Evaluation of the Biostimulant Effect of Sinorhizobium meliloti on Grapevine Under Rational and Deficit Irrigation ^†

Abstract

Agricultural irrigation management is considered more necessary than ever, as climate change directly threatens the growth of important Mediterranean crops in Greece. In this work, the metabolic characteristics of grapevine plants were assessed under rational 100% of available water (AW) and irrigation-deficient (57% of AW) conditions in 9 L pots, with the application of a Sinorhizobium meliloti biostimulant. Leaf area, proline, and total phenolic and chlorophyll content were assessed during the experiment as indicators of abiotic stress. The data of the experiment showed that the use of S. meliloti could act as a biotic stress inhibitor due to the irrigation deficit caused in the grapevine cultivation. This case study complements the literature on grapevine cultivation management practices in the scenario of imposing irrigation regimes due to climate change.

1. Introduction

Grapevine cultivation of Vitis vinifera L. var. “Debina” (Vitales: Vitaceae) is one of the most important crops of Greece, famous for its wine products in the Ioannina region (Epirus, Greece). Grapevine is considered a drought-resilient species, and there are cases where a small amount of water regime could improve the quality of berries [1,2]. However, the rising temperatures and escalating drought rates in the Mediterranean region due to climate change could further lead to irrigation deficits for many crops, as well as for grapevine [3,4,5,6]. High irrigation deficit fractions have a negative impact on grapevine growth and dry weight [5,7,8], noted by the deterioration of the qualitative traits of berries [9]. Crop abiotic stress relief is an urgent need in the face of climate change, and this could be achieved by water management and the application of biostimulants that enhance plant growth, even in harsh conditions [10,11].

Plant growth-promoting rhizobacteria (PGPR) have been widely tested for their beneficial contributions to plant metabolism under abiotic stress conditions, increasing the proline levels and regulating the reactive oxygen species in a wide range of plants [12,13]. Sinorhizobium meliloti (Hyphomicrobiales: Rhizobiaceae) is a non-famous PGPR used in agriculture for its beneficial impact, mostly on legume growth under irrigation deficit conditions [14]. Its application may reduce the utilization of nitrogen fertilizers [15] because it fixes the gaseous nitrogen into usable forms, such as ammonia [16]. S. meliloti has also been referred to as a biostimulant in V. vinifera [17], but its action has been mainly studied in the Fabaceae family [18,19,20]. This research aims to investigate the potential of S. meliloti on the growth and metabolic traits of V. vinifera L. var. “Debina” under normal and deficit irrigation conditions, as a biostimulant. This research aims to provide additional insights into the implementation of S. meliloti on grapevines, as only a limited number of studies have been conducted to explore its biostimulant properties on plant metabolism.

2. Methods

2.1. Experimental Design

Grapevine cuttings of V. vinifera L. var. “Debina” were collected from the Zitsa viticultural zone (Ioannina, Greece) and placed under misting conditions until three leaves were developed at the greenhouse of the Agriculture Department of the University of Ioannina in Kostakioi (Arta, Greece), where the experiment was carried out for 122 days. Then, the young grapevines were planted in 9 L pots, containing a vineyard soil derived from the plain of Arta, as a growth substrate, with the addition of a granular fertilizer (N12-P12-K17). The pots were irrigated with a drip irrigation system, and the irrigation amount and frequency were arranged from a central computer program connected with temperature and moisture sensors, to ensure rational irrigation based on climatic data. Furthermore, to manage optimal irrigation, the evapotranspiration of the plants was calculated in randomly selected plants at 14-day intervals to estimate their water loss. In addition, soil moisture was monitored using a ΔT-SM150 Kit soil moisture meter to provide additional confirmation of irrigation adequacy for each treatment. Based on these data, two different irrigation categories were arranged: the optimal irrigation as 100% of available water (AW) and the deficit irrigation (57% of AW). Achieving a balanced-level water deficit of 57% of AW, on the one hand, stressed grapevine plant metabolism, and, on the other hand, ensured their viability under extreme conditions. The biostimulant formulation Hydromaat, Futureco Bioscience^® containing S. meliloti cepa B2352 (2% w/w), was used in the experiment, and the following treatments were organized in a completely randomized design on the greenhouse benches: Treatment B (100% of AW and S. meliloti application), treatment SB (57% of AW and S. meliloti application), treatment C (100% of AW), and treatment SC (57% of AW) (Figure 1). Each treatment consisted of 27 plants. Leaf sampling for chemical analysis was always performed on the same plants, and each leaf was extracted separately. In each sampling of each treatment, 5 leaves were taken from each plant.

2.2. Determination of Metabolic and Growth Parameters

The total chlorophyll content (TCHL) was monitored in the V. vinifera leaves by the non-destructive method of the SPAD-502 portable device (Minolta Co., Ltd., Osaka, Japan). In order to linearly correlate the SPAD values with the actual chlorophyll concentration, the chemical determination protocol of Uddling et al. 2007 [21] was used. A total of 10 mL of pure acetone was used as an extraction solvent for 0.02 g of fresh grapevine leaf tissue, crushed in a mortar. The mixture was poured into glass tubes, vortexed for 10 sec, and then left overnight in the dark (4 °C). The supernatant solution was measured in a spectrophotometer (Jasco-V630 UV-VIS, Jasco International Co., Ltd., Tokyo, Japan) at 644.8 and 661.6 nm, and the TCHL was calculated by the equations of Lichtenthaler and Buschmann 2001 [22] in µg of fresh leaf (FL) per cm² of leaf area:

Ca (μg/mL) = 11.24 × A_661.6 − 2.04 × A_644.8

Cb (μg/mL) = 20.13 A_644.8 − 4.19 A_661.6

The total phenolic content (TPC) of V. vinifera leaves was determined by the protocol of Katalinic et al. 2013 [23]. A total of 0.1 g of dry leaf (DL) tissue was extracted in a mortar by adding 10 mL of 80% ethanol, and the mixture was centrifuged at 3000× g for 15 min at 12 °C. Then, 250 mL of the supernatant was diluted in a volume of 10 mL diH₂O, and in a new test tube, 1 mL of the diluted extract solution, 4.5 mL of diH₂O and 500 µL of Folin–Ciocalteu 2N reagent were added. After 3 min, 4 mL of dehydrated Na₂CO₃ solution, 7.5% w/v, was placed in the tube. After a vortex of the final mixture, and a 20 min water bath incubation (40 °C in the dark), the absorbance was recorded at 765 nm against the blank. TPC results were reported as mg of Gallic Acid Equivalent (GAE) g⁻¹ of DL.

Proline determination of grapevine leaves was performed by the protocol of Papantzikos et al. 2024 [24]. For 0.1 g of FL tissue, 4 mL of 70% ethanol was used as an extraction solvent. Then, the homogenized mixture was centrifuged at 4000× g (10 min at 4 °C), and 1 mL of the supernatant solution was added to a new test tube with 2 mL of freshly prepared acid–ninhydrin solution. The final mixture was vortexed and incubated in a water bath at 95 °C for 25 min. At the final step, an ice bath was used to cool the reaction mixture until room temperature was achieved. The absorbance was determined at 520 nm in a spectrophotometer (Jasco-V630 UV-VIS, Jasco International Co., Ltd., Tokyo, Japan). A calibration curve was established to quantify proline solutions ranging from 0.025 to 0.8 mM, in the same medium as the one used for the extraction, and the data were reported in µmol of proline g⁻¹ of FL.

At the end of the experiment, leaf area (cm²) was determined using the Image J (v. 1.54 g) protocol [25] for the total number of leaves of all of the plants used in the experiment.

2.3. Statistical Analysis

Statistical analysis was performed by using the software SPSS v.26 (IBM-SPSS Statistics, Armonk, NY, USA) with one-way ANOVA variance analysis. To compare the means, the Bonferroni test was utilized for statistical significance (p ≤ 0.05). The statistically significant differences in each figure are represented by different letters between treatments according to the Bonferroni test.

3. Results and Discussion

3.1. Leaf Area

Under optimal irrigated conditions, PGPR treatment (B = 3449.37 ± 89.44 cm²) showed a larger leaf area, with a statistically significant difference to the control (C = 1707.60 ± 207.36 cm²) (F = 48.83, df = 5, p < 0.001) (Figure 2). An analogous effect has also been observed in treatment B, with a statistically significant difference in C (p < 0.001). Interestingly, the results also show that treatments with the biostimulant under deficit irrigation (SB) promote a larger leaf area than the control group (C) in normal irrigated conditions (p < 0.001). The beneficial effect of PGPR on the grapevine’s leaf biomass has also been showcased in the study by Rolli et al. 2015 [26] in drought stress conditions.

3.2. Proline Content

The proline accumulation on grapevine leaf tissues was higher at irrigation deficit conditions in the S. meliloti treatment (SB) compared to the SC, with a statistically significant difference, both on day 56 (F = 201.400, df = 5, p < 0.001) and day 122 (F = 321.13, df = 5, p < 0.001), as shown in Figure 3. This fact may portray the prevention of grapevine oxidative damage in abiotic conditions, with proline acting as a defense mechanism. The increased proline levels in grapevine leaves as a result of PGPR application have been shown in the work of Theocharis et al. 2012 in cold stress conditions [27]. The deficiently irrigated control KC noted higher proline content compared to the regular irrigated control C (p < 0.05). Proline functions as an osmolyte and ROS scavenger in stress conditions. While the accumulation of proline is usually reported as a plant’s response to abiotic stress, studies suggest that it may be elevated in the application of PGPR, even in non-stress conditions, likely reflecting enhanced assimilation of nitrogen and modulation of signaling pathways that influence proline biosynthesis [28,29].

3.3. Total Phenolic Content

PGPR applications in plants may moderate the oxidative damage by modulating the antioxidant content and reducing ROS levels, enhancing the plant metabolism [30]. In this research, TPC was more enriched in the biostimulant treatments. As shown in Figure 4, the higher total phenolic content on treatment SB was at day 56 (46.99 ± 1.17 mg GAE g⁻¹) compared to SC (33.69 ± 0.28 mg GAE g⁻¹) with a statistically significant difference (F = 68.37, df = 5, p < 0.001). Total phenolic components act as a metabolic defense shield [31], improving antioxidant enzyme production [32], in abiotic conditions such as the irrigation deficit. An analogous outcome of antioxidant enzyme production has been displayed in the study of Bianco & Defez 2009, where S. meliloti was applied in salt-stressed barrel clover plants [33].

3.4. Total Chlorophyll Content

Grapevine TCHL was higher in the case of optimal conditions compared to deficit irrigation. But in the case of deficit irrigation, the TCHL on SB treatment was higher, especially on day 74 (35.16 ± 0.19 µg cm⁻²) compared to SC (20.84 ± 0.45 µg cm⁻²), with a statistically significant difference (F = 430.75, df = 5, p < 0.001) (Figure 5). The biostimulant effect of S. meliloti may be based on its contribution to biological nitrogen fixation [34] and the regulation of cytokinins under abiotic stress [35], acting in the physiological processes such as chlorophyll accumulation [36,37]. Although gas exchange measurements could provide a better perspective on the chlorophyll levels of the grapevine.

4. Conclusions

Oxidative damage from abiotic stresses in plants can lead to major deficiencies in crop yield and agricultural production, while also causing substantial economic damage in rural areas. Deficit irrigation is not a distant concept from our times, in which we are significantly aware of the influence of climate change on the environment. A framework that could contribute to compensating for some crop abiotic stress relief may be the PGPR-based biostimulants, which can be integrated into agricultural water management practices. In this study, the growth and metabolic characteristics of the grapevine with the addition of a PGPR-based biostimulant, both under optimal and irrigation deficit conditions, displayed a statistically significant improvement. The picture of proline and total phenolic compounds was improved, and the increase in chlorophyll content and leaf area may indicate the robustness of the grapevine plants with the application of the S. meliloti biostimulant. Moreover, it is important to mention that more studies need to be carried out on S. meliloti application on grapevine in field conditions, with the intention to investigate the same metabolic parameters in real cultivation conditions.