An innovative microbial derivative has been developed to protect vines from the negative effects of water stress. The application of this product has been the subject of an in-depth study showing that the treatment enables faster and more effective osmotic regulation, limiting production and quality losses in increasingly frequent water stress situations.
Vineyard water levels are a key aspect to be managed during the season to ensure a good vegetative-production balance and optimal production results. Against the backdrop of global warming, dry winters are rising, with a poor accumulation of soil water reserves. Spring and summer are characterised by frequent extreme events or intense rain events that bring no benefits to water balance in the vineyard.
It is estimated that 99% of the water used by vines is transpired to allow photochemical and thermoregulation tissue processes. “Constitutional water”, namely water stored in the plant and is essential for maintaining the right cell turgor, is necessary for growth, stomata movement and root system functioning. With the same amount of rainfall, high temperatures increase vineyard evapotranspiration, and the incidence of water stress rises even in the wettest districts. The amount of water required by the vine varies considerably during the different phenological phases (Figure 1). This means that the consequences of water shortage differ considerably according to the development stage. The most delicate moment for water shortage is between fruit set and veraison, during the grape’s vegetative growth phase. Water stress occurring when cell division and development processes are active impacts final yield much more than stresses of the same intensity occurring at veraison or post-veraison. Furthermore, loss of product caused by premature stress is irreversible, even if the amount of water replenished during the rest of the season increases. If water deficit is moderate and controlled in the final phase of ripening, it could also have positive effects on quality, especially for red grapes, whereas the first reaction of the plant to a substantial deficit is a slowdown in photosynthetic activity.

FIGURE 1. Distribution of water needs during the vine’s vegetative cycle, with the total requirement being 100. Water stress during the fruit set-veraison period severely compromises seasonal production. Hence treatments with LalVigne ProHydro™ are recommended during this phase.
When stress lasts several days, the stomata close and photosynthesis ceases, increasing leaf temperature as there is no transpiration thermoregulation. If these conditions continue for long periods, cell turgor is lost, with the accumulation of reactive oxidising species (mainly hydrogen peroxide) in the tissues, which is the cause of classic leaf yellowing and necrosis that can cause permanent damage to the photosynthesis system.
How to fight water stress with a new foliar treatment
A dual synergistic approach is required to manage extended or unpredictable water stress. On the one hand, planning of long-term strategies to be implemented during the planting phase; on the other, identifying flexible and timely techniques applicable during the season. The use of new natural foliar applications based on the action of specific microbial derivatives, such as inactivated yeasts and bacterial extracts, is a state-of-the-art cultivation strategy.

PHOTO 1. Impact of water stress; even if water becomes available later, the plant is no longer able to recover pre-stress photosynthesis rates.
Extensive R&D conducted by Lallemand (patent pending) has led to the development of several specific formulations that can optimise vineyard performance in terms of improving tolerance to abiotic stresses (Giordano et al., 2021) and phenolic and aromatic maturation (Pastore et al., 2020).
Specifically, LalVigne ProHydro™, based on the selected wine yeast derivative (Saccharomyces cerevisiae) and L-proline of natural origin (Corynebacterium glutamicum), was developed to improve vine response to water stress. Its preventive use achieves a dual action, ensuring high photosynthetic activity and avoiding excessive slowdown of basic plant metabolism, while preparing the plant to cope with water stress consequences.
How the new foliar treatment works
The water inside the plant moves, because of the negative water potential gradient, shifting from the soil to the atmosphere through roots, stems, shoots and leaves.
In the event of water shortage, resources may be insufficient to ensure transpiration, photochemical reaction and cell turgor, three aspects that will be inhibited to varying degrees and dynamics. In these situations, the vine puts strategies in place to adapt to limiting conditions, such as increasing leaf angle and folding leaf margin, to escape exposure to direct light, or various chemical-physical rearrangements to regulate the water and biochemical balance of its tissues. These include the accumulation of proline, an osmotically active amino acid which, in the case of stress, is synthesised in chloroplasts and accumulated in the cytoplasm at concentrations equal to 10 times those of the pre-stress situation. Proline fosters osmotic adjustments necessary for maintaining cell turgor and draws water from the organelles with higher water potential. In addition, day-night proline turnover encourages reducing power (NADP+) accumulation and prevents the formation of reactive oxygen species, toxic molecules that cause yellowing and necrosis of stressed leaves.

FIGURE 2. Percentage of endogenous proline accumulation in response to treatment with LalVigne ProHydro™ at 1 kg/ha. A single treatment induced a constant accumulation of over 170% for the next 14 days, equal to a constant increase of 1.26 micromoles per day per g of weight.
In 2020, potted Pinot noir vines were treated with LalVigne ProHydro™ and compared with an untreated control sample. From one day before treatment and at regular intervals after treatment, leaves were sampled, on which proline concentration was determined. After sampling, the leaves were cleaned with distilled water to remove any exogenous proline deposits so that only endogenous proline was measured (i.e., contained in leaf tissues). One hour after treatment, no significant differences between the treatments were observed. Starting from three days after treatment, the percentage variation of proline compared to pre-treatment levels gradually and steadily increased from +68% to +170% at 14 days from treatment, while untreated plants in the same period recorded a proline increase of 50%. This means that preventive treatment with LalVigne ProHydro™ prepares the plant for water stress and osmotic imbalances, with a priming effect that leads to earlier, more abundant proline biosynthesis.
In addition to its osmolytic function, proline acts as a free radical scavenger, preventing permanent tissue damage caused by oxidising chemical species the vine produces when under stress. From a practical point of view, this translates into better use of the water resource for photochemical processes and thermoregulation, favouring preservation of low foliage temperature and more effective prevention of excess energy that causes yellowing and foliar abscission.
Experimental trial set up
Perugia University, in collaboration with Università Cattolica del Sacro Cuore (Piacenza), evaluated the use of LalVigne ProHydro™ on the physiological and production functions of seven-year-old vines (cv. Sangiovese, clone VCR30) grown in pots and artificially subjected to water stress conditions in a period of high temperatures.


PHOTO 2. Potted vines subjected to water stress. Left, untreated vines with evident chlorosis caused by water stress; right, vines treated when ripe with LalVigne ProHydro™, where no damage from water stress is evident.
Three foliar treatments of LalVigne ProHydro™ were applied at the equivalent dose of 1 kg/ha at these phenological stages: fruit set (19 June), pea-size (3 July), and cluster closure (17 July). Control vines were treated with water only at the same time point.
All the plants were kept under full irrigation conditions (90% of pot capacity) for up to three days after the last treatment (20 July), when half of the vines from each sample were subjected to water stress, keeping maximum water capacity at 40%. On 8 August (a few days before veraison), 90% of maximum water availability was restored for all plants until the end of the season.
Photosynthesis (Pn), stomatal conductance (gs), water use efficiency (WUE), photochemical efficiency of PSII (Fv/Fm), and chlorophyll content (SPAD units), as well as quantitative and compositional parameters of the grapes at harvest (12 September), were recorded.

FIGURE 3. Physiological parameters recorded in plants treated and not treated with LalVigne ProHydro™, indicated by the arrows at a dose of 1 kg/ha: plants always irrigated (A, C) and subjected to a period of pre-veraison water stress (B, D). A and B: photosynthesis and maximum air temperature; C and D: water use efficiency (WUE) calculated as the ratio between photosynthesis (Pn) and stomatal conductance (gs). *: indicates significant differences between control and treatment.
Results
The data that emerged show how treatment with LalVigne ProHydro™ was able to limit the effects of water and heat stress during the year. From a climatic perspective, the study was conducted in a period with maximum temperatures (T max) above 35°C for over 22 days (Figure 3A-B). It can be seen that regardless of the water regime implemented, all the plants in the trial were subjected to significant thermal stress during the season. The physiological data and, in particular, photosynthesis data show how, in response to the first and second treatment with LalVigne ProHydro™, there was increased photosynthesis compared to the untreated control. These differences decreased when lower temperatures were recorded on 15 July (Figure 3A-B). In the samples not subjected to water stress, after the third treatment, following seven consecutive days with T max above 35°C, treated vines showed better photosynthesis than control vines (Figure 3A). In the same period, with vines subjected to water stress, the treated plants showed a higher level of photosynthesis than the control, respectively +48% on 25 July (five days from the beginning of water stress) and +21% on 1 August (12 days of stress) (Figure 3B). When full water volumes were restored, the treated plants responded promptly with a fast and consistent recovery of Pn (+56%) and WUE (+40%), which returned to pre-stress levels, while the untreated plants never recovered full photosynthetic rates (Figure 3B-D).
This suggests that during the stress period, the treated plants did not suffer permanent or irreversible damage to the photosynthesis system, as shown by the photochemical efficiency data evaluated through the Fv/Fm fluorescence ratio (Figure 4A). This parameter has a threshold value of 0.65, below which there is an irreversible loss of efficiency of chloroplast photosystem II, evident as leaf yellowing, chlorosis and necrosis (Photo 2), the result of hydrogen peroxide and other phytotoxic molecules accumulation under severe stress conditions. The plants treated with LalVigne ProHydro™ maintained a higher photochemical efficiency than the control, staying above the threshold value of 0.65 for the entire trial. Conversely, the control vines fell below this value, triggering chronic photoinhibition processes. No photoinhibition phenomena occurred in the irrigated plants, either in the treated or control plants (data not shown).
In addition to rapidly recovering photosynthetic efficiency after the period of stress, the treated vines maintained their foliar system in activity longer, ensuring good photosynthesis levels until harvest. This observation is also confirmed by the higher content of chlorophyll found in the treated vines both during the hottest days in the irrigated trials (data not reported) and in those subjected to water stress (Figure 4B).

FIGURE 4. Photochemical efficiency of the photosynthetic system (A) and chlorophyll content in SPAD units (B) of LalVigne ProHydro™ treated and control plants at times indicated by the arrows at a dose of 1 kg/ha in plants subjected to a period of pre-veraison water stress. The photochemical efficiency is calculated as the ratio between Fv fluorescence (difference between the minimum and maximum fluorescence) and Fm (maximum fluorescence); values below 0.65 indicate that the photosynthesis system suffered permanent damage. *: indicates significant differences between control and treated plants.

The better physiological performance of the treated vines allowed a better allocation of dry matter and reduction of grape dehydration phenomena, as shown by the average weight of the berries and yield per vine, higher than the control, both in the irrigated and in the stressed vines (Table 1). At the same time, treatment supported sugar accumulation and concentration of polyphenols in plants subject to water stress (Table 2).

Conclusions
LalVigne ProHydro™ is a new microbial derivative capable of ensuring greater photosynthesis and fostering a faster plant recovery in case of water stress. The treatment stimulates the natural biosynthesis of endogenous proline in the leaves, which allows a higher level of cell turgor and avoids the biosynthesis of phytotoxic molecules, such as hydrogen peroxide and other reactive oxygen species.
In this study, carried out in a semi-controlled environment in potted Sangiovese vines, it was shown that foliar treatments carried out between fruit set and veraison with LalVigne ProHydro™ can help to avoid production losses linked to water stress and, at the same time, maintain the accumulation of sugars and phenolic compounds. These effects are linked to the enhanced physiological performance of the treated plants when stressful conditions arise. The treated plants do not suffer permanent damage to the photosynthetic system, allowing complete functional integrity of the foliage to be retained, with a greater allocation of photosynthates until harvest. These data confirm that the maximum effectiveness of LalVigne ProHydro™ is obtained with preventive treatments undertaken in the period in which water stress can severely compromise yield (fruit set-veraison period).
Acknowledgement
Our thanks to Professor Alberto Palliotti of Perugia University for the important scientific contribution made to this work.
* This article was first published in Italy in L’Enologo – Number 4, April 2022.
– For more information, please contact Piet Loubser at ploubser@lallemand.com.
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