Effect of vineyard soil variability on chlorophyll fluorescence, yield and quality of table grape as influenced by soil moisture, grown under double cropping system in protected condition

Experimental site, design and double cropping viticulture practices

The study was performed during the 2016–2017 growing season in a 7-year-old commercial vineyard of “Summer Black” (V. vinifera L. × V. labruscana L.), owned by Fujian Jiujiu Tianchen Eco-Agri LTD located in the county of Anxi (Fujian, China) (25°00′N, 117°59′E) usually characterized by humid monsoon with abundant sunshine. The formal approval was obtained before the experiments were conducted in the field. In south China, grape cultivation is mainly dominated by double cropping system, where the vines are pruned two times and grapes are harvested twice within a year. At first, to get crop in the summer season cycle, grapevine was pruned and forced with 2.5–3.0% hydrogen cyanamide in early February when the temperature was above 10 °C. Around late March flowering was initiated, berry formation and veraison were around late-April and mid-May, respectively. Summer crop was harvested in June–July. After that, the vines were again pruned and forced in early-August, which flowered in September, followed by the berry formation and veraison stage during October and November leading to harvest in December–January. Different growth stages during two different seasons are illustrated in Table 1. The vineyard is established on a sloppy land. Vines in the vineyard were maintained under a tunnel covered with plastic polyethylene film to act a shelter on top. Water was being supplied through drip irrigation system with drip emitters at a rate of 4 L m⁻² h⁻¹ prior to bud break and fruit setting stage. No irrigation was supplied after veraison up to harvest. In this study, the vineyard was divided vertically into three sites at different height: the first site was selected at 192 m above sea level (“lower” site), the second site was around 10 m above first site (“middle” site) and the third site was at around 20 m above the first site (“upper” site). The vineyard soil is almost similar in characters and classified as sandy loam. Each site (upper, middle and lower) was considered as one treatment and each site was divided into three blocks with 10 vines from each block, that is, in total 30 plants were selected from each of the upper, middle and lower sites for data collection. Areal temperature and rainfall pattern data for the study year have been illustrated in Fig. 1.

Determination of soil moisture

Soil moisture was determined by Gravimetric methods. The soil samples were collected from 0 to 20 cm and soil from 20 to 40 cm depth with the help of a soil auger at 15 days after veraison. Each sample was a composite of two sub-samples taken from both sides of vine at 0.30 m far from emitters. It is reported that much of the grapevine root system represents the soil volume covers 0.4 m depth (Bassoi et al., 2003), hence, soil collecting depths were chosen to represent the biologically active top soils and the subsoil containing most of the grapevine roots. Soils were collected from the field with airtight aluminum containers and taken to the laboratory. The soil samples were weighed and then dried in an oven at 65 °C until all the moisture was driven off. After being removing from the oven, they were cooled down slowly to room temperature and weighed again. The difference between initial and final weight is the amount of moisture in the soil. The moisture content in the soil was calculated by the following formula: moisture content (%) = ((initial weight−oven dry weight)/oven-dry weight)) × 100.

Measurement of leaf OJIP transients

The transients were measured on fully expanded grapevine leaves by a portable plant efficiency analyzer, Handy-Pea (The Hansatech Instruments Ltd, Norfolk, UK) at 15 days after veraison. Randomly nine leaves from each replicate were selected for measurements of chlorophyll a fluorescence. Before the measurements, all the leaves were dark adapted for 3 h at room temperature. The OJIP transient was induced by the red light with maximum intensity of >3,000 μmol m⁻² s⁻¹ at leaf surface provided by an array of three light-emitting diodes at peak wavelength 650 nm, spectral-line half width 22 nm in a time scan from 20 μs to 1 s after the onset of irradiation with a data acquisition rate of 100 readings with a record length of 1 ms–300 s. Measured OJIP transients were analyzed according to the JIP-test equations (Smit et al., 2009; Strasser, Tsimilli-Michael & Srivastava, 2004). From the original measurements the following values were used: the minimal fluorescence intensity at 20 μs when all PSII reaction centers (RCs) are open (the O step, considered as F_o); the intensity at 300 μs which is used for calculation of the initial slope (M_o), defined as the net ratio of RCs closure; the intensity at 2 ms (the J step, F_J); the intensity at 30 ms (the I step, F_I), and the maximal fluorescence intensity when all PSII RCs are closed (the P step considered as F_m).

Berry sampling and fruit analysis

To determine the optimum harvesting time, grapes from different experimental plot were periodically tested with a digital hand refractometer and harvesting time was considered when the TSS content of berries attained approximately 16 °Brix. Physicochemical analysis of the grapes was accomplished by collecting 10 clusters from each replication of a treatment, and 12 berries were collected from each cluster (four from each upper, middle and lower portion) in total 120 berries per replication. Vine yield per tree (kg) was expressed as the multiplication of the total number of cluster per tree and the total weight of the cluster (g).

The collected 120 samples were divided into two parts. The first 60 berries were again divided into three biological replicates of 20 berries each, which were then used to measure the physical characters of the berries by recording diameter of berries, berry length and individual berry weight and after that the berries were crushed into juice for measuring the TSS, pH and Titratable acidity (TA). For harvesting berry, increased concentration of total soluble solid is a reliable indicator (Coombe & McCarthy, 2010). TSS was measured using a digital refractometer (DR301-95, Krüss Optronic, Hamburg,Germany) and the result was expressed in °Brix. pH was measured by pH meter. TA was measured by the potentiometric method, where the prepared juice was being titrated with 0.1 N NaOH solutions until the pH value reached at 8.2 on the pH meter. The results were calculated in percentage of tartaric acid. Skin from the rest 60 berries were manually peeled and frozen immediately in liquid N₂ and kept at −80 °C until further analysis.

Extraction and estimation of total phenolics, flavonoid and antioxidant activity

The berry skin samples at first were ground in liquid N₂ with the help of a mortar and pestle, from that powdery sample 0.5 g was homogenized in 10 mL of 80% chilled ethanol. The extracts were centrifuged at 12,000 rpm at 4 °C for 20 min. Then the supernatant was filtered on a filter paper, collected and used for the estimation of total phenolics, flavonoid and total antioxidant capacity.

Total phenolics content in the sample was determined using the Folin–Ciocalteu colorimetric method according to Huang et al. (2009) with minor modification. Briefly, 0.5 mL of aliquots and standard Gallic acid of different concentrations (20, 40, 60, 80, 100 μg/mL) was added into a 10 mL tube in which 4 mL of distilled water and 0.5 mL of Folin–Ciocalteu’s reagent (FCR; Sigma Chemical, St. Louis, MO, USA) was added previously. The tube was shaken. About 5 min later, one mL of 7% sodium carbonate (Na₂CO₃) was added to the mixture. The mixture was kept in dark condition at room temperature (25 °C) for 120 min. After that, an intense blue color was developed. The absorbance was then measured against a blank at 765 nm using UV–VIS spectrophotometer (TU-1810; Beijing Beifen-Ruili Analytical Instrument (Group) Co., Ltd., China). The measurement was performed in triplicates. The data for total phenolics contents were expressed milligram GAE 100 g⁻¹ berry skin fresh weight (FW) (Gallic acid equivalent).

Total flavonoid content was measured by a colorimetric assay according to Huang et al. (2009). A 0.5 mL of aliquots and 0.5 mL standard quercetin solution (100, 200, 400, 600, 800, 1,000 μg/mL) was added into a 10 mL tube containing four mL of water and 0.3 mL of 5% NaNO₂ was added into it, then the mixture was allowed to stand for 5 min at room temperature. After that, 0.3 mL of 10% AlCl₃ was added to the mixture. After 5 min, two mL of 1M NaOH was added. Distilled water was added to make up the total volume up to 10 mL and mixed well. Orange yellowish color was developed. The absorbance was measured spectrophotometrically at 510 nm against a blank. All the procedures were performed in triplicate. The calibration curve was made using standard quercetin. The result of total flavonoid was expressed as milligrams quercetin equivalents (QEs) per 100 g berry skin FW (mg QE/100 g of FW).

The total antioxidant capacity was measured by the Ferric Reducing Antioxidant Power (FRAP) assay according to Benzie & Strain (1996). FRAP reagent includes acetate buffer (30 mM, pH 3.6), TPTZ (10 mM in HCl 40 mM) and FeCl₃·6H₂O (20 mM). The FRAP reagent solution was prepared by adding acetate buffer, FeCl₃·6H₂O and TPTZ using 10:1:1 (v/v/v) ratio. We mixed 30 μL of Skin extracts with 2,900 μL of the FRAP solution and 70 μL Mili-Q water then allowed to stand for 30 min in the dark condition at room temperature (25 °C). After that, the absorbance of the colored solution was determined at 593 nm against a blank. The standard curve was prepared by using one mM Trolox and result was expressed as millimolar TE/g berry skin FW.

Extraction and estimation of total and individual anthocyanin

Anthocyanin extractions were performed with a protocol similar to that of Liang et al. (2011) with slight modifications. Briefly, the frozen berry skin samples were ground in liquid nitrogen with a mortar and pestle. From there, a 0.5 g powdery sample was homogenized in 10 mL of 1% HCl-methanol solution and further mixed. It was vortex for 1 min and then kept in a shaker at 250 rpm for 2 h at 4 °C in the dark. Then, centrifuged at 4 °C for 20 min at 12,000 rpm. Then the supernatant was collected and kept at −40 °C until the time of analysis. This extract was used for the estimation of total and individual anthocyanin.

The total anthocyanin content was measured by pH differential method using a protocol similar to that of Giusti & Wrolstad (2001). The total required amount of skin extract was added into two tubes, and these extracts were diluted 10× with KCl (0.025M) buffer at pH 1.0 and CH₃CO₂Na·3H₂O (0.4M) buffer at pH 4.5. The absorbance was measured at 520 and 700 nm against distilled water as a blank, respectively, and calculated using the equation A = (A₅₂₀–A₇₀₀) pH1.0 − (A₅₂₀–A₇₀₀) pH 4.5. Each anthocyanin extract was diluted to get the sample in the buffer at pH 1.0 with an absorbance <1. The resultant anthocyanin content was expressed as milligrams of malvidin-3-monoglucoside equivalence per killogram of berry FW.

The chromatographic analysis of individual anthocyanin, delphinidin-3-O-glucoside (DP-3-G), cyanidin-3-O-glucoside (CG-3-G), petunidin-3-O-glucoside (PT-3-G), peonidin-3-O-glucoside (PN-3-G) and malvidin-3-O-glucoside (MV-3-G) were measured by an High-performance liquid chromatography (HPLC) system (LC-100; Wufeng series, Shanghai, China) equipped with LC-P100 pump and operated by LC-WS100 software, according to Simoes et al. (2009) with modification. Skin extracts were filtrated by a membrane filter (0.45 μm, MillexHV; Millipore, Bedford, MA, USA) and a volume of 20 μL of solution was injected directly into the HPLC system. The samples were analyzed using a BRISA LC2 C18 column (5 μm particle size, 250 × 4.6 mm I.D.). The gradients profile consisted of two eluents: (A) acetonitrile/water/formic acid at a ratio of 50:45:5 (v/v/v) and (B) water/formic acid at a ratio of 95:5 (v/v). The gradients used were as follows: 0–3% A at 1 min, 3–15% A at 11 min, 15–25% A at 12 min, 25–30% A at 4 min, 30% A at 7 min, and constant for 5 min before the mobile phase returned to the initial conditions. The flow rate was 1.0 mL/min at 30 °C and detection were at 520 nm. Peaks were identified according to each peak of UV-Vis spectra corresponding to the spectra of standard and comparing with their retention times. Anthocyanin content was quantified using peak areas of external standards. Standard curves were constructed using peak area vs. concentration (linear calibration curves). The resultant individual anthocyanin was expressed as milligram per killogram berry FW.

Statistical analysis

Statistical analyses were done by SPSS v19.0 (SPSS Inc., Chicago, IL, USA). Data were subjected to one-way analysis of variance. When there was a significant difference (p < 0.05), the mean separation was performed using Tukey’s significance test set at p < 0.05. Correlations between variables were performed by Pearson’s correlation coefficients in SPSS.

Soil moisture at different sites of soil during winter and summer seasons

Significant differences in the soil moisture at three different sites of soil were observed during both winter and summer seasons (Table 2). In case of top soil (0–20 cm depth), the highest soil moisture (30.78% and 41.62%) in winter and summer seasons were recorded from the lower site which was significantly higher than the other two sites. The lowest soil moisture was recorded from the upper site (22.33% and 33.02%) in both growing seasons. Available soil moisture recorded from 20 to 40 cm depth also followed the similar trends like top soil, where the lower site had the highest soil moisture (42.32% and 55.81%) which was significantly higher than the middle (34.27% and 49.33%) and upper site (27.89% and 45.10%). Significant difference in soil moisture also found between winter and summer cropping cycle. Soil moisture was higher in the summer season compared to the winter season as expected.

Effects of different sites of soil on chlorophyll a fluorescence

OJIP transients from leaves of grapevine from three different sites shown in Fig. 2. All OJIP transients of leaves from three sites resulted in a typical polyphasic rise with basic steps of O-J-I-P in both winter and summer seasons. In the winter season, the fluorescence induction curve from the upper and middle site showed a considerable increase in J, I and P phases where there was low moisture compared to vines from lower sites (Fig. 2) indicating water stress due to the lower moisture content in the soil from upper and middle sites. A similar curve was also observed in the summer season although the overall fluorescence intensity value were lower than the winter season curve.

Chlorophyll a fluorescence provides several parameters about photosynthetic fluxes which are considered as a reliable indicator of plant stress monitoring. Table 3 and Fig. 2 show different technical parameters obtained from chlorophyll a fluorescent transient under three different soil moisture at the lower, middle and upper sites during winter and summer seasons. In this study, a significant gradual increase in minimal fluorescence (F_o) was observed from lower site to upper site (592.94, 728.55 and 816.89, respectively) in the winter cropping cycle. Similar trend was also recorded for the summer season (368.18, 457.33 and 576.55). Significant differences were observed in case of F_v/F_m value, in the winter season which were 0.80, 0.75 and 0.71 for the lower, middle and upper sites, respectively. Similarly, in the summer season, F_v/F_m value gradually decreased from lower sites to upper sites (0.83, 0.81 and 0.75, respectively). When comparing the two seasons, there was significant difference between summer and winter in all the JIP parameters like F_o, F_m, F_v and F_v/F_m.

Effects of different sites of soil on yield and berry physicochemical characteristics

Analysis of the data indicated that during the winter season, grapevine from the middle site provided the highest value regarding cluster per tree, cluster weight, thereby yielding per vine (8.87 kg), which was statistically higher than upper and lower sites. The lowest yield per vine (5.45 kg) was recorded from the lower site (Table 4). Data from the summer season also followed the similar trend with higher values in terms of the above same three parameters (Table 4). Significant difference was observed between winter and summer seasons regarding cluster weight and yield per vine but not the cluster per tree. Moreover, during both growing seasons, there was no significant difference among the sites of soil regarding berry width, length and individual berry weight although data from the summer season was significantly higher than winter.

As shown in Table 4, the highest TSS (°Brix) value during both growing seasons was obtained from the upper site which was statistically similar to the middle site. While in both seasons, the lowest TSS value was recorded from the lower site. Overall, TSS value was higher in the winter season compared to the summer season grape. There was no significant difference in pH value in different sites; however, pH was higher in winter grapes.

Effects of different sites of soil on phenolics, flavonoid and antioxidant properties

Table 5 shows the differences those were observed in determination of total phenolics, flavonoid and antioxidant properties of table grape as influenced by the soil levels. Comparatively, significantly higher contents of total phenolics, flavonoid and antioxidant properties were recorded from the middle site of the vineyard compared to upper and lower sites during both the seasons. Comparing both growing seasons, significantly higher values were recorded from winter for all three parameters (Table 5).

Effects of different sites of soil on individual and total anthocyanin

The chromatograms of table grape obtained from skin extract eluted in the following order: DP-3-G, CG-3-G, PT-3-G, PN-3-G and MV-3-G (Fig. 3). The middle site provided the highest values regarding DP-3-G, CG-3-G, PT-3-G, PN-3-G and MV-3-G contents in our study during both winter and summer seasons (Table 6). Among the five studied individual anthocyanin MV-3-G was obtained in a higher amount than the rest of the anthocyanin in both the seasons. Total anthocyanin content varied between 791.20–893.89 mg kg⁻¹ for this variety in winter and 587.6–771.71 mg kg⁻¹ in the summer season. The middle site provided the highest total anthocyanin content during both growing seasons (Table 6). However, grapes from the winter season had a higher total and individual anthocyanin compared to the summer season.

Correlation between different parameters

Pearson’s correlation coefficient was computed to identify the relationship between different variables. As shown in Tables 7 and 8, significant positive correlations were found between TSS and TSS/acidity (r = 0.959, p = 0.01; r = 796, p = 0.05, respectively), also TSS and pH (r = 0.783, p = 0.05; r = 0.915, p = 0.01, respectively) during winter and summer seasons; while significant negative correlation was observed between TSS and acidity (r = −0.821, p = 0.01; r = −0.626,) in both growing seasons. Yield per vine was positively correlated with antioxidant and total anthocyanin in the winter season, and phenolics, flavonoid, antioxidant and total anthocyanin in the summer season. A positive correlation was found between soil moisture and acidity, individual fruit weight, berry width in both the seasons. However, negative correlation was observed between soil moisture and TSS, TSS/acidity, phenolics and flavanoid, antioxidant and total anthocyanin during winter and summer cropping cycle.