Foliar Application of Calcium and Growth Regulators Modulate Sweet Cherry (Prunus avium L.) Tree Performance.

: Cracking of sweet cherry (Prunus avium L.) fruits is caused by rain events close to harvest. This problem has occurred in most cherry growing regions with significant economic losses. Several orchard management practices have been applied to reduce the severity of this disorder, like the foliar application of minerals or growth regulators. In the present study, we hypothesized that preharvest spray treatments improve the physiological performance of sweet cherry trees and could also mitigate environmental stressful conditions. Effects of repeated foliar spraying of calcium (Ca), gibberellic acid (GA3), abscisic acid (ABA), salicylic acid (SA), glycine betaine (GB), and the biostimulant Ascophyllum nodosum (AN) on the physiological and biochemical performance of 'Skeena' sweet cherry trees during two consecutive years (without Ca in 2015 and in 2016 with addition of Ca) were studied. Results showed that in general spray treatments improved the physiological performance and water status of the trees. AN and ABA sprays were demonstrated to be the best compounds for increasing yield and reducing cherry cracking as well as improving photosynthetic performance and leaf metabolites content. In conclusion, AN and ABA might be promising tools in the fruit production system.

1. Introduction

The moss="Chemical">st recent climate projections [1] point to a decrease in <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Chemical">water availability, ss="Chemical">span class="Chemical">Species">an increase in air temperature, ss="Chemical">span class="Chemical">Species">and the occurrence of extreme phenomena, such as excessive rainfall near the harvest period, which may increase the incidence of fruit cracking in ss="Chemical">span class="Chemical">Species">an class="Species">sweet cherry (Prunus avium L.). Consequently, significant economic losses occur, due to a strong reduction of the commercial value of the cherries. Under the current climate changing scenario [1] and also due to the increase of global trade in fruit to meet consumer demand for regular supply of high quality fruit, it is important to understand the relationship between preharvest treatments with calcium (Ca) and growth regulators and the physiological parameters of sweet cherry trees. This information can provide new insights into the putative potential measures to mitigate environmental stressful conditions.

Ca is <span class="Species">an import<ss="Chemical">span class="Species">ant macronutrient, which is involved in the regulation of the main physiological processes in plants, contributing to the strength of the cell walls and membranes and reducing cherry cracking [2,3,4,5,6]. Under drought conditions, growth and physiological performance are improved by Ca sprays of <span class="Species">Zoysia japonica Species">and Species">an class="Species">Zea mays plants [7,8]. Gibberellic acid (GA3) has been used as a compound to promote growth, which regulates plant growth processes, like seed germination, flower, and fruit development [9,10,11]. However, the impact of GA3 spray treatment on fruit cracking incidence is sometimes contradictory [12,13,14,15,16]. Other plant hormones, like salicylic acid (SA) and abscisic acid (ABA), are signaling phytohormones with different regulatory roles in plant metabolism and adaptation to abiotic stresses [17,18]. The yield increase in olive [19], peach [20], and strawberry [21] is associated with SA application, as well as the quality improvement of cherry fruits [22,23]. ABA stimulates stomatal closure and minimizes water loss by transpiration [24]. Additonally, Balbontín et al. [25] mentioned that ABA foliar sprays reduced cracking in ‘Bing’ cherries. Therefore, exogenous ABA application can have a great interest in water conservation in agricultural settings.

Although no consi<span class="Chemical">stent literature is available about the effect of preharve<ss="Chemical">span class="Chemical">st substances, such as <span class="Chemical">glycine betaine (Species">an class="Chemical">GB) and Ascophyllum nodosum (AN), on the physiological performance of sweet cherry trees, these compounds might be a new and innovative solution to increase the crop ability to tolerate stressful environments. The accumulation of osmolytes such as GB (quaternary ammonium compound) in cells can stabilize the structures by maintaining the integrity of membranes against the damaging effects of abiotic stresses via osmoregulation or osmoprotection [26]. Seaweed based biostimulants, like AN, are composed of several components, such as plant hormones, proteins, sugars, vitamins, humic substances, and phenolic compounds [27,28]. Several published reports suggest that biostimulants improve plant productivity by increasing the minerals assimilation and the photosynthetic rate, reducing the transpiration and the fruit cracking incidence [27,29,30,31]. Despite these well-documented effects, no consistent results are yet available, at least to our knowledge, about the influence of plant growth regulators, with the addition or no addition of Ca, on the performance of sweet cherry trees.

Therefore, the objective of this ss="Chemical">study was to assess the effect of pl<ss="Chemical">span class="Species">ant growth regulators, with the addition or no addition of Ca, on the plant physiological and biochemical responses, namely plant <span class="Chemical">water status, photosynthetic performspan class="Chemical">Species">ance, Species">and leaf metabolites, as well as on the yield of Species">an class="Species">sweet cherry trees and cherry cracking incidence.

2. Material and Methods

2.1. Experimental Site and Plant Material

Experiments were conducted in Carrazedo de Montenegro, Portugal (latitude 41°33′ N, longitude 7°17′ W, altitude 682 m), in 2015 ss="Species">and 2016, on a six-year-old late-maturing ‘Skeena’ <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Species">sweet cherry orchard grafted on ‘Gisela 6’ rootstock. The soil characteristics were: 13 g kg−1 of orgspan class="Chemical">Species">anic matter content, high Species">an class="Chemical">K2O (125 mg kg−1) and medium P2O5 (75 mg kg−1) contents, medium texture, and pH 5.5. Trees were trained under a vertical axis system with a spacing of 4.5 m between rows and 2.0 m in the row (about 864 trees ha−1) [32]. Between May and September, trees were daily drip-irrigated for 4 h per day (drippers 1 m apart in line with a 4 L h−1 flow rate) and summer pruned. According to recommendations provided by a certified soil analysis laboratory of University of Trás-os-Montes e Alto Douro (UTAD), trees were also periodically fertilized.

Meteorological data [air temperature (°C), rainfall (mm), ss="Species">and solar radiation (W m−2)] for both years (Figure 1) were recorded by a <ss="Chemical">span class="Chemical">standard weather station (IMT280, iMETOS, Weiz, Austria) located near the experimental site. In 2015, the mean air temperature between March and June was, on average, about 2.6 °C higher than 2016. Additionally, the mean solar radiation until June was also higher in 2015 than 2016. Annual rainfall in 2015 was 470 mm against the long-term rainfall (30 years) of 923 mm. The year 2016 experienced higher annual rainfall (1140 mm), mainly in spring (Figure 1), corresponding to the final phase of the flower development and the fruit development of cherry trees.

Figure 1

Mean air temperature (°C), rainfall (mm), and mean solar radiation (W m−2) in 2015 and 2016 measured at Carrazedo de Montenegro.

2.2. Experimental Design and Treatments

Six trees from each treatment were selected, a total of 42 trees. In 2015, the experiment included the following treatments: 0.5 mL L−1 bioss="Chemical">stimul<ss="Chemical">span class="Species">ant <span class="Species">Ascophyllum nodosum (AN), 10 mg L−1 Species">an class="Chemical">gibberellic acid (GA3), 10 µM abscisic acid (ABA), 1 mM salicylic acid (SA), 1 mL L−1 glycine betaine (GB), 5 g kg−1 calcium (CaCl2), and control (distilled water). In 2016, the same seven treatments were applied to the same trees selected in the previous year, but now including 5 g kg−1 CaCl2, except for the control treatment. All cherry trees were sprayed with 2.5 L of spraying solution per tree. Wetting agent (1 mL L−1) was mixed in control and treatment solutions. Foliar treatments, except CaCl2, were applied 30, 49, and 56 days after full bloom (DAFB), corresponding to the shuck split (beginning of fruit development), the transition from green to yellow color, and from yellow to orange color. CaCl2 was added at 56, 62, and 69 DAFB, corresponding to the first application at the transition from yellow to orange color and the other applications were applied one week later.

The gas exchss="Species">ange <ss="Chemical">span class="Species">and relative <span class="Chemical">water content (RWC) determinations were performed at midday on 08 July 2015 Species">and 15 July 2016 (corresponding to the harvest date of the cherries) in healthy, fully expspan class="Chemical">Species">anded mature leaves that were well exposed to the sun. For each treatment, fifty fruits were harvested per tree to determine cracking index, Species">and yield was determined for each tree. Furthermore, leaves were collected for biochemical Species">analyses (photosynthetic pigments, total Species">an class="Chemical">soluble sugars, starch, and soluble proteins) and immediately frozen in liquid nitrogen and then stored at −80 °C until analysis.

2.3. Leaf Gas Exchange

Leaf gas exchss="Species">ange measurements were performed using a portable LCpro+ Infrared Gas <ss="Chemical">span class="Species">Analyzer System (IRGA) (ADC Bioscientific Ltd, Hoddesdon, England), with a 2.5 cm2 leaf chamber (ADC-PLC), operating in the open mode, at midday (13:00–14:30 h) in both years. Incident photosynthetic photon flux density (PPFD) on the leaves was always greater than 1500 μmol m−2 s−1. Net <span class="Chemical">CO2 assimilation rate (A, μmol m−2 s−1), trSpecies">anspiration rate (E, mmol m−2 s−1), Species">and stomatal conductSpecies">ance (g, mmol m−2 s−1) were calculated using the equations developed by von Caemmerer Species">and Farquhar [33]. Intrinsic Species">an class="Chemical">water-use efficiency was calculated as the ratio of A to g (A/g, µmol mol−1), according to Düring [34]. All results are expressed as the average of six replicates with standard error (SE) shown.

2.4. Leaf Water Status

After the midday gas exchss="Species">ange measurements, <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Species">sweet cherry leaves were detached Species">and immediately placed into air-tight tubes, Species">and the following parameters were studied: fresh weight (FW in g), weight at full turgor (TW in g, measured after immersing the leaf petioles in deionized Species">an class="Chemical">water for 24 h at 4 °C in the dark), and dry weight (DW in g, measured after drying at 70 °C to a constant weight). The relative water content (RWC in %) was calculated as follows: RWC = (FW−DW)/(TW−DW) × 100. Results are expressed as the average of six replicates with SE shown.

2.5. Metabolite Composition Determination

2.5.1. Photosynthetic Pigments

For <sp<span class="Species">an class="Chemical">chlorophyllss="Chemical">span> (ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">Chl) and carotenoid (Carot) determination, leaf discs (0.8 cm diameter) were ground with mortar and pestle using acetone/distilled-water (80/20, v/v) as extraction solvent. Analyses were performed under the dim light to avoid chlorophyll degradation. Determination of total chlorophyll (Chltotal) and total carotenoids (Carottotal) were performed according to Šesták et al. [35] and Lichtenthaler [36], respectively. The results were expressed as mg g−1 DW as the mean ± SE of six replicates.

2.5.2. Total Soluble Sugars and Starch

Total <sp<span class="Species">an class="Chemical">soluble sugarsss="Chemical">span> (SS) quss="<span class="Chemical">Species">anss="Chemical">span>tification was performed using the methodology of Irigoyen et al. [37], by heating foliar discs in ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">ethanol/distilled-water (80/20, v/v) for 1 h, at 80 °C. After the reaction of the alcoholic extract with fresh anthrone in a boiling water bath for 10 min, SS were quantified by recorded absorbance values at 625 nm. Afterwards, starch (St) was extracted from the same solid fraction by heating leaf discs in 30% perchloric acid at 60 °C for 1 h, using the methodology of Osaki et al. [38]. The St concentration was determined by the anthrone method described above. Glucose was used as a standard for both SS and St quantification. The results were expressed as mg g−1 DW as the mean ± SE of six replicates.

2.5.3. Soluble Proteins

Total soluble proteins (SP) determination was performed using <sp<span class="Species">an class="Chemical">phoss="Chemical">sphatess="Chemical">span> buffer (pH 7.5), 0.1 mM ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">ethylenediaminetetraacetic acid (EDTA), 100 mM phenyl-methylsulfonyl fluoride (PMSF), and 20 g L−1 polyvinylpyrolli-done (PVP). The absorbance values were recorded at 595 nm, using bovine serum albumin (BSA) as standard [39]. The results were expressed as mg g−1 DW as the mean ± SE of six replicates.

2.6. Yield Determination

Sweet cherries were harvess="Chemical">sted at optimum maturity <ss="Chemical">span class="Chemical">stage and yield per tree was recorded in kilograms as the mean ± SE of six replicates.

2.7. Fruit Cracking Index Determination

The cracking index (CI in %) was determined according to Chriss="Chemical">stensen [40]. Fifty fruits without defects were selected <ss="Chemical">span class="Species">and immersed in 2 L plastic containers filled with distilled <span class="Chemical">water (20 ± 1 °C) for 6 h. Cracked fruits were removed, counted, Species">and fruits without cracks were reincubated. After 2, 4, Species">and 6 h, the fruits were observed for macroscopic cracks, with the CI calculated according to the following formula: CI = [(5a + 3b + c) × 100]/250, where a, b, Species">and c represent the number of cracked fruits after 2, 4, Species">and 6 h, respectively. The measurements are presented as average values (n = 3) with SE.

2.8. Statistical Analysis

The ss="Chemical">stati<ss="Chemical">span class="Chemical">stical analysis was carried out using the statistical software program SPSS V.25 (SPSS-IBM, Orchard Road-Armonk, New York, NY, U<span class="Chemical">SA). Statistical differences were evaluated by one-way Species">analysis of varispan class="Chemical">Species">ance (ANOVA) followed by the post hoc Duncspan class="Chemical">Species">an’s multiple rSpecies">ange test (P < 0.05), establishing treatment effect. The ANOVA requirements, namely the normal distribution of the residuals Species">and the homogeneity of varispan class="Chemical">Species">ance, were evaluated by meSpecies">ans of the Shapiro–Wilk’s test Species">and Bartlett’s tests, respectively. Dependent variables were Species">analyzed using ANOVA with or without Welch correction, depending if homogeneity of varispan class="Chemical">Species">ances was observed or not. For the relationship between parameters, Species">an class="Species">Pearson’s correlation was performed.

3. Results

3.1. Leaf Gas Exchange Parameters

The results show that for both years, moss="Chemical">st of the gas exch<ss="Chemical">span class="Species">ange parameters were significantly affected (P < 0.05) by the spray treatments (Figure 2), except the transpiration rate (E) (data not shown).

Figure 2

Net CO2 assimilation (A) (A,B), stomatal conductance (gs) (C,D), and intrinsic water-use efficiency (A/gs) (E,F) of fully exposed leaves of ‘Skeena’ cherry treated after spray treatments application in 2015–2016. Each column is expressed as mean ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among treatments by Duncan’s test.

In 2015, <sp<span class="Species">an class="Chemical">GA3ss="Chemical">span> sprays increased (P < 0.05) photosynthetic rate (A) compared to the control (ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">H2O) (Figure 2a). In turn, SA and GB spray treatments decreased (P < 0.01) g (Figure 2c). All spray treatments increased (P < 0.01) intrinsic water-use efficiency (A/g) compared to the control (Figure 2e).

In 2016, spray treatments increased (P < 0.05) A, with the highess="Chemical">st value for <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Chemical">SA+Ca-treated cherry trees (13.85 µmol m−2 s−1), whereas control trees recorded the minimum rate (Figure 2b). ss="Chemical">span class="Chemical">Species">an class="Chemical">GA3+Ca-, SA+Ca-, and GB+Ca-treated cherry trees presented significantly lower (P < 0.05) g values than control and Ca-treated cherry trees (Figure 2d). AN+Ca, GA3+Ca, ABA+Ca, SA+Ca, and GB+Ca spray treatments increased (P < 0.05) A/g compared to the control (Figure 2f).

3.2. Leaf Water Status

In both years, relative <sp<span class="Species">an class="Chemical">waterss="Chemical">span> content (RWC) was affected by the spray treatments (P < 0.05) (Figure 3). In 2015, treatments with ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">GA3, AN, and GB increased (P < 0.05) RWC up to 3%, 3.4%, and 4.8%, respectively, compared with the control (Figure 3A). In 2016, all spray treatments presented higher (P < 0.001) RWC values in comparison with control plants. The highest value was obtained in cherry trees treated with ABA+Ca, about 4% higher than control (Figure 3B).

Figure 3

Relative water content (RWC, %) of ‘Skeena’ leaf cherry after spray treatments application in 2015 (A) and 2016 (B). Each column is expressed as mean ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among treatments by Duncan’s test.

3.3. Leaf Photosynthetic Pigments and Metabolites

The content of photosynthetic pigments <sp<span class="Species">an class="Chemical">Chltotalss="Chemical">span> ss="<span class="Chemical">Species">anss="Chemical">span>d ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">Carottotal was affected by spray treatments (P < 0.05) in 2015, while in 2016 significant differences (P < 0.05) were only found for the content of Chltotal (Table 1). Compared with the control, the highest Chltotal content (10.56 mg g−1 DW) was observed with the ABA treatment in 2015 and both AN+Ca and GB+Ca treatments in 2016. In relation to the Carottotal content in 2015, cherry trees treated with Ca, AN, GA3, SA, and GB presented higher (P < 0.001) values than the control. Both years presented similar Chltotal contents for control and Ca-treated plants. In turn, Carottotal concentration seems to be influenced by the year, being higher (P < 0.01) in 2016.

Table 1

Total chlorophyll content (Chltotal), total carotenoids content (Carottotal), soluble sugars content (SS), starch content (St), and soluble protein content (SP) of ‘Skeena’ leaf cherry after spray treatments application in 2015–2016.

Year	Treatment(T)	Chltotal(mg g−1 DW)	Carottotal(mg g−1 DW)	SS(mg g−1 DW)	St(mg g−1 DW)	SP(mg g−1 DW)
2015	Control	7.95 ± 0.42 a	0.93 ± 0.04 a	70.35 ± 1.16 a	42.98 ± 0.56 a	10.34 ± 0.79 a
	Ca	8.37 ± 0.86 a	1.15 ± 0.05 cd	75.04 ± 3.87 abc	41.67 ± 1.94 a	12.30 ± 0.53 ab
	AN	9.34 ± 0.27 ab	1.23 ± 0.05 de	85.81 ± 3.00 bc	47.41 ± 1.89 a	15.08 ± 0.43 cd
	GA3	9.10 ± 0.32 ab	1.35 ± 0.04 e	75.24 ± 4.85 abc	45.39 ± 2.59 a	13.81 ± 0.91 bcd
	ABA	10.56 ± 0.68 b	1.01 ± 0.02 ab	87.44 ± 4.65 c	48.13 ± 3.00 a	12.90 ± 1.08 abc
	SA	8.56 ± 0.33 a	1.19 ± 0.05 cd	78.31 ± 6.03 abc	49.94 ± 1.39 a	15.75 ± 1.06 d
	GB	8.35 ± 0.26 a	1.10 ± 0.03 bc	72.79 ± 4.63 ab	48.06 ± 2.91 a	15.26 ± 1.11 cd
	P (T)	*	***	*	ns	**
2016	Control	7.51 ± 0.26 ab	1.19 ± 0.14 a	75.85 ± 2.12 a	101.10 ± 2.10 a	10.79 ± 0.69 a
	Ca	7.24 ± 0.40 a	1.25 ± 0.05 a	80.84 ± 3.74 ab	116.49 ± 6.77 ab	10.87 ± 0.85 a
	AN+Ca	8.76 ± 0.44 c	1.35 ± 0.04 a	85.89 ± 5.36 ab	126.44 ± 3.28 bc	11.90 ± 0.79 a
	GA3+Ca	7.97 ± 0.78 abc	1.30 ± 0.07 a	91.60 ± 1.85 b	110.58 ± 5.14 ab	12.90 ± 1.41 a
	ABA+Ca	8.22 ± 0.40 abc	1.32 ± 0.05 a	93.92 ± 5.09 b	121.41 ± 8.12 bc	13.29 ± 0.54 a
	SA+Ca	8.50 ± 0.43 bc	1.34 ± 0.05 a	93.27 ± 6.69 b	137.61 ± 7.71 c	11.64 ± 0.92 a
	GB+Ca	8.86 ± 0.39 c	1.43 ± 0.06 a	87.67 ± 2.71 ab	125.77 ± 7.27 bc	12.32 ± 1.42 a
	P (T)	*	ns	*	**	ns

Values are means ± standard error (n = 6). Means flanked by the same letter are not significantly different at P < 0.05 (Duncan’s test). * P < 0.05, ** P < 0.01, *** P < 0.001 by Duncan’s test, ns—not significant. DW: Dry Weight.

The results also indicated that spray treatments increased leaf metabolites in cherry leaves (Table 1). <sp<span class="Species">an class="Chemical">Soluble sugarsss="Chemical">span> (SS) content was significss="<span class="Chemical">Species">anss="Chemical">span>tly affected by the spray treatments (P < 0.05) in both years. In 2015, AN ss="<span class="Chemical">Species">anss="Chemical">span>d ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">ABA sprays increased (P < 0.05) SS content in relation to the control. On the other hand, addition of Ca to GA3, ABA, and SA usually resulted in higher SS concentration in 2016. Significant differences were also observed among spray treatments for soluble proteins (SP) and starch (St) concentration in the first and second year of the experiment, respectively. In 2015, the highest SP concentration was recorded in cherry trees sprayed with AN, GA3, SA, and GB. In 2016, the combined treatments AN+Ca, ABA+Ca, SA+Ca, and GB+Ca showed a higher (P < 0.01) St content.

3.4. Yield of Sweet Cherry Trees

Significss="Species">ant yield differences were found among ss="Chemical">spray treatments (P < 0.05) in both years (Figure 4). In 2015, cherry trees ss="Chemical">sprayed with <ss="Chemical">span class="Species">AN and <span class="Chemical">ABA exhibited the highest (P < 0.05) yield, averaging 45% Species">and 41%, respectively, compared to the control (Figure 4A). This behavior was also observed in 2016, although the highest production was obtained for Species">an class="Chemical">GB+Ca-treated cherry trees, up to 40% compared to the control (Figure 4B).

Figure 4

Yield (kg tree−1) of ‘Skeena’ cherry trees after spray treatments application in 2015 (A) and 2016 (B). Each column is expressed as mean ± SE (n = 6). Different letters indicate significant differences (P < 0.05) among treatments by Duncan’s test.

3.5. Cracking Incidence

ss="Chemical">Spray treatments did not affect signific<ss="Chemical">span class="Species">antly (P > 0.05) the fruit cracking index (CI) in both years (Figure 5). However, cherries treated with Ca, AN, <span class="Chemical">ABA, Species">an class="Chemical">SA, and GB spray treatments showed a decreasing trend of CI in both years, compared to the control. In contrast, GA3/GA3+Ca sprays increased the CI, in both years.

Figure 5

Cracking index (%) of ‘Skeena’ fruits after spray treatments application in 2015 (A) and 2016 (B). Each column is expressed as mean ± SE (n = 3, each with 50 fruits).

4. Discussion

4.1. Spray Treatments Modulate Leaf Gas Exchange and Water Status of Sweet Cherry Trees

Overall, spray treatments improved the physiological behavior of <spss="Species">an class="<ss="Chemical">span class="Chemical">Species">sweet cherry trees in both years (Figure 2). A presented a positive correlation with g (r = 0.54, P < 0.001), as observed previously by Gonçalves et al. [41] in ss="Chemical">span class="Chemical">Species">an class="Species">sweet cherry. Higher g values were noticed in 2016 compared to 2015, which may be due to the higher solar radiation recorded in July 2016, near the harvest, and consequently, the values of A were also higher in 2016 (Figure 1 and Figure 2). Our findings are in agreement with previous studies, which demonstrated an increase of A in broad beans (Vicia faba) and grapevine (Vitis vinifera L.) treated with GA3 [42]. Grapevine treated with GA3 also resulted in favorable A/g [43]. Other studies with Brassica juncea, corn, and soybean treated with SA also showed an improvement of A, A/g and g adjustment [44,45,46], which agrees with our results. The first sign of plant defense for maintaining the water status is the stomatal closure [47]. Indeed, our results indicate that GB sprays improved A and also increased stomata closure by the reduction of g (Figure 2), which might be considered a strategy for enhancing tolerance to various abiotic stresses [48]. Moreover, GB-treated olive trees under drought showed an enhancement of A [49]. Tradescantia virginiana plants treated with ABA and grown under well-watered conditions had lower g and an improvement in A/g, while the A was unaffected [50]. Although, in both years of our study, the application of ABA increased A/g, g was not affected, and an improvement in A was observed, mainly in 2016. Additionally, A and A/g values also increased in response to AN+Ca treatment (in 2016) (Figure 2). This finding is in agreement with previous works, which revealed an increase of A, a reduction of E, and g parameters in plants treated with biostimulant products [29,30].

The leaf RWC was around 90% in both years, suggess="Chemical">sting sufficient drip irrigation. Nevertheless, in treated-pl<ss="Chemical">span class="Species">ants with foliar compounds, the RWC values were mostly higher in both years (Figure 3). The <span class="Chemical">water balSpecies">ance in plSpecies">ants is estimated by calculation of RWC, which is positively correlated with the photosynthetic efficiency of plSpecies">ants [51]. This relationship is established by the correlation between RWC Species">and A, mainly in 2016 (r = 0.54, P < 0.05). Similarly with our results, a significspan class="Chemical">Species">ant improvement of RWC in different crops exposed to several biotic Species">an class="Disease">stresses and treated with biostimulant [29,52,53,54], GA3 [55], ABA [56], SA [57], and GB [49,58] was previously reported.

4.2. Photosynthetic Pigments’ and Metabolites’ Behavior in Response to Spray Treatments

<sp<span class="Species">an class="Chemical">Chltotalss="Chemical">span> exhibited the ss="<span class="Chemical">Species">anss="Chemical">span> class="Chemical">same tendency as A, displaying higher contents compared to the control in both years (Table 1). ABA sprays increased Chltotal content, which is in accordance with a previous study that revealed that the exogenous application of ABA increases the synthesis of chlorophylls in ‘micro’ tomato leaf tissue [59]. Photosynthetic pigments contents, Chltotal and Carottotal, also increased in AN-, SA-, and GB-treated cherry trees (Table 1). Similarly, Kabiri et al. [60], mentioned that the SA application in Nigella sativa increased the content of chlorophylls and carotenoids. According to Hayat and Ahmad [61], this increase improved the antioxidant capacity of plants and it was related to the synthesis of protective compounds. Additionally, overaccumulation of GB due to the introduction of the betaine aldehyde dehydrogenase (BADH) gene can increase the protection of chlorophylls and carotenoids and enhance the photosynthetic rate [62]. Several biostimulants, like AN, have been stated to stimulate plant growth by increasing photosynthetic pigments [28]. These findings can be related to the preservation of carotenoids as a mechanism of photoprotection [63] and amelioration of the leaf water retention. Our study indicates that spray treatments, mainly AN, SA, and GB induced the increased chlorophylls and carotenoids levels in the leaf tissue, which can improve the antioxidant capacity of plants to abiotic stress.

The spray treatments also affected the SP content in cherry leaves, showing higher levels in <span class="Species">AN-, <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Chemical">GA3-, ss="Chemical">span class="Chemical">Species">an class="Chemical">SA-, and GB-treated trees, up to 34% with SA spray treatment (Table 1). These treatments might induce the growing of antioxidant responses as described for SA [18,61]. The higher accumulation of SS in leaves observed in response to AN and ABA sprays in 2015 and to GA3+Ca, ABA+Ca, and SA+Ca sprays in 2016 (Table 1) can be a protective mechanism to preserve cell homeostasis, indicating that these spray treatments provided a better cherry tree photosynthetic performance. Interestingly, the highest SS accumulation in cherry leaves was found for ABA spray treatment for around 20% in both years (Table 1). Exogenous application of ABA is reported to increase the maturity index and anthocyanin content in cherries [64] and the soluble sugars in grapes [65]. Although our previous works reported that ABA application increased anthocyanin content in cherry, no significant effect was observed on the maturation [66]. The highest St content observed in 2016 might be related to the higher photosynthetic efficiency determined in the same year (Table 1). Overall, the present study suggests a relation between acclimation of photosynthesis (A) and St accumulation (r = 0.46, P < 0.05). Indeed, several authors have suggested that a decrease of St in leaf was correlated with the acclimation of photosynthesis [67,68]. In 2016, the higher accumulation of St in mainly AN+Ca-, ABA+Ca-, SA+Ca-, and GB+Ca-treated cherry trees may have a positive effect on tree production, since St is a crucial storage carbohydrate that is frequently mobilized in the form of SS [69]. The St accumulation in leaves of ABA+Ca-, SA+Ca-, and GB+Ca-treated cherry trees could be associated with the increase in weight observed in the fruits collected from the plants treated with these spray treatments [23]. In addition, ABA might be related to control of the enzymes responsible for St degradation, regulating the St accumulation during osmotic stress in plants [70].

4.3. Effect of Spray Treatments on Yield of Sweet Cherry Trees and Fruit Cracking Incidence

Yield ss="Species">and cracking incidence evaluation are import<ss="Chemical">span class="Species">ant parameters in the effectiveness of spray treatments. However, fruit yield is a function of several factors, such as meteorological conditions, rootstock vigor, irrigation, and pruning [71]. Our previous work reported that on average, fruit weight was reduced in 2016 by 33% compared to 2015 [23]. On the other hand, yield was higher in 2016 compared to 2015 (Figure 4), which was due to a higher crop load rather than larger fruit size. This may also be because of a winter pruning in 2014, which will have reduced the number of flowers in the following year (2015); in turn, in 2015 no pruning was performed. Nevertheless, AN and <span class="Chemical">ABA sprays were related to the increase in yield in both years, while in 2016 also Species">an class="Chemical">GA3- and GB-treated cherry trees showed a significant increase of yield (Figure 4). Although cherry cracking incidence did not significantly decrease with spray treatments in both years, it was observed that also the AN- and ABA-treated fruits showed the least cracking index in 2015 and 2016 (Figure 5). Reduced cracking index for ABA-treated ‘Bing’ cherry was reported by Balbontín et al. [25]. An additional promising strategy to mitigate cherry cracking is AN, as had been reported by our research group for ‘Skeena’ and ‘Sweetheart’ cherry fruits [31]. On the other hand, foliar application of GA3 increased ‘Skeena’ cherry cracking, and this finding corroborates with previous studies [12]. Biostimulants, like AN, have offered a potentially novel approach in plants to stimulate growth and to increase yield, as reported by Basak [72], Colavita et al. [73], and Jannin et al. [30]. Quiroga et al. [74] reported that ABA foliar application in the field-grown grapevine could improve yield per plant. Our data suggest that the application of GA3 and GB seems to benefit with the combination of Ca in increasing of cherry yield. A positive impact of exogenous GB application on plant growth and final crop yield has been reported on sunflower (Helianthus annuus L.) and maize (Zea mays) under drought [75,76]. As observed in our study, foliar application of GA3 increased fruit yield in several crops, such as in tomato [77,78] and cucumber (Cucumis sativus L.) [79]. Our data also indicated a positive correlation between yield and A (r = 0.54, P < 0.01), as observed by Parry et al. [80] and Zhu et al. [81] in other species. Positive correlations were also observed between yield and SS (r = 0.50, P < 0.001), St (r = 0.83, P < 0.001), SP (r = 0.46, P < 0.01), and RWC (r = 0.66, P < 0.001). Indeed, other studies have also reported that yield is correlated positively with SS and RWC in Sorghum bicolor L. [82] and in several cultivars of banana fruits [83].

5. Conclusions

Foliar spraying of growth regulators ss="Species">and <ss="Chemical">sp<ss="Chemical">span class="Species">an class="Chemical">calcium was associated with ss="Chemical">span class="Chemical">Species">an enhss="Chemical">span class="Chemical">Species">ancement of the physiological performss="Chemical">span class="Chemical">Species">ance ss="Chemical">span class="Chemical">Species">and yield of the ‘Skeena’ ss="Chemical">span class="Chemical">Species">an class="Species">sweet cherry trees. Among the treatments evaluated, foliar application of AN and ABA were more effective in increasing yield and in reducing the incidence of cherry cracking. Therefore, these two foliar sprays are attractive compounds to improve physiological performance and yield of sweet cherry trees and might be a promising cherry-cracking mitigation approach. Although AN and ABA foliar application might be a cultural practice in the near future, further studies on the influence of environmental conditions, concentrations, and time of foliar compounds application in order to develop new cultivar-specific farming strategies will be required.