Effect of different Concentrations of Salicylic acid on some Physicochemical Properties of Grape cv. Shahroodi

 Grape (Vitis vinifera L.) is one of the most important fruits cultivated in many areas with different climates all over the world. Grape, which is rich in antioxidants, flavonoids, anthocyanins and phenolic acids, is highly regarded in terms of economic value. Salicylic acid is one of the compounds which have been used in recent years for improving the physicochemical properties of many fruits. The application of salicylic acid increases the shelf life of crops by inhibiting ethylene production and respiration, as well as by reducing the activity of some enzymes which are responsible for softening the cell wall. Furthermore, salicylic acid results in the enhancement of phenolic compound production by increasing the activity of some enzymes such as phenylalanine ammonia-lyase.

Materials and Methods:

 The present study was conducted in 2014 on 13-year-old Shahroodi grapes that were grown using a head-training system with a spacing of 2m × 2m in "Shahrood" County, Semnan Province. A total of 60 grapevines with uniform age and growth characteristics were selected for the experiment. Salicylic acid was obtained from Merck and solutions were prepared using a small amount of organic solvent and surfactant to reduce the surface tension of the solutions. For the purpose, the Salicylic acid powder was dissolved in a few drops of ethanol and a few drops of Tween 20 for improving solution penetration to the plants, The solution was prepared at concentrations of 0 (control), 1, 2, 3 mM. Then the plants were sprayed two weeks after the fruit set stage (when berries were at the pea-sized stage with a diameter of 4 to 5 mm). The experimental design used in this experiment was a randomized complete block design (RCBD) with three replications. After harvesting and transporting of fruits to the laboratory, different characteristics were measured:
Chlorophyll and carotenoid Content
Chlorophyll a, b, and total Chlorophyll and carotenoid content measured according to Arnon (1967) method in grape leaves. The extraction steps were carried out with full precision to minimize contact with heat and light. In order to stabilize the pigments, the samples taken from the central part of the mature leaves were immediately ground in liquid nitrogen. The samples were vortexed two times with an interval of one minute and stored for 1.5 hours at a temperature of 4 degrees Celsius in order to dissolve the pigments in cold acetone solvent. After that, they were centrifuged for 15 minutes at a speed of 6000 rpm. The supernatant after centrifugation of the samples was used to measure chlorophyll and total carotenoids. The absorbance of the samples was read at three wavelengths of 470, 645, and 663 nm using a Shimadzu UV (160) spectrophotometer. Using the absorbance of the samples in these three wavelengths and the equations below, the amounts of chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids were calculated in terms of milligrams per gram of tissue sample weight.
Fruit characteristics
 Fruit characteristics such as fruit yield, fruit firmness (with magnets-Taylor device),  sugar and acid content, and content of phenolic compounds were also measured. The fruit tissue firmness was measured using a firmness tester (model FDKA 32, Wagner) and expressed in Newtons per square centimeter. The characteristics of soluble solids (TSS) were expressed using a handheld refractometer (Ataga, Japan) and expressed in terms of Brix, the pH of the extract was measured using a digital pH meter (Sartorius PP-20, Germany). Titratable acidity or TA was done through the titration of the extract using 0.1 normal sodium until pH 1.8-2.8 and its results were reported using the following formula and under the title of tartaric acid percentage.
Total phenol content
 The amount of total phenol was measured by Folin-Ciocalteu method and spectrophotometry. The absorbance of the reaction mixture was measured after 120 minutes of storage in the absence of light at a wavelength of 765 nm using a Shimadzu UV spectrophotometer (160). The amount of total phenol was expressed from the standard curve in terms of mg of gallic acid per 100 grams of extract in three replicates for each sample and standard.
Total flavonoid
 The amount of total flavonoid was measured by aluminum chloride calorimetric method. In this experiment, 50 microliters of methanolic extract from the flesh and fruit skin were combined with 10 microliters of 10% aluminum chloride and 10 microliters of 1 M potassium acetate. Then, 280 microliters of double distilled water were added to the mixture. After the samples were mixed, they were left at room temperature for 40 minutes. The absorbance of the reaction mixture was measured at a wavelength of 415 nm using a Shimadzu UV spectrophotometer (160) in three replicates. A blank containing double distilled water was used for comparison. The amount of total flavonoids was determined based on the standard curve of quercetin and the results were expressed in micrograms of quercetin per gram of fruit skin and flesh weight.
Total anthocyanin
 The amount of total anthocyanin was measured using the difference in pH method. In this method, the absorbance was measured using a spectrophotometer at wavelengths of 520 and 700 nm along with potassium chloride and sodium acetate buffers with different pH values of 1 and 4.5.

 The results of the data analysis of variance showed that the application of salicylic acid resulted in significant differences in the measured characteristics. Therefore, the use of this compound improved the physical and biochemical quality of the treated grape clusters.Comparison of treatments means showed that using 2 mM salicylic acid brought about a significant effect on the amount of chlorophyll (a, b and total) and carotenoids compared to other treatments, especially control. Salicylic acid application caused a reduction in pH and soluble solids, and an increase in firmness, 100-berry weight and yield. The results of the study indicated a significant increase in organic acids and a decrease in reducing sugars in the grape clusters treated with salicylic acid. The highest amount of tartaric acid was found in the treatment with 2 mM salicylic acid, with an average of 0.66, while the control treatment had the lowest amount of tartaric acid, with an average of 0.52. This suggests that the use of salicylic acid led to a reduction in transpiration and consumption of organic acids. In terms of reducing sugars, the highest and lowest amounts were observed in the control treatment (14%/74) and the treatment with 2 mM salicylic acid (13%/60), respectively. Increasing the concentration of salicylic acid up to 2 mM resulted in the enhancement of anthocyanins and phenolic compounds contents, but higher concentrations (more than 2 mM) reduced the amounts of these compounds in treated fruits.

 Pre-harvest use of salicylic acid could have a positive effect on the improvement of qualitative and quantitative characteristics of grape cv. Shahroodi. However, further comprehensive studies are needed to be conducted to recommend this compound to the growers of fruit trees.