24-epibrassinosteroid and jasmine oil improve vegetative growth and productivity of Flame Seedless grapevines under abiotic stresses

Plant materials and experimental procedure

During two seasons in 2023 and 2024, 10-year-old Flame Seedless grapevines grafted on Freedom grape rootstocks were studied in clay soil with a 1.5-m groundwater table and a flood irrigation system on the El-Baramon experimental farm in the Dakahlia Governorate (31°11′98′′ N, 31°45′13′′ E). Grapevines were cultivated using a quadrilateral cordon training system of short spurs in a Spanish baron trellis. All grapevines were spur pruned by the third week of January in both seasons, retaining 68 buds per vine (four cordons, each with three five-bud spurs and one two-bud renewal spur).

The irrigation supply was the Nile River, water samples were taken for analysis, the quality was determined, and referred to in Ali et al. (2014). Soil samples under each treatment were-collected from the root zone (0–90 cm) and assessed as a clay soil with an average of 7.3 pH, 1.06% organic material, 0.61 dS m⁻¹ E.C., 1.7% CaCO₃, 0.35 meq 100 g⁻¹ HCO³⁻, 3.78 meq 100 g⁻¹ SO₄²⁻, 1.0 meq 100 g⁻¹Cl⁻, 0.85 meq 100 g⁻¹ Na⁺, 0.75 meq 100 g⁻¹ Ca²⁺, 0.82 meq 100 g⁻¹ Mg²⁺, 24.6 mg kg⁻¹ N, 16.0 mg kg⁻¹ P, and 247.0 mg kg⁻¹ K across seasons. The flood irrigation system was applied, and the irrigation water amount was based on climatic data collected from the nearest meteorological station. The applied irrigation requirement (IR) for each irrigation interval during the two growing seasons was calculated using the plant’s phonological stages and the percentage of growth shaded by the tree canopy. The total amount of water used was 1,090.9 m³/ha for each of the 11 irrigations over the season. During the growing season (March–October), the overall rate is around 12,000 m³/ha. Irrigation was done once a month during both seasons, except for May, June, and July, when it was done twice a month (Gaser, Abo El-Wafa & Abd El-Hameed, 2018). The control group consisted of grapevines at the study site under climate stresses, whose weather data are shown in Table 1, which received no treatments other than water spraying.

Jasmine essential oil (100%) was obtained from EL-Masrayia Company for Natural Oils in Cairo, Egypt, and stored in opaque bottles at 4 °C until used. GC-MS was used to identify the essential oil components at the National Research Center’s central laboratory in Giza, Egypt (Fig. 1). The source of brassinosteroid was 24-epibrassinosteroid, which was purchased from the Sigma Aldrich Company, St. Louis, MO, USA.

Experimental design and treatments

This experiment included 63 grapevines as uniform as possible in vigor, canopy structure, and trunk diameter, planted at a spacing of 2 m × 3 m and free of any physiological diseases or nutrient deficiencies. The grapevines selected were foliar-sprayed with seven spray treatments, including the control treatment at the study site, whose weather data are shown in Table 1, which was sprayed with water. In both seasons, the same nine vines were subjected to the same treatment as follow: the control, distilled water-sprayed vines (T1), 24-epibrassinosteroid at 1 mg/L (T2), 24-epibrassinosteroid at 2 mg/L (T3), 24-epibrassinosteroid at 3 mg/L (T4), jasmine oil 500 µL/L (T5), jasmine oil 1,000 µL/L (T6) and jasmine oil 1,500 µL/L (T7). The vine had received ≈2.5 L until run-off at three different times: 2 weeks after the beginning of vegetative growth, when shoot length reached about 25–30 cm, after berry set, and the veraison stage.

Measurement and determinations

In mid-February during seasons 2023 and 2024, on each side of the vine, five non-fruiting shoots from the renewal spurs were selected at random and labeled to measure vegetative growth. The number of leaves per shoot, average shoot length using a measuring tape to the nearest centimeter (cm) in three separate readings and the average shoot diameter were calculated using a digital caliper. Two mature leaves on each marked shoot (i.e., the 6th and 7th from the shoot tip) were collected to measure leaf area (cm²) using a LI-3100 leaf area meter. The average leaf area was then determined. During dormant seasons, internode length (cm) was measured in five shoots per vine from the third base internode using a digital caliper with an accuracy of 0.01. Pruning products were weighed in kilograms per vine using a digital bench scale (Rotation Scales, Batavia, IL, USA) model PC-500. Wood ripening was recorded by labeling twelve shoots of the current season’s growth of each replicate to follow up on the average of wood ripening. According to Elsayed & El-Shewaikh (2023), five ripe cane samples were collected during the dormant season during the first week of November, and the coefficient of wood ripening was calculated by dividing the length of the ripened part of the shoot by the total shoot length.

The nutritional status of grapevines was assessed by determining the chlorophyll content of leaves, including chlorophyll a, b, and total chlorophyll, the mineral content of leaf petioles, and the cane total carbohydrate content. Two weeks following fruit set, samples of twenty fresh and mature apical fifth and sixth leaves from the leaves opposite the basal clusters on each shoot were collected from each side of the vine to determine the levels of chlorophyll a and b, as well as total chlorophyll, as defined by Lichtenthaler & Wellburn (1994). Chlorophyll b and chlorophyll a were measured at wavelengths of 646 and 663 nm, respectively, by a spectrophotometer (UV/Visible spectrophotometer, Libra SS0PC, Thermo Fisher Scientific, Waltham, MA, USA). Total chlorophyll contents (mg g⁻¹ fw) were calculated using the following equations:

(1) $$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l}=[((\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l}\mathrm{a}+\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l}\mathrm{b})\times \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e})/(1000\times \mathrm{f}\mathrm{w}).$$

At dormant seasons, four non-fruiting shoots off the renewal spurs, two shoots at each side of the vine, were randomly selected by the end of the growing season in late December to assess total carbohydrates. Total carbohydrates as a percentage of dry weight were calculated using a spectrophotometer set (UV/Visible spectrophotometer, Libra SS0PC, Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm. To determine macro- and micronutrient content, samples of 15 leaf petioles per replicate were dried at 60 °C for 72 h to constant weight. Following the pulverization of dried leaf petioles with the mortar and pestle equipment, concentrated sulfuric acid with repeated additions of 30% hydrogen peroxide was used to digest the powder (Wolf, 1982). Total nitrogen (g 100 g⁻¹dw) and phosphorus (g 100 g⁻¹dw) were measured colorimetrically using the generated solution and a spectrophotometer (UV/Visible spectrophotometer, Libra SS0PC, Thermo Fisher Scientific, Waltham, MA, USA) (Jones, Wolf & Mills, 1991). Potassium (g 100 g⁻¹dw) was determined using the flame photometer (Tendon, 2005). Magnesium (mg g⁻¹dw), calcium (g g⁻¹dw), iron (mg g⁻¹dw), manganese (mg g⁻¹dw), and zinc (mg g⁻¹dw) were determined using an atomic absorption spectrometer according to Carter (1993).

A sample of 27 clusters per treatment (three clusters/vine) was harvested in the second week of May during the 2023 and 2024 seasons, when the berries reached full color. Average yield/vine (cluster weight multiplied by the number of clusters/vine) was calculated for each treatment (kg/vine). To determine the physical qualities, ten clusters from each vine were randomly harvested. Each cluster was weighed individually, and the average cluster weight (g) was recorded. The average berry weight (g) was calculated by weighing 100 randomly selected berries from each cluster. Cluster length (cm) and width (cm) were measured from the uppermost berry to the lowest berry. A digital caliper with 0.01 mm accuracy was used to measure the diameter and length of the berries (mm). Berry firmness was measured and quantified in newtons (N) using a handheld digital penetrometer (FT-02) with a 2 mm plunger tip.

To determine the chemical properties of the berry juice, another random sample of vine berries was collected. At an air-conditioned room temperature (about 20–22 °C), the soluble solids content (°Brix) was measured using a handheld refractometer model N-1E (Atago Co., Ltd., Tokyo, Japan). Using phenol-phthalein as an indicator and NaOH (0.1 N), titratable acidity (TA) was estimated as a percentage of tartaric acid in 10 mL of juice (Association of Official Analytical Chemists (AOAC), 2019), and the SSC/TA ratio was calculated. The total sugars were measured colorimetrically using the phenol and sulfuric acid technique reported by Dubois et al. (1956). The spectrophotometer was used to measure the absorbance at 490 nm, and the concentration of total sugars was computed as g glucose 100/g fw (as a percentage).

The total anthocyanin, flavonoids, and total phenols content in berry skin (2 g) was determined using a methanolic HCL extraction solvent according to the method of Lee & Francis (1972) using a UV-Vis Spectrophotometer at wavelengths of 760, 510, and 535 nm, respectively. Total phenolic and total flavonoid contents were evaluated according to the protocol of Slinkard & Singleton (1977). The Folin-Ciocalteau reagent was used to measure a calibration curve for gallic acid concentrations to determine the total phenolic content as gallic acid equivalents (mg/100 g dry weight). A measurement of mg catechin equivalents/100 g (constant weight) was used to express total flavonoids by measuring a calibration curve for known catechin concentrations. Anthocyanin pigment was expressed as mg/100 g fresh weight.

The phenylalanine ammonia-lyase (PAL) contents of the grape juice extract were measured according to Jones (1984), using an aspectrophotometer at a wavelength of 290 nm at room temperature. Calculated PAL activity using the following equation:

$$\mathrm{P}\mathrm{A}\mathrm{L}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{U}\mathrm{m}\mathrm{i}\mathrm{n}{\mathrm{g}}^{-1}\mathrm{F}\mathrm{W})=(\mathrm{\Delta }\mathrm{A}290/6.22)\times (1/30)\times (1/1),$$where ΔA290 is the change in absorbance at 290 nm, and 6.22 is the extinction coefficient of L-Phe. The peroxidase enzymatic activity was determined using a Hitachi U-2000 spectrophotometer (l = 460 nm) by the procedure outlined by Clemente & Pastore (1998). The polyphenoloxidase activity (l = 420 nm) was measured using the method described by Siddiq, Sinha & Cash, 1992. POX and PPO enzyme activity (U min g⁻¹ FW). Catalase activity was determined using the method of Aebi (1984), and absorbance was measured at a wavelength of 240 nm using a spectrophotometer. Catalase activity was calculated using the following equation:

$$\mathrm{C}\mathrm{a}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{U}\mathrm{m}\mathrm{i}\mathrm{n}{\mathrm{g}}^{-1}\mathrm{F}\mathrm{W})=(\mathrm{\Delta }\mathrm{A}240/0.0436)\times (1/30)\times (1/1),$$

where ΔA240 is the change in absorbance at 240 nm, and 0.0436 is the extinction coefficient of hydrogen peroxide according to Bergmeyer (1983). Units of catalase (U), which are the amount of enzyme needed to break down 1 μmol of hydrogen peroxide per minute at 25 °C and pH 7.0, are used to evaluate catalase activity.

Statistical analysis

Treatments were organized in a randomized complete block design (RCBD) system with three replicates, each replicate consisting of three vines. Data were first examined using the Shapiro-Wilk and Levene tests for normality and variance homogeneity, respectively. Before analyzing variance (ANOVA), the percentage data were first converted to the values of the Arcsine square root. The outcomes were then shown as back-transformed means. The ANOVA was performed using the CoStat program, version 6.311 (CoHort software, Monterey, CA, USA). Probability (p) < 0.05 was used for mean comparisons using Tukey’s honest significant difference (HSD) test. The score and loading plot for vegetative growth and biochemical parameters were generated using a principal component analysis (PCA) (Jolliffe, 2011). The two-way hierarchical cluster analysis (HCA) and heat map were generated using the means of the data matrices (Michie, 1982). Both PCA and HCA were performed using JMP Pro 16 (SAS Institute, Cary, NC, USA).

Vegetative growth

Data presented in Table 2 indicate that spraying Flame Seedless grapevines three times with Br and JO significantly enhanced the vegetative growth characteristics as compared with the control. These characteristics include shoot length, diameter, leaf area, number of leaves/shoots, internode length (cm), pruning wood weight (Kg/vine), and coefficient of wood ripening during the 2023 and 2024 seasons. In both seasons, the highest significant values for the above traits were obtained by foliar with Br at 3 mg/L; the control recorded the lowest values. Data revealed non-significant differences between foliar JO at 1,000 and 1,500 µL/L on shoot length, leaf area, shoot diameter, number of leaves/shoot, internode length, coefficient of wood ripening, and pruning wood weight in both seasons of the study. No significant differences were found between foliar Br at 1 and 2 mg/L on shoot diameter, leaf area, and number of leaves per shoot during the 2024 season. Data also showed non-significant differences between foliar with Br at 1 and 2 mg/L on internode length and wood ripening in both seasons of the study.

The nutritional status

After the application of Br and JO, there were statistically significant differences in the effect of all spraying via the leaves on the macro and micronutrient content of the Flame Seedless’ grapevines (p < 0.05). The results in Figs. 2A-2D and 3A-3D indicate that JO and Br treatments had a significant effect on P, N, Ca, K, Mg, Zn, Fe, and Mn contents in leaf petioles compared to untreated vines during both seasons. The data indicate that spraying Br was more effective than jasmine oil in improving the macroelements and microelements content in leaf petioles of vines. The greatest values of mineral contents were obtained due to spraying the vines with Br at 3 mg/L. Moreover, no significant differences were observed in the nitrogen, phosphorus, potassium, calcium, and magnesium contents of leaf petioles when Br was applied at concentrations of 1 and 2 mg/L during the two study seasons. Non-significant differences were also observed in the phosphorus content in leaf petioles in the two seasons, on the one hand, during the two study seasons, and the calcium content in the second year, on the other hand, when spraying with JO at concentrations of 500 and 1,000 µl/L. Moreover, no significant differences were observed between the 1,000 µL JO treatment and the 1 mg/L Br treatment for their effects on leaf petiole manganese and magnesium content during the first study season. In addition, no significant difference was observed between the 1 and 2 mg/L Br treatment and the 1,000 and 5,000 µL JO treatment on leaf petiole zinc content during the first study season.

The foliar application repercussions of JO and Br on chlorophyll a, chlorophyll b, and total chlorophyll contents in leaves, and total carbohydrates in cane contents of Flame Seedless grapevines grown in the 2023 and 2024 seasons are presented in Figs. 4A-4D. The foliar applications of JO and Br significantly improved leaf chlorophyll a, chlorophyll b, and total chlorophyll contents and cane carbohydrate content compared with the control. Foliar application with 3 mg/L Br was the most effective treatment in this study. Non-significant differences were found between foliar Br at 1 and 2 mg/L on leaf chlorophyll a, chlorophyll b, total chlorophyll contents, and cane carbohydrates content in both study seasons. Foliar spray data also showed no significant differences between different jasmine oil concentrations on total carbohydrates and chlorophyll a in the two seasons of the study, and different Br concentrations on chlorophyll a in the first season of the study. There were also no significant differences between spraying jasmine oil at concentrations of 1,000 and 1,500 µL/L on leaf chlorophyll b and total chlorophyll in the second season of the study.

Total yield

Data on the influence of the two applied treatments, Br and JO, on the yield of Flame Seedless grapevines in both seasons are shown in Fig. 5. The data show that, in the 2023 and 2024 seasons, all applied treatments significantly improved yield per vine as compared to the control. The results revealed that the effect of Br concentrations was superior to that of jasmine oil concentrations on yield, which gradually improved with increasing Br and JO concentrations. After careful analysis of the data, it was found that, compared to all treatments in the seasons, the application of Br at 3 mg/L produced the highest significant yield/vine, while the control group had the lowest yield/vine. The data also showed no significant differences between JO at concentrations of 1,000 and 1,500 µl/L in vine productivity in both study seasons.

Physical characteristics

In the experiment, the physical characteristics of the clusters and berries in Flame Seedless grapevines changed in both seasons as a result of the foliar treatments of Br and JO (Table 3). The data show that, in the 2023 and 2024 seasons, all applied treatments significantly improved physical properties, including cluster weight, length, width, and weight of 100 berries per cluster, as well as berry length, berry width, and berry firmness, compared to the control in both seasons. As compared with the other treatments in both seasons, the physical characteristics increased at a Br dose of 3 mg/L in both experimental years. Also, the results revealed that the effect of Br concentrations is better than that of JO concentrations on the physical properties of the clusters and berries. There were no significant differences in cluster weight, length, width, and 100-grain weight per cluster between JO at concentrations of 1,000 and 1,500 µl/L in the first and second seasons. The data are also clear, with no significant differences between the Br treatments at concentrations of 2 and 3 mg/L in cluster weight during the two study seasons. Also, data showed that no significant differences between foliar Br at 1, 2, and 3 mg/L on berry firmness in the first season of the study.

Chemical characteristics

The data shown in Table 4 revealed that SSC, SSC/TA ratio, total sugars, total phenols, total anthocyanin, and flavonoids were improved gradually while decreasing acidity in both seasons, with increasing concentrations of JO and Br compared to the control. Also, the results revealed that the effect of JO concentrations is better than that of Br concentrations on the TA, SSC, SSC/TA ratio, total sugars, total anthocyanin, total phenols, and flavonoids of berries. According to additional data analysis, the application of JO at 1,500 µL/L in this study was the most effective in raising the SSC, SSC/TA ratio, total sugars and decreasing TA in comparison to the other treatment, while control reduced SSC, SSC/TA ratio and total sugars and increasing TA. Also, data showed that no significant differences between foliar JO at 500, 1,000, and 1,500 µL/L on total phenols in both seasons of the study.

Enzyme activity

The data shown in Figs. 6A–6D indicate that the two treatments used, Br and JO, affected the POX, PAL, PPO, and CAT activities of Flame Seedless grapevines throughout both seasons. According to additional data analysis, the application of JO at 1,500 µL/L in this study was the most effective in raising the POX, PAL, PPO, and CAT activities in comparison to the other treatments, while the control reduced POX, PAL, PPO, and CAT activities in both seasons. Also, data showed that non-significant differences between foliar jasmine oil at 1,000 and 1,500 µL/L on POX activity in the both seasons of study and data showed that non-significant differences between foliar Br at 1 and 2 mg/L on POX activity and pectin methyl in the two seasons of study and data showed that no significant differences between foliar Br at 2 and 3 mg/L on PAL enzyme activities.

Principal component analysis and hierarchical cluster analysis

The purpose of applying PCA and HCA was to give a more comprehensive image of the sprayed Br and JO on vegetative growth parameters and biochemical attributes of Flame Seedless grapevines during the 2023 and 2024 seasons. As for the PCA (Fig. 7), the PCA score plots demonstrated a clear separation among treatments, with samples treated with Br (1, 2 and 3 mg/L) distinctly clustering on the positive side of the first principal component (PC1), reflecting their substantial positive influence on most measured traits in both seasons (Figs. 7A and 7C). The first two principal components accounted for 64.5% and 29.0% of the total variance in 2023, and 67.4% and 24.7% in 2024, respectively, confirming the efficiency of PCA in capturing the major sources of variability. Treatments with JO at 500, 1,000, and 1,500 µL/L were positioned intermediately between Br and the control. While the control treatment consistently aligned on the negative side of PC1, indicating comparatively lower effectiveness.

Across both seasons, Br treatments were strongly associated with the positive side of PC1, reflecting their pronounced positive impact on parameters such as shoot diameter, pruning weight, total chlorophyll content, chlorophyll a and b, leaf surface area, cluster weight, and concentrations of essential macro- and micronutrients (N, P, K, Ca, Mg, Fe, Zn, Mn). Meanwhile, the control treatment consistently clustered on the negative side of PC1, corresponding to reduced performance in most studied traits. Notably, variables such as total anthocyanins, total flavonoids, total phenols, and total sugars contributed substantially to the variation along PC2 in both seasons, further discriminating between treatments (Figs. 7B and 7D).

Complementarily, HCA (Figs. 8A and 8B) accompanied by heatmap visualization further validated these findings. The highest and lowest values are represented with blue and white colors, respectively. The heatmaps revealed that Br treatments exhibited the highest values across most evaluated physiological and biochemical traits in both seasons. JO treatments showed moderate values, whereas the control consistently presented the lowest values. This clustering pattern confirmed the differentiation observed in PCA, emphasizing the superior efficacy of Br in enhancing grapevine growth and biochemical performance.