Introduction

Harvested apples in Egypt span across an area of 28,085 ha producing 70,4727 ton/ha (FAO and UNICEF 2018). The “Anna” apples (Malus domestica L. Borkh) belong to the family of Rosacea. In previous publications, it was suggested that amino acids enhance fresh and dry matter (Fawzy et al. 2012), increase growth behavior (Bahari et al. 2013; Romero et al. 2014; Rouphael et al. 2017), and stimulate plants’ root growth, which may enhance both water and nutrient uptake capability, resulting in an increased yield productivity (Colla et al. 2017; Rouphael et al. 2017). When amino acids are applied through the root system or foliar feeding, the uptake and concentrations of leaf nutrients are improved (Mohammadipour and Souri 2019a, 2019b; Pranckietienė et al. 2015), including iron and zinc (Souri et al. 2018). The exogenous application of amino acids promotes growth, induces leaf pigmentation, stimulates chlorophyll biosynthesis, and reduces chlorophyll degradation in many crops (Fahimi et al. 2016; Kazem Souri et al. 2017; Mohammadipour and Souri 2019a, 2019b). Amino acids have several major biological functions in plant cells, such as detoxification (Bashir et al. 2018; Hussain et al. 2018; Rizwan et al. 2017), optimization of nutrient uptake, translocation and metabolism, vitamin biosynthesis, growth biostimulation, and increasing tolerance to environmental stresses (drought, salinity, and cold conditions). Also, they play a vital role in the synthesis and production of amino chelate fertilizers (Souri and Hatamian 2019). The foliar application of amino acids was found to increase the biomass and crop productivity in plants (Basanth and Mahesh 2018; Kazem Souri et al. 2017). Radkowski et al. (2018) declared that the content of the investigated macro elements was increased by the foliar fertilization with the amino acid preparation. Khan et al. (2019) reported that amino acids are natural plant growth stimulators. As a result, they are utilized extensively for enhancing the yield and quality of crops. Noroozlo et al. (2019) stated that amino acids play various roles in plant metabolism; the exogenous application of amino acids may have benefits and stimulation effects on plant growth and quality. Souri and Hatamian (2019) declared that amino acids have an affinity for nutrient elements and some amino acids can make chelates with nutrients. They also mentioned that these properties assist them in enhancing the uptake and delivery of micronutrients, mainly Fe, in plants. These micronutrients are good sources of nitrogen for plant uptake and utilization as well as having stimulating effects on plant root and shoot growth. Mataffo et al. (2020) stated that foliar application of amino acid–enriched urea fertilizer is considered a promising environmentally friendly strategy for enhancing the yield and nutrient efficiency of plants. Matysiak et al. (2020) reported that the exogenous application of some amino acids has been proven to positively affect plant growth and development under stressful conditions. Sheng et al. (2020) examined the effects the amino acid-Fe compound fertilizer spraying has on leaf development in peaches (Prunus persica (L.) Batsch) at different developmental stages. The results demonstrated that the foliar spraying increased leaves’ fresh weights considerably. Fe concentration, photosynthetic parameter, and Fe-S protein analyses were used to measure Fe accumulation, total chlorophyll content, net photosynthetic rate, and stomatal conductance. Furthermore, the same authors observed that amino acid-Fe compound fertilizer significantly increased the leaf content from nitrite reductase and succinate dehydrogenase’s activities, over control. This study is aimed at utilizing some alternative fertilizers from amino acids, such as tryptophan and glycine, in apple orchards to decrease environmental hazards and increase apple production and quality.

Materials and methods

The present experiment was performed during the two successive seasons of 2018 and 2019 on 10-year-old “Anna” apple trees (Malus domestica L. Borkh), planted 4 × 4 m apart in a clay soil under flood irrigation in a private orchard located at Kafr El-Dawar, Beheira governorate, Egypt. Table 1 presents the physiochemical analysis of experimental soil, which was conducted as per Cottenie and Verlo (1982). Sixty uniform trees were chosen for this study and were subjected to the same cultural practices in both seasons. They were sprayed three times as follows: before flowering, during the full bloom, and 1 month later with the following treatments: control (water only); tryptophan ((S)-2-amino-3-(3-indoly) propionic acid—C11H12N2O2—ROTH Besteffen Sie Zum Nufftarif—China ) at 25, 50, and 100 ppm; glycine (aminoethanoic; glycocoll C2H5NO2—Anhui BBCA Pharmaceutical Company Ltd.—China) at 25, 50, and 100 ppm; and their combinations, tryptophan at 25 ppm + glycine at 25 ppm, tryptophan at 50 ppm + glycine at 50 ppm, and tryptophan at 100 ppm + glycine at 100 ppm.

Table 1 Physiochemical properties of the experimental soil
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The previously applied treatments were arranged in a randomized complete block design where each treatment consisted of sex trees/sex replicate. The following parameters were utilized to investigate the impact of the treatments mentioned above.

Vegetative parameters

At the beginning of the vegetative growth, eight shoots from each side of each replicate/tree were selected and labeled, whereas at the end of each growing season, the shoot length and diameter were measured in centimeters and the leaf area was measured in centimeters squared. Total chlorophyll was also measured by Spad 502 (μ Mol/m2).

Fruit set and fruit drop percentage

Four branches from each side from each tree/replicate were selected carefully and labeled in March from 2018 and 2019, and the number of flowers on each branch was calculated. The fruit set percentage was also calculated as per Westwood (1988) equation:

$$ \mathrm{Fruit}\ \mathrm{set}\%=\left(\mathrm{number}\ \mathrm{of}\ \mathrm{set}\ \mathrm{fruitlets}/\mathrm{number}\ \mathrm{of}\ \mathrm{opened}\ \mathrm{flowers}\right)\times 100. $$

Estimation of preharvest fruit drop was done by calculating the number of dropping fruit after fruit setting until the harvesting date in each season (June). Then, the fruit drop was calculated as a percentage as follows:

$$ \mathrm{Fruit}\ \mathrm{drop}\%=\left(\mathrm{number}\ \mathrm{of}\ \mathrm{drop}\mathrm{ped}\ \mathrm{fruitlets}/\mathrm{number}\ \mathrm{of}\ \mathrm{set}\ \mathrm{fruitlets}\right)\times 100. $$

Fruit yield

Estimation of yield was conducted in kilograms per tree and ton per hectare at harvest time (June).

Leaves chemical composition

A random selection of samples of thirty leaves was made from the middle part of the shoots according to Abdelsalam et al. (2018) and Chuntanaparb and Cummings (1980) from each replicate following harvest time in June to determine their content of N%, P%, K%, and Ca%, and Fe, Zn, Mn, and B at ppm. The leaf samples were washed first with tap water, then with distilled water, and dried at a temperature of 70 °C until it maintained a constant weight; finally, they were ground and acid digested using H2SO4 and H2O2 until the solution became clear (Wilde et al. 1985). The digested solution was used for determining each of the following: nitrogen using micro Kjeldahl method, phosphorus using vanadomolybdo method, and potassium using flame photometer following the method presented by Chapman and Pratt (1961). Zinc, manganese, calcium, and iron were determined using an atomic absorption spectrophotometer (2-8200 Series Polarized Zeeman, Hitachi, Tokyo, Japan) through specific lamps for specific nutrients. By dry ashing, boron was estimated using the method described by Chapman and Pratt (1961), and, subsequently, it was measured through colorimetry, Azomethine-H, as indicated by Bingham (1983).

Fruit quality

At harvest time, samples of thirty fruit were randomly selected from each replicate/tree to determine the physical and chemical characteristics of fruit.

Fruit physical characteristics

Fruit weight (g), fruit length (cm), fruit diameter (cm), and fruit firmness (Ib/inch2) were determined via a Magness and Taylor pressure tester with a 7/18-inch plunger.

Fruit chemical characteristics

Total soluble solids were estimated by using a hand refractometer. The percentage of titratable acidity in fruit juice was determined according to Resio et al. (2005). Total sugars, reducing and nonreducing sugars, were estimated as per Malik and Singh (1980). Anthocyanin was determined at the stage of coloration (mg/100 g fresh weight peel) according to Rabino (1977).

Statistical analysis

The obtained data were subjected to one-way ANOVA as per Snedecor and Cochran (1980), and least significant difference (LSD) instances at 0.05% were utilized for comparing the means of treatments.

Results

Vegetative growth parameters

Data listed in Table 2 indicated that a significant improvement was observed in shoot length and diameter by the foliar spraying of glycine and tryptophan at 50 and 100 ppm and their combinations of 50 ppm glycine + 50 ppm tryptophan and 100 ppm glycine + 100 ppm tryptophan in comparison with the usage of 25 ppm and control in both seasons. Furthermore, 50 ppm glycine + 50 ppm tryptophan was shown to be the best treatment which gave the best results in the two seasons in comparison with control and the other applied treatments. Regarding leaf area, it was considerably increased by 100 ppm tryptophan sprays and by the combinations of tryptophan at 50 ppm + glycine at 50 ppm as well as tryptophan at 100 ppm + glycine at 100 ppm compared with control in both seasons. Moreover, remarkable improvement is observed in the leaf area by spraying glycine at 50 and 100 ppm and tryptophan at 50 ppm in both seasons compared with control. Total chlorophyll was notably enhanced by foliar spraying of glycine and tryptophan at 25, 50, and 100 ppm and their combinations compared with control in the two seasons. Spraying the combination of tryptophan + glycine at 50 ppm and glycine at 100 ppm and tryptophan at 50 ppm showed the highest increment in comparison with control in the two experimental seasons.

Table 2 The effect of the foliar application of tryptophan and glycine on length and diameter of shoots, leaf area, and total chlorophyll during 2018 and 2019 seasons
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Fruit set percentage and fruit yield

The data in Table 3 showed that spraying “Anna” apple trees by tryptophan or glycine and their combinations at the different concentrations of 25, 50, and 100 ppm significantly improved the fruit set percentage and the fruit yield per tree and per hectare, whereas they reduced the fruit drop percentage in comparison with control in both seasons. The foliar spraying of glycine or tryptophan with a concentration of 50 or 100 ppm gave better results than that with a concentration of 25 ppm in the two seasons. Furthermore, the combination of tryptophan + glycine at 50 ppm was the best treatment, yielding the highest fruit set and fruit yield percentages and the lowest fruit drop percentage in comparison with the other applied treatments in both seasons.

Table 3 The effect of the foliar application of tryptophan and glycine on fruit set %, fruit drop %, and fruit yield during 2018 and 2019 seasons
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Fruit quality

The results in Table 4 indicated that spraying tryptophan and glycine at 25, 50, and 100 ppm, whether each one alone or in combination with the other, exhibited great improvement in weight, size, length, and diameter of the fruit of “Anna” apple tree in the two experimental seasons in comparison with control, and the foliar spraying of 100 ppm tryptophan was superior in these terms. Besides, the foliar application of tryptophan with glycine combinations at 50 and 100 ppm provided the highest increase in the fruit weight and size, whereas the foliar spray of tryptophan and glycine at 50 and 100 ppm gave the highest increment in fruit length and diameter in the two seasons compared with control.

Table 4 The effect of the foliar application of tryptophan and glycine on fruit weight, fruit size, fruit length, and fruit diameter during 2018 and 2019 seasons
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Data in Table 5 demonstrated that fruit content of “Anna” apple trees from TSS percentage, fruit firmness, total sugars, and reduced sugars was noticeably increased by spraying glycine and tryptophan amino acids at 25, 50, and 100 ppm and their combinations in the two seasons. The highest increment in TSS percentage was obtained by spraying tryptophan at 50 ppm and glycine at 50 and 100 ppm in both seasons in comparison with the other applied treatments and control. The foliar spray of glycine and tryptophan and their combinations at 50 and 100 ppm provided the best increase in fruit firmness compared with the other applied treatments and control in both seasons. The foliar application of glycine at 100 ppm and the combination of 100 ppm glycine + 100 ppm tryptophan gave the highest increase in total and reduced sugar percentages compared with the used concentrations and control in both experimental seasons. A significant increase in nonreduced sugar percentage was indicated through applying glycine at 100 ppm and the combinations of glycine and tryptophan at 50 and 100 ppm compared with the other applied treatments and control in the two seasons. The foliar spraying of tryptophan or glycine at 25, 50, and 100 ppm individually or in combinations exhibited a significant reduction in fruit acidity in comparison with control in both seasons. Spraying tryptophan or glycine and their combination at 50 ppm showed the lowest values compared with the other applied treatments and control in the two seasons.

Table 5 The effect of the foliar application of tryptophan and glycine on TSS, acidity, total sugar, reduced sugar, nonreduced sugar percentages, and firmness of fruit during 2018 and 2019 seasons
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Leaves chemical composition of macro and micronutrients

The results in Table 6 indicated that obvious improvement was observed in leaf composition of N, P, K, and Ca after spraying “Anna” apple trees with glycine or tryptophan at 25, 50, and 100 pm and by their combinations compared with control in both seasons. Spraying tryptophan or glycine at 50 and 100 ppm and their combinations at 50 ppm exhibited the highest increment in comparison with the other applied concentrations in the two experimental seasons. Additionally, the combination of 100 ppm glycine + 100 ppm tryptophan raised also the leaf mineral composition of K and Ca over control.

Table 6 The effect of the foliar application of tryptophan and glycine on leaf mineral composition of N, P, K, and Ca during 2018 and 2019 seasons
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Data in Table 7 demonstrated that leaf mineral composition of Fe, Zn, Mn, and B was significantly increased by spraying “Anna” apple trees with glycine or tryptophan at 25 and 50 compared with control in both seasons. Spraying glycine and tryptophan at 50 or 100 ppm and their combination of 50 ppm glycine + 50 ppm tryptophan gave the best results in comparison with the other applied treatments in both seasons. Additionally, the combination of 100 ppm glycine + 100 ppm tryptophan provided good results in enhancing the leaf mineral composition of Fe and B over control.

Table 7 The effect of the foliar application of tryptophan and glycine on leaf mineral composition of Fe, Zn, Mn, and B during 2018 and 2019 seasons
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Discussion

The obtained results indicated that foliar spraying of glycine and tryptophan and their combinations had positive effects on shoot length, shoot diameter, total chlorophyll, yield, and fruit quality of “Anna” apple cultivar. Our obtained results were previously explained by Ahmed et al. (2012) and Talaat et al. (2014) who observed that foliar spraying of “Valencia” orange trees with 25, 50, and 100 mg/l of tryptophan increased shoot length, shoot thickness, leaves number, leaves area, yield, fruit quality, leaf mineral composition of N, P, K, Mg, Zn, Cu, Fe, and Mn leaf content of cytokines, gibberellins, auxins, and chemical elements. On the other hand, it reduced the concentration of abscisic acid. Tryptophan amino acid foliar application increased the yield and its components of chickpea (Abbas et al. 2013), lupine (Amin et al. 2014), and onion (Abd El-wahed et al. 2016; Mustafa et al. 2016). Furthermore, El Sayed et al. (2014) and Ghasemi et al. (2013) illustrated that glycine amino acid plays a vital role in forming the total chlorophyll and vegetative growth and in increasing the viability of Fe, Zn, Mn, and Cu to the plants. Spraying “Manfalouty” pomegranate cultivar with 50, 75, and 100 mg/l tryptophan at full bloom and 4 weeks later increased the vegetative growth, yield, and fruit quality. Meanwhile, the best results were provided by the concentration of 100 mg/l compared with control (El Sayed et al. 2014). Glycine has been incorporated into nutrient fertilizer formulation in the form of a chelate or a simple complex, to improve how the plant efficiently uses applied fertilizers and facilitate nutrient uptake and translocation in plants (Fahimi et al. 2016; Souri and Hatamian 2019). It was noticed that spraying apple trees with tryptophan amino acid at the stages of pre- and post-bloom increased the yield greatly in comparison with control (Sharma 2016). Recent studies by Khan et al. (2019), Souri and Hatamian (2019), and Weiland et al. (2016) showed that amino acids are widely utilized for improving the yield and quality of crops and the better root development supported by the addition of amino acids which can boost nitrogen fixation and induce the root-enhancing surface for nutrient uptake. The highest values of fruit set percentage, yield, and vegetative growth characteristics such as shoot length, shoot diameter, number of leaves, and leaf area of “Washington” navel orange were recorded with 50 ppm tryptophan treatment, followed by 25 ppm tryptophan treatment in comparison with control (Ahmed et al. 2017). Furthermore, Teixeira et al. (2017) stated that L-tryptophan and L-glycine are signal-transducing molecules and make the nutrients ready-made by the plants. Additionally, Khedr (2018) reported that spraying tryptophan at 50 and 100 ppm twice, at full bloom stage and the beginning of fruit set stage (21 days after full bloom stage), exhibited positive results in terms of Le-Conte pear’s productivity and fruit quality. Moreover, 100 ppm tryptophan greatly increased the percentage of fruit set, yield, fruit weight, TSS, and total sugars of Le-Conte pear fruit when crosschecked against the use of water as a control. Application of different concentrations of glycine at 0, 5, 10, 20, or 40 mgL−1 to coriander plants (Coriandrum sativum L.) showed that leaf SPAD value, fresh and dry weights of shoots, roots, and leaf concentrations of N, K, Mg, and Zn were significantly increased by 10 mgL−1 glycine in comparison with the control plants (Mohammadipour and Souri 2019a; Noroozlo et al. 2019). Noroozlo et al. (2019) investigated the effect of foliar spraying of Romaine lettuce (Lactuca sativa subvar Sahara) with glycine at 0, 250, 500, and 1000 mgL−1. The obtained results indicated that significant increase in leaf total chlorophyll content and root fresh weight was observed by 250 mgL−1 concentration compared with control. Besides, the highest shoot fresh and dry weights and leaf vitamin C were indicated with a concentration of 500 mgL−1. Besides, 1000 mgL−1 glycine spray significantly increased leaf Fe concentration compared with control plants. Moreover, Souri and Hatamian (2019) demonstrated that in many plants, especially horticultural crops, glycine plays a vital role in controlling nutrition. Elkhatib et al. (2020) reported that foliar spraying of common bean cv. Nebraska with tryptophan amino acid at 0.5 and 1 mgL−1 concentrations increased vegetative growth, leaf chlorophyll, and N, P, and K contents, yield, and its components. Hancı and Tuncer (2020) investigated the effects of exogenous L-tryptophan at 125, 250, and 375 ppm on some growth parameters of lettuce plants grown under salt stress. They noticed that foliar spray of high concentrations gave the announced impact on the leaf number, salinity, weight of fresh leaf and fresh root, and surface area of lettuce plants under salinity at 200 mM.

Conclusions

  • The foliar spraying of “Anna” apple trees with glycine and tryptophan at 25, 50, and 100 ppm and their combinations increased significantly shoot length, shoot diameter, leaf area, total chlorophyll, percentages of fruit set and fruit yield, fruit physical and chemical characteristics, and leaf mineral composition of N, P, K, Ca, Fe, Zn, Mn, and B, whereas it reduced the fruit drop percentage in the two seasons in comparison with control.

  • The concentrations of 50 and 100 ppm gave better results and were more effective than 25 ppm in both seasons.

  • Spraying glycine or tryptophan at a concentration of 50 or 100 ppm provided good results. However, better effects and higher increment were displayed when both glycine and tryptophan were combined at the same mentioned concentrations in the two seasons.

  • The best treatment was the combination of 50 ppm glycine + 50 ppm tryptophan, providing the highest results in comparison with the other applied treatments and control in both experimental seasons.

  • The obtained results indicated that amino acids, such as tryptophan and glycine, may be good alternatives to chemical fertilizers for reducing the environmental hazards and pollution.