Eudesmanolides for use as mycorrhization stimulant

Abstract

The present invention relates to the use of eudesmanolides sesquiterpene lactones as a biostimulant of fungal spore germination and / or of plant mycorrhization. More specifically, it relates to the use of eudesmanolides sesquiterpene lactones to stimulate spore germination and hyphae development in arbuscular mycorrhizal fungi and / or to increase mycorrhization capacity in plants.

Description

[0001]Eudesmanolides for Use as a Mycorrhization Stimulant DESCRIPTION The present invention relates to the use of eudesmanolide-type sesquiterpene lactones as biostimulants. More specifically, it relates to the application of eudesmanolide-type sesquiterpene lactones to stimulate spore germination and hyphal development of arbuscular mycorrhizal fungi and / or to increase mycorrhization capacity in plants. Therefore, the present invention falls within the fields of agriculture and microbiology, more particularly, the development of microbial inoculants as biostimulants. BACKGROUND OF THE INVENTION The growing human population requires a considerable increase in food production, leading to the overexploitation of resources. Currently, crop varieties with higher yields and greater resistance to environmental stresses and diseases are being developed.However, the massive use of chemical fertilizers and pesticides is still necessary to supplement essential nutrients and reduce disease damage in agricultural production systems. The use and overuse of chemical fertilizers and pesticides in agriculture is generating an enormous environmental impact, contaminating soils and aquifers, and contributing to climate change, which negatively affects people, ecosystems, and species worldwide. Therefore, there is an urgent need to find more sustainable and environmentally friendly alternatives that reduce the use of these harmful agrochemicals and protect our soils, our environment, and human health.A strategy gaining significant traction worldwide is the use of beneficial microorganisms with biostimulant properties that establish symbiotic relationships with plants, significantly improving agroecosystems and their productivity. Among these beneficial microorganisms, arbuscular mycorrhizal fungi (belonging to the phylum Glomeromycota) stand out. They form beneficial associations with more than 80% of terrestrial plant species, including most species of agronomic and industrial interest (cereals, vegetables, fruit trees, cotton, etc.), as well as ornamental and forestry species. These mutualistic symbioses are known as arbuscular mycorrhizae and date back approximately 450 million years. Arbuscular mycorrhizae are characterized by the formation of specific structures within the roots of host plants known as arbuscules.Nutrient exchange between the fungus and the host plant takes place in the arbuscules. In arbuscular mycorrhizae, the arbuscular mycorrhizal (AM) fungus also develops a large network of hyphae, known as extraradical mycelium, which explores a large area of ​​soil and constitutes the assimilative structure for water and mineral nutrients, functioning as pseudoroots. In addition to nutrition, arbuscular mycorrhizae offer other benefits to the host plant, including improved defense responses against pathogens and greater tolerance to environmental stresses such as drought and salinity. Due to all these properties, AM fungi have enormous potential as biostimulants (biofertilizers and bioprotection agents) in more sustainable and balanced agriculture.However, despite their potential, their application in agriculture remains challenging due to the variability of results when applied to production systems, hindering their commercialization and implementation. Mycorrhizal fungi (MFFs) are obligate biotrophs, meaning they depend on a host plant to develop and complete their life cycle. This makes it extremely difficult to produce quantifiable and homogeneous inoculants. Furthermore, these inoculants are non-sterile and susceptible to contamination problems. Therefore, most MMF products on the market are spore-based, which are easier to quantify and store, offer greater homogeneity, and have a lower risk of contamination. However, spore production is expensive, and spores are slower to colonize and form mycorrhizae, thus reducing the effectiveness of the inoculants.Furthermore, their implementation as biostimulants in agronomy and forestry is being hampered by the variability of their results in the field. The variability in results obtained with inoculants based on mycorrhizal fungi (MA) stems primarily from three factors: a) environmental conditions, b) the quality and effectiveness of the inoculants used, and c) management techniques, especially chemical fertilization. In short, it would be desirable to have commercial products based on MA fungi that could reliably and stably establish mycorrhizal symbiosis with plants, thereby improving their effectiveness in production systems. Given this, the current state of the art indicates a need for new compounds capable of stimulating the development and / or growth of MA fungi to enhance their mycorrhization capacity and their use in intensive crops in a more environmentally friendly manner.DESCRIPTION OF THE INVENTION Through trials aimed at improving the development of plants of agronomic and / or forestry interest in production systems, the inventors have observed that the application of eudesmanolide-type sesquiterpene lactones improves plant productivity, favoring the establishment of beneficial plant-arbuscular mycorrhizal (AM) symbiosis and the efficiency of photosystem II. The examples in this description demonstrate, using in vitro and in vivo models, that the compounds defined in the claims improve spore germination and subsequent hyphal development of AM fungi (Example 1) and the mycorrhization capacity of the AM fungus (Examples 2, 3, 4, and 5). Therefore, the present invention describes the use of eudesmanolide-type sesquiterpene lactones as biostimulants for the growth of AM fungi and the formation of arbuscular mycorrhizae.Thus, in a first aspect, the present invention relates to the use of a composition, hereinafter referred to as the “composition of the invention”, comprising a compound of general formula (I) (hereinafter referred to as the “compound of the invention”):. where: R1 is selected from H, -OH, =O and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond, with the condition that: - when the 11,13 bond is single, then R1 is H, –OH, =O or, - when link 11,13 is double, then - when a bond selected from 3,4; 4,15; or 4,5 is a double bond, the other two are single bonds; preferably bond 4,15 is a double bond and the other two are single bonds; as a biostimulant for fungal germination and mycorrhization. In a preferred embodiment, when bond 1,2 is a double bond, bond 4,15 is a double bond and R2 is H. In another preferred embodiment, R2 is at position 1 of the ring and bond 1,2 is a single bond. The term “alkyl (C1-C5)” refers, in the present invention, to saturated hydrocarbon chains, linear or branched, having from 1 to 5 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl. The radical R1 of the compound of general formula (I) can be any element or chemical group within the indicated list, and The bond in the compound of general formula (I) can be either single or double under the stated conditions. However, in a preferred embodiment, the compound has formula (Ia): where: R1 is selected from H, and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond in one of the three positions. In a more preferred embodiment, R2 of the compound of formula (Ia) is in position 1 of the ring, more preferably R2 of the general formula (Ia) is H. In another preferred embodiment, alone or in combination with any and all of the foregoing preferred embodiments, of the compound of formula (Ia) is a double bond at position 4,15. In another preferred embodiment, alone or in combination with any and all of the foregoing embodiments, the compounds of formula (Ia) are selected from: M2 M18 In another preferred embodiment of the use of the compound of general formula (I), the compound is of formula (Ib): where: R1 is selected from H and the group: In another preferred embodiment of the use of the compound of general formula (I), the compound is of formula (Ic): where: R1 is selected from H, -OH, =O and the group: represents a single or double bond in one of the three positions. In a more preferred embodiment, R1 of the compound of formula (Ic) is selected from =O and the group: In another preferred embodiment, alone or in combination with any and all of the preceding preferred embodiments of the compound of formula (Ic), it is a double bond at position 4,15. In another preferred embodiment, alone or in combination with any and all of the preceding preferred embodiments, the compound of formula (Ic) is selected from: The compound of the invention forms part of a composition, giving rise to the composition of the invention, which may comprise one or more compounds falling within the general formula (I) and preferably those of general formula (Ia), (Ib), (Ic), or any combination thereof. Likewise, the composition of the invention may comprise an agriculturally acceptable carrier. In the present invention, "agriculturally acceptable carrier" means all adjuvants, inert components, dispersants, surfactants, adhesives, binders, etc., commonly used in the formulation technology of agricultural compositions and well known to those skilled in the art.Furthermore, the composition of the invention may also include, in addition to the compound of the invention, other compounds useful in the growth and / or development of plants and fungi, such as macro- / micronutrients, phytohormones, fertilizers, pesticides, herbicides, etc. As understood by those skilled in the art, the composition of the invention may also include macro- and / or microelements that provide nutrients to plants. Thus, in a preferred embodiment, the composition of the invention further comprises macroelements and / or microelements. Macroelements are those elements expressed as a percentage in the plant (g / 100g). The main ones are nitrogen, phosphorus, potassium, calcium, magnesium, and sulfur. Microelements are expressed as ppm (parts per million) = mg / kg = mg / 1000g, and the main ones are iron, zinc, copper, manganese, molybdenum, boron, and chlorine.It is known that there are approximately 27 chemical elements that have functions in plants, and because plants require them at specific concentrations, they must be added to the soil. There are also other beneficial nutrients, such as silicon, sodium, and cobalt, that enhance certain characteristics of plants in different species. Experts in the field know which macro- and micronutrients are appropriate to add to a plant of interest to meet its needs. Thus, in a preferred embodiment, the macro-elements of the composition of the invention are selected from the group consisting of nitrogen salts, phosphorus salts, potassium salts, calcium salts, magnesium salts, sulfur salts, and combinations thereof.In another preferred embodiment, the microelements of the composition of the invention are selected from the group consisting of iron salts, zinc salts, copper salts, manganese salts, molybdenum salts, boron salts, chlorine salts, and combinations thereof. Phytohormones are substances produced by plant cells that participate in numerous biological processes and are capable of regulating the physiological phenomena of plants. Examples of phytohormones that can be used in the composition of the invention include, but are not limited to, auxins, gibberellins, cytokinins, and ethylene. Auxins are a group of phytohormones that function as regulators of plant growth (essentially causing cell elongation) and can be natural (synthesized by a plant cell in the wild) or artificial (synthesized in the laboratory).Examples of natural auxins include, but are not limited to, indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), and phenylacetic acid. Examples of synthetic auxins include, but are not limited to, indolebutyric acid (IBA) and naphthaleneacetic acid (NAA). Gibberellin is a phytohormone produced in the apical zone, fruits, and seeds, whose main functions are breaking seed dormancy, inducing germination, inducing bud and fruit development, and regulating stem longitudinal growth. Examples of gibberellins include, but are not limited to, gibberellic acid (GA3), gibberellin A1 (GA1), and gibberellin A4 (GA4). Cytokinins are phytohormones that promote cell division and differentiation. Examples of cytokinins include, but are not limited to, cis- and trans-zeatin, isopentenyladenine, dihydrozeatin (with their respective glycosylated derivatives), benzyladenine, kinetin, and topolin.Other compounds of non-plant origin and synthetic diphenylurea derivatives such as CPPU and thidiazuron (TDZ), which act as structural analogues of the natural molecule, are also considered cytokinins. In the present invention, "fertilizer" refers to the compound(s) necessary for plant growth and development, including microorganisms (or substances produced by them) that provide or improve nutrient availability when applied to the plant, thus promoting its development and growth. The composition of the invention may also include organisms that can be beneficial to plants, such as bacteria (including actinomycetes), fungi, algae, and protozoa. Examples of such microorganisms include, but are not limited to, fungi of the genus Trichoderma (such as T. harzianum Rifai, T. viride Pers., T. polysporum Link fr., T. reesei EG Simmons, T. virens, T. longibrachatum Rifai, T.parceromosum , T. pseudokoningii , T. hamatum , T. lignorum, T. citroviride, etc.), mycorrhizae (Glomus sp., etc.), bacteria of the genus Rhizobium (such as R. cellulosilyticum, R. daejeonense, R. etli, R. gallicum, R. hainanense, R. indigoferae, R. leguminosarum, R. loessense, , R. lusitanum, , R. mongolense, R. phaseoli, R. rhizogenes, R. sullae, R. tropici, R. yanglingense, etc.), of the genus Neorhizobium (N. galegae, N. huautlense, etc.), Allorhizobium (A. undicola), Pararhizobium (P. giardini), Mesorhizobium (M. loti, M, ciceri, M. mediterraneum, M. huakuii, M. tianshanense, etc.), Ensifer (E. fredii, E. meliloti, etc.), Bradyrhizobium (B. japonicum, B. lupini, etc.)The compounds of the invention can be present within the composition of the invention at a very wide concentration range, which may vary depending on the plant, the season in which it is administered, and the presence of other components in the composition. A person skilled in the art, based on the needs of the crop and their general knowledge, can determine the concentration necessary for the compound of the invention to exert its effect of stimulating fungal germination and mycorrhization. However, in a preferred embodiment of the composition of the invention, the compound of the invention is at a concentration between 10 and 1000 nM. In an even more preferred embodiment of the composition of the invention, the compound of the invention is at a concentration between 100 and 500 nM.As explained at the beginning of this description, the compound of the invention improves plant productivity by promoting the establishment of the beneficial plant-fungus symbiosis (MA) and the efficiency of photosystem II. As previously mentioned, the compound of the invention enhances the efficiency of photosystem II (PSII). PSII is a complex of membrane proteins present in the thylakoid membranes of higher plants and algae, and it plays a fundamental role in photosynthesis. Therefore, quantifying the photochemical efficiency of PSII is a variable used to detect stress levels in plants and is related to promoting plant growth.Thus, the composition of the invention, comprising the compound of the invention, can be administered to a plant to enhance its productivity by stimulating the germination of mycorrhizal fungi and / or stimulating mycorrhization. Therefore, in a preferred embodiment of the composition of the invention, the composition is administered to a plant. Any mycorrhizal plant, more than 80% of terrestrial plants, including plants of agronomic interest, can benefit from the effects of the composition of the invention. In the present invention, "plant" means any autotrophic and photosynthetic living organism whose cells have a wall composed primarily of cellulose and lack locomotor capacity. In a preferred embodiment of the use of the invention, the plants are selected from cultivated and ornamental plants.Examples of cultivated plants include, but are not limited to, cereals (such as rice, corn, wheat, einkorn, barley, oats, quinoa, wheat, rye, spelt, etc.), vegetables (such as carrot, chard, spinach, beetroot, celery, celeriac, parsnip, parsley, eggplant, potato, pepper, tomato, garlic, shallot, onion, asparagus, leek, chard, borage, endive, escarole, lamb's lettuce, lettuce, mushroom, pea, broad bean, green bean, courgette, pumpkin, cucumber, artichoke, sweet potato, broccoli, white cabbage, Chinese cabbage, Brussels sprouts, Savoy cabbage, red cabbage, cauliflower, swede, kohlrabi, turnip, radish, etc.), tobacco plants, and sunflower plants. Other types of plants whose growth can be increased are fruit trees, such as orange, apple, and pear trees, or berry-bearing trees, such as strawberries, raspberries, blueberries, and currants. Examples of ornamental plants include, but are not limited to, carnations, roses, tulips, poinsettias, and geraniums.In a preferred embodiment of the invention, the cultivated plants are non-leguminous plants. In the present invention, "non-leguminous plant" means any plant that does not belong to the Fabaceae or Leguminosae family within the Fabales order. Among non-leguminous plants, the Solanaceae family, which includes the tomato, is of particular agronomic interest. However, in another preferred embodiment, the plant is a tomato plant (Solanum lycopersicum) or a lettuce plant (Lactuca sativa). As explained in previous paragraphs, the concentration of the compound of the invention in the composition of the invention may vary depending on the plant.Thus, in a preferred embodiment, the concentration of compound M2 is 10, 100, or 1000 nM when the composition is administered to tomato plants as a biostimulant for fungal germination and mycorrhization, thereby promoting tomato plant growth. In another preferred embodiment, the concentration of compound M2 is 10 nM when the composition is administered to lettuce plants as a biostimulant for fungal germination and mycorrhization, thereby promoting lettuce plant growth. In yet another preferred embodiment, the concentration of compound M12 is 10 or 100 nM when the composition is administered to tomato plants as a biostimulant for fungal germination and mycorrhization, thereby promoting tomato plant growth.In another preferred embodiment, the concentration of the compound of formula M12 is 10 or 100 nM when the composition is applied to lettuce plants as a biostimulant for fungal germination and mycorrhization, thereby promoting lettuce plant growth. The composition of the invention can be in liquid or solid form, depending on the method of application. It can be applied directly to the soil before or after sowing, by spraying, in the sowing furrow, or over the entire surface. Alternatively, the composition can be applied to the seed before sowing. The application rates and timing of the composition during the crop cycle will depend on the concentration of the compounds of the invention and / or the type of crop.Techniques for administering compositions and the quantity to be administered are widely known in the prior art and their use is routine practice for those skilled in the art. Thus, in a preferred embodiment of the invention, the composition is administered to the soil of the plant. In a preferred embodiment, the composition of the invention is formulated in liquid or solid form. The liquid forms are suitable for spraying onto the soil, the plant, or the plant material, or for creating a solution in which the plants or plant material are immersed. Alternatively, the compositions can be obtained in solid form by freeze-drying and subsequent pelletizing, which can then be applied directly to the soil or resuspended in solutions, preferably aqueous.Analogous to the use of the invention, and due to the properties of the compounds of the invention, the present description also contemplates a method for stimulating fungal germination and / or mycorrhization of plants by fungi, resulting in greater plant growth and improved yield, i.e., increased biomass per plant, fruit size and number per plant, etc. Thus, in another aspect, the present invention relates to a method for stimulating fungal germination and / or mycorrhization of plants by fungi, hereinafter referred to as the “method of the invention,” comprising: (a) contacting the fungus or the plant root with a composition comprising a compound of general formula (I), previously described in the first aspect of the invention. where: R1 is selected from H, -OH, =O and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond, with the condition that: - when the 11,13 bond is single, then R1 is H, –OH, =O or - when link 11,13 is double, then - if a selected bond from among 3,4; 4,15; or 4,5 is double, the other two are single bonds; and (b) developing the fungus and / or plant of step (a). In a first step, the method of the invention comprises bringing a plant or the seed from said plant into contact with the composition of the invention as defined in the preceding paragraphs.The plant (or the seed thereof) can be brought into contact with the composition of the invention by any known technique, such as, but not limited to, through hydroponics, through a solution applied to the soil, by applying the strain or composition by spraying, misting, coating, fumigating, or impregnating any aerial part of the plant or seed, by introducing the composition of the invention into irrigation water, by germinating seeds of the plant in the presence of said composition, by cultivating plant material in vitro in contact with the composition of the invention, or by immersing the root of the plant in a solution comprising the composition of the invention.Thus, in a preferred embodiment of the method of the invention, contact between the plant (or the seed derived therefrom) and the composition of the invention is effected by means of irrigation water, spraying, misting, coating, fumigation, immersion, or impregnation. In a second step, the method of the invention comprises developing the plant (or seed) from step (a). This development comprises providing the plant (or seed) with suitable conditions of light, temperature, humidity, nutrients, etc., for the plant to grow (or for the seed to germinate and produce a plant). Suitable growing conditions for various cultivated plants can be found in manuals widely known to those skilled in the art and available to the public. In another preferred embodiment, when bond 1,2 is a double bond, bond 4,15 is a double bond, and R2 is a H bond.In another preferred embodiment, R2 is in position 1 of the ring and the 1,2 bond is a single bond. In a preferred embodiment of the method of the invention, the compound has formula (Ia):. (Ia) where: R1 is selected from H and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond in one of the three positions. In another preferred embodiment of the method of the invention, R2 of the compound of formula (Ia) is in position 1 of the ring, more preferably R2 of the general formula (Ia) is H. In another preferred embodiment of the method of the invention, alone or in combination with any and all of the foregoing preferred embodiments, The compound of formula (Ia) has a double bond at position 4,15. In another preferred embodiment of the method of the invention, the compounds of formula (Ia) are selected from: In another preferred embodiment of the method of the invention, the compound has formula (Ib): where: R1 is selected from H and the group: In another preferred embodiment of the method of the invention, the compound has formula (Ic): R1 is selected from H, -OH, =O and the group: represents a single or double bond in one of the three positions. In a more preferred embodiment of the method of the invention, R1 of the compound of formula (Ic) is selected from the group =O and the group: In another preferred embodiment of the method of the invention, alone or in combination with any and all of the preceding preferred embodiments of the compound of formula (Ic), it is a double bond at position 4,15. In another preferred embodiment of the method of the invention, alone or in combination with any and all of the preceding preferred embodiments, the compound of formula (Ic) is selected from: In another preferred embodiment of the method of the invention, the compound is at a concentration of between 10 and 1000 nM. In another preferred embodiment of the method of the invention, the compound is at a concentration of between 100 and 500 nM. In another preferred embodiment of the method of the invention, the plant is a lettuce plant or a tomato plant. In another preferred embodiment of the method of the invention, the composition is administered to the soil surrounding the plant. In another preferred embodiment of the method of the invention, the composition is formulated in liquid form, solid form, or hydroponic form. All terms employed in the method of the invention, as well as all preferred embodiments of the method of the invention, have been previously explained in connection with the use of the invention and are applicable to the method of the invention. DESCRIPTION OF THE FIGURES Figure 1.Molecular structures of eudesmanolide M2 ​​(A), and of several compounds derived from eudesmanolide M2: M12 (B), M18 (C), and M26 (D). And the structure of the synthetic strigolactone 2'-epi-GR24 (E), used in the present invention. Figure 2. Quantification of the germination levels of spores of the fungus Rhizophagus irregularis in vitro. Effect of the exogenous application of the sesquiterpene lactones of the eudesmanolide type M2, M12, M18, and M26 (10, 100, and 1000 nM). The bars represent the relative increase in germination compared to the untreated control. The application of the synthetic strigolactone 2'-epi-GR24 was used as a positive control. The bars correspond to the mean of 5 independent replicates (15 spores / replicate) ± the standard error. Statistical analysis T-test between each treatment compared with the untreated control. *p<0.05, **p<0.01, ***p<0.001. Figure 3.Quantification of mycorrhization levels of the fungus Rhizophagus irregularis in lettuce (cv. Maureen) (A) and tomato (cv. MoneyMaker) (B) plants. Effect of exogenous application of eudesmanolides M2 and M12 (10 and 100 nM). Bars represent the relative increase in mycorrhization compared to untreated control plants (C). Application of the synthetic strigolactone 2'-epi-GR24 was used as a positive control. Bars correspond to the mean of 10 independent replicates ± the standard error. Statistical analysis: T-test between each treatment compared to the untreated control. *p<0.05, **p<0.01, ***p<0.001. Figure 4. Quantification of the efficiency of photosystem II [Y(II)] in lettuce (cv. Maureen) (A) and tomato (cv. MoneyMaker) (B) leaves, in plants mycorrhized with the MA fungus Rhizophagus irregularis. Effect of the exogenous application of the eudesmanolides M2 and M12 (10 and 100 nM).The bars represent the increase in the effective quantum yield of photosystem II (PSII) compared to non-mycorrhizal control plants (Nm) and untreated non-mycorrhizal control plants (C). The application of the synthetic strigolactone 2'-epi-GR24 was used as a positive control. The bars correspond to the mean of 10 independent replicates ± the standard error. Statistical analysis: T-test between each treatment compared to the untreated control. *p<0.05, **p<0.01, ***p<0.001. Figure 5. Quantification of the mycorrhization levels of the mycorrhizal fungus Rhizophagus irregularis in commercial tomato plants under production conditions. Two different varieties were used: the Chocolate Pear tomato (A) and the Kardia rootstock (B). Effect of the exogenous application of eudesmanolide M2 ​​(10, 100, and 1000 nM). The bars represent the relative increase in mycorrhization compared to untreated control plants. The application of synthetic strigolactone 2'-epi-GR24 was used as a positive control.The bars represent the mean of 10 independent replicates ± the standard error. Statistical analysis: T-test between each treatment compared to the untreated control. *p<0.05, **p<0.01, ***p<0.001. EXAMPLES The invention will now be illustrated by means of experiments carried out by the inventors, which demonstrate the ability of eudesmanolide-type sesquiterpene lactones to stimulate the germination of spores of the fungus MA R. irregularis in vitro, as well as mycorrhization in lettuce and tomato plants grown in trays and pots. Example 1. Studies of stimulation of spore germination of the fungus MA R. irregularis in vitro. MATERIALS AND METHODS A series of in vitro experiments were carried out on the germination of spores of the fungus MA R. irregularis (MUCL 57021), after the exogenous application of different eudesmanolide-type sesquiterpene lactones in order to determine their germination-stimulating capacity.Different concentrations of the compounds were used to evaluate their effect and determine the most effective dose. Experiments were conducted in 90 mm diameter Petri dishes containing 35 ml of agar medium (2%) in deionized water under sterile conditions. Stock solutions (1 mM) of the different eudesmanolides were prepared by dissolving the compounds in 100% acetone. Prior to application to the Petri dishes, serial dilutions of the eudesmanolides were prepared in sterile deionized water to a final acetone concentration of 1%. Before addition to the Petri dishes, all eudesmanolide dilutions M2, M12, M18, and M26 (Figure 1) were sterilized using 0.22 µm filters. Next, in a laminar flow hood, 50 µl of each dilution were added per plate and spread homogeneously over the entire surface of the agar using a seeding loop.To rule out the possible effect of acetone on spore germination, all trials included controls with 1% acetone in sterile deionized water. As with the treatments, 50 µl was added to the control plates. The plates were left open for 1 hour to allow absorption of the added compounds. Subsequently, a solution of 15 axenic (sterilized) spores was added to each R. irregularis plate. The plates were sealed and incubated at 25°C in the dark. Spore germination was assessed daily. Three different concentrations of the various eudesmanolides were tested, always within the physiological concentration range in which they are found in the plant, along with their corresponding control. The concentrations used were 0, 10, 100 and 1000 nanomolar (nM) and 5 independent replicates were used per treatment [5 plates with 15 spores per plate (75 spores per treatment)].Since strigolactones have the ability to stimulate branching and hyphal development in MA fungi, the synthetic strigolactone 2'-epi-GR24 was included as a positive germination control in all studies (Figure 1E). Spore germination was examined daily starting on the third day. In this first trial, germination levels were quantified 7 days after the application of the different eudesmanolides. RESULTS: 2'-epi-GR24 induced spore germination at all three concentrations used, indicating that the spores were viable (Figure 2). The four eudesmanolides tested (M2, M12, M18, and M26) also significantly stimulated R. irregularis spore germination (p < 0.01) compared to the untreated control. In the case of M2, an induction of 6.5 times (650%) was observed after the application of 100 nM compared to the untreated control (Figure 2A).In the case of compound M12, a 3.2-fold (320%) induction was detected after the application of 10 nM compared to the control (Figure 2A). For compound M18, a 4.0-fold (400%) increase in germination levels was observed after the application of 100 nM compared to the untreated control (Figure 2B). And after the application of compound M26, a 3.0-fold (300%) increase was detected after the application of 100 nM compared to the untreated control (Figure 2B). The results demonstrate that the four sesquiterpene lactones studied have the capacity to promote the germination of spores of the fungus MA R. irregularis (MUCL 57021) by more than 300%, although not all four exhibit the same induction capacity. Differences were also observed between the different concentrations used, with the lowest concentrations (10 and 100 nM) being the most efficient in all cases.These results show that eudesmanolide-type sesquiterpene lactones M2, M12, M18, and M26 (Figure 1) function as signaling molecules in the rhizosphere, used by the plant to promote colonization by MA fungi. Example 2. Evaluation of the stimulation of mycorrhization by the MA fungus R. irregularis in potted lettuce and tomato plants. MATERIALS AND METHODS Following the in vitro results, a series of experiments were carried out to determine whether the increased germination capacity of the MA fungal spores observed after the treatments resulted in greater mycorrhization of plants of agronomic interest. Lettuce (Lactuca sativa) and tomato (Solanum lycopersicum) plants were used as hosts, along with spores of the MA fungus R. irregularis (MUCL 57021). The objective of this trial was to evaluate the ability to stimulate mycorrhization of the eudesmonolide-type sesquiterpene lactones tested in the previous study.For this purpose, compounds M2 and M12 were selected (Figure 2) because they showed the best germination data. Lettuce (L. sativa cv. Maureen) and tomato (S. lycopersicum cv. MoneyMaker) seeds were surface-sterilized with 50% commercial bleach for 10 min and washed three times with sterile distilled water. The seeds were then sown on sterilized vermiculite and incubated at 25°C in a phytotron for germination. Ten-day-old seedlings were individually transplanted into 100 ml cells containing sterile sand:vermiculite (1:1). For each genotype, the plants were inoculated with 675 spores of the mycorrhizal fungus R. irregularis (Ri plants). As a mycorrhization control, a set of plants not inoculated with the mycorrhizal fungus was included. Ri plants were treated with the eudesmanolides M2 and M12, at two different concentrations 10 and 100 nM. As a positive control, the treatment with the synthetic strigolactone 2'-epi-GR24 was included (Akiyama et al., 2005).Negative controls with untreated Ri plants (Control) were also included. For the application of the different compounds (eudesmanolides and 2'-epi-GR24), stock solutions (1 mM) were prepared in 100% acetone. Prior to their addition, corresponding serial dilutions of the different compounds were prepared in Hewitt nutrient solution, at a final acetone concentration of 1‰. To promote mycorrhization, a modified Hewitt solution containing 25% of the standard phosphate levels was used. The plants were treated twice a week with the different compounds, using a volume of 10 ml. The control (untreated) treatments were also irrigated twice a week with 10 ml of 1‰ acetone. Ten independent replicates were used per treatment. Mycorrhization levels were evaluated 6 weeks after transplanting.RESULTS No negative effects on plant growth or development were observed after the addition of the different compounds. The treatment with 2'-epi-GR24 (positive control) at 10 nM slightly improved the mycorrhization levels of R. irregularis spores compared to the untreated control, although not significantly (Figures 3A and B), in both lettuce and tomato. Regarding the eudesmanolid treatment, the two compounds studied (2 and 12) also significantly improved the mycorrhization levels of R. irregularis spores (p < 0.01) compared to the untreated control in both cultivars. In lettuce, compound M2 increased mycorrhization levels 2.4 times (240%) after the application of 10 nM (Figure 3A). No change in mycorrhization levels was observed at the 100 nM concentration. Treatment with compound M12, treatment with 100 nM stimulated mycorrhization 2.5-fold (250%) (p < 0.05) (Figure 3A). Treatment with 10 nM also stimulated mycorrhization by 1.9-fold (190%), although this increase was not statistically significant (Figure 4A). Clearer results were obtained in tomato plants. Eudesmanolide M2 ​​stimulated mycorrhization levels by 4.2-fold (420%) and 4.1-fold (410%) when applied at 10 (p < 0.001) and 100 nM (p < 0.01), respectively (Figure 3B). Compound M12 significantly increased mycorrhization by 2.8-fold (280%) when applied at 10 nM (Figure 3B). No effect on mycorrhization was observed at the 100 nM concentration. These results show that eudesmanolide-type sesquiterpene lactones function as signaling molecules in the rhizosphere and can therefore be applied exogenously at physiological concentrations to stimulate the germination of MA fungal spores and promote mycorrhization. Example 3.Study of the benefits of eudesmanolide application and mycorrhization in lettuce and tomato plants. Several physiological parameters were analyzed in plants treated with compounds M2 and M12, and mycorrhized with the fungus MA R. irregularis, from the experiment described in Study 2. One of the parameters analyzed was the photosynthetic efficiency of photosystem II [Y(II)], a measure used to assess the plant's stress level. Y(II) levels were measured in 6-week-old lettuce and tomato leaves using the miniPAM II photosynthesis performance analyzer (Walz). RESULTS Mycorrhization with the fungus MA R. irregularis improved photosynthetic efficiency in all cases, indicating a lower stress level in the mycorrhized plants. In the case of lettuce, a 12% increase was observed compared to the unmycorrhized control condition (C) (Figure 4A).Treatment with 2'-epi-GR24 (positive control) at 10 and 100 nM improved Y(II) levels by 25% (p < 0.001), respectively. Eudesmanolide treatments also improved results compared to untreated mycorrhizal plants. Treatment with compound M2 at 10 nM induced a 33% increase in photosynthetic efficiency, and treatment with M12 at 10 nM induced a 27% increase (p < 0.001) compared to control plants (Figure 5A). For compound M12, treatment with M12 at 10 nM induced a 23% increase in Y(II) (p < 0.001) compared to control plants, and treatment with M12 at 100 nM induced a 17% increase (p < 0.01) compared to control plants (Figure 4A). In the case of tomato plants, greater increases in photosynthetic efficiency were observed in plants mycorrhized with the fungus MA R. irregularis, with a 35% improvement (Figure 5B). Treatments with eudesmanolides also improved Y(II) levels to a greater extent than in the case of lettuce.Treatment with 2'-epi-GR24 (positive control) at 10 and 100 nM improved Y(II) levels by 50% and 61% (p < 0.001), respectively. Treatment with compound M2 at 10 nM induced a 57% increase in photosynthetic efficiency, and treatment with M12 at 10 nM induced a 65% increase (p < 0.001) compared to control plants (Figure 5B). For compound M12, treatment with M12 at 10 nM induced a 59% increase in Y(II) compared to control plants, and treatment with M12 at 100 nM induced a 32% increase (p < 0.01) compared to control plants (Figure 4B). These results show that exogenously applied eudesmanolide-type sesquiterpene lactones at physiological concentrations improve mycorrhization levels in lettuce and tomato plants. Furthermore, their addition enhances the efficiency of photosystem II in the plant, thus reducing stress levels. Example 4. Evaluation of the stimulation of mycorrhization by the fungus *R. irregularis* in potted commercial tomato plant cultivars.Based on the in-planta mycorrhization results obtained in lettuce and tomato, a new mycorrhization experiment was conducted with a commercial tomato variety. Tomato plants of the 'Chocolate Pear' variety (Albenga) (Unigen Seeds), supplied by the Hortoventas cooperative, and spores of the fungus MA R. irregularis (MUCL 57021) were used. The tomato seeds were surface-sterilized with 50% commercial bleach for 10 minutes and washed three times with sterile distilled water. The seeds were then sown in sterilized vermiculite and incubated at 25°C in a phytotron for germination. Ten-day-old seedlings were individually transplanted into 100 ml cells containing sterile sand:vermiculite (1:1). The plants were inoculated with 300 spores of the fungus MA R. irregularis (Ri plants). As a mycorrhization control, a set of plants not inoculated with the MA fungus was included.The Ri plants were treated with M2 (10 and 100 nM), as it yielded the best results in previous trials. Thirty-day-old plants were individually transplanted into 0.5 L pots containing sterile sand:vermiculite (1:1). Negative controls with untreated Ri plants (Control) were included. No negative effects on plant growth or development were observed after the addition of the compound. For the application of eudesmanolide 2, a stock solution (1 mM) in 100% acetone was prepared. Prior to treatment, corresponding serial dilutions of the compound were prepared in distilled water to a final acetone concentration of 1‰. The plants were treated twice a week, using a volume of 10 ml per plant. The control (untreated) treatments were also watered once a week with 10 ml of 1‰ acetone. Ten independent replicates were used per treatment.Mycorrhization levels were assessed 7 weeks after transplanting. RESULTS Treatment with M2 increased mycorrhization levels in the commercial variety *Chocolate Pear* by 1.7-fold (170%; p < 0.05) after application of 10 nM (Figure 5A). A similar increase of 1.5-fold (150%; p < 0.05) was observed after application of 100 nM (Figure 5A). These results show that eudesmanolide-type sesquiterpene lactones can be applied exogenously at physiological concentrations to stimulate the germination of mycorrhizal fungi spores and promote mycorrhization in commercially important tomato plants. EXAMPLE 5. Experiments to stimulate mycorrhization of the mycorrhizal fungus *R. irregularis* under tomato plant production conditions in a nursery. MATERIALS AND METHODS In Spain, 90% of tomato plants are marketed grafted onto a vigorous variety resistant to different diseases and pests (rootstocks) (Thies, 2021).Therefore, in this trial, tomato plants of the Kardia variety (Syngenta), commonly used as rootstock, were grown in a nursery and inoculated with spores of the mycorrhizal fungus R. irregularis (MUCL 57021). Tomato seeds were sown in 35 ml cells with peat and incubated at 25°C in a germination chamber at a commercial nursery. The plants were inoculated with 300 spores of the mycorrhizal fungus R. irregularis (Ri plants). As a mycorrhization control, a set of plants not inoculated with the mycorrhizal fungus was included. The Ri plants were treated with eudesmanolide 2 (1000 nM). Thirty-day-old plants were individually transplanted into 0.5 L pots containing sterile sand:vermiculite (1:1). As a positive control, treatment with the synthetic strigolactone 2'-epi-GR24 was included. Negative controls were also included with Ri plants untreated with any compound (Control).For the application of the different compounds (eudesmanolide M2 ​​and 2'-epi-GR24), stock solutions (1 mM) were prepared in 100% acetone. Prior to treatment, corresponding serial dilutions of the compounds were prepared in distilled water to a final acetone concentration of 1‰. The plants were treated once a week with the different compounds, using a volume of 10 ml per plant. The control (untreated) treatments were also irrigated once a week with 10 ml of 1‰ acetone. Ten independent replicates were used per treatment. Mycorrhization levels were evaluated after 8 weeks. RESULTS No negative effects on plant growth or development were observed after the addition of the compound. Treatment with 2'-epi-GR24 (positive control) 100 nM improved the mycorrhization levels of R. irregularis spores 2.0 times (200%; p < 0.05) compared to the untreated control treatment (Figure 5B).Treatment with M2 (1000 nM) increased the mycorrhization levels of R. irregularis spores by 3.2-fold (320%; p < 0.001) compared to the untreated control (Figure 5B). These results show that eudesmanolide-type sesquiterpene lactones stimulate the germination of mycorrhizal fungi spores and promote mycorrhization in commercial tomato varieties used as rootstocks under nursery production conditions.

Claims

CLAIMS 1. Use of a composition comprising a compound of general formula (I): where: R1 is selected from H, -OH, =O and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond, with the condition that: - when the 11,13 bond is single, then R1 is H, –OH, =O, or - when one bond selected from 3,4; 4,15; or 4,5 is a double bond, the other two are single bonds; as a biostimulant for fungal germination and / or mycorrhization.

2. Use according to claim 1, wherein the compound is of formula (Ia): where: R1 is selected from H and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond in one of the three positions.

3. Use according to claim 2, wherein R2 is H.

4. Use according to any of claims 2 or 3, wherein it is a double bond in position 4,15.

5. Use according to any of claims 2 to 4, wherein the compounds of formula (Ia) are selected from:

6. Use according to claim 1, wherein the compound is of formula (Ib): where: R1 is selected from H and the group:

7. Use according to claim 1, wherein the compound is of formula (Ic): where: R1 is selected from H, -OH, =O and the group: represents a single or double bond in one of the three positions.

8. Use according to claim 7, wherein R1 is selected from =O and the group:

9. Use according to any of claims 7 or 8, wherein is a link double in position 4,15.

10. Use according to any of claims 7 to 9, wherein the compounds are selected from:

11. Use according to any one of claims 1 to 10, wherein the compound is at a concentration of between 10 and 1000 nM.

12. Use according to claim 11, wherein the compound is at a concentration of between 100 and 500 nM.

13. Use according to any one of claims 1 to 12, wherein the composition is administered to a plant.

14. Use according to claim 13, wherein the plant is a lettuce plant or a tomato plant.

15. Use according to any one of claims 1 to 14, wherein the composition is administered to the soil of the plant.

16. Use according to any one of claims 1 to 15, wherein the composition is formulated in liquid form, solid form, or hydroponic form.

17. Method for stimulating fungal germination and / or mycorrhization of plants by fungi, comprising: (a) bringing the fungus or the plant root into contact with a composition comprising a compound of general formula (I): where: R1 is selected from H, -OH, =O and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond, with the condition that: - when the 11,13 bond is single, then R1 is H, –OH, =O, or - when link 11,13 is double, then - when a bond selected from 3,4; 4,15; or 4,5 is double, the other two are single bonds; and (b) developing the fungus and / or plant from step (a).

18. Method according to claim 17, wherein the compound is of formula (Ia): where: R1 is selected from H and the group: R2 is selected from H, -OH, -OCO-alkyl (C1-C5); and represents a single or double bond in one of the three positions.

19. Method according to claim 18, wherein R2 is H.

20. Method according to any one of claims 18 or 19, wherein is a double bond at position 4,15.

21. Method according to any one of claims 18 to 20, wherein the compounds of formula (Ia) are selected from: M2 M18 22. Method according to claim 17, wherein the compound is of formula (Ib): where: R1 is selected from H and the group:

23. Method according to claim 17, wherein the compound has formula (Ic): where: R1 is selected from H, -OH, =O and the group: represents a single or double bond in one of the three positions.

24. Method according to claim 23, wherein R1 is selected from =O and the group:

25. Method according to any one of claims 23 or 24, wherein is a double bond at position 4,15.

26. Method according to any one of claims 23 to 25, wherein the compounds are selected from: M12 M26 27. A method according to any one of claims 17 to 26, wherein the compound is at a concentration of between 10 and 1000 nM.

28. A method according to claim 27, wherein the compound is at a concentration of between 100 and 500 nM.

29. A method according to any one of claims 17 to 28, wherein the plant is a lettuce plant or a tomato plant.

30. A method according to any one of claims 17 to 29, wherein the composition is administered to the soil surrounding the plant.

31. A method according to any one of claims 17 to 30, wherein the composition is formulated in liquid form, solid form, or hydroponic form.

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