# Effect of Seawater Irrigation on the Sugars, Organic Acids, and Volatiles in 'Reliance' Grape
## 2 Materials and Methods  
### Plant Materials and Treatments  
Five-year-old 'Reliance' grapes (_V. labrusca \(\times\) V. vinifera._, Ontario \(\times\) Suffolk Red) grown in a vineyard in Tai'an, Shandong province, China (average annual rainfall was 638.4 mm between 2014 and 2019, and meteorological data for the vineyard in 2018 is available in Table S1), were irrigated with seawater in 2018. The vines were planted in a vertical trellis system and spaced at 2.5 m \(\times\) 1 m in a north-south row orientation. A furrow was made 40 cm away from the main trunk on both sides to ease treating diluted seawater. All treatments were replicated thrice in a randomized block design. Three blocks with similar texture and fertility in each experimental block were selected and randomly assigned S1, S2, and S3. A control group was also included. Fifty vines selected in every two rows formed a replicate. Guard rows were set between treatments to reduce the marginal effect. Irrigation treatment was followed according to our published method with little modification [2]. Briefly, the vines were irrigated with 10% seawater every seven days starting at the pea-size period (S1), stage EL 32 (S2), and pre-veraison period (S3), seawater was irrigated six times for S1, five times for S2, and four times for S3. Seawater was drawn from Penglai city, China. The physical and chemical properties of the undiluted seawater are outlined in Table 1. The initial irrigation time is presented in Table 2. The control group was irrigated with fresh water (EC = 50 us/cm). Each irrigation was based on weather forecasts approximately two days before the rain. The experiment used the method of furrow irrigation; the furrow was 40 cm wide and 100 cm long for each vine, the average irrigation amount per plant was 20 L. Vines were slightly hoed and covered with soil after irrigation to reduce the evaporation of water. Samples were collected at six growth stages: EL 31 (pea-size berries), EL 33 (still hard and green berries), EL 35 (berries begin to color and enlarge), EL 36 (berries with intermediate Brix values), EL 37 (berries not quite rip), and EL 38 (harvest-ripe berries) ([PERSON], 1995) (Table 2). Samples were used to determine berry maturity parameters, sugars, organic acids, volatiles, and gene expression. At each stage, 600 berries of each replicate were manually collected in iceboxes and transported to the laboratory within 30 min. Fifty replicates in each group were used to determine the berry hardness within 2 h after harvesting. The berries were immediately frozen in liquid nitrogen and stored at \(-80\)\({}^{\circ}\)C after weighing to determine sugars, organic acids, volatiles, and RT-qPCR analysis. Soil samples were collected from the soil horizon (0-40 cm) 40 cm away from grapevines at the EL 38 stage for the determination of soluble salt, chloride (Cl\({}^{-}\)), and sulfate (SO4\({}^{2-}\)) content, and the pH level. The analyzed samples were a mixture of soils from twelve grapevines from each replicate. All treatments were carried out in triplicate and results presented as the average of three analyses (\(n=3\)). Then, the determination of berry weight, hardness, total soluble sugar, total soluble solids (TSS), and tittatable acidity (TA) was conducted. A scale of one percent accuracy was used to measure the average berry weight of 100 berries. Texture parameters of the berries (skin hardness, frangibility, firmness, and average flesh hardness) were determined using a Universal Testing Machine Texture Analyser (TA.XT Plus, Stable Micro Systems, Surrey, UK) equipped with a P/2n (d = 2 mm) needle probe and an HDP/90 platform. The penetration speed was 1 mm s-1, the puncture depth was 7 mm, and the minimum perceptive power was 5 g. Fifty fresh berries were randomly selected from each replicate for these analyses. The equatorial center of the berry was selected as the puncture point [21].