraying impact of Put, Brs, and Chit on the shoot number, shoot length and shoot thickness of mango cv.Keitt during 2021-2022.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.

Table 4 .
The spraying impact of Put, Brs, and Chit on the leaf chlorophyll content, leaf area and trunk girth of mango cv.Keitt during 2021-2022.

Table 5 .
The spraying impact of Put, Brs, and Chit on the fruit set and drop percentages and fruit yields of mango cv.Keitt during 2021-2022.
The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.

Table 6 .
The spraying impact of Put, Brs, and Chit on the fruit weight, size, length, and diameter of mango cv.Keitt during 2021-2022.
The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.

Table 7 .
The spraying impact of Put, Brs, and Chit on fruit firmness and the weights of the peel, seed and pulp of mango cv.Keitt during 2021 and 2022.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.

Table 8 .
The spraying impact of Put, Brs, and Chit on the fruit content from TSS, carotene, acidity and TSS-acidity of mango cv.Keitt during 2021 and 2022.

Table 9 .
The spraying impact of Put, BRs, and Chit on the fruit content from VC, including the total, reduced and non-reduced sugars of mango cv.Keitt during 2021-2022.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.

Table 10 .
The spraying impact of Put, Brs, and Chit on the leaf mineral content from nitrogen, phosphorous and potassium of mango cv.Keitt during 2021 and 2022.The differences between the treatments that have the same letters in the same column are not significant.*** means high significance.