Preharvest Foliar Applications of Citric Acid, Gibberellic Acid and Humic Acid Improve Growth and Fruit Quality of ‘Le Conte’ Pear (Pyrus communis L.)

A two-year (2020-21) study was conducted to investigate the possibility of relying of tenyears old pear trees grown on sandy loam soil irrigated by drip on citric acid (CA), gibberellic acid (GA3) and humic acid (HA). The CA was applied at the concentrations of 500, 1000 and 1500 ppm, GA3 at 50, 100 and 150 ppm and HA at 3, 4 and 5%, whereas water spray was used as the control. The results of our study proved that CA, GA3 and HA improved the shoot length, shoot thickness, leaf area and leaf chlorophyll of pear as compared with the control. Moreover, they also positively increased the fruit set percentage and final yield of ‘Le Conte’ pear. The fruit weight, size and firmness were also improved under the influence of aforementioned treatments. The fruit soluble solids, total sugars, leaf nitrogen, leaf phosphorus and leaf potassium of pear were also enhanced as compared with the control. Additionally, spraying of GA3 at 150 ppm, as well as, HA at 5 and 4% were the superior treatments and showed the most significant impact on plant growth, yield, fruit quality and leaf mineral content of pear. This study provides a basis for the future elucidation of HA-, GA3and CA-modulated molecular mechanisms in pear, which can make a significant contribution in the scientific community.


Introduction
Pear (Pyrus communis L.) belongs to family Rosaceae [1] and is cultivated on an area of 1,379,387 hectares in the world with the production of approximately 23,919,075 tons [2]. The exogenous application of plant bio-stimulants, such as citric acid (CA), gibberellic acid (GA 3 ) and humic acid (HA), has emerged as an effective approach to improve plant growth and yield and thus sustain food production.
Research reported that the pear soil characteristics are preferably light acidic soil, which a pH of 6-7. However, soil outside of that range will not have an undesirable impact on growth. The ideal environmental conditions are of cold winter, which is required to stimulate the productivity of the fruit, and the majority of cultivars require approximately 0.2%, 0.3% and 0.4% significantly improved the parameters of vegetative traits, fruit set percentage, yielding and fruit physicochemical characteristics, such as sugars, anthocyanin and firmness. Foliar application of HA caused to improvement of nutrient efficiency and an increase in leaf Zn and Fe contents, which raised carbohydrate and protein production [58].
It was noticed that HA enhanced the weight, size, TSS, and V.C. in grape [59], apricots [60] and Guava paluma [56]. Moreover, mango performance via the vegetative yield and fruit chemical or physical quality were markedly improved as a result of the foliar spray of HA on mango cv. Zebda as mentioned by El-Hoseiny et al. [61]. Hence, considering the aforementioned beneficial aspects of CA, GA 3 and HA on plant performance, this study was aimed to investigate their impacts on vegetative growth, yield and fruit quality of 'Le Conte' pear as a good nutritional alternative to lessen the reliance on chemical fertilizers in fruit nutrition. Although the current study provided ample information to signify the roles of CA, GA 3 and HA in improving pear performance, the molecular mechanisms behind the improved yield and fruit quality remain to be explored.

Plant Material, Treatments, Experimental Site and Design
The current study was carried out during the 2020 and 2021 seasons to examine the impact of the exogenous applications of Citric acid (CA), Gibberellic acid (GA 3 ) and Humic acid (HA) on the performance of ten-years-old 'Le Conte' pear trees budded on Pyrus communis rootstock. The trees were cultivated on the distances of 3.5 × 3.5 m in a private orchard in Burg EL-Arab, Alexandria governorate, Egypt, at 29.55 longitude and 30.90 latitude in sandy loam soil under drip irrigation.
The experiment was composed from ten treatments; control (water only); CA (Sunshine Biotech International Co., Ltd., Prachin Buri, Thailand) at 500, 1000 and 1500 ppm; GA 3 (Shijiazhuang Awiner Biotech Co., Ltd. Shijiazhuang, China) at 50, 100 and 150 ppm; HA (X-humate 100% super powder potassium humate manufacturing-Humate (Tianjin) International Ltd., Tianjin, China) at 3, 4 and 5%. Each treatment had eight replicates (trees), which were arranged in a randomized complete block design (RCBD). The trees were sprayed three times; the first spray was in February (before starting of the bloom), the second was during full bloom (March), and the third spray was performed two weeks later than the second spray. The pear trees received the same horticultural treatment in the orchard. The analysis of the soil composition is illustrated in Table 1.

Vegetative Growth Attributes
Six healthy branches on each tree were selected and tagged, and the average of their shoot length was measured in cm at the end of each vegetative season. The thickness of shoots was measured two times i.e., at the start of the vegetative season (March) and after harvesting (July) to measure the increase in shoot thickness in cm by using a Digital Vernier Caliper in both seasons.
The leaf total chlorophyll was measured using a digital chlorophyll meter (SPAD-502Plus, Konica Minolta, Inc., Tokyo, Japan). Thirty leaves were randomly picked from each tree in each treatment to determine the leaf area in cm 2 using a CI-203 laser area meter (CID Inc., Washington, DC, USA). The average leaf area (cm 2 ) was determined using the following equation as reported by Demirsoy [62].
Fruit yield was estimated in kg per replicate/tree at harvesting time (July).

Fruit Physical Characteristics
Fruit fresh weight, length (from maximum vertical point) and diameter (from maximum horizontal point) were calculated by taking the average of thirty fruits from each replication of same treatment. Fruit weight was measured with digital weighing balance (MJ-W176P, Panasonic, Kadoma, Japan), whereas the length and diameter were measured with digital Vernier callipers (DR-MV0100NG, Ningbo Dongrun Imp. & Exp. Co., Ltd., Ningbo, China). The length was divided by diameter of each fruit to calculate the lengthto-width ratio (fruit shape index). Fruit firmness was calculated using a Magness and Taylor pressure tester with a 7/18-inch plunger (mod. FT 02) (0-2 Lb., Via Reale, 63, 48011 Alfonsine, Italy).

Fruit Biochemical Characteristics
The total soluble solids were determined with a hand-held digital refractometer (Atago, Hybrid PAL-BXIACID F5, Japan). The total titratable acids were determined with NaOH-based titrimetric method and expressed as percent citric acid. The sugar-acid ratio was calculated by dividing the total soluble solids to total titratable acids within the same sample. Sugars were determined as detailed earlier by Ali et al. [63]. Briefly, an aliquot of fruit pulp, treated with 25% lead acetate and 20% potassium oxalate, was hydrolysed with HCl and kept overnight to convert non-reducing sugars into reducing sugars. Then, the HCl-hydrolysed aliquot was neutralized with 0.1 N NaOH and titrated against Fehling solutions.

Leaf Minerals
In July, and after harvesting the fruit, thirty leaves were taken from the middle part of branches of the trees, and then they were washed, cleaned, well-crushed and digested with H 2 SO 4 and H 2 O 2 to assess their nitrogen, phosphorus and potassium contents. Nitrogen was assessed using the micro-Kjeldahl method [64], phosphorus was determined using the vanadomolybdate method [65], and potassium was determined using a flame photometer (SKZ International Co., Ltd., Jinan, China) [66]. Iron, zinc and manganese were determined using inductively coupled plasma-mass spectrometry (ICP-MS) [67][68][69].

Statistical Analysis
One-way analysis of variance (ANOVA) was utilized to analyze the obtained results statistically, and the least significant difference (LSD) at 0.05% was used to make a comparison between the means of treatments, and it was measured using the statistical software 'Statistix v8.1 . Variables with a statistically significant impact of treatments (p ≤ 0.05) were further subjected to principal component analysis (PCA) using XLSTAT ver. 2018. Correlation coefficient values were determined with the Pearson (n) method. The clustering of variables with associated treatments was determined with their highest squared cosine values corresponding to factor, F1 or F2.

Vegetative Growth Attributes
The exogenous application of CA, GA 3 and HA greatly enhanced the vegetative performance of 'Le Conte' pear plants in terms of their shoot length, shoot thickness, leaf area and leaf total chlorophyll content in both growing seasons (Table 2). Among all treatments, the foliar application of HA at 5% or 4% and GA 3 at 150 ppm had the most notable impact on the previously listed vegetative measurements. Furthermore, it was noticed that the application of GA 3 at 100 ppm also highly increased the shoot length and thickness as well as the leaf area and chlorophyll content over untreated plants.

Fruit Set Percentage, Fruit Drop and Yield
The foliar application of CA, GA 3 and HA raised the percentage of fruit set and fruit yield per tree over control. On the other hand, the fruit drop percentages were lowered by these treatments (Figure 1). The maximum fruit set (19.22% "2020" and 21.51% "2021") and minimum fruit drop (55.89% "2020"and 51.59% "2021") was obtained by the foliar application of 5% HA. The most remarkable yield was resulted from the application of HA at 5% and 4% and GA 3 at 150 or 100 ppm over untreated plants. Moreover, GA3 also improved the fruit set percentage and lessened the percentage of fruit drop, and thus HA and GA3 improved the productivity of the trees over the usage of CA during the studied seasons. It was clear from the data that the impact of HA, GA3 or CA was enhanced gradually in parallel to the increase of the applied concentration.

Fruit Set Percentage, Fruit Drop and Yield
The foliar application of CA, GA3 and HA raised the percentage of fruit set and frui yield per tree over control. On the other hand, the fruit drop percentages were lowered by these treatments (Figure 1). The maximum fruit set (19.22% "2020" and 21.51% "2021" and minimum fruit drop (55.89% "2020"and 51.59% "2021") was obtained by the folia application of 5% HA. The most remarkable yield was resulted from the application o HA at 5% and 4% and GA3 at 150 or 100 ppm over untreated plants. Moreover, GA3 also improved the fruit set percentage and lessened the percentage of fruit drop, and thus HA and GA3 improved the productivity of the trees over the usage of CA during the studied seasons. It was clear from the data that the impact of HA, GA3 or CA was enhanced grad ually in parallel to the increase of the applied concentration.

Fruit Physical Characteristics
The results shown in Table 3 clearly indicated that, in comparison to the control, th spraying of HA at 4% or 5%, as well as the application of GA3 at the rate of 150 ppm resulted in remarkable increments in fruit weight, size, length, diameter and fruit firmnes of 'Le Conte' pear. Additionally, the application of 100 ppm GA3 and 1500 ppm CA also had a positive impact on boosting the same attributes compared to the control in both growing seasons. Moreover, the fruit shape index was significantly increased by the folia application of CA at 1500 ppm and 100 or 150 ppm GA3 as well as HA at 4% or 5% com pared with other applied treatments in both seasons.

Fruit Physical Characteristics
The results shown in Table 3 clearly indicated that, in comparison to the control, the spraying of HA at 4% or 5%, as well as the application of GA 3 at the rate of 150 ppm resulted in remarkable increments in fruit weight, size, length, diameter and fruit firmness of 'Le Conte' pear. Additionally, the application of 100 ppm GA 3 and 1500 ppm CA also had a positive impact on boosting the same attributes compared to the control in both growing seasons. Moreover, the fruit shape index was significantly increased by the foliar application of CA at 1500 ppm and 100 or 150 ppm GA 3 as well as HA at 4% or 5% compared with other applied treatments in both seasons.

Fruit Biochemical Characteristics
From the results demonstrated in Table 4, the spraying of CA, GA 3 and HA had a beneficial impact on fruit biochemical characteristics as compared to the control. The exogenous application of 5% or 4% HA and 150 ppm GA 3 markedly increased the soluble solids, total sugars and the ratio between TSS and acidity compared to the control. Additionally, the foliar application of HA at 5% and GA 3 at 150 ppm remarkably improved the content of reducing and non-reducing sugars in pear fruits. Additionally, fruit acidity was noted higher in untreated pears as compared with CA-, GA 3 -and HA-treated pear plants. The clearest effect on decreasing the fruit acidity percentage was noticeably related to the application of HA at 4% and 5% and also GA 3 at 150 and 100 ppm.  Same letters indicate non-significant difference among treatments according to Fisher's LSD method (p ≤ 0.05).

Leaf Minerals
The foliar supplied CA, GA 3 and HA considerably increased the leaf nitrogen, phosphorus, potassium, iron, zinc and manganese of pear plants as compared to the control (Table 5). Furthermore, during both growing seasons, the high content of macro and micronutrients was accompanied to the foliar sprays of HA at 5% or 4%, as well as GA 3 at 150 ppm, rather than the other treatments. Additionally, it was noticed that the influence of CA, GA 3 and HA on the measured parameters was increased in dose-dependent manner, where the highest applied concentration gave the most increase in the leaf mineral content and this reflected on improving the final obtained productivity and fruit quality.

Discussion
There were differences between CA, GA 3 and HA treatments in terms of their impact on plant growth and fruit quality of 'Le Conte' pear. However, the results are in line with the findings of Mansour et al. [70], who reported that spraying CA on pear trees enhanced the fruit production, fruit weight, fruit firmness, as well as the percentages of total soluble solids and total sugars, while diminishing the fruit acidity. El-Badawy [71] reported that CA enhanced the shoot length, leaves/shoot ratio, leaf area index, TSS percent, vitamin C and fruit firmness in apricot cv. Canino. Ca raises the intake of elements' rate and diminishes the adverse impacts of Pb under environmental stressors; thus, it can ameliorate photosynthetic rate [72,73], photosynthetic pigment production [74] and plant growth [75].
Osama et al. [76] noticed that spraying of CA at 400 and 800 ppm on 'Keitt' mango trees increased the percentages of fruit set and fruit retention, fruit number, fruit production, fruit physical attributes in terms of fruit and peel weight and pulp/fruit ratio in comparison with the control. Additionally, they added that CA also increased the fruit chemical features such as total soluble solids and sugars, while reduced the fruit acidity in comparison to the control.
Treating 'Washington' navel orange trees with CA at 1 g/L enhanced the total chlorophyll content in the leaves, number of shoots, shoot length and thickness, leaf number per shoot, leaf area and leaf mineral content of N, P, K, Ca, Mg, Fe, Mn and Zn [24]. Mohamed [77] reported that the foliar application of CA at 1000 ppm on grapevines increased yield, number of clusters per vine, cluster weight, length, width, berry weight, berry length, berry diameter, berry content from reducing sugars, anthocyanin and total soluble solids, while it reduced the acidity of berries in the two seasons compared to the control.
Iqbal et al. [78] documented that GA 3 reduced flower drop, while increasing the flower retention and the yield and fruit quality in many fruit crops such as mango, citrus, apple and guava. In relative to the control treatment, treating apples with 20 mg/L GA 3 enhanced the number of buds, fruit setting, fruit number, weight and yield, as well as the total sugar content, while it lessened the buds dropping before anthesis [79]. Nkansah et al. [80] found that the application of mango trees cv. Keitt with 25 mg/L GA 3 at full bloom raised the fruit set, fruit retention, number of fruits, fruit weight and yield. Treating apricot trees with 50 or 100 mg/L from gibberellic acid in mid-May had a positive effect on improving the fruit weight, length and diameter [81].
Our results are also in line with the findings of Mosa et al. [82], who noticed that the application of 50 and 100 mg/L GA 3 on apple trees (cv. Anna) obviously increased the shoot length, shoot thickness, leaf area, fruit set % and fruit yield, while it lessened the fruit drop percentage. Furthermore, it also increased the fruit weight, length, diameter and firmness, as well as TSS %, ascorbic acid and leaf nitrogen, phosphorus, potassium, calcium and magnesium compared to the untreated plants.
In the same trend, it was noticed by Beerappa et al. [83] that, when GA 3 at the rate of 50 and 100 ppm was applied on pomegranate trees (cv. Bhagwa), it raised the fruit length, diameter, number of arils, total aril weight, fruit weight, peel weight and peel thickness. In addition, it also improved the fruit number and consequently the fruit yield, TSS, ascorbic acid content, total sugars, reducing sugars, non-reducing sugars, juice content and sugar/acid ratio. Similarly, the exogenous application of GA 3 at 10, 20 and 30 mg/L on pear tree (cv. Le-Conte) increased shoot length and diameter, leaf area and fruit length, diameter and size. Additionally, they also boosted the number and weight of fruits, as well as the yield in comparison to untreated plants [84].
In case of HA application, our results are in corroboration with the earlier findings [85][86][87], when HA was applied to grapevines at full bloom, it enhanced the leaf chlorophyll content, berry weight, size, width, yield, sugars content and the sugar/acidity ratio while it lessened the fruit titratable acidity comparing with untreated vines. Moreover, the same authors explained the increase in the size of berries was as result of raising the mineral nutrient uptake, and thus HA's effect seemed to be similar with the influence of auxin, gibberellin and cytokinin-like activity.
As HA includes multiple minerals, it could improve the soil fertility and raise the nutrient availability by adsorbing them on mineral surfaces, resulting in improving the plant development and yield of broad bean [88]. In grapes, HA increased the leaf chlorophyll content and photosynthesis rate; therefore, it led to increasing the growth [85,89]. Treating olive cultivars with HA 100 mg/L increased the fruit content from the oil, protein, total chlorophyll and phenol contents [90]. Under water stress, it was noted that HA increased the leaf water retention, photosynthetic rate, the roots density and absorbed elements; thus, it could improve the growth, yield and quality of fruit [91,92].
Application of HA encourages the growth of plants and crop production [93] as well as microbial activities [92,94,95], and it significantly affects the soil physicochemical characteristics, microbe number and activity; thus, it can raise the availability and absorption of macro and micro nutrients and the prolongation of roots [86,92,[96][97][98]. The foliar spraying of HA ameliorated the shoot length, shoot diameter and leaf area, fruit set percentage and fruit yield and decreased the fruit drop percentage. Furthermore, it also enhanced the fruit weight, length, diameter, firmness, TSS and TSS-acid ratio, while decreasing the fruit acidity. In addition, it increased the concentration of sugars in the fruit and leaf contents of macro and micronutrients, such as N, P, K, Ca, Fe, Zn, Mn and B when compared with untreated plants in apple [82] and apricot [99].
The efficacy of CA, GA and HA to modulate plant physiology depends on its concentration, application method and plant [63,100]. The results from this study also showed that response of pear plant growth and fruit development to CA, GA 3 and HA applications changed with change in their concentrations. Thus, principal component analysis was conducted to delineate treatment and concentration-dependent effects (Figure 2). Based on the highest squared cosine value corresponding to factors F1 or F2, the plant growth, yield and fruit quality attributes were clustered around CA, GA 3 and HA treatments. The efficacy of CA, GA and HA to modulate plant physiology depends on its concentration, application method and plant [63,100]. The results from this study also showed that response of pear plant growth and fruit development to CA, GA3 and HA applications changed with change in their concentrations. Thus, principal component analysis was conducted to delineate treatment and concentration-dependent effects (Figure 2). Based on the highest squared cosine value corresponding to factors F1 or F2, the plant growth, yield and fruit quality attributes were clustered around CA, GA3 and HA treatments.
Factor F1, covering 90.29% variability in data (eigenvalue 22.573), showed clustering of the shoot thickness, shoot length, leaf area, leaf chlorophyll, fruit weight, fruit size, fruit firmness, fruit length, fruit diameter, leaf nitrogen, leaf phosphorus, leaf potassium, leaf iron, leaf zinc, leaf manganese, fruit TSS, fruit TTA, sugar-acid ratio, total sugars, reducing sugars, non-reducing sugars, fruit set (%) and fruit yield of pear with 100-150 ppm GA3 and 4-5% HA suggesting pa ositive influence of GA3 and HA applications on these parameters.
Factor F1, covering 90.29% variability in data (eigenvalue 22.573), showed clustering of the shoot thickness, shoot length, leaf area, leaf chlorophyll, fruit weight, fruit size, fruit firmness, fruit length, fruit diameter, leaf nitrogen, leaf phosphorus, leaf potassium, leaf iron, leaf zinc, leaf manganese, fruit TSS, fruit TTA, sugar-acid ratio, total sugars, reducing sugars, non-reducing sugars, fruit set (%) and fruit yield of pear with 100-150 ppm GA 3 and 4-5% HA suggesting pa ositive influence of GA 3 and HA applications on these parameters.
The fruit drop (%) and total titratable acidity showed a positive association with the control, 500-1000 ppm CA, 50 ppm GA 3 and 3% HA applications. This cluster was located opposite to 100-150 ppm GA 3 and 4-5% HA on the F1 axis depicting a strong negative correlation of 100-150 ppm GA 3 and 4-5% HA applications on these parameters compared to the control. The second factor, covering 5.28% variability in data (eigenvalue 1.320), showed clustering of fruit shape index with 1500 ppm CA. Thus, principal component analysis helped to delineate the individual roles of CA, GA 3 and HA treatments in regulating various aspects of plant growth, yield and fruit quality of pears.

Conclusions
This study suggests that foliar applications of CA, GA 3 and HA can be used to increase the yield and nutritional quality of 'Le Conte' pear-two important factors contributing to economic profit and consumer health, respectively. Since foliar applications of CA, GA 3 and HA at different levels differentially regulate distinct aspects of plant growth and development, specific concentrations of these bio-stimulants may help to achieve specific objectives of fruit production. Overall, 1500 ppm CA, 150 ppm GA 3 and 4-5% HA may be used as an effective preharvest application strategy to improve plant health, increase yield and improve the quality of pears. Citric, gibberellic and humic acids are effective tools to improve fruit vegetative growth, yield and physiochemical characteristics under stress conditions; thus, they could be used as alternatives to the chemical fertilizers to alleviate undesirable impacts on the soil composition, microbe activity and nutritional value of the produced crops.

Data Availability Statement:
The data used to support the findings of this study are included within the article.