IT201900019739A1 - Biostimulant composition and its use in agriculture - Google Patents

Description

Description of the industrial invention entitled: "Biostimulant composition and its use in agriculture"

DESCRIPTION

The present invention relates to a biostimulant composition obtainable from natural sources, effective in regulating plant development, in particular in the flowering transition phase, and in increasing the efficiency of water use (WUE: Water Use Efficiency).

In the field of agriculture, it is of fundamental importance to promote the growth, health and productivity of plants. Traditionally, this purpose has been achieved through the use of fertilizers and pesticides, and by introducing physical changes to the soil. However, the use of agrochemicals has long-term environmental consequences such as resource depletion, environmental damage and effects on public health. In order to limit the use of dangerous strategies, in an attempt to improve crop productivity by promoting plant growth and their resistance to stress, numerous efforts have been made to develop and implement ecologically cautious approaches based on the use of biostimulant products. Biostimulants are defined as any substance or microorganism that is applied to plants with the aim of improving their nutritional efficiency, resistance to abiotic stress and / or quality traits of the crop, regardless of the nutrient content. By extension, biostimulants therefore also include a large variety of commercial products containing mixtures of these substances and / or microorganisms (Du Jardin P, 2015. "Plant biostimulants: definition, concept, main categories and regulation"; Sci Hort 196: 3- 14). The role that we would like to attribute to biostimulants in modern agriculture involves the support or even in some cases the replacement of conventional synthetic products, which today are widely used in the management of most crops. Several biostimulants already on the market are focused on solving or mitigating a single specific problem affecting one or more crops. Others, on the other hand, are described as able to guarantee more beneficial effects to the plant, leading to a general improvement in yield in terms of quantity and / or quality.

Biostimulants can be useful in preventing damage from environmental stress in plants, such as extreme temperatures, lack of water, excess salts in the soil or irrigation water, nutrient deficiency. Furthermore, one of the most important and fundamental aspects on which a biostimulating product can affect is the regulation of the reproduction of a plant. This complex physiological process can be divided into two fundamental stages of development: the acquisition of the competence to bloom (passage from the juvenile phase of development to the adult one) and the conversion of the apical meristem from vegetative to floral, with the production of flower buds instead of leaves.

Several scientific publications have described how these important steps are determined by a complicated molecular genetic mechanism that responds to endogenous signals, such as hormones, sugars, other signal molecules such as small RNAs, as well as environmental stimuli, such as photoperiod, temperature, light intensity and nutrient availability. (Bäurle I, Dean C, 2006. "The timing of developmental transitions in plants", Cell 125: 655–664; Poethig RS, 2003. "Phase change and the regulation of developmental timing in plants", Science 301: 334–336 ). Obviously, the management of the reproductive phase can have crucial repercussions on the yield of crops whose marketable product is the fruit.

The activity of genetic improvers can also benefit from this, because these often resort to crosses between parental genotypes to obtain varieties of particular commercial interest, and therefore have an interest in stimulating the production of flowers and then of fruits resulting from crosses. In particular, the anticipation of the floral transition is a trait increasingly pursued in modern agriculture. In fact, this characteristic can favor the yield, quality and health of the plants, depending, of course, on the species and the conditions in which the plant is bred. A plant that blooms earlier must compete in the ecosystem with fewer flowers to receive visits from pollinating insects, thus increasing the probability of fruit development from them. Furthermore, early flowering, at certain latitudes and for certain species, guarantees the plant to intercept the greatest possible amount of solar radiation, thus obtaining an optimal harvest in terms of production. Early flowering also allows crops to have more time for seed development and maturation. Finally, the health situation of the crop is also relevant in this sense, because the anticipation of flowering can prevent some crops from being attacked by specific pathogens and / or insects. A classic example concerns corn, which in our latitudes is particularly subject to the attack of a moth called Ostrinia nubilalis. Its larvae attack the small fruits (caryopses) on the ear, digging inside small tunnels in which a microclimate is subsequently created, suitable for colonization by toxigenic fungi, in particular belonging to the genus Fusarium.

One of the measures used today to partially prevent this situation consists in the early sowing of corn hybrids, in order to anticipate the second generation of O. nubilalis by decreasing production losses and the load of toxins in the grain (Alma A, et al, 2005. "Relationships between Ostrinia nubilalis (Lepidoptera: Crambidae) feeding activity, crop technique and mycotoxin contamination of corn kernel in northwestern Italy", Int J Pest Manage 51: 165-173; Long N, et al, 2017. "Maize yield and planting date relationship: a synthesis-analysis for US high-yielding contestwinner and field research data ", Front Plant Sci https://doi.org/10.3389/fpls.2017.02106).

However, the early sowing procedure, exposing the crop early in the season, is accompanied by the risk of not optimal conditions for the development of the plant. Therefore, an early flowering induced on plants sown at standard times represents a highly desirable alternative approach. Similarly, on arboreal plants such as cherry, apricot, peach and olive, the early and uniform flowering results in more uniform productions and higher yields, as well as in the possibility of placing first fruits on the fruit market, thus increasing prices and revenues. For example, the importance of flowering at the maximum seasonal solar radiation available leads the olive tree to maximize its production. Olive fruits grown in well-lit conditions are larger than those grown with low light intensity, a higher percentage of oil and a lower water content (Caruso G, et al, 2017. "Irrigation and fruit canopy position modify oil quality of olive trees (cv. Frantoio) ”, J Sci Food Agric 97: 3530–3539). Furthermore, oils obtained from olives grown in well-lit conditions show a higher content of polyphenols and better organoleptic characteristics than those extracted from less illuminated fruits.

Another primary goal of modern agriculture is to achieve greater water use efficiency (WUE) by plants. A higher WUE means that for the same amount of water used, the plant is able to produce more biomass; in particular, the so-called “agronomic WUE” focuses on the production of biomass of commercial interest (for example, the fruits in tomato cultivation). This objective is very important from an agronomic and commercial point of view, especially for areas at risk in the context of global warming and climate change such as semi-arid regions with a Mediterranean climate including the countries of southern Europe, North Africa and Western Asia bordering the Mediterranean basin itself, but also Western Australia and parts of South Africa and Chile.

As will be illustrated in more detail in the experimental part that follows, the present inventors have observed that the treatment of crops with a mixture derived from the exudate of the root structure of plants subjected to aeroponic cultivation in the presence of particular nutrients, influences the transition from the vegetative phase. to the reproductive one by exerting a biostimulating activity on the development of the flower buds, and is surprisingly able to induce an early flowering in the treated plants. The experiments conducted by the present inventors have also shown that the mixture derived from root exudate advantageously exerts a specific action on the ability of the treated plants to use the available water more efficiently, both in irrigation conditions and in water and nutritional stress. .

In order to identify the components of the mixture derived from root exudate that are most likely to contribute in concert to the beneficial effects described above, the inventors have subjected the radical exudate to chemical analysis using High Performance Liquid Chromatography , HPLC) followed by mass analysis (HPLC ‐ MS) and ion chromatography. The results of the study revealed the presence of different components, among which the organic oxalic and succinic acids and at least one plant hormone of the strigolactone group are essential.

Therefore, the subject of the invention is a biostimulating composition as defined in the attached claim 1.

The invention also relates to a process for producing a biostimulating composition as defined above, a biostimulating composition obtainable by means of said process, the use of the biostimulating composition as defined above to anticipate the flowering transition of a plant and / or to improve efficiency. use of water by a plant, all as defined in the attached claims, which form an integral part of the present description.

In the composition of the present invention a synergistic action takes place between the oxalic and succinic organic acids and the at least one hormone of the strigolactones group which results in the achievement of a specific bioactivity which allows to regulate in a controlled manner the development of the treated plants and their water balance, thus affecting fruit ripening and cumulative yield during the growing season, leading to a significant increase. This therefore allows the biostimulant composition according to the invention to be used effectively in agronomic practices aimed at improving the development and crop yield of plants while allowing correct management of the soil and the environment.

Strigolactones are a class of molecules produced mainly but not exclusively by plant roots, and whose chemical structure is characterized by a tricyclic lactone (rings A, B and C) connected through an enol-ethereal bridge to a butyrolactone (ring D) (Figure 1) (Prandi C, McErlean CSP, 2019, “The Chemistry of Strigolactones. In: Koltai H, Prandi C (eds) Strigolactones - Biology and Applications. Springer, Cham). For these molecules, only an exogenous function was initially identified, i.e. action in the rhizosphere where strigolactones are released in minimal quantities from the roots. In their exogenous function, strigolactones play different roles, ranging from stimulation of beneficial microorganisms such as arbuscular mycorrhizal fungi to induction to germination of parasitic plant seeds (Rochange S, et al, 2019. "The Role of Strigolactones in Plant– Microbe Interactions "; in: Koltai H, Prandi C (eds) Strigolactones - Biology and Applications. Springer, Cham; Vurro M, et al, 2019." Strigolactones and Parasitic Plants ", in: Koltai H., Prandi C. (eds ) Strigolactones - Biology and Applications. Springer, Cham).

Subsequent studies have also highlighted important endogenous functions of strigolactones in the plants where they are produced, revealing their nature as plant hormones, or phytohormones. In this capacity, strigolactones modulate various development processes in the plant, including the morphology of the stem and, more limitedly, of the root, leaf senescence, and the response to environmental stress. In particular, both the lack of mineral nutrients, especially phosphorus, and the lack of water cause an increase in the synthesis of strigolactones at the root and stem level, respectively (Cardinale F, et al, 2018. "Strigolactones: mediators of osmotic stress responses with a potential for agrochemical manipulation of crop resilience ". J Exp Bot 69: 2291-2303; Rameau C, et al, 2019." Strigolactones as Plant Hormones ", in: Koltai H, Prandi C (eds) Strigolactones - Biology and Applications. Springer, Cham).

Furthermore, in the species of the Solanaceae family tested up to now (tomato, petunia, potato) and in some legumes such as Lotus japonicus, the genetic impairment of synthesis or perception of strigolactones heavily disturbs the reproductive process, significantly reducing the production of flowers, seeds. and fruits (Kohlen W, et al, 2012. "The tomato CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis", New Phytol 196: 535-547; Liu J, et al, 2013. "Carotenoid cleavage dioxygenase 7 modulates plant growth, reproduction, senescence, and certain nodulation in the model legume Lotus japonicus", J Exp Bot 64: 1967-1981; Pasare SA, et al, (2013) "The role of the potato (Solanum tuberosum) CCD8 gene in stolon and tuber development ", New Phytol 198: 1108-1120).

Without wishing to be bound by any theory, the present inventors believe that the hormones of the strigolactones group are involved in the reproductive transition and in the ability of the plant to tolerate states of water shortage by reducing the loss of water from the stomata, small openings with regulated closure. which are found in the epidermis of the leaves. In turn, these variations in stomatal closure seem to depend at least in part on an altered sensitivity / transport of the abscisic acid phytohormone, which is a powerful stimulator of stomatal closure.

In a preferred embodiment, the at least one plant hormone of the strigolactone group is present in the biostimulant composition of the present invention in a concentration by weight in the range from 0.0000015% to 0.0000030% w / v on the total volume of the composition, more preferably in the range from 0.0000020% to 0.0000028% w / v on the total volume of the composition, for example in a concentration by weight of 0.0000020%, 0.0000021%, 0.0000022%, 0.0000023%, 0.0000024%, 0.0000025%, 0.0000026%, 0.0000027% and 0.0000028% w / v on the total volume of the composition.

Preferably, the at least one plant hormone of the strigolactone group in the biostimulating composition of the invention is the solanacle, present in the root exudates of Solanum lycopersicum (tomato) plants.

Alongside the at least one strigolactone hormone as described above, the biostimulating composition according to the invention is characterized by also comprising the oxalic and succinic organic acids.

Plant cells contain numerous organic acids, which vary mainly in the length of the carbon chain and in the number of carboxylic groups. These molecules are involved in cellular metabolism as intermediates of the tricarboxylic acid cycle, and consequently in the production of metabolic energy in the process of cellular respiration. Furthermore, organic acids can be precursors for the synthesis of other important biomolecules (Tcherkez G. et. Al, 2012 “Respiratory carbon fluxes in leaves”, Curr Opin Plant Biol, 15: 308-314).

In a preferred embodiment, oxalic acid is present in the biostimulating composition of the present invention in a concentration by weight in the range from 0.0025% to 0.0040% w / v on the total volume of the composition, more preferably in the 'range from 0.0030% to 0.0038% w / v on the total volume of the composition, for example in a concentration by weight of 0.0030%, 0.0031%, 0.0032%, 0.0033%, 0 , 0034%, 0.0035%, 0.0036%, 0.0037% and 0.0038% w / v on the total volume of the composition.

In another preferred embodiment, succinic acid is present in the biostimulant composition of the present invention in a concentration by weight in the range from 0.004% to 0.008% w / v on the total volume of the composition, more preferably in the range from 0.005% to 0.007% w / v on the total volume of the composition, for example in a concentration by weight of 0.005%, 0.006% and 0.007% w / v on the total volume of the composition.

As previously indicated, the aforementioned plant hormone of the strigolactones group, the aforementioned oxalic acid and / or the aforementioned succinic acid are contained in the secretions of root systems of different plants, preferably of a plant of the Solanaceae family, preferably Solanum lycopersicum.

According to the present invention, the biostimulating composition can be in the form of a lyophilisate.

In an alternative embodiment, the composition of the present invention can be in the form of a water-based liquid composition further comprising a eutectic solvent.

In the context of this description, the term "eutectic solvent" means a solvent suitable for the production of a eutectic mixture, that is, a mixture characterized by a melting point temperature that is far below the individual substances that compose it, thus remaining fluid at room temperature.

Preferably, but not as a limitation, the eutectic solvent present in the biostimulating composition of the invention is a natural deep eutectic solvent (NADES).

As is known in the art, NADES solvents are characterized by a high solubilizing capacity of both polar and non-polar compounds. The strong hydrogen bond between NADES and solutes not only results in this enormous increase in solubility, but also contributes to the stability of secondary metabolites under various conditions such as high temperature, light and storage time.

In this manufacturing mode, predetermined quantities of biostimulant are formulated in eutectic mixtures of solvents (or DES acronym for Deep Eutectic Solvents), said eutectic mixtures of solvents having by way of example and not limiting a composition chosen from:

1. “LAP” mixture: lactic acid: 1,2-propanediol, in molar ratio 2: 1; And

2. “PCH” mixture: 1,2-propanediol: choline chloride: water, in molar ratio 1: 1: 1 or 1: 1: 3.

A first preferred biostimulating composition of the present invention comprises the components 0.0000020% by weight of the at least one plant hormone of the strigolactone group, 0.0030% by weight of succinic acid and 0.005% by weight of oxalic acid in a "LAP "As solvent on the total volume of the composition.

A second preferred biostimulating composition of the present invention comprises the components 0.0000028% by weight of the at least one plant hormone of the strigolactone group, 0.0035% by weight of succinic acid and 0.006% by weight of oxalic acid in a "PCH "As a solvent.

According to the invention, the biostimulating composition of the invention may include additional active compounds or additives.

For example, the biostimulating composition according to the invention can also comprise at least one polyphenolic compound, preferably gallic acid.

In another embodiment, the biostimulating composition according to the invention also comprises at least one organic acid selected from the group consisting of citric acid, malic acid, glycolic acid, lactic acid and acetic acid.

Within the scope of the present invention there is also a process for the preparation of the biostimulant composition described above, which includes the steps of:

(i) cultivating one or more plants by aeroponic cultivation for at least 14 days, preferably for at least 21 days, using a first nutrient solution comprising potassium dihydrogen phosphate (KH2PO4);

(ii) continue the aeroponic cultivation of one or more plants for at least a further 3 days, preferably for at least a further 7 days, using a second nutrient solution comprising potassium chloride (KCl) and not comprising KH2PO4, thus obtaining the production of an exudate by the roots of said one or more plants;

(iii) collect the exudate produced by the roots in phase (ii);

(iv) separate the lipid component of the root exudate from the aqueous phase, and collect said aqueous phase;

(v) dissolving the lipid component in a solvent, thus obtaining a first homogeneous mixture, and adding to said first homogeneous mixture a predetermined volume of the aqueous phase separated in step (iv), thus obtaining the biostimulating composition in the form of a second homogeneous mixture .

In the context of this description, the term "aeroponic cultivation" refers to a soilless cultivation, that is carried out in the absence of soil, in which the roots of the plants remain aerial, without any type of soil to support them, improving in a way their degree of oxygenation is remarkable. For the process to work, it is necessary that the roots constantly receive the correct doses of water and nutrients through nebulization that allow the correct development and growth of the plant.

According to the present invention, in plants in aeroponic cultivation, the replacement of the first nutrient solution containing KH2PO4 with the second nutrient solution without KH2PO4 but containing KCl results in a stimulation of the synthesis and radical exudation of the molecules of a hormone of the strigolactone group.

The separation of the lipid component of the radical exudate is preferably carried out by chromatography on a column designed to retain molecules of a hydrophobic nature, although other types of separation methods can be used which are in themselves known to the average technician in the field. The selection of the most appropriate method to be used in the context of the present invention falls largely within the capabilities of the average technician in the sector.

In the previously described embodiment, for the elution step from the chromatographic column, for example, a solution comprising an apolar solvent, preferably acetone, more preferably a solution comprising water and acetone, can be used.

Preferably, a predetermined volume of the aqueous phase in the range from 1/2 to 1/10 is used in the process of the invention, for example 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9 and 1/10, of the total volume of said aqueous phase separated from the lipid component of the root exudate.

More preferably, in the process according to the invention, the first mixture containing the lipid component of the radical exudate is added to the separated aqueous phase in a volume ratio of about 5 to 1.

In order to increase the stability and duration of storage times, the biostimulating composition of the invention can be subjected to dehydration and drying. Therefore, the process according to the invention can optionally include the additional step of subjecting the biostimulating composition obtained in the form of a homogeneous mixture to a freeze-drying process. The freeze-drying techniques are consolidated and the expert in the sector is able to apply them, without having to resort to any inventive step.

Preferably, in the process according to the invention, one or more plants subjected to aeroponic cultivation belong to the Solanaceae family, preferably Solanum lycopersicum.

Another aspect of the present invention is a biostimulating composition obtainable by means of the process as defined above, said composition comprising oxalic acid, succinic acid and at least one plant hormone of the strigolactone group.

As previously described, the biostimulating composition according to the invention is particularly suitable for use in regulating the development of treated plants and their water balance, promoting physiological processes such as growth, absorption of water and nutrients from the soil, development and therefore productivity.

Therefore, a further aspect of the present invention is the use of the biostimulant composition according to the invention to anticipate the floral transition of a plant and / or to improve the efficiency of water use by a plant.

The following examples are provided for illustrative and non-limiting purposes of the scope of the invention as defined in the attached claims.

EXAMPLE 1: Chemical characterization of the biostimulating composition

The presence of the at least one strigolactone hormone in the biostimulating composition of the invention, in particular the solanacle, was determined by carrying out a sample extracted in a 50:50 acetonitrile / H2O solution of high performance liquid chromatography ("High Performance Liquid Chromatography ”, HPLC) followed by mass analysis (HPLC ‐ MS). For the analysis a QTRAP3200 developed by Sciex was used interfaced through an ESI TurboIonSpray source to a UHPL Shimadzu Nexera. The elution was performed with a solution of 0.1% formic acid in water and 0.1% formic acid in methanol, using a flow of 0.4 ml min-1 and a gradient of 15 minutes from 50/50. at 0/100. The injected sample volume was 20 μl. The chromatographic column used was a Kinetex C18 (Phenomenex) 2.6 μm, 100 × 4.6 mm, 100 Å maintained at a constant temperature of 40 ° C. The parameters for the mass spectrometry analysis have been optimized through a direct infusion of the sample into the spectrometer to maximize the MRM signal (multiple reaction monitoring): ion spray 5000 V, temperature 500 ° C, declustering potential 100 V, entrance potential 10 V). Qualifying Transition: 343> 201 (CE 12 eV, CXP 23 V); Quantifying Transition: 343> 183 (CE 10 eV, CXP 23 V). Quantification was performed using a 5-point calibration line (1-10-25-50-100 ppb). Limit of detection, LOD 1 ppb.

The determination of the presence of succinic and oxalic organic acids in the biostimulant composition of the invention and their subsequent quantification was performed using an ion chromatography procedure. Briefly, the aqueous phase remaining at the passage on the C18 column was passed through anion exchange columns (type Dionex IonPac AS4A-SC 250 × 4 mm) and subsequently eluted with aqueous solutions of NaHCO3 and Na2CO3 (7.5 mM, pH 10.2, Flow Rate: 1.7ml / min). Optimal retention times were 5.12 min and 6.29 min for succinic acid and oxalic acid, respectively.

In conclusion, the analytical procedures as described above to which the biostimulant composition was subjected highlighted the presence of the following components:

- solanacle (plant hormone belonging to the strigolactone family), present at a concentration by weight in the range from 0.0000015% to 0.0000030% w / v on the total volume of the composition, or at a concentration from 70 pM to 80 pM;

- oxalic acid, present at a concentration by weight in the range from 0.0025% to 0.0040% w / v on the total volume of the composition, (31 mg / l on average), or at a concentration of 0.25 mM to 0.3 mM;

- succinic acid, present at a concentration by weight in the range from 0.004% to 0.008% w / v on the total volume of the composition, (50 mg / l on average), or at a concentration from 0.35 mM to 0, 4 mM.

EXAMPLE 2: Production of the biostimulant composition

In order to produce the biostimulant composition according to the invention, the present inventors have set up the procedure described below. The plants, more specifically tomato plants (Solanum lycopersicum), were grown for about 4 weeks in aeroponic systems, in which bioactive substances exuded by their roots can be extracted. Plants belonging to any variety can be used for the process, but the use of plants genetically insensitive to strigolactones increases their production.

As shown in Figure 2, the plants in aeroponics were continuously hydrated at the root by nebulizing an optimized circulating nutrient solution in order to maximize their production and release of biostimulating substances. The composition of the nutrient solution that was used in the experiments conducted by the inventors is as follows:

Every 3 weeks the nutrient solution containing KH2PO4 was replaced with a solution containing the same components, except for KH2PO4 which was replaced with KCl. Subsequently, this solution was replaced every 3 days. After collecting the exudate produced by the root systems of plants in culture thanks to the collection of the liquids deposited in the growth tank, the latter was subjected to a filtration process using sieves with pores of 2 �m in diameter in order to eliminate contaminants coarse. The filtrate thus obtained was subjected to a chromatographic step on C18 columns (GracePure SPE C18-Fast 5000 mg / 20 m) capable of retaining the hydrophobic component. The columns were subsequently eluted through a solution consisting of water and acetone in the ratio of 1: 1. Subsequently, a volume equal to one fifth of the aqueous solution containing the hydrophilic components not bound to the chromatographic column was recovered and joined to the hydrophobic component eluted therefrom. The volumetric ratio between the solutions that provided the hydrophobic component of radical exudates (elution solution of the C18 column) and the hydrophilic one was 1 to 60. Finally, the composition was lyophilized and stored at -20 ° C.

EXAMPLE 3: Characterization of the biological activity of the biostimulating composition - Early flowering

In order to characterize the biological activity of the composition of the invention, the present inventors have examined different batches of the composition for their ability to induce the germination of the seeds of the Phelipanche aegyptica plant. In particular, the bar graph of Figure 3 shows the activity of 5 serial dilutions of the biostimulant composition (1: 1, 8 nM; 1:10, 800 pM; 1:10 <2>, 80 pM; 1:10 < 3>, 8 pM; 1:10 <4>, 0.8 pM) in comparison with two different concentrations (10 µM and 1 µM) of a synthetic analogue of strigolactones, racemic GR24 (StrigoLab Srl).

In order to study the physiological and agronomic effects of the biostimulating composition on plant development, the inventors designed a series of experiments in which tomato plants were treated with said composition by spraying the leaves. The experiments conducted in the greenhouse with 10-liter potted plants concerned both aspects of development, in particular the transition from the vegetative phase to the reproductive phase, and the evaluation of the response of the treated plants to situations of water and nutritional stress. For the first tests, the treatments were carried out during the vegetative phase (plants with 4-5 leaves, about 6 weeks from sowing), in the budding phase (plants with 6-7 leaves, about 9 weeks from sowing) and on the flowers. recently opened (plants with 7-8 leaves, about 11 weeks from sowing). Five plants were used for each type of treatment. The flower transition and the development of the flowers were evaluated every two-three days by counting the flower buds and assigning to each of them a specific coefficient as exemplified in Figure 4. Each plant therefore had a total score given by the sum of the coefficients assigned to each. of their buds and flowers in the inflorescences.

The present inventors have conducted comparative studies by measuring the effectiveness of the biostimulant composition of the invention compared to that of strigolactone alone, using the synthetic molecule GR24 as indicated above. More specifically, for their studies, the inventors used a biostimulating composition comprising the strigolactone solanacle hormone at a concentration of 0.0000020% w / v (corresponding to a concentration of about 80 pM), succinic acid at a concentration of 0.003% w / v and oxalic acid at a concentration of 0.005% w / v, on the total volume of the composition. The synthetic molecule of strigolactone was instead used as a solution at a concentration of 5 µM and at a concentration of 0.1 µM. The graph of Figure 5A shows the effects of the biostimulating composition and of the synthetic strigolactone GR24 on the transition from the vegetative to the reproductive phase expressed through the arbitrary score on the different phenological phases of the flower / fruit normalized on the average value obtained by the control plants at the date of the same survey. phenological. The results obtained show that the plants treated with the biostimulating composition of the invention actually anticipated flowering. In particular, after 4-5 days from the first treatment, the plants treated with the biostimulating composition showed a higher score than the untreated control plants but similar to the score of the plants treated with GR24 5 µM. The plants treated with GR240.1 µM instead showed flower development indices comparable to those of the untreated controls. This difference between plants treated with the biostimulant of the invention or with pGR24 5 µM compared to untreated plants was maintained up to about 3-4 weeks after the first treatment, when the relationship between the flower development scores of the treated plants and the untreated controls was reported to around 1, due to the onset of flowering of the control plants. In the same period the values of plants treated with GR24 0.1 µM were slightly higher than those of untreated plants, and significantly lower than those of plants treated with the biostimulating composition or GR24 5 µM. This suggests that the treatment with the biostimulating composition of the invention is more effective in a specific phase of development, that is, on adult plants (reproductively competent), but whose apical meristems are not yet producing flower buds.

The pure hormone in the form of the synthetic analogue GR24 proved effective at concentrations equal to or greater than 5 µM, since at 0.1 µM the effect is almost nil; the biostimulating composition of the invention instead showed a significant effect in a formulation in which strigolactone solanacle is present in a concentration of 80 pM. This highlights how the biostimulant composition has emerging properties linked to the synergy of its various components.

In the context of this description, the term "emergent property" means a property that is owned by a complex system but not by the individual components of that system. Failure to recognize a property as emergent can lead erroneously to attribute this property to one or more of the components of the system (so-called "fallacy of division").

This specific bioactivity of the biostimulating composition on the development of flower buds also has implications for fruit development and ripening, and leads the treated plants to increase the cumulative yield of fruits during the growing season, especially at the beginning. The graph in Figure 5B shows the cumulative yield per plant expressed in fresh weight of the fruit after treatment with the synthetic molecule GR24 or with the biostimulating composition, and demonstrates a markedly higher effect for the latter.

Therefore, as shown by Figures 3 and 5A-B, the comparative studies conducted by the present inventors and illustrated previously have surprisingly revealed that the biostimulating composition in which the solanacle is present at a very low concentration (about 80 pM) is endowed with a bioactivity significantly higher, equal to about 4-5 orders of magnitude, compared to that of the strigolactone molecule GR24 only (used at the 5 µM concentration, and no longer active at the 0.1 µM concentration), demonstrating a synergistic effect, and not simply additive, of the different components.

EXAMPLE 4: Characterization of the biological activity of the biostimulant composition - Efficiency of water use

The present inventors have also conducted experimental tests to evaluate the effect of the biostimulant composition of the invention on the water use efficiency (WUE) of tomato plants in conditions of water stress, phosphorus deficiency and a combination of both stresses. The WUE parameter, widely used at the agronomic level, indicates the plant's ability to use the available water and is typically calculated as the ratio between the dry biomass of the plant and the volume of water supplied to it during cultivation. Probably the higher this ratio is, the better the plant has used the available water for its own development and growth.

For these studies, a biostimulating composition and a solution of synthetic strigolactone GR24 (5 µM) were used as described in Example 3 above. Since in the flowering tests described above the late treatments of the plants had not shown clear effects, the first nebulization was anticipated (plants with 1-2 leaves, 3 weeks after sowing), and then continued during the vegetative phase (plants with 4 -5 leaves, about 6 weeks from sowing), to finish in the budding phase (plants with 6-7 leaves, about 9 weeks from sowing).

Figure 6A shows the effects of the biostimulant composition and GR24 on the water use efficiency (WUE) of the whole plant in irrigated, water stressed, nutritional or combined conditions and shows that the WUE of plants tends to increase under stress. The same parameter is clearly higher for plants treated with the biostimulant composition in non-stressed conditions, apparently due to a greater allocation of resources to the root systems (Figure 6B). The data reported in the latter figure also indicate a higher efficacy in regulating the water balance by the biostimulant composition (solanacle concentration equal to 80 pM) compared to the synthetic strigolactone GR24 (use concentration equal to 5 µM).

Plants subjected to water and nutritional stress conditions treated with the biostimulant composition maintained a tendency to increase root WUE (Figure 7A), and to productivity, i.e. the allocation of resources in fruits (Figure 7B), compared to controls. and to plants treated only with synthetic strigolactone GR24.

Claims (14)

CLAIMS 1. Biostimulating composition comprising oxalic acid, succinic acid, and at least one plant hormone of the strigolactone group. 2. Biostimulant composition according to claim 1, comprising at least one plant hormone of the strigolactone group in a concentration by weight in the range from 0.0000015% to 0.0000030% w / v on the total volume of the composition. 3. Biostimulant composition according to claim 1 or 2, comprising oxalic acid in a concentration by weight in the range from 0.0025% to 0.0040% w / v on the total volume of the composition. 4. Biostimulant composition according to any one of claims 1 to 3, comprising succinic acid in a concentration by weight in the range from 0.004% to 0.008% w / v on the total volume of the composition. 5. Biostimulating composition according to any one of claims 1 to 4, in which the at least one plant hormone of the strigolactone group is the solanacle. Biostimulant composition according to any one of claims 1 to 5, wherein the aforementioned at least one plant hormone of the strigolactones group, the aforementioned oxalic acid and / or the aforementioned succinic acid are present in the form of a root exudate of a plant of the family of the Solanaceae, preferably Solanum lycopersicum. 7. Biostimulant composition according to any one of claims 1 to 6, which is in lyophilized form. 8. Biostimulant composition according to any one of claims 1 to 6, which is a water-based liquid composition also comprising a eutectic solvent. 9. Process for the preparation of a biostimulating composition, comprising the steps of: (i) cultivating one or more plants by aeroponic cultivation for at least 14 days, preferably for at least 21 days, using a first nutrient solution comprising potassium dihydrogen phosphate (KH2PO4 ); (ii) continue the aeroponic cultivation of one or more plants for at least a further 3 days, preferably for at least a further 7 days, using a second nutrient solution comprising potassium chloride (KCl) and not comprising KH2PO4, thus obtaining the production of an exudate by the roots of said one or more plants; (iii) collect the exudate produced by the roots in phase (ii); (iv) separate the lipid component of the root exudate from the aqueous phase, and collect said aqueous phase (v) dissolving the lipid component in a solvent, thus obtaining a first homogeneous mixture, and adding to said first homogeneous mixture a predetermined volume of the aqueous phase separated in step (iv), thus obtaining the biostimulating composition in the form of a second homogeneous mixture . 10. Process according to claim 9, which further comprises subjecting the biostimulating composition obtained in step (v) to lyophilization. 11. Process according to claim 9 or 10, in which the separation of the lipid component of the radical exudate from the aqueous phase is carried out by column chromatography. 12. Process according to any one of claims 9 to 11, in which the one or more plants are a plant belonging to the Solanaceae family, preferably Solanum lycopersicum. 13. Biostimulating composition obtainable by the process according to any one of claims 9 to 12, the biostimulating composition comprising oxalic acid, succinic acid and at least one plant hormone of the strigolactone group. 14. Use of the biostimulant composition according to any of claims 1 to 8 and 13 to anticipate the floral transition of a plant and / or to improve the efficiency of use of water by a plant.