WO2025061271A1 - Exogenous addition of specific phytosterol blends to stimulate target plant species - Google Patents

Abstract

A method of manufacturing an agricultural composition for application to a target plant species, said method comprising the steps of: a. Identifying the relative concentration of n phytosterols in target plant species selected from the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, and selecting the two phytosterols present in the highest relative concentration, b. Providing an agricultural composition comprising: i. a mixture of phytosterols obtained by blending at least one natural source of phytosterols, comprising at least 2 phytosterols being identical in nature to the at least 2 phytosterols identified in step a., wherein the the natural source of phytosterols is obtained from the group consisting of trees, sunflower, rapeseed extracts, ii. at least one surfactant.

Description

EXOGENOUS ADDITION OF SPECIFIC PHYTOSTEROL BLENDS TO STIMULATE TARGET PLANT SPECIES

FIELD OF THE INVENTION

The invention relates to agriculture and, in particular, to cultivated plants, notably field-grown plants, to the prevention of adverse effects linked to exposure to abiotic and/or biotic stresses in said cultivated plants, including the loss of dry matter, and to any biostimulating effect on such plants. Hence, the invention involves a method of manufacture of a phytosterol-based agricultural composition, the composition as such, a slurry comprising said composition in a diluted form, and its uses to substantially enhance crop performance. In particular, this preventive treatment process targets the onset of deleterious effects induced by exposure to an abiotic and/or biotic stress or induces a biostimulant effect on the plant.

STATE OF THE ART

Plants, i.e., crop plants and especially ornamental plants, are subjected to various forms of stress. In particular, plants are constantly exposed to their environment and cannot escape abiotic stress factors (e.g. drought, heat, cold, frost, nutrient deficiency, pollution, high salinity, etc.). In addition, they can also be exposed to biotic stress factors, i.e., stresses resulting from the harmful action of a living or bio-aggressive organism (viruses, fungi, bacteria, insects, pests, etc.) and more generally of a plant pathogen.

In general, these abiotic and biotic stresses cause morphological, physiological, biochemical, and molecular changes in plants, resulting in a decrease in the per-hectare crop yield, i.e., a decrease in the production or the quality of dry matter.

In otherwords, a cultivated plant, for example a field-grown plant, is subjected to these various forms of stress, which, among other effects, provokes a decrease in the production of dry matter by the plant compared to a plant which is cultivated under optimal conditions (controlled conditions with regard to water supply, daylight/nighttime period, absence of exposure to abiotic and/or biotic stresses, etc.). To combat abiotic stress, especially water stress (or drought), farmers have adapted by simplifying their crop rotations and giving precedence to winter crops. The first consequence of this simplification is not only an increased risk that plants growing wild (weeds) and pests will develop resistance to phytopharmaceutical products, but also an increased risk of water pollution due to large applications of products at the same time of the year. The second consequence is the disproportionate cultivation of starch-p reducing plants (notably straw cereals) compared to protein-producing plants (legumes). In addition, to mitigate drought conditions, farmers resort to extensive crop irrigation, which leads to environmental and economic problems.

Concerning the fight against biotic stresses, farmers use chemical or biocontrol products that rely on natural mechanisms. The use of chemicals in agriculture is controversial, however, given their potential toxicity for human health and for the environment. It is therefore necessary to minimize the use of these products, while optimizing their effects.

In conditions when no stress occurs, biostimulants can also be used on crops to help them develop their full growing potential and productivity, notably by influencing the metabolism of the plant.

To combat these different types of stress, both curative and preventive treatments are known from the skilled artisan. Some treatments consist in applying to the plants a mixture of surfactants, such as sucrose stearate, with 3-sitosterol, a plant sterol, following exposure to a biotic or an abiotic stress. This is the case, for example, of document W02019/030442 of the Applicant, which describes the curative application of a composition containing 80% sucrose stearate by weight and 20% 3-sitosterol by weight, diluted to 3% in water. A preventive treatment using sucrose stearate and beta-sitosterol present at least at 30% by weight in a global composition of plant sterols, in the form of a suspo-emulsion can also be found in the document W02023/057640 of the Applicant.

Similarly, document WO2018/229710 describes a composition for stimulating plant growth, where applicable in the presence of a stress factor, this composition being in the form of a concentrated suspension comprising a mixture of phytosterols in an amount greater than 25% of the suspension by weight, wherein beta-sitosterol is present in an amount ranging from 40% to 45% in the said mixture of phytosterols. The proportion range of campesterol, stigmasterol and brassicasterol is also mentioned, pointing the sourcing of the phytosterols towards soybean-originated deodorizer distillate.

All the cited documents disclose, as a source of beta-sitosterol, the use of isolated betasitosterol or the use of an extract of soybean including beta-sitosterol.

Moreover, the Applicant has observed that a soybean extract as source of phytosterols including beta-sitosterol may not be suitable as an exogenous addition from specie to specie.

Therefore, there is still a need to improve the yield that is to say to improve the efficiency of such compositions, notably from specie to specie, depending on the species of plant, or possibly the variety of the plant, and to optimize the quantity of raw materials which is used, notably for economic and environmental purposes.

DISCLOSURE OF THE INVENTION

Definitions

As used therein, the expression "abiotic stress" refers to a non-living stimulus on living vegetal organisms, for instance a climate hazard on a crop.

For the purpose of the invention, "plant pathogen" refers to a pathogen capable of infecting and/or invading a plant part and causing disease therein.

As used therein, the expressions "drought stress" or "water stress" can be used interchangeably and refer to an external phenomenon where the plant is lacking supply of water in an amount which would be sufficient for the plant to grow normally if such drought stress would not occur.

As used therein, the expression "at an early stage of the plant's growth" refers to the germination and vegetative phases, that is an interval between the beginning of germination and before the plant flowers. In particular, it refers to the stage during which the leaves of the plant cover the inter-row (row sowing crop cultivation). The skilled artisan knows that this early stage of the plant's growth is crop-dependent.

As used herein, the expressions "treated plant" and "treated crop" may be used interchangeably and refer to a plant or crop which is preventively treated (i.e., treated at an early stage, before exposure to an abiotic or biotic stress) with the composition of the invention and which undergoes growth by being exposed to soil and climatic conditions and stresses.

As used herein, the expressions "control plant", "control crop" and "untreated control" may be used interchangeably and refer to a plant or crop which is untreated with the composition of the invention and which undergoes growth by being exposed to the same soil and climatic conditions and stresses, notably at least one abiotic stress, preferably a multi-abiotic stresses sequence, as the treated candidate plant.

As used therein, the expressions "prior to the onset of abiotic stress" and "prior to the exposure to an abiotic stress" particularly with regard to drought stress, may be used interchangeably and refer to the period during which the useful soil water reserve is properly filled, i.e., the time elapsed from the moment when the useful soil water reserve is sufficiently or completely full (field capacity) to the moment when the wilting point is reached.

As used therein, the expression "biotic stress" refers to a damage performed on a cultivated crop by other living organisms, such as bacteria, viruses, fungi, parasites, insects, and weeds.

As used therein, the expressions "prior to the onset of a biotic stress" and "prior to the exposure to a biotic stress", may be used interchangeably and refer to the period during which the plant has not been attacked thus far by any living organism, i.e., before the first symptoms appear, for example before the first spots or degraded material appear on the leaves and/or stems of the cultivated plant.

When used in relation to biotic and/or abiotic stress, "resistance" may be replaced by tolerance, defense, protection, strength, robustness, vigor, resilience and the like. As used therein, PS means phytosterol.

As used therein, PSi (composition) refers to a phytosterol present in the composition of the invention, from highest (PSi with i=l) to lowest relative concentration. Therefore PSI (composition) designates the PS present in the highest relative concentration in the composition, PS2 (composition) designates the PS present in a lower relative concentration compared to PSI (composition) but still in a higher relative concentration compared to PS3 (composition), etc.

As used therein, PSi (plant) refers to a phytosterol present in the constitutive balance of the plant, from highest (PSi with i=l) to lowest relative concentration. Therefore PSI (plant) designates the PS present in the highest relative concentration in the plant, PS2 (plant) designates the PS present in a lower relative concentration compared to PSI (plant) but still in a higher relative concentration compared to PS3 (plant), etc.

As used therein, the expressions "plant sterol" and "phytosterol" may be used indifferently and refer to any vegetally-occurring or chemically-synthesized sterol.

As used therein, the expression "sterol" refers to any vegetally-, animally-occurring, or chemically-synthesized sterol. The expression "sterol" therefore embraces the expression "plant sterol".

As used therein, cholesterol can be indifferently considered as a vegetally- or an animally- occurring sterol, since this sterol can be found both in some vegetals as well as in some animals.

As used herein, the term "easily usable soil water reserve" (or EUSWR) refers to the proportion of the usable soil water reserve (USWR) that a cultivated plant can extract without reducing its transpiration (or evapotranspiration), experiencing drought stress, or limiting its growth. The EUSWR generally represents 40% to 80% of the USWR depending on the depth of the soil and the species of plants cultivated. As used herein, the term "soil survival reserve" (or SSR) refers to the proportion of the USWR that a cultivated plant cannot extract. The plant is consequently in a state of drought stress because its transpiration (or evapotranspiration) is not reduced. Therefore, the cultivated plant limits its growth or even wilts.

As used herein, the term "wilting point" (or WP) refers to the soil water status below which the plant can no longer draw the water necessary for its growth, i.e., the point below which the tension between the roots and the plant is high and the roots can no longer extract water from the soil. It is therefore the threshold below which the cultivated plant has entirely consumed the EUSWR and will wilt, albeit reversibly, but with an impact on the yield. This parameter is determined in particular through the measurement of soil humidity, for example by means of a neutron probe, a tensiometer or a time-domain reflectometry (TDR) moisture meter.

As used herein, the term "dry matter" refers to the matter derived from organic matter that remains after removal of all the water it contains.

As used herein, the terms "yield" or "harvest yield" refer to the amount of product harvested, whether seeds or fruits, dry matter or green matter, or wine, over a given cultivation area.

As used herein, the expression "preserving yield" refers to the maintenance of the per-hectare yield, i.e., the yield of the crop treated with the composition of the invention is essentially the same as the yield of the untreated crop, both treated and untreated crops being exposed to the same stress or multiple stresses climatic scenario.

As used herein, the term "cultivated plant," in contrast to a naturally existing plant, refers to all plants that can be cultivated, i.e., sown, planted, and exploited by man.

As used therein, the terms "crops" and "plants" may be used indifferently and refer to a II plants and plant populations such as desirable and undesirable wild plants, cultivars, and plant varieties (whether or not protectable by plant variety or plant breeder's rights). Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods which can be assisted or supplemented by one or more biotechnological methods such as by use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods.

As used therein, the term "plant" includes whole plants and parts thereof, including, but not limited to, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruits (the mature ovary), plant tissues (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. "Fruit" and "plant produce" are to be understood as any plant product which is further utilized after harvesting, e.g., fruits in the proper sense, nuts, wood etc., that is anything of economic value produced by the plant.

As used therein, the expression "effective amount" is an amount sufficient to affect beneficial or desired results.

As used therein, the term "slurry" refers to the composition of the invention diluted in water or in a solution containing water and possibly one or more active ingredients.

As used therein, the term "bactericide" refers to the ability of a substance to increase mortality or inhibit the growth rate of bacteria.

As used therein, the terms "insecticide" and "insecticidal" refer to the ability of a substance to increase mortality or inhibit growth rate of insects.

As used herein, the term "insects" comprises all organisms in the class "Insecta".

As used therein, the terms "nematicide" and "nematicidal" refer to the ability of a substance to increase mortality or inhibit the growth rate of nematodes. In general, the term "nematode" comprises eggs, larvae, juvenile and mature forms of said organism. As used therein, the terms "acaricide" and "acaricidal" refer to the ability of a substance to increase mortality or inhibit growth rate of ectoparasites belonging to the class Arachnida, sub-class Acari.

As used herein, "biocontrol product" is defined as an agent or product that uses natural mechanisms. They form a set of tools that can be used, alone or in combination with other plant protection methods, to combat crop enemies in integrated pest management.

As used therein, the term "biostimulant" is defined as a substance which stimulates the nutrition processes of plants regardless of the nutritional substances that they contain, with the sole aim of improving one of more of the following properties of the plants or of their rhizosphere.

As used therein, the expressions "foliar spraying" or "foliar spray" refer to a pressurized projection of a slurry that forms a large number of microdroplets that cover the upper and/or lower surfaces of the treated leaf.

As used therein, the term "seed imbibition" refers to an immersion of a seed in a solution containing the composition.

The use of "a" or "an" for a molecule does not exclude, unless otherwise stated, the presence of plurality of molecules, in such a way that the expression "one or more" can be substituted for it.

As used therein, the expression "insoluble in the aqueous phase" refers to a compound which presents the inability to form with water a homogenous, one-phase solution at the microscopic or the macroscopic level, at a given temperature and atmospheric pressure.

By contrast and as used therein, the term "solubility" refers to a compound which leads to a homogenous solution without remaining insoluble particles when it is added to a liquid, at a given temperature and atmospheric pressure. Any percentage by weight of a molecule of the invention refers to the total weight of the said invention, which means relative to the sum of all ingredients giving a hundred. Weight percent can be symbolized as wt%.

Various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and conciseness and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

To the extent to which any of the above-mentioned definitions cannot be correlated with previous definitions provided in any patent, scientific literature, or non-patent prior art (referenced herein or even not referenced herein), then it is understood that these above- mentioned definitions will be used herein.

As used therein, the term "healthy plant" refers to a plant's situation or period of time wherein the plant does not substantially suffer from any deleterious impact of any stress. It can be, for example and in a non-exhaustive way, in mild climatic conditions, when the EUSWR is conveniently filled, when the field is conveniently fed with nutrients and/or treated against pests.

Detailed description of the invention

It has thus been surprisingly found that when the balance of phytosterols aligns with the constitutive blend of a plant, preferably in healthy situation, the composition obtained from the method of manufacture can then be applied in the lowest possible quantity while exhibiting high effectiveness with regard to abiotic and biotic stresses, resulting in only a small loss of dry matter. To the knowledge of the Applicant, no variations were ever reported on a specific, exogenous phytosterol balance pointing towards a given crop.

In other words, it was observed that phytosterol-based compositions aiming at enhancing crop performance work best if they mimic the phytosterol composition of the target species.

The Applicant has ascertained that, most surprisingly, aligning the phytosterol balance of an agricultural composition substantially similarto the phytosterol profile of a target plant species may result in an improved efficiency into that species, in particular to improve the plant's resistance towards abiotic and/or biotic stress phases, thereby limiting the decrease in per- hectare yield which is the usual consequence of said abiotic and/or biotic stress on a cultivated crop.

It was observed that the method of manufacture of phytosterol-based composition from at least one natural source of phytosterols, when applied to the crop plant as a preventive care, i.e., prior to the onset of a stress, enabled a further reduction in the harmful effects of abiotic and/or biotic stresses, notably the loss of dry matter and the resulting decrease in per-hectare yields.

It was also observed that the method of manufacture leading to a phytosterol-based composition from at least one natural source of phytosterols was efficient to help the plant growing even in the absence of stress when applied at an early stage of the target plant’s growth.

To further enhance the exogenous supply of phytosterols to plants, the Applicant has formulated these sterols with at least one surfactant which role is to help increasing the concentration of sterols in the slurry and to improve the foliar uptake of the active ingredient, notably through a solubilization of the epicuticular waxes of the leaves.

Consequently, it is possible to reduce the quantity of phytosterols and to specifically adapt the balance of phytosterols contained in the composition in view of the balance of same phytosterols constitutive of the target species, in order to obtain beneficial effects for the plant that are at least similar to, and usually better than, those provided by the compositions of the prior art.

According to a first aspect, the invention concerns a method of manufacturing an agricultural composition for application to a target plant species, said method comprising the steps of: a. Identifying the relative concentration of n phytosterols (PSi (plant)) with n > 2 present from highest (PSi with i=l) to lowest relative concentration (PSi with i =n) in target plant species at a phenological stage, said phytosterols being selected from the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, and selecting the two phytosterols present in the highest relative concentration, b. Providing an agricultural composition comprising: i. a mixture of phytosterols obtained from at least one natural source of phytosterols, comprising at least 2 phytosterols (PSi (composition)) being identical in nature to the at least 2 phytosterols identified (PSi (plant)) in step a., wherein the mixture of phytosterols represents between 0.2% and 10% of the agricultural composition by weight, and wherein the natural source of phytosterols is obtained from the group consisting of trees, sunflower, rapeseed extracts, ii. at least one surfactant.

According to an advantageous embodiment, the relative concentration of each phytosterols among n phytosterols with n 2s 2 in an organ of said target plant species at a phenological stage mentioned in step a. is calculated according to the following formula: PSi (plant) = (PSi (plant)/(sum of n PS (plant)), and in that step b. requires the calculation of the relative concentration of the same phytosterols than the ones of step a. in the mixture of phytosterols according to the following formula: PSi (composition) = (PSi (composition)/(sum of n PS (composition)). Finally, the mixture for which 0.75 [PSi (composition)]/[PSi (plant)]

1.25 (with i being identical between PSi (composition) and PSi (plant)) for at least two phytosterols is selected.

In other words, the method of the invention, advantageously requires the steps of: a. Calculating the relative concentration of each phytosterols among n phytosterols with n > 2 in an organ of said target plant species at a phenological stage according to the following formula: PSi (plant) = (PSi (plant)/(sum of n PS (plant)), b. Identifying the at least two (2) phytosterols present in the highest relative concentration in the result of calculation of step a., c. Providing a mixture of phytosterols obtained from at least one natural source of phytosterols, said mixture comprising at least 2 phytosterols PSi (composition) being identical in nature to the at least 2 phytosterols identified in step b., d. Calculating the relative concentration of each phytosterols among n phytosterols with n > 2 in the mixture of phytosterols according to the following formula: PSi (composition) = (PSi (composition)/(sum of n PS (composition)), e. Selecting the mixture for which 0.75 < [PSi (compo)]/[PSi (plant)] < 1.25 (with i being identical between PSi (composition) and PSi (plant)) for at least two phytosterols, f. Providing at least one surfactant, g. Blending the mixture of phytosterols of step e. with the at least one surfactant of step f., wherein the mixture of phytosterols represents between 0.2% and 10% of the agricultural composition by weight, preferably between 0.5% and 6%, advantageously between 1% and 5%.

According to the invention, the step a. involves determining the relative concentration of phytosterols in the natural composition of the species of the plant to be treated. Step b. involves identifying the at least two phytosterols which are present in the natural composition of the species of the plants to be treated, in a result of a calculation of step a. These steps can be performed by isolating a target organ of the target plant, extracting the phytosterols according to known methods such as solid-liquid extraction, liquid-liquid extraction, supercritical CO2 soxhlet extraction and analyzing the intrinsic composition of the phytosterols of the chosen organ of the plant according to known methods.

Qualitative and/or quantitative determination of phytosterols in plant material can be performed in either or in combination of the following analytical procedures: capillary gas chromatography, flame ionization detection, mass spectrometry, thin layer chromatography, high-performance or ultrahigh-performance liquid chromatography, near-infrared spectroscopy or chemometric methods. Preferably, the determination of phytosterols in plant materials is performed by chromatography, by integrating for example the peak attributed to an identified phytosterol, and expressing its relative concentration as the integral of this peak (PSi plant) over the sum of all integrals (sum of n PS (plant)).

Practically, the organs of the target plant which can be isolated to be further analyzed are chosen among the roots, stem, tubers, flowers, leaves, seeds, bracts, sepals, petals, stamens, carpels, anthers and ovules of the plant. Preferably, the organs of the plant which are isolated to be further analyzed are the leaves.

The organs of the target plant which can be isolated to be further analyzed are chosen when the plant is exposed to or not exposed to any kind of biotic and/or abiotic stress. Preferably, the organs of the plant which can be isolated to be further analyzed are chosen when the plant is in a healthy situation, which means in the absence of any biotic and/or abiotic stress.

The organs of the plant are isolated and further analyzed at a phenological stage, preferably chosen between BBCH 0 and the time of application of said agricultural composition.

Steps a. and b. allow determining the main phytosterols which are present in the constitutive balance of the plant, both in nature (i.e., chemical structure) and in relative composition. The said relative composition is determined according to the following equation: PSi (plant) = (PSi (plant)/(sum of n PS (plant)), wherein PS is a phytosterol, i is the a phytosterol analyzed in the constitutive balance of the plant, n the number of phytosterols identified in the balance of the plant, with n 2 2, and the sum of n phytosterols being equal to 100%. As an example, the constitutive sterol mixture of a target plant is determined by HPLC, from extraction of fresh, healthy leaves. Table 1 summarizes the constitutive phytosterol blends for both monocotyledon and dicotyledon crops. The balance is expressed in percentage for each phytosterols, with 100% belonging to the sum of these five phytosterols, at a given phonological stage.

Table 1. Sterol balance of various crops, in percentage of the total mentioned sterols (zero means that the values of the said sterol was under the detection threshold)

As mentioned above, the constitutive composition is expressed at a particular phenological stage. Indeed, this constitutive composition changes during the lifecycle of the plant. For instance, Table 2 mentions the phytosterols profile of rapeseed according to three different stages. It is seen that the overall balance differs from one stage to another. Table 2. Phytosterol balance (in percentage of the total mentioned sterols) of rapeseed leaves at three different phenological stages in healthy situation (zero means that the values of the said sterol was under the detection threshold)

According to a specific embodiment of the invention, - when the target plant is wheat, the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 30 and BBCH 52;

-when the target plant is barley, the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 30 and BBCH 52;

- when the target plant is vine, the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 13 and BBCH 77;

Step b. of the invention requires identifying the at least 2 phytosterols present in the highest quantity in an organ of said target plant species at a phenological stage.

The expression "highest quantity of at least 2 phytosterols" refers to the 2 phytosterols among the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, possibly further comprising cholesterol, which appear as the most concentrated phytosterols in said organ after being extracted from the organ and analyzed by analytical techniques such as HPLC. The quantity of each phytosterol is expressed in a relative way according to the total sum of the analyzed phytosterols being equal to 100%, that is to say its concentration divided by the sum of the concentrations of the phytosterols.

Step c. of the method of the invention, requires providing a mixture of phytosterols obtained from at least 1 natural source of phytosterols which is obtained from the group consisting of trees, sunflower and rapeseed extracts, said mixture comprising at least 2 phytosterols being identical in nature to the at least 2 phytosterols identified in step b., said at least 2 phytosterols of the mixture comprising a first phytosterol (PSI composition) and a second phytosterol (PS2 composition). The proportion of each phytosterol is obtained as mentioned above, which means by dividing the concentration of the said phytosterol to the sum of the concentrations of the phytosterols.

In the following of the description, the expression "2 phytosterols being identical in nature means 2 phytosterol having the same chemical formula. According to an embodiment, said at least 1 natural source of phytosterols is a tree's extract chosen from the group consisting of pine, palm and eucalyptus extracts, preferably pine extract.

Advantageously, said pine extract is originated from tall oil.

According to another embodiment, said at least 1 natural source of phytosterols is selected from the group consisting of sunflower and rapeseed extracts, said extracts being deodorization distillates from oils.

According to another embodiment, tree's extract is a palm extract, said palm extract being a deodorization distillate from oils.

According to an embodiment, the mixture of phytosterols further comprises another natural source of phytosterols different from the natural source in step c., wherein said other natural source of phytosterols is selected from the group consisting of pine extract originated from tall oil, and deodorization distillates from oils of palm, soybean, sunflower and rapeseed.

According to an embodiment, the mixture of phytosterols further comprises isolated cholesterol and/or isolated stigmasterol.

Advantageously, the cholesterol and/or stigmasterol are from a natural or a synthetic source.

According to an another embodiment, when the mixture of phytosterols comprises at least 2 natural sources of phytosterols, the first natural source of phytosterols represents at least 20%, at least 30%, at least 40% or even at least 50%, the 100% complement being provided by the at least second natural source of phytosterols, possibly in presence of further sources of phytosterols selected from the group consisting of isolated cholesterol and/or isolated stigmasterol.

As above mentioned, palm, soybean, sunflower and rapeseed extracts are deodorization distillates from vegetable oils originating respectively from palm, soybean, sunflower and rapeseed. A simplified process includes refining the vegetable oil, giving access to deodorization distillates. The latter are further purified to remove notably free fatty esters and tocopherols, leading to phytosterols.

As above mentioned, extract of pine is originated from tall oil as a by-product of the kraft process of wood pulp in the paper industry. This tall oil is also refined to give access to unsaponifiable phytosterols.

As an example, Table 3 describes the distribution of the five main phytosterols contained in different extracts as sources of phytosterols (in percentage of the total of these five phytosterols).

Table 3. Composition of naturally-occurring phytosterols according to the main five phytosterols, expressed in percentage of the total of these five phytosterols (zero means that the values of the said sterol was under the detection threshold)

As mentioned before, the Applicant has discovered that the application of a composition having a phytosterol profile similar to the one of the target species had several advantages in comparison with the application of a composition not having the same profile.

The aim of the invention was then to manufacture compositions reproducing a similar profile the one of the target species by using available raw materials. In practice, the phytosterol mixture comprising the composition of phytosterols which is substantially similar to the natural composition of the target plant is prepared from at least 1 natural source, preferably by mixing at least 2 sources or even 3 sources in a given ratio, to fit to the natural composition of the plant.

Step d. of the method of the invention, requires calculating the relative concentration of each phytosterols among n phytosterols with n

2 in the mixture of phytosterols according to the following formula: PSi (composition) = (PSi (composition)/(sum of n PS (composition)).

Step e. requires selecting the mixtures from step d. The similarity of the profile is characterized in the invention by the calculation of the quotient between at least 2 phytosterols present in the mixture of phytosterols (PSI (composition), PS2 (composition)) with same said 2 phytosterols identified in the target species (PSI (plant), PS2 (plant)). For each phytosterol, its concentration is also calculated relative to the total concentration of all phytosterols which have been identified.

In practice, the similarity profile is calculated following the equation: quotient = [PSi (composition)]/[PSi (plant)] (with i being identical between PSi (composition) and PSi (plant)) for at least two phytosterols, the said quotient ranging from 0.75 to 1.25.

As examples, the Applicant has developed a method of manufacturing an agricultural composition comprising:

- 100% of pine extract originated from tall oil, this mixture reproducing a profile of phytosterols, especially relating to beta-sitosterol, campesterol, brassicasterol and stigmasterol similar to the one of healthy leaves of vine at a BBCH 55 phenological stage; or

- between 65% and 80% of pine extract, between 20% and 35% of soybean extract, this mixture reproducing a profile of phytosterols, especially relating to beta-sitosterol, campesterol, brassicasterol and stigmasterol similar to the one of healthy leaves of wheat at a BBCH 32 phenological stage. The advantages of such a composition are illustrated in the following description in comparison to a composition containing soybean extract only as source of phytosterols which is not adjusted to the phytosterol profile of the plant at a specific phenological stage. As other examples, the Applicant has developed a method of manufacturing an agricultural composition comprising:

- between 65% and 75% of pine extract and between 25% and 35% of soybean extract, this mixture reproducing a profile of phytosterols, especially relating to beta-sitosterol, campesterol, brassicasterol, stigmasterol similar to the one of healthy leaves of barley at a BBCH 51 phenological stage;. The advantages of such a composition are illustrated in the following description in comparison to a composition containing soybean extract only, as source of phytosterols.

Step f. of the method of the invention, requires providing at least one surfactant.

The at least one surfactant aims at playing three roles: first is to help stabilizing the sterol- based formulation, second is to improve the wetting performance of the slurry, third is to increase the uptake of phytosterols through the leaves when the said phytosterols are delivered to the target plant by foliar spray, and/or through the tegument when the said phytosterols are delivered by seed imbibition.

In practice, the surfactant is selected from the group comprising: anionic surfactants, advantageously anionic surfactants whose polar head group is a carboxylate, a sulfonate or a sulfated alcohol; cationic surfactants, advantageously cationic surfactants whose polar head group is an amine, a quaternary amine or a quaternary ammonium ester; amphoteric surfactants, advantageously betaine derivatives or phospholipids; neutral surfactants, advantageously ethoxylates, alkanolamines, alkylglucamides, polyol esters, alkyl monoglucosides or alkyl polyglucosides, or polyol ethers, polyoxyethylene sorbitan esters (especially Tween 20, Tween 21, Tween 22, Tween 23, Tween 24, Tween 28, Tween 40, Tween 60, Tween 61, Tween 65, Tween 80, Tween L-0515, Tween L-1010, Tween L- 1505), or sorbitan esters (especially Span 20, Span 40, Span 60, Span 65, Span 80, Span 83, Span 85, Span 120); natural surfactants, advantageously lecithins, preferably soy lecithin or other lecithins, or surfactants derived from amino acids; and surfactants synthesized from natural raw materials, advantageously polyol derivatives, preferably fatty acid sugar esters; the preferred fatty acid sugar esters are saccharose stearate, saccharose palmitate and their polyesters, or mixtures thereof.

In a preferred embodiment, the surfactant is a sugar fatty acid ester, preferably sucrose stearate and/or sucrose palmitate.

In the description and in claims the terms "sucrose stearate" and "saccharose stearate" are used indifferently. As well, "sucrose palmitate" and "saccharose palmitate" are used indifferently.

In accordance with the invention, the expression "saccharose stearate" designates pure saccharose stearate or a mixture of saccharose esters of fatty acids containing mostly saccharose stearate. Example of pure saccharose stearate corresponds to CAS number [136152-91-5], Example of a mixture of saccharose ester of fatty acids containing mostly saccharose stearate corresponds for example to CAS number [25168-73-4] or [84066-95-5].

In accordance with the invention, the expression "saccharose palmitate" designates pure saccharose palmitate or a mixture of saccharose esters of fatty acids containing mostly saccharose palmitate. Example of pure saccharose palmitate corresponds to CAS number [110539-62-3], Example of a mixture of saccharose ester of fatty acids containing mostly saccharose palmitate corresponds to CAS number [26446-38-8],

According to a specific embodiment, the at least one surfactant is a mixture containing: between 20% and 80% by weight, advantageously 70% saccharose stearate with a monoester content ranging between 20% and 80% by weight of saccharose stearate, advantageously 70%, with the balance being a mixture of di-, tri- and/or polyesters; and between 20% and 80% by weight, advantageously 30% saccharose palmitate with a monoester content ranging between 20% and 80% by weight of saccharose palmitate, advantageously 70%, with the balance being a mixture of di-, tri- and/or polyesters. According to a specific embodiment, the at least one surfactant is sucrose stearate (preferably CAS number [25168-73-4] or [84066-95-5]).

The invention also relates to an agricultural composition for application to an organ of a target plant species, which is the one obtained by the method mentioned above with the same features, and also recited in claims.

Accordingly, the agricultural composition comprises: a. between 0.2% and 10% of the agricultural composition by weight of a mixture of phytosterols obtained from at least 1 natural source of phytosterols, said mixture comprising at least 2 phytosterols (PSi (composition)) selected from the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, said at least 2 phytosterols (PSi (composition)) being identical in nature to at least 2 phytosterols (PSi (plant)) present in the highest quantity in said organ of the target plant species at a phenological stage, and wherein the natural source of phytosterols is obtained from the group consisting of trees, sunflower, rapeseed extracts, b. at least one surfactant.

The agricultural composition can take the form of a homogenous or heterogenous aqueous solution as disclosed for example in W02019/030442, an oil-in-water or a water-in-oil emulsion as disclosed for example in WO2021/214406, an oil-in-water or a water-in-oil suspo- emulsion as disclosed for example in W02023/057640, or any other formulation the skilled artisan may find suitable for the invention purpose.

Preferably, the agricultural composition obtained by the method of manufacture of the invention is an aqueous solution, an emulsion or a multiphase composition having the form of a suspo-emulsion.

When the composition has the form of an aqueous solution, said aqueous solution comprises:

0.2 to 10 wt% of the mixture of phytosterols,

2 to 20 wt% of at least one surfactant, 70 to 97.8 wt% of water.

Advantageously, the aqueous solution comprises:

0.5 to 6 wt% of the mixture of phytosterols,

4 to 10 wt% of at least one surfactant,

84 to 95.5 wt% of water.

Even more advantageously, the composition of the invention has the form of an aqueous solution comprising:

1 to 5 wt% of the mixture of phytosterols,

5 to 9 wt% of at least one surfactant,

86 to 94 wt% of water.

When the composition has the form of an emulsion, said emulsion comprises, comprises: at least one surfactant representing between 2 and 20 wt% of the composition, preferably between 4 and 10 wt% of the composition, more preferably between 5 and 9 wt% of the composition; an oily phase, wherein the phytosterol mixture represents between 0.2 and 10 wt% of the composition, advantageously between 0.5 and 6 wt% of the composition, preferably between 1 and 5 wt% of the composition.

According to an advantageous embodiment, the composition is a multiphase agricultural composition in the form of a suspo-emulsion, i.e. a composition comprising lipophilic droplets containing the above mentioned mixture of phytosterols, said lipophilic droplets being dispersed in an aqueous phase to form an oil-in-water emulsion.

The composition further comprising: at least one surfactant located at the interface of the lipophilic droplets and of the aqueous phase and selected from among the surfactants that are soluble in the aqueous phase and/or the surfactants that are soluble in the lipophilic droplets; and at least one surfactant suspended in the oil-in-water emulsion, said second surfactant having the form of particles insoluble in the said emulsion. In other words, the suspo-emulsion comprises two distinct oil and water phases, but it cannot be described as an emulsion, given that the aqueous phase also includes a third phase made of solid particles. These solid particles are composed of molecules which are insoluble in the aqueous phase, whose aim is to further improve the uptake of the sterols within the plant.

According to a first embodiment of the multiphase composition, the surfactant which is located at the interface of the lipophilic droplets and of the aqueous phase and the surfactant which is suspended in the oil-in-water emulsion are identical.

Therefore, the multiphase composition comprises only one surfactant located at two different places, respectively at the interface between the lipophilic droplets and the aqueous phase, and in the aqueous phase under the form of suspended particles which are insoluble in the aqueous phase.

Practically, the part of the surfactant which is located at the interface of the lipophilic droplets and of the aqueous phase represents between 2 and 20 wt% of the composition, preferably between 4 and 10 wt% of the composition, more preferably between 5 and 9 wt% of the composition;

- the oily phase includes the phytosterol mixture, said phytosterol mixture representing between 0.2 and 10 wt% of the composition, advantageously between 0.5 and 6 wt% of the composition, preferably between 1 and 5 wt% of the composition,

- The part of the surfactant which is suspended in the oil-in-water emulsion represents between 0.5 and 2 wt% of the composition, more preferably between 1 and 1.5 wt% of the composition.

According to a second embodiment of the multiphase composition, the surfactant which is located at the interface of the lipophilic droplets and of the aqueous phase and the surfactant which is suspended in the oil-in-water emulsion are not identical.

In that embodiment, the composition comprises: one first surfactant located at the interface between the lipophilic droplets and the aqueous phase, which represents between 2 and 20 wt% of the composition, preferably between 4 and 10 wt% of the composition, more preferably between 5 and 9 wt% of the composition; and one second surfactant suspended in the aqueous phase, which represents between 0.5 and 2 wt% of the composition, more preferably between 1 and 1.5 wt% of the composition.

In both embodiments, the surfactant located at the interface between the lipophilic droplets and the aqueous phase may present a preferential oil solubility, a preferential water solubility or can also present both oil and water solubility, the solubilities between oil and water being here different or identical. This last feature allows the possibility that surfactants which are soluble in the aqueous phase and surfactants which are soluble in the lipophilic droplets are the same molecule. If different, surfactants which are soluble in the aqueous phase and surfactants which are soluble in the lipophilic droplets differ from each other by the proportion of their hydrophobic and hydrophilic parts. Practically, the hydrophilic/lipophilic balance (HLB) of surfactants which are soluble in the aqueous phase is higher than the one of surfactants which are soluble in the lipophilic droplets. In contrast, the HLB of surfactants which are soluble in the lipophilic droplets is lower than the one of surfactants which are soluble in the aqueous phase.

Advantageously, the surfactant is soluble in water heated at 80 °C at a concentration of at least 2 g/L.

Advantageously, the surfactant is soluble in the oil phase heated at 110 °C at a concentration of at least 2 g/L.

Advantageously, the limit of solubility in water of the surfactant suspended in the aqueous phase observed practically at 25 °C is less than 10 mg/L, preferably less than 5 mg/L, more preferably, less than 2 mg/L. According to the invention, the majority of the lipophilic droplets present in the multiphase composition before adding surfactant to the emulsion, advantageously at least 90% of the lipophilic droplets (also named Dv90 emulsion) have a diameter comprised between 0.01 and 70 pm, preferably between 0.1 and 50 pm, most preferably between 0.1 and 20 pm, advantageously between 0.5 to 7 pm, preferably between 2 and 6 pm as determined by laser diffraction.

Advantageously at least 90% of the particles of the multiphase composition (also named Dv90 suspo-emulsion) have a diameter comprised between 1 and 1000 pm, advantageously between 10 and 250 pm with a peak maximum preferably of between 10 pm and 100 pm as determined by laser diffraction.

In practice, surfactants are selected according to the desired solubility in lipophilic droplets or in water, from the group comprising: anionic surfactants, advantageously anionic surfactants whose polar head group is a carboxylate, a sulfonate or a sulfated alcohol; cationic surfactants, advantageously cationic surfactants whose polar head group is an amine, a quaternary amine or a quaternary ammonium ester; amphoteric surfactants, advantageously betaine derivatives or phospholipids; neutral surfactants, advantageously ethoxylates, alkanolamines, alkylglucamides, polyol esters, alkyl monoglucosides or alkyl polyglucosides, polyol ethers, polyoxyethylene sorbitan esters (especially Tween 20, Tween 21, Tween 22, Tween 23, Tween 24, Tween 28, Tween 40, Tween 60, Tween 61, Tween 65, Tween 80, Tween L- 0515, Tween L-1010, Tween L-1505), or sorbitan esters (especially Span 20, Span 40, Span 60, Span 65, Span 80, Span 83, Span 85, Span 120); natural surfactants, advantageously lecithins, preferably soy lecithin, or surfactants derived from amino acids; and surfactants synthesized from natural raw materials, advantageously polyol derivatives, preferably fatty acid sugar esters; the preferred fatty acid sugar esters are saccharose stearate, saccharose palmitate and their polyesters, or mixtures thereof. Preferably, the surfactant which is located at the interface of the lipophilic droplets and of the aqueous phase and the surfactant which is suspended in the oil-in-water emulsion is selected from among the group comprising the fatty acid sugar esters.

Indeed, fatty acid sugar esters may be used as both in the water phase or in the oil phase as surfactant since they are soluble in oil and in water at elevated temperatures, typically more than 80 °C, with different solubility constants. Indeed, sucrose esters can become soluble in water, but at high temperature only. It concerns for example sucrose stearate which is soluble in water at around 80 °C. Fatty acid sugar esters are also soluble in the oil droplets, but only to the condition that they undergo prior heating to their melting temperature, which can easily be determined by a person skilled in the art.

Also, these esters are solids at ambient temperature. Being naturally lipophilic compounds, they are insoluble in the aqueous phase at a standard temperature of 20 °C and are therefore potent candidates for the role of surfactant being suspended in the aqueous phase of the suspo-emulsion.

Advantageously, the fatty acid sugar esters are saccharose stearate, saccharose palmitate and their polyesters, or mixtures thereof.

According to a specific embodiment, the surfactant contains sucrose stearate or, advantageously, a mixture containing saccharose stearate and sucrose palmitate; in particular is a mixture containing: between 20% and 80% by weight, advantageously 70% saccharose stearate with a monoester content ranging between 20% and 80% by weight of saccharose stearate, advantageously 70%, with the balance being a mixture of di-, tri- and/or polyesters; and between 20% and 80% by weight, advantageously 30% saccharose palmitate with a monoester content ranging between 20% and 80% by weight of saccharose palmitate, advantageously 70%, with the balance being a mixture of di-, tri- and/or polyesters. In any embodiment, the phytosterols/surfactant mass ratio in the agricultural composition containing the phytosterol mixture and surfactant is practically between 0.01 and 5, advantageously between 0.1 and 2.5, preferably between 0.2 and 1.

The method of manufacture of the suspo-emulsion above mentioned comprises the steps of:

1. Providing a mixture of phytosterols according to step a. of the process of manufacturing of the composition of the invention,

2. Preparing a lipophilic phase comprising the mixture of phytosterols, optionally heating the mixture of phytosterols at high temperature, preferably in a range between about 70 °C to about 140 °C, preferably between about 90 °C to about 120 °C, more preferably about 100 °C;

3. Preparing an aqueous phase, optionally heating the aqueous phase at high temperature, preferably in a range between about 50 °C to about 90 °C, preferably between about 70 °C to about 90 °C, more preferably about 80 °C;

4. Simultaneously, adding of a surfactant to the lipophilic phase of step 2 and /or adding of a surfactant to the aqueous phase of step 3;

5. Stirring the lipophilic phase and the aqueous phase together until an emulsion of lipophilic droplets is obtained, preferably when at least 90% of said lipophilic droplets have a diameter comprised between 0.01 and 70 pm, preferably between 0.1 and 50 pm, most preferably between 0.1 and 20 pm, advantageously between 0.5 to 7 pm, preferably between 2 and 6 pm as determined by laser diffraction,

6. Cooling the emulsion until a temperature of between 20 °C and 30 °C, preferably between 20 °C and 25 °C is reached.

7. Adding a surfactant having the form of solid particles to the stirred emulsion, advantageously at a temperature of between 20 °C and 30 °C, preferably between 20 °C and 25 °C, until at least 90% of the particles of the multiphase composition have a diameter comprised between 1 and 1000 pm, advantageously between 10 and 250 pm, as determined by laser diffraction.

According to some embodiments, the composition of the invention, whether being formulated under the form of an aqueous solution, an emulsion or a suspo-emulsion or any kind of formulation known by the one skilled in the art, also contains at least one component selected from among the group including: at least one fluidifying agent selected from among the group comprising glycerol, ethanol, propylene glycol, polyethylene glycol with an average molecular weight between 100, preferably between 200 and 8000 Da, advantageously between 200 and 1000 Da, preferably equal to 200 Da and more preferably equal to 400 Da; with the fluidifying agent advantageously representing between 1% and 15% of the composition by weight, and advantageously between 2% and 8%; and/or at least one solubilizing agent for phytosterols (or fatty substances) selected from among the group comprising lecithins, fatty alcohols as for example oleyl alcohol; fatty acids as for example oleic acid, linoleic acid ; glycerides, triglycerides, plant oils, advantageously soybean oil, grapeseed oil, sea buckthorn oil, corn oil, rapeseed oil or sunflower oil ; with the solubilizing agent advantageously representing between 1% and 30% of the composition by weight, and advantageously between 3% and 10 %; and/or at least one wetting agent selected from among the group comprising silanes, siloxanes, triglycerides, a mixture of fatty acids, a mixture of fatty acid methyl esters, advantageously including methyl tetradecanoate, methyl hexadecanoate and methyl octadecanoate, or a mixture thereof with the wetting agent advantageously representing between 0.1% and 5% of the composition by weight; and/or at least one chelating agent, selected from among the group comprising natural chelating agents, advantageously sodium phytate or amino acid-based chelating agents; and synthetic chelating agents, advantageously 2,2'-bipyridine, dimercaptopropanol, ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) or its salts, ethylenediaminetetraacetic acid (EDTA) or its salts, nitrilotriacetic acid, iminodiacetic acid, salicylic acid ortriethanolamine, and preferably EDTA; with the chelating agent advantageously representing between 0.01% and 5% of the composition by weight; and/or at least one preservative, advantageously selected from among the group comprising benzyl alcohol, benzoic acid and its salts, especially sodium benzoate, dehydroacetic acid and its salts, especially sodium dehydroacetate, salicylic acid and its salts, sorbic acid and its salts, especially potassium sorbate, 2-phenylethanol, phenoxyethanol, phenylpropanol, and preferably benzyl alcohol; with the preservative advantageously representing between 0.01% and 5% of the composition by weight; and/or at least one penetrating agent, advantageously selected from alkoxylated polyol esters, alkoxylated alcohols, polyoxyethylene sorbitan esters, polyoxyethylene alkylamines; and/or at least one rheological modifier, advantageously selected from agar, xanthan gum, carrageenan, guar gum, alginates, polyacrylics, polyamides polyesters polymers and clays; and/or at least one anti-drift adjuvant, selected from alkylamines or their salts, preferably isopropylamine, or high molecular weight polymers; and/or at least one antioxidant, selected from the group comprising citric acid and its salts, tartaric acid and its salts, sodium lactate, potassium lactate, calcium lactate, lecithins, tocopherols, polyphenols, butylhydroxyanisole, dibutylhydroxytoluene, octyl gallate, dodecyl gallate, lycopene.

Of course, all the above components can display more properties than the main property in which they were sorted in.

According to a specific embodiment, the mixture of sterols and the at least one surfactant of the invention are combined with at least one active ingredient.

For the purpose of the invention, the term "active ingredient" refers to a product that allows the plant to combat preferably abiotic and/or biotic stresses, advantageously selected from the group comprising:

- a phytopharmaceutical product such as a plant growth regulator, a fungicide, a fungistatic agent, a bactericide, a bacteriostatic agent, an insecticide, an acaricide, a parasiticide, a nematicide, a mole toxicant or an herbicide;

- a biocontrol product based on natural mechanisms that enables plants to combat fungal infections, bacterial infections, viral infections, pest attacks and/or competition with weeds; and/or

- a nutrient, organic or inorganic such as a micronutrient or a fertilizer.

Plant growth regulator may be selected from the group consisting of:

Antiauxins: clofibric acid, 2,3,5-tri-iodobenzoic acid; Auxins: 4-CPA, 2,4-D, 2,4-DB, 2,4-DEP, dichlorprop, fenoprop, IAA (indole-3-acetic acid), IBA, naphthaleneacetamide, a-naphthaleneacetic acid, 1-naphthol, naphthoxyacetic acid, potassium naphthenate, sodium naphthenate, 2,4,5-T;

Cytokinins: 2iP, 6-benzylaminopurine (6-BA), 2,6-dimethylpyridine, kinetin, zeatin;

Defoliants: calcium cyanamide, dimethipin, endothal, merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos, tributyl phosphorotrithioate;

Ethylene modulators: aviglycine, 1-methylcyclopropene (1-MCP), prohexadione (prohexadione calcium), trinexapac (trinexapac-ethyl);

Ethylene releasers: ACC, etacelasil, ethephon, glyoxime; Gibberellins: gibberelline, gibberellic acid;

Growth inhibitors: abscisic acid, ancymidol, butralin, carbaryl, chlorphonium, chlorpropham, dikegulac, flumetralin, fluoridamid, fosamine, glyphosine, isopyrimol, jasmonic acid, maleic hydrazide, mepiquat (mepiquat chloride, mepiquat pentaborate), piproctanyl, prohydrojasmon, propham, 2,3,5-tri-iodobenzoic acid;

Morphactins: chlorfluren, chlorflurenol, dichlorflurenol, flurenol;

Growth retardants: chlormequat (chlormequat chloride), daminozide, flurprimidol, mefluidide, paclobutrazol, tetcyclacis, uniconazole, metconazole;

Growth stimulators: brassinolide, forchlorfenuron, hymexazol;

Unclassified plant growth regulators/classification unknown: amidochlor, benzofluor, buminafos, carvone, choline chloride, ciobutide, clofencet, cloxyfonac, cyanamide, cyclanilide, cycloheximide, cyprosulfamide, epocholeone, ethychlozate, ethylene, fenridazon, fluprimidol, fluthiacet, heptopargil, holosulf, inabenfide, karetazan, lead arsenate, methasulfocarb, pydanon, sintofen, triapenthenol.

Fungicides and fungistatics may be selected among the group:

Respiration inhibitors: o Inhibitors of complex III at Qo site like for example azoxystrobin, coumethoxystrobin, coumoxystrobin, dimoxystrobin, enestroburin, fenaminstrobin, fenoxystrobin/flufenoxystrobin, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, pyrametostrobin, pyrao-xystrobin, trifloxystrobin, pyribencarb, triclopyricarb/chlorodincarb, famoxadone, fenamidone; o inhibitors of complex III at Qi site: cyazofamid, amisulbrom; o inhibitors of complex II: flutolanil, benodanil, bixafen, boscalid, carboxin, fenfuram, fluopyram, flutolanil, fluxapyroxad, furametpyr, isopyrazam, mepronil, oxycarboxin, penflufen, penthiopyrad, sedaxane, tecloftalam, thifluzamide; o other respiration inhibitors (e.g. complex I, uncouplers): diflumetorim; o nitrophenyl derivates: binapacryl, dinobuton, dinocap, fluazinam; ferimzone; organometal compounds: fentin-acetate, fentin chloride or fentin hydroxide; ametoctradin; and silthiofam;

Sterol biosynthesis inhibitors (SBI fungicides): o C14 demethylase inhibitors (DMI fungicides): triazoles: azaconazole, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole; o imidazoles: imazalil, pefurazoate, prochloraz, triflumizol; pyrimidines, pyridines and piperazines: fenarimol, nuarimol, pyrifenox, triforine; Deltal4-reductase inhibitors: aldimorph, dodemorph, dodemorph-acetate, fenpropimorph, tridemorph, fenpropidin, piperalin, spiroxamine; Inhibitors of 3-keto reductase: fenhexamid;

Nucleic acid synthesis inhibitors: o Phenylamides or acyl amino acid fungicides: benalaxyl, benalaxyl-M, kiral-axyl, metalaxyl, ofurace, oxadixyl; o others: hymexazole, octhilinone, oxolinic acid, bupirimate, 5-fluorocytosine;

Inhibitors of cell division and cytoskeleton: o tubulin inhibitors: benzimidazoles, thiophanates: benomyl, carbendazim, fuberidazole, thiabendazole, thiophanate-methyl; triazolopyrimidines; o cell division inhibitors: diethofencarb, ethaboxam, pencycuron, fluopicolide, zoxamide, metrafenone, pyriofenone;

Inhibitors of amino acid and protein synthesis: o methionine synthesis inhibitors (anilino-pyrimidines): cyprodinil, mepanipyrim, pyrimethanil; o protein synthesis inhibitors: blasticidin-S, kasugamycin, kasugamycin hydrochloridehydrate, mildiomycin, streptomycin, oxytetracyclin, polyoxine, validamycin A; Signal transduction inhibitors: o MAP/histidine kinase inhibitors: fluoroimid, iprodione, procymidone, vinclozolin, fenpiclonil, fludioxonil; G protein inhibitors: quinoxyfen;

Lipid and membrane synthesis inhibitors: o Phospholipid biosynthesis inhibitors: edifenphos, iprobenfos, pyrazophos, isoprothiolane; lipid peroxidation: dicloran, quintozene, tecnazene, tolclofos-methyl, biphenyl, chloroneb, etridiazole; phospholipid biosynthesis and cell wall deposition: dimethomorph, flumorph, mandipropamid, pyrimorph, benthiavalicarb, iprovalicarb, valifenalate; o compounds affecting cell membrane permeability and fatty acid: propamocarb, propamocarb-hydrochloridfatty acid amide;

Inhibitors with Multi Site Action: o Inorganic active substances: Bordeaux mixture, copper acetate, copper hydroxide, copper oxychloride, basic copper sulfate, sulfur; o thio- and dithiocarbamates: ferbam, mancozeb, maneb, metam, metiram, propineb, thiram, zineb, ziram; o organochlorine compounds (e.g. phthalimides, sulfamides, chloronitriles): anilazine, chlorothalonil, captafol, captan, folpet, dichlofluanid, dichlorophen, hexachlorobenzene, pentachlorphenole and its salts, phthalide, tolylfl uanid; o others: guanidine, dodine, dodine free base, guazatine, guazatine-acetate, iminoctadine, iminoctadine-triacetate, iminoctadinetris(albesilate), dithianon;

Cell wall synthesis inhibitors: o Inhibitors of glucan synthesis: validamycin, polyoxin B; o melanin synthesis inhibitors: pyroquilon, tricyclazole, carpropamid, dicyclomet, fenoxanil;

Plant defense inducers: o acibenzolar-S-methyl, probenazole, isotiani I, tiadinil, prohexadione-calcium; o phosphonates: fosetyl, fosetyl-aluminum, phosphorous acid and its salts;

Other mode of action: o bronopol, chinomethionat, cyflufenamid, cymoxanil, dazomet, debacarb, diclomezine, difenzoquat, difenzoquat-methylsulfate, diphenylamin, fenpyrazamine, flumetover, flusulfamide, flutianil, methasulfocarb, nitrapyrin, nitrothal-isopropyl, oxine-copper, picarbutrazox, proquinazid, tebufloquin, tecloftalam, triazoxide. Insecticidal compounds may be selected from the group consisting of:

Acetylcholine esterase inhibitors from the class of carbamates: aldicarb, alanycarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, trimethacarb, XMC, xylylcarb, and triazamate;

Acetylcholine esterase inhibitors from the class of organophosphates: acephate, azamethiphos, azinphos-ethyl, azinphosmethyl, cadusafos, chlorethoxyfos, chlorfenvinphos, chlormephos, chlorpyrifos, chlorpyrifos-methyl, coumaphos, cyanophos, demeton-S-methyl, diazinon, dichlorvos/DDVP, dicrotophos, dimethoate, dimethylvinphos, disulfoton, EPN, ethion, ethoprophos, famphur, fenamiphos, fenitrothion, fenthion, fosthiazate, heptenophos, imicyafos, isofenphos, isopropyl O-(methoxyaminothio-phosphoryl)salicylate, isoxathion, malathion, mecarbam, methamidophos, methidathion, mevinphos, monocrotophos, naiad, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phenthoate, phorate, phosalone, phosmet, phosphamidon, phoxim, pirimiphos-methyl, profenofos, propetamphos, prothiofos, pyraclofos, pyridaphenthion, quinalphos, sulfotep, tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, trichlorfon, vamidothion;

GABA-gated chloride channel antagonists:

Cyclodiene organochlorine compounds: endosulfan; or M-2.B fiproles (phenylpyrazoles): ethiprole, fipronil, flufiprole, pyrafluprole, or pyriprole;

Sodium channel modulators from the class of pyrethroids: acrinathrin, allethrin, d-cis- trans allethrin, d-trans allethrin, bifenthrin, bioallethrin, bioallethrin S-cylclopentenyl, bioresmethrin, cycloprothrin, cyfluthrin, betacyfluthrin, cyhalothrin, lambda-cyhalothrin, gamma-cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, theta- cypermethrin, zeta-cypermethrin, cyphenothrin, deltamethrin, momfluorothrin, empenthrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, tau- fluvalinate, halfenprox, imiprothrin, meperfluthrin,metofluthrin, permethrin, phenothrin, prallethrin, profluthrin, pyrethrin (pyrethrum), resmethrin, silafluofen, tefluthrin, tetramethylfluthrin, tetramethrin, tralomethrin, transfl uthrin, DDT and methoxychlor; Nicotinic acteylcholine receptor agonists from the class of neonicotinoids: acteamiprid, chlothia nidin, cycloxaprid, dinotefuran, flupyradifurone, imidacloprid, nitenpyram, sulfoxaflor, thiacloprid, thiamethoxam;

Allosteric nicotinic acteylcholine receptor activators from the class of spinosyns: spinosad, spinetoram;

Chloride channel activators from the class of mectins: abamectin, emamectin benzoate, ivermectin, lepimectin or milbemectin;

Juvenile hormone mimics: hydroprene, kinoprene, methoprene, fenoxycarb or pyri proxyfen;

Non-specific multi-site inhibitors: methyl bromide and other alkyl halides, chloropicrin, sulfuryl fluoride, borax or tartar emetic;

Selective homopteran feeding blockers: pymetrozine, flonicamid, pyrifluquinazon;

Mite growth inhibitors: clofentezine, hexythiazox, diflovidazin or etoxazole;

Inhibitors of mitochondrial ATP synthase: diafenthiuron, azocyclotin, cyhexatin, fenbutatin oxide, propargite, or tetradifon;

Uncouplers of oxidative phosphorylation: chlorfenapyr, DNOC, or sulfluramid; M-13 nicotinic acetylcholine receptor channel blockers: bensultap, cartap hydrochloride, thiocyclam, thiosultap sodium;

Inhibitors of the chitin biosynthesis type 0 (benzoylurea class): bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, teflubenzuron, triflumuron;

Inhibitors of the chitin biosynthesis type 1: buprofezin;

Moulting disruptors: cyromazine;

Ecdyson receptor agonists: methoxyfenozide, tebufenozide, halofenozide, fufenozide or chromafenozide;

Octopamin receptor agonists: amitraz;

Mitochondrial complex III electron transport inhibitors: hydramethylnon, acequinocyl, flometoquin, fluacrypyrim or pyriminostrobin;

Mitochondrial complex I electron transport inhibitors: fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad, flufenerim, or rotenone;

Voltage-dependent sodium channel blockers: indoxacarb, metaflumizone; Inhibitors of the lipid synthesis, inhibitors of acetyl CoA carboxylase: spirodiclofen, spiromesifen or spirotetramat;

Mitochondrial complex II electron transport inhibitors: cyenopyrafen, cyflumetofen or pyflubumide;

Ryanodine receptor-modulators from the class of diamides: flubendiamide, chloranthraniliprole (rynaxypyr), cyanthraniliprole (cyazypyr); and

Others: afidopyropen.

Unexpectedly, the Applicant has noted that when the combination of the agricultural composition obtained from the method of manufacture of the invention is combined with at least one active ingredient, this facilitates the uptake of the active ingredient into the plant cell by means of the cuticle and plant cell membrane passage mechanisms which were described previously. The composition or the slurry described in the invention therefore allows the presence of a higher concentration or quantity of active ingredient in the plant. On condition that the composition or slurry obtained from the method of manufacture of the invention is applied preferably prior to the onset of the stress, a systemic action of the active ingredient in the plant is observed, leading in turn to an enhanced fight against abiotic and/or biotic stresses. In addition, the composition or slurry allows decreasing the doses of active ingredients used while guaranteeing the improved effectiveness of these active ingredients.

According to another aspect, the invention also relates to a slurry resulting from the dilution of the composition obtained from the method of manufacture as previously described.

The dilution of the slurry is performed to increase the volume of the composition of the invention while maintaining the benefit of the exogenous addition of sterols to the plant as previously discussed.

Advantageously, the composition of the invention is diluted with water in a range going from 0.001% (1 part of composition and 99999 parts of water) to 90% (9 parts of composition and 1 part of water), more precisely from 0.01% to 70%, more advantageously from 0.1% to 50%, even more advantageously from 0.5% to 20%. This slurry can be used as a foliar spray to treat plants, as a bath to prime seeds, and/or as the irrigation medium to provide water to plants from the roots.

Another object of the invention is also an agricultural kit containing separately the composition of the invention (before dilution) and at least one active ingredient as described above.

In use, the composition of the invention may be mixed by the farmer with an effective amount of the active ingredient and then diluted in order to obtain a slurry which is applied on the plant.

Another option is to dilute the composition of the invention in order to obtain the slurry and only then, to add to the slurry the at least one active ingredient.

The Applicant has noted that the method of manufacturing an agricultural composition for application to a target plant species according to the invention enabled a reduction in the harmful effects of abiotic and/or biotic stresses, notably the loss of dry matter and the resulting decrease in per-hectare yields.

It is important to note that the composition may be applied on the plant at any moment, which means irrelevant from the phenological stage at which the phytosterol profile of the target plant is determined, provided that this moment occurs prior to the onset of any biotic and/or abiotic stress. If such biotic and/or abiotic stress does not occur, then an early stage of the application of the composition displays biostimulation properties during the lifecycle of the plant.

The invention also concerns a preventive treatment for a target plant to limit the loss of dry matter related to an abiotic and/or biotic stress consisting in applying to the target plant, prior to the onset of said abiotic and/or biotic stress the slurry as above disclosed.

In addition to the loss of dry matter, several other physiological parameters benefit from the exogenous addition of sterols, such as an increase of the chlorophyll content in leaves, a reduction of the leaves rolling and an increase of the activity of the organogenesis (apex growth).

Indeed, the composition obtained from the method of manufacture of the invention presents the advantages of improving plant growth and of reducing the number of days during which the cultivated plant is below the wilting point. The plant is thus more able to combat the deleterious effects of exposure to abiotic and/or biotic stress.

In other words, the application of the composition of the invention to a cultivated plant prior to the onset of an abiotic and/or biotic stress, increases the time spent in the easily usable soil water reserve (EUSWR) and reduces the time spent below the wilting point, i.e., the time spent in the soil survival reserve (SSR). The result is improved dry matter production and/or harvest yield.

In addition to this advantageous of biotic and/or abiotic stress resilience, several other advantages are offered by the agricultural composition obtained from the method of manufacture of the invention to the plant when grown under no particular stress situation. In other words, applying the agricultural composition allows the plant growing better by biostimulation in standard, non-stressed situation, for example when the EUSWR is sufficiently filled.

Therefore, the invention also concerns a biostimulation treatment process consisting in applying to the plant the slurry as above mentioned.

As above mentioned, the method of the invention may also include treating a plant prior to the exposure of an abiotic and/or biotic stress so as to improve its resilience against such stresses. In case such stress may not occur, the method of the invention may help the plant growing.

The method of the invention thus also relates to a preventive treatment process for a cultivated plant that aims to limit the loss of dry matter related to an abiotic and/or biotic stress; it consists in applying to the plant, prior to the onset of said abiotic and/or biotic stress, the composition or the slurry previously described.

In general, abiotic stress is responsible for a decrease in yield or in production of dry matter and results from drought (a lack of water, or water stress), extreme temperatures (thermal stress), excess water (flooding), frost, wind, soil salinity (salt stress), ultraviolet radiation, insufficient access to certain nutrients, soil with stress-inducing characteristics (chemical composition, pollution, redox potential, etc.) or physical damage, and advantageously drought and/or extreme temperatures.

According to a specific embodiment, abiotic stress corresponds to drought stress.

According to a specific embodiment, as regards biotic stress resulting in a decrease in yield or in dry matter production, this can be caused by the harmful action of a plant pathogen living on the cultivated plants, whether a fungal infection and/or a bacterial infection and/or a viral infection and/or a pest attack and/or competition with weeds.

For example, a fungal infection of the plant can be mildew on grapes, septoria on wheat, rynchosporium on barley, or powdery mildew on straw cereals and grapes; a bacterial infection of the plant can be crown gall, bacterial canker or fire flight; a viral infection of the plant can be mosaic diseases or yellow dwarf viruses; pests capable of attacking a cultivated plant include aphids, flea beetles or weevils.

In particular, the method of the invention helps to reduce the intensity of a fungal disease, advantageously without affecting its frequency. It means that the composition or slurry of the invention, when applied to the plant prior to the onset of an infection, particularly a fungal infection, leads to a decrease in the surface area of the spotting or discoloration of the leaf compared to a plant that has not received the preventive treatment of the invention.

In practice, the composition or slurry resulting from the method of manufacture of the invention is applied by spraying the leaves, by sprinkling, irrigation, seed imbibition, seed coating, drip irrigation or gravity irrigation of the cultivated plant, by incorporation into the soil, by addition to a hydroponic crop medium or by plant immersion.

Practically, the composition of the invention may not be applied directly on the plant and needs to be diluted to form a slurry. Advantageously, the composition is applied to the cultivated plant by foliar spraying at a dose of composition of 0.1 L/ha (hectare) to 15 L/ha, preferably 1 L/ha to 5 L/ha. Practically, the required dose of the composition is diluted in water in order to obtain a slurry. The slurry is then applied on the plant at a volume of between 30 and 400 L/ha, advantageously between 50 and 200 L/ha.

In a specific embodiment, the composition contains 2.5% by weight of the mixture of phytosterols and the required dose of composition ranges from 1 L/ha to 5 L/ha.

Advantageously, the slurry of the invention is applied only once by foliar spraying and/or irrigation and/or seed imbibition.

As a foliar spraying, the slurry is preferably applied during a stage when the plant leaves cover the soil.

The Applicant has also noted that the composition or slurry of the invention improves the growth and development of the plant, and particularly that of the young seedling when the slurry is applied prior to the onset of the stress. In particular, these improvements are even more advantageous when the slurry is applied via seed imbibition.

Another object of the invention is also the use of a mixture of phytosterols originating from at least one natural source of phytosterols, said source being chosen from the group consisting of trees, sunflower and rapeseed extracts, for producing an agricultural composition.

According to an embodiment, the trees extract is chosen from the group consisting of pine, eucalyptus and palm extracts, preferably pine extract.

Advantageously, the pine extract is originated from tall oil. According to another embodiment, sunflower and rapeseed extracts are deodorization distillates from oils.

According to an embodiment, said mixture of phytosterols further comprises another natural source of phytosterols different from the natural source in step b., wherein said other natural source of phytosterols is selected from the group consisting of pine, palm, soybean, sunflower and rapeseed extracts.

When the mixture of phytosterols comprises at least 2 natural sources of phytosterols, the first natural source of phytosterols represents at least 20%, at least 30%, at least 40% or even at least 50%, the 100% complement being provided by the at least second natural source of phytosterols, possibly in presence of further sources of phytosterols selected from the group consisting of isolated cholesterol and/or isolated stigmasterol.

The invention therefore also relates to a process for stimulating the growth and development of the young seedlings that consists in applying the composition or slurry previously described prior to the onset of any abiotic and/or biotic stress, preferably via seed imbibition. The method of manufacture of the invention therefore limits the period of time during which the young seedling is exposed to abiotic and/or biotic stresses. Furthermore, the effect of the product applied via seed imbibition lasts over time, since the plants treated with the composition or slurry of the invention are more tolerant to an abiotic and/or biotic stress.

The invention also relates to the use of the composition or slurry as described previously: to promote plant growth; to increase the tolerance of a cultivated plant to an abiotic stress and/or; to decrease the intensity of a biotic stress affecting a cultivated plant.

The invention also relates to the use of the composition or slurry as described previously as a biostimulant for a cultivated plant. The invention also relates to the use of the composition or slurry as described previously to improve these parameters on a cultivated plant: it improves the yield or dry matter production; it promotes deeper root development; it controls the opening or closing of the stomata; it improves the vegetative development and/or the flowering it strengthens the stem of the crop plant and improve its tolerance to physiological lodging, the adverse effects of lodging, which can include lower seed filling, loss of quality, yield loss and harvesting difficulties; it improves the effectiveness of phytopharmaceutical fungicide or biocontrol products.

The Applicant has noted that the composition was especially efficient for this specific effect on plants, irrelevant from the fact that the plant is monocot or a dicot provided that the composition which is applied have a phytosterol's profile similar to the one of the target plant.

The invention and the benefits it produces are more visible in the following figures and examples, which are given in order to illustrate the invention in a non-exhaustive way.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: particle size distribution and Dv90 of a suspo-emulsion prepared according to the method of manufacture of the invention (example 1).

Figure 2: daily rainfalls (black histograms) and mean air temperature (grey curve). Sowing is at day 0. Treatment of the crop with the product is at day 171 (black arrow). The black star is the harvest. The figure starts in October.

Figure 3: daily rainfalls (black histograms), irrigation (grey histograms) and mean air temperature (grey curve). Sowing is at day -7 (i.e., 7 days before the beginning of measurements). Treatment of the crop with the product is at day 98 (black arrow). The black star is the harvest. The figure starts in February.

Figure 4: daily rainfalls (black histograms) and mean air temperature (grey curve). Sowing is at day 0. Treatment of the crop with the product is at day 177 (black arrow). The black star is the harvest. The figure starts in October. Figure 5: daily rainfalls (black histograms) and mean air temperature (grey curve). The black arrows indicate the three applications of the product obtained with the method of manufacture of the invention. The grey star is the date of Apex measurements. The white star is the date of pressure chamber measurements. The black star is the harvest. The figure starts in March.

EXAMPLES OF APPLICATION OF THE INVENTION

1. Preparation of the compositions according to the invention

1.1. Mixture of phytosterols according to a target plant

Any percentage by weight of a compound or a molecule of the invention refers to the total weight of the said invention, which means relative to the sum of all ingredients giving a hundred percent. This percentage by weight can be symbolized as wt%.

The method of manufacture of the invention involves the step of blending phytosterols from at least two naturally-occurring sources, the said sources being either already mixed phytosterols and/or isolated phytosterols.

The method of manufacture of the invention involves the step of blending phytosterols from at least one naturally-occurring source.

In the following examples, two different sources of phytosterols has been used respectively: a deodorization-distillate-originated from soybean, a tai l-oil-originated from pine.

The repartition of the main five phytosterols, expressed in percentage of the total of these five phytosterols of the deodorization-distillate-originated from soybean, and the tail-oil- originated from pine are reminded in Table 4. Table 4. Composition of naturally-occurring phytosterols according to the main five phytosterols, expressed in percentage of the total of these five phytosterols (zero means that the values of the said sterol was under the detection threshold)

The method of manufacture of the invention involves the step of blending these sources to adequate the blending to the constitutive balance of the target plant. Table 5summarizes the appropriate blending to be performed from the sources according to the target plant.

Table 5. Blending of phytosterols according to a target specie, expressed in percentage of the blending composition

This blending allows obtaining phytosterol-based compositions according to the invention where the proportion of the five phytosterols facing the target crop can be decomposed as in Table 6.

Table 6. Mixture of phytosterols pointing towards a target plant species, in percentage of the total sterols, according to the method of manufacture of the invention

SUBSTITUTE SHEET (RULE 26)

Table 7 indicates the calculation of the quotient for each phytosterol and for each crop, for two cases: one with a soybean-originated phytosterol-based composition and another with an optimized phytosterol-based composition. The quotient is calculated according to the following equation:

Quotient = [PSi (composition)]/[PSi (plant)], with the quotient ranging from 0.75-1.25. It shows that the calculation of the quotient leads to more acceptable values when the optimized phytosterol-based composition is used compared to the soybean-originated phytosterol- based composition.

Table 7. Values of the quotient for each phytosterol, for two different composition and for wheat, barley and vine

n.d.: not determined because the denominator was equal to zero. 1.2. Formulas of the composition

SUBSTITUTE SHEET (RULE 26) Table 8 summarizes the composition of the invention which are used as plant treatment in the examples given below. Example 0 corresponds to example disclosed in W02023/057640.

Table 8. Compositions of the invention

1.3. Method of manufacture of a composition

The various composition according to the invention are manufactured comprising the following steps: (i) Preparation at about 100 °C of a lipophilic phase comprising the mixture of phytosterols, the at least one surfactant, the at least one fatty acid methyl ester and the solvent,

(ii) Preparation at about 80 °C of a hydrophilic phase comprising water, and benzyl alcohol if any,

(iii) Mixing the lipophilic phase of step (i) with the hydrophilic phase of step (ii) and stirring until at least 90% of the volume of lipophilic droplets with a diameter comprised between 0.1 and 20 pm are obtained, with a maximum peak between 2 and 6 pm as determined by laser diffraction,

(iv) Cooling the emulsion to ambient temperature of about 20 °C, and

(v) Adding a water-insoluble surfactant to the emulsion, at ambient temperature of about 20 °C and stirring until at least 90% of the particles with a diameter comprised between 10 and 250 pm are obtained and suspended in the aqueous phase, with a maximum peak between 10 pm and 1000 pm as determined by laser diffraction.

The particle size distribution of a realization example is presented on figure 1. As shown, at least 90% of the particles of the suspo-emulsion have a diameter comprised between 10 and 250 pm with a peak maximum preferably of between 10 pm and 100 pm as determined by laser diffraction, herein 33 pm.

2. Evaluation of the capacity of the slurry of the invention to reduce the sensitivity of soft winter wheat to drought stress

The main objective was to investigate the ability of the slurry of example 1 of Table 8 to improve the resistance of soft winter wheat against drought stress. The experiment was benchmarked against a control experiment (non-treated crop) and a treated crop with a phytosterol-based composition whose phytosterol balance is originated from soybean deodorizer distillates.

2.1. Equipment and methods

2.1.1. Description of the experimental setup

The description of the experimental setup is presented in Table 9. Table 9

2.1.2. Modalities considered

The description of the modalities considered is presented in Table 10.

Table 10

The slurry was obtained by diluting each composition in water at 1.25%. It was then applied only once, by foliar spray, at a volume of 1 L/ha, under the following climatic conditions: temperature of 14 °C, wind of 15 km/h and relative humidity of 65%.

2.1.3. Pedoclimatic context

During its lifecycle, the plant was exposed to a total of 42 days under hydric stress, following the climate pattern exposed in Figure 2. The experiment was not irrigated. The total available water of the soil was 134 mm, leading a moderate impact of any period of hydric stress. 2.1.4. Data collection method

Leaf rolling is a visible phenomenon that expresses the drought stress situation of wheat. It is estimated by visual inspections of crops and comparison with an abacus, from 0% of rolling of the upper canopy leaf (last leaf) indicating no hydric stress, to 100% of rolling of the same leaf indicating very severe hydric stress. The measurement is incremented by 10% stepwise. 60 measurements were performed for each modality.

The harvest yield is measured at harvest on a cutting width of 9.1 m. The measure is taken every single second.

The climatic data over the entire crop cycle are obtained from the data gathered by Copernicus CDS (Boogaard, H. et al. Copernicus Climate Change Service (C3S) Climate Data Store (CDS). DOI: 10.24381/cds.6c68c9bb), according to the GPS coordinates of the field.

2.2. Results

2.2.1. Leaf rolling

Table 11 summarizes the mean value of leaf rolling per modality, expressed at two different crop stages.

Table 11. Mean leaf rolling values

2.2.2. Harvest yield

Table 12 summarizes the impact of the composition of the invention on the harvest yield. Measurements were performed 261 days after sowing and show that an 5.7% increase of the harvest yield is observed when soft winter wheat has been treated by foliar spray with the composition 2 of the invention. Table 12. Harvest yield of soft winter wheat

2.3. Conclusion

This example demonstrates that when soft winter wheat is treated prior to the onset of a drought stress by foliar spray with sterols, both physiological parameters such as leaf rolling and the harvest yield can be improved. Moreover, there is a significant difference for these two parameters between a non-optimized balance (i.e., soybean-originated) and an optimized sterol balance (mixture of the invention), in favour to the latter, in favour to the latter, with an increase of the harvest yield of 5.7%.

3. Evaluation of the capacity of the slurry of the invention to stimulate the growing and to reduce the sensitivity of spring barley to drought stress

The main objective was to investigate the ability of the slurry of example 3 of Table 8 to improve the stimulation of the growing of spring barley, and to improve its resistance against drought stress, depending on the irrigation conditions. The experiment was benchmarked against a control experiment (non-treated crop) and a treated crop with a phytosterol-based composition whose phytosterol was originated from soybean deodorizer distillates. The experiment was conducted both in irrigated and non-irrigated situations.

3.1. Equipment and methods

3.1.1. Description of the experimental setup

The description of the experimental setup is presented in Table 13.

Table 13

3.1.2. Modalities considered

The description of the modalities considered is presented in Table 14. Table 14

The slurry was obtained by diluting each composition in water at 1.25%. It was then applied only once, by foliar spray, at a volume of 1 L/ha, under the following climatic conditions: temperature of 23 °C, wind of 15 km/h and relative humidity of 60%.

3.1.3. Pedoclimatic context

The climatic pattern to which the spring barley has been exposed to is shown in Figure 3. The field was split into two sections: irrigated and non-irrigated. The total available water of the soil was 90 mm, leading a strong impact of any period of hydric stress. From application to harvest, the field received a total of 117.5 mm of rainfalls (ca. 2.2 mm/day). In the irrigated section, a total of 62 mm was added. This total of 179.5 mm of added water is sufficient to consider that this modality did not suffer from any drought stress.

3.1.4. Data collection method

Normalized Difference Vegetation Index (NDVI) is performed using a GreenSeeker (Trimble Agriculture) device.

N-tester Index values is obtained using a N-tester (Yara) device.

The harvest yield is measured at harvest on a cutting width of 9.1 m. The weight is measured each one second.

The climatic data over the entire crop cycle are obtained from the data gathered by Copernicus CDS, according to the GPS coordinates of the field.

3.2. Results

3.2.1. NDVI

Table 15 summarizes the mean value of a hundred measurements per non-irrigated modality.

Table 15. NDVI

3.2.2. N -tester

Table 16 summarizes the N-tester index value for each non-irrigated modality. It represents, by a chlorophyll index measurement, the plant's nitrogen supply status, which is directly correlated with the intensity of drought stress.

Table 16. N-tester

3.2.3. Number of grains per spike

Table 17 summarizes the mean number of grains per spike of 30 measurements for each nonirrigated modality. The results show an increase in the number of grains per spike of 4.6% and 6.5% when the spring barley was treated with a standard and an optimized sterol balance, respectively, compared with an untreated spring barley modality.

Table 17. Number of grains per spike

3.2.4. Harvest yield Table 18 summarizes the impact of the composition of the invention on the harvest yield, for both irrigated and non-irrigated modalities. Measurements were performed 156 days after sowing and show that an 9.0% increase of the harvest yield is observed when spring barley has been treated by foliar spray with the composition 2 of the invention. Table 18. Harvest yield of spring barley. Exceeding values compared to control are given in parentheses

3.3. Conclusion This example demonstrates that when spring barley is treated by foliar spray with sterols, physiological parameters such as vegetation health, numberof grains perspike and the harvest yield can be improved, in the case a drought stress occurs after the application of the product. Moreover, there is a significant difference for these two parameters between a non-optimized and an optimized sterol balance, in favour to the latter, irrelevant from the fact if the field is irrigated or non-irrigated. Thus, when irrigated spring barley is treated with the compositions 2 or 3 of the invention, the yield is improved by 7.5% and 4.6%, respectively, compared to untreated control, values which are even better than a composition containing soybean- originated-only phytosterols. Under a situation of hydric stress (non-irrigated field), the composition 2 of the invention allows a 9.0% increase of the harvest yield against the control. Thus, the composition of the invention features both biostimulation and drought stress reduction effects. Ultimately, the harvest yield of the non-irrigated modality, treated with composition 2 approaches by 0.8% the harvest yield of the untreated but irrigated modality, allowing thus to avoid any irrigation of the field.

4. Evaluation of the capacity of the slurry of the invention to reduce the sensitivity of winter barley to drought stress

The main objective was to investigate the ability of the slurry of the invention to improve the resistance of winter barley against drought stress. The experiment was benchmarked against a control experiment (non-treated crop) and a treated crop with a soybean-originated phytosterol composition formulated the same way.

4.1. Equipment and methods

4.1.1. Description of the experimental setup

The description of the experimental setup is presented in Table 19.

Table 19

4.1.2. Modalities considered

The description of the modalities considered is presented in Table 20.

Table 20

The slurry was obtained by diluting each composition in water at 1.25%. It was then applied only once, by foliar spray, at a volume of 1 L/ha, under the following climatic conditions: temperature of 16 °C, wind of 10 km/h and relative humidity of 40%.

4.1.3. Pedoclimatic context

After application of the product, the plant was exposed to a total of 49 days under hydric stress, following the trend exposed in Figure 4. The experiment was not irrigated. The total available water of the soil was 40 mm only, leading a strong impact of any period of hydric stress.

4.1.4. Data collection method

The harvest yield is measured at harvest on a cutting width of 9.1 m. The measure is taken every single second.

The climatic data over the entire crop cycle are obtained from the data gathered by Copernicus Climate Data Store. 4.2. Results of harvest yield

Table 21 summarizes the impact of the composition of the invention on the harvest yield. Measurements were performed 261 days after sowing and show that an 5.7% increase of the harvest yield is observed when winter barley has been treated by foliar spray with a pine- originated composition of the invention.

Table 21. Harvest yield of winter barley

4.3. Conclusion

This example demonstrates that when winter barley is treated by foliar spray with phytosterols priorto the onset of a hydric stress such as drought stress, the harvest yield is improved against an untreated control. In addition, when the composition of the phytosterols is obtained from the composition according to the example 1 in Table 8, the yield is even better and increased by 5.7% compared with the control experiment.

5. Vineyard trial

The goal of the experiment is to compare the benefits of a preventive treatment of vine against hydric stress by spraying on leaves a composition of the invention before flowering.

5.1. Equipment and methods

5.1.1. Description of the experimental setup

The description of the experimental setup is presented in Table 22.

Table 22

5.1.2. Modalities considered

The description of the modalities considered is presented in Table 23.

Table 23

The slurry was obtained by diluting each composition in water at 1%. It was then applied at three different BBCH stages, by foliar spray, at a volume of 1 L/ha, under the following climatic conditions: temperature of < 22 °C, no wind and relative humidity of < 76%.

5.1.3. Pedoclimatic context

The climatic data are obtained from the data gathered by Copernicus CDS, according to the GPS coordinates of the field. The data are summarized in the figure 5. During the last 50 days, very low rainfalls were accumulated, with only 20.6 mm recorded, giving a mean value of 0.40 mm/day.

5.1.4. Data collection method

The apex method developed by Pichon et al. (Pichon, L. et al. Precis. Agric. 2021, 22 (2), 608- 626. DOI: 10.1007/sllll9-021-09797-9) is an efficient method to characterise the vegetative growth of vines. It is based on the observation of the tips of the shoots, known as apexes. It involves observing around fifty apexes and classifying them into three categories (full growth, slow growth or no growth). A synthetic index, called the apex growth index (iC-Apex), is then calculated to characterise the vegetative growth of the area observed.

The hydric situation of vine is estimated according to a pressure measurement, performed as a basis potential according to Van Leeuwen et al. (Van Leeuwen, C. et al. J. Int. Sci. Vigne Vin 2009, 43 (3), 121-134. DOI: 10.20870/oeno-one.2009.43.3.798). The pressure measurement allows qualifying the level of the hydric stress, according to the scale given in Table 24.

Table 24. Hydric situation of vine stems according to the measured pressure

5.2. Results

5.2.1. Apex measurement

Apex measurements were performed at day 140. The results are presented in Table 25.

Table 25. Apex method

5.2.2. Pressure chamber

The hydric situation of vine was estimated at day 145, according to basis potential measurement (see Table 24). The pressure measurement allows qualifying the level of the hydric stress, according to the scale given in Table 26. Table 26. Hydric situation of vine stems according to the measured pressure

Despite the fact that all modalities suffer from severe hydric deficit, it is less pronounced for soybean-originated modality and even less pronounced for pine-originated modality.

5.2.3. Harvest

Harvest was performed at day 158. The results are shown in Table 27. Table 27. Harvest

5.3. Conclusions

This trial shows that composition 1 of the invention with an improved balance of sterols gives better vigour with keeping the vine growing than other solutions and a untreated condition. The composition of the invention also helps reducing the negative effect of hydric stress on the plant, even in severe conditions of hydric stress.

Thus, compared to a control experiment, the yield is improved by 0.9% and 6.8% when vine is treated with soybean-originated and pine-originated compositions of the invention, respectively.

Claims

1. A method of manufacturing an agricultural composition for application to a target plant species, said method comprising the steps of: a. Identifying the relative concentration of n phytosterols (PSi (plant)) with n > 2 present from highest (PSi with i=l) to lowest relative concentration (PSi with i =n) in target plant species at a phonological stage, said phytosterols being selected from the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, and selecting the two phytosterols present in the highest relative concentration, b. Providing an agricultural composition comprising: i. a mixture of phytosterols obtained from at least one natural source of phytosterols, comprising at least 2 phytosterols (PSi (composition)) being identical in nature to the at least 2 phytosterols identified (PSi (plant)) in step a., wherein the mixture of phytosterols represents between 0.2% and 10% of the agricultural composition by weight, and wherein the natural source of phytosterols is obtained from the group consisting of trees, sunflower, rapeseed extracts, ii. at least one surfactant.

2. The method according to claim 1, characterized in that the relative concentration of each phytosterols among n phytosterols with n 3= 2 in an organ of said target plant species at a phenological stage mentioned in step a. is calculated according to the following formula: PSi (plant) = (PSi (plant)/(sum of n PS (plant)), and in that step b. requires the calculation of the relative concentration of the same phytosterols than the ones of step a. in the mixture of phytosterols according to the following formula: PSi (composition) = (PSi (composition)/(sum of n PS (composition)), and in that the mixture for which 0.75

[PSi (composition)]/[PSi (plant)]

1.25 (with i being identical between PSi (composition) and PSi (plant)) for at least two phytosterols is selected.

3. The method according to any of claims 1 to 2, characterized in that trees extract is chosen from the group consisting of pine and palm extracts, preferably pine extract.

4. The method according to claim 3 characterized in that said pine extract is originated from tall oil.

5. The method according to any of claims 1 to 2, characterized in that said sunflower and rapeseed extracts are deodorization distillates from oils.

6. The method according to any of claims 1 to 5, characterized in that the mixture of phytosterols further comprises another natural source of phytosterols different from the natural source, wherein said other natural source of phytosterols is selected from the group consisting of pine, palm, soybean, sunflower and rapeseed extracts.

7. The method according to any of claims 1 to 6, characterized in that the mixture of phytosterols further comprises another natural source of phytosterols different from the natural source in step b., wherein said natural source of phytosterols is selected from the group consisting of pine extract originated from tall oil, and deodorization distillates from oils of palm, soybean, sunflower and rapeseed.

8. The method according to any of claims 1 to 7, characterized in that group of phytosterols in step a. further comprises cholesterol.

9. The method according to claim 8, characterized in that the mixture of phytosterols further comprises isolated cholesterol.

10. The method according to any of claims 1 to 9, characterized in that the mixture of phytosterols further comprises isolated stigmasterol.

11. The method according to claim 9 or 10, characterized in that the cholesterol and/or stigmasterol are from a natural or a synthetic source.

12. The method according to any of claims 1 to 11, characterized in that the surfactant is a sugar fatty acid ester, preferably sucrose stearate and/or sucrose palmitate.

13. The method according to any of claims 1 to 12, characterized in that the phonological stage is chosen between BBCH 0 and the time of application of said agricultural composition.

14. The method according to any of claims 1 to 13, characterized in that:

- when the target plant is wheat, the phonological stage the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 30 and BBCH 52;

- when the target plant is barley, the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 30 and BBCH 52;

- when the target plant is vine, the phenological stage at which the organs of the plant are analyzed is chosen between BBCH 13 and BBCH 77;

15. The method according to any of claims 1 to 14, characterized in that the organ is leaves or seeds, preferably leaves.

16. The method according to any of claims 1 to 15, characterized in that:

- when the target plant is wheat, the agricultural composition comprises between 65% and 80% of pine extract and between 20% and 35% of soybean extract;

- when the target plant is barley the agricultural composition comprises between 65% and 75% of pine extract and between 25% and 35% of soybean extract;

- when the target plant is vine, the agricultural composition comprises 100 % of pine extract.

17. The method according to any of claims 1 to 16, wherein the organ is leaves.

18. An agricultural composition for application to an organ of a target plant species, wherein said agricultural composition comprises: a. between 0.2% and 10% of the agricultural composition by weight of a mixture of phytosterols obtained from at least 1 natural source of phytosterols, said mixture comprising at least 2 phytosterols (PSi (composition)) selected from the group of phytosterols consisting of beta-sitosterol, campesterol, brassicasterol, and stigmasterol, said at least 2 phytosterols (PSi (composition)) being identical in nature to at least 2 phytosterols (PSi (plant)) present in the highest quantity in said organ of the target plant species at a phenological stage, and wherein the natural source of phytosterols is obtained from the group consisting of trees, sunflower, rapeseed extracts, b. at least one surfactant.

19. The agricultural composition according to claim 18, characterized in that the quotient [PSi (composition)]/[PSi (plant)] is between 0.75 and 1.25 (with i being identical between PSi (composition) and PSi (plant)) for at least two phytosterols.

20. The agricultural composition according to claim 18 or 19, characterized in that trees extract is chosen from the group consisting of pine and palm extracts, preferably pine extract.

21. The agricultural composition according to claim 20, characterized in that said pine extract is originated from tall oil.

22. The agricultural composition according to claim 18 or 19, characterized in that sunflower and rapeseed extracts are deodorization distillates from oils.

23. The agricultural composition according to any of claims 18 to 22, characterized in that the mixture of phytosterols further comprises another natural source of phytosterols different from the natural source in step b., wherein said other natural source of phytosterols is selected from the group consisting of pine, palm, soybean, sunflower and rapeseed extracts.

24. The agricultural composition according to any of claims 18 to 23, characterized in that the group of phytosterols in step a. further comprises cholesterol.

25. The agricultural composition according to claim 24, characterized in that the mixture of phytosterols further comprises isolated cholesterol.

26. The agricultural composition according to any of claims 18 to 25, characterized in that the mixture of phytosterols further comprises isolated stigmasterol.

27. The agricultural composition according to claim 25 or 26, characterized in that the cholesterol and/or stigmasterol are from a natural or a synthetic source.

28. The agricultural composition according to any of claims 18 to 27, characterized in that the surfactant is a sugar fatty acid ester, preferably sucrose stearate and/or sucrose palmitate.

29. The agricultural composition according to any of claims 18 to 28, characterized in that:

- when the target plant is wheat, the agricultural composition comprises between 65% and

80% of pine extract and between 20% and 35% of soybean extract;

- when the target plant is barley the agricultural composition comprises between 65% and

75% of pine extract and between 25% and 35% of soybean extract;

- when the target plant is vine, the agricultural composition comprises 100 % of pine extract.

30. A slurry resulting from the dilution of the composition according to any of claims 18 to 29.

31. Biostimulation treatment process consisting in applying to the plant the slurry according to claim 30.

32. Preventive treatment process for a target plant to limit the loss of dry matter related to an abiotic and/or biotic stress consisting in applying to the target plant, prior to the onset of said abiotic and/or biotic stress the slurry according to claim 30.

33. Preventive treatment process according to claim 32, wherein the abiotic stress is drought stress.

34. Use of a mixture of phytosterols originating from at least one natural source of phytosterols, said natural source being chosen from the group consisting of trees, sunflower, rapeseed extracts, for producing an agricultural composition.

35. The use according to claim 34, characterized in that trees extract is chosen from the group consisting of pine and palm extracts, preferably pine extract.

36. The use according to claim 35, characterized in that said pine extract is originated from tall oil.

37. The use according to claim 34 characterized in that said at least one natural source of phytosterols is selected from the group consisting of sunflower and rapeseed extracts in a form of deodorization distillates from oils.

38. The use according to any of claims 34 to 37, characterized in that said mixture of phytosterols further comprises another natural source of phytosterols different from the natural source in step b., wherein said other natural source of phytosterols is selected from the group consisting of pine, palm, soybean, sunflower and rapeseed extracts.